Bacterial Toxins: Tools in Cell Biology and Pharmacology

Bacterial Toxins Edited by Klaus Aktories Bacterial Toxins Tools in Cell Biology and Pharmacology Edited by Klaus Ak...

Author: Klaus Aktories

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Bacterial Toxins Edited by Klaus Aktories

Bacterial Toxins Tools in Cell Biology and Pharmacology

Edited by Klaus Aktories

WILEY-VCH Verlag GmbH & Co. KGaA

All books published by Wiley-VCH are carefully produced. Nevertheless, authors, editors, and publisher do not warrant the information contained in these books, including this book, to be free of errors. Readers are advised to keep in mind that statements, data, illustrations, procedural details or other items may inadvertently be inaccurate. Library of Congress Card No.: applied for British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library. Bibliographic information published by the Deutsche Nationalbibliothek Die Deutsche Nationalbibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data are available in the Internet at .

 1997 Chapman & Hall GmbH, Weinheim

 2002 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

All rights reserved (including those of translation into other languages). No part of this book may be reproduced in any form – by photoprinting, microfilm, or any other means – nor transmitted or translated into a machine language without written permission from the publishers. Registered names, trademarks, etc. used in this book, even when not specifically marked as such, are not to be considered unprotected by law.

Printed in the Federal Republic of Germany Printed on acid-free paper ISBN: 978-3-527-30881-1

Preface

Remarkable progress has been made in the field of bacterial protein toxins: the molecular mechanisms of several toxins (e.g., clostridial neurotoxins and cytotoxins) whose modes of action were obscure until recently, have been elucidated during the last years. This progress in "cellular and molecular toxinology" not only provides insights into the mode of action of important virulence factors and pathomechanisms of diseases, but also has a maior impact on the understanding of regulation and mechanisms of eukaryotic cell functions which are disturbed by the toxins. These spectacular advances mainly depend on a bidirectional approach: cell biological methods are successfully applied for elucidation of the molecular mechanisms of bacterial toxins and, on the other hand, the bacterial toxins are used as powerful, extremely valuable tools to unravel mechanisms in molecular cell biology. The reasons for using toxins as tools are evident. First, they are very potent agents, a fact which is often based on their enzymatic activity (e.g., clostridial neurotoxins, which act as endoproteases, are the most potent agents known). Second, the high selectivity and specificity of the toxins are most important for their use as tools. Cell selectivity may be due to specific receptor binding (e.g., selectivity of neurotoxins). The high specificity of action depends on the extremely specific recognition of target proteins. Finally, bacterial protein toxins seem to be maximally efficient agents. In most cases the toxins strike the eukaryotic cell at a crucial site indicating modification of "important" eukaryotic components or signaling pathways. Therefore, the toxins are excellent tools to recognize the biological importance of a cellular component, or the biological significance of a signal pathway altered by the toxins. This book is intended to establish and facilitate the use of bacterial protein toxins as tools in cell biology and pharmacology. The volume provides a review and an update of recent developments in the field of those bacterial protein toxins which are most often used as cell biological and pharmacological tools. Moreover, the volume gives detailed methodological protocols and advice for application of the toxins, and is intended to introduce these bacterial protein toxins as cell biological tools to a broad audience of scientists. Therefore, it was decided to have for each toxin or toxin family one chapter as a special review to give the general properties of the toxins, and up to three Preface

additional chapters which focus on methodological aspects of toxin usage. Seven toxins or toxin families are covered by this volume. These include the "classical" G-protein-ADP-ribosylating toxins cholera toxin and pertussis toxin, which are established tools to study signal transduction processes (chapters 1-4).Properties and application of C3-like transferases, which specifically ADP-ribosylate Rho proteins, are described in chapters 5-7. Chapters 8-11 deal with the actinADP-ribosylating toxins, which are the most effective agents to depolymerize cellular F-actin and to inhibit actin polymerization. The Rho-glucosylating C. difficile toxins, whose molecular mechanism has been elucidated only recently, are discussed in chapters 12 and 13. Reviews of recent studies on the basic properties of clostridial neurotoxins and their application in cell and neurobiology are given in chapters 14 to 16. Chapters 17 and 18 describe in detail the general properties, new developments and methodological aspects of poreforming toxins. In chapter 19, the application of bacterial protein toxins as transporting tools is discussed and application protocols are given. Finally, chapter 20 is a brief safety guide, which should help to deal with general problems in handling of biological toxins. Last not least, I would like to thank all the authors for their outstanding effort. I am convinced that their excellent contributions will inspire research in the field of protein toxins and facilitate the use of toxins as tools in biological sciences. February 1997

Preface

Klaus Aktories

A note on the layout of this book: In order to facilitate the use of this book as a methodological source for your bench work, a wide page format has been chosen. Due to the type of durable binding used, the book has the advantage of lying flat on your bench top for convenient use. In addition, a wide margin leaves room for your own notes and provides some key notes and pictograms to assist you in finding the relevant information: a pipette symbol marks the start of a step by step protocol section, a grey bar runs down the margin of the whole protocol section

this symbol draws your attention to potential hazards and safety suggestions

comments on the key steps in methodology are highlighted by a key symbol

a “good idea” symbol marks useful hints for optimization of methodology

this pictrogram indicates discussions of alternative approaches

the tool indicates troubleshooting guides that should help you in finding out what could or did go wrong and in solving and avoiding problems

suggestions for monitoring quality and reliability of the experimental procedure are highlighted by the magnifying glass

Layout Features

Contents

.

CHAPTER 1 Cholera Toxin: Mechanism of Action and Potential Use in Vaccine Development

G.-F. ZHANG. W. A . PATTON. J . MOSS and M.VAUGHAN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1.1 1.2 1.2.1 1.2.2 1.2.3 1.2.4 1.3 1.3.1 1.3.2 1.4

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Molecular Aspects of Cholera Toxin Action . . . . . . . . . . . . Structure and Relationship to Other Toxins . . . . . . . . . . . . . Toxin Entry into Cells and Events Leading to Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Enzymology of Cholera Toxin . . . . . . . . . . . . . . . . . . . . . . . In Vitro Stimulation of Cholera Toxin Activity by ARF . . . . . . Practical Aspects of Cholera Toxin Use . . . . . . . . . . . . . . . . Vaccine and Vaccine Development . . . . . . . . . . . . . . . . . . . Cholera Toxin as a Molecular Tool . . . . . . . . . . . . . . . . . . . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 1 2 2

3 4 5 5 5 7 8

.

CHAPTER 2 Cholera Toxin and Escherichia coli Heat-labile Enterotoxin: Biochemical Methods for Assessing Enzymatic Activities W. A . PATTON. G.-F. ZHANG. J . MOSS and M. VAUGHAN . . .

15

21 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 General Information on CT. LT. ARF and Reagents . . . . . . . 2.2.1 Sources. Purification. and Activation of CTA and LTA . . . . . 2.2.2 Sources and Purification of ARF . . . . . . . . . . . . . . . . . . . . . . 2.2.3 Reagents and Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.4 Stock Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Assay 1 : The G, Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.1 Additional Reagents and Materials Required . . . . . . . . . . . 2.3.2 Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4 Assay 2: The Agmatine Assay . . . . . . . . . . . . . . . . . . . . . . . 2.4.1 Additional Reagents and Materials Required . . . . . . . . . . . 2.4.2 Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 Assay 3: Auto-ADP-ribosylation Assay . . . . . . . . . . . . . . . . 2.51 Additional Reagents and Materials Required . . . . . . . . . . . 2.5.2 Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

15 16 16 17 19 19 20 20 21 22 22 23 24 24 24 Contents

2.6 2.6.1 2.6.2 2.7 2.7.1 2.7.1.1 2.7.1.2 2.7.2 2.7.2.1 2.7.2.2 2.7.2.3 2.7.3 2.7.3.1 2.7.3.2

Assay 4: NAD Glycohydrolase Assay . . . . . . . . . . . . . . . Additional Reagents and Materials Required. . . . . . . . . . Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Comments and Considerations . . . . . . . . . . . . . . . . . . . . Appropriate Controls and Analysis of Data . . . . . . . . . . . Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Interfering Substances, Troubleshooting, and Assay Optimization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Interfering substances . . . . . . . . . . . . . . . . . . . . . . . . . . . . Troubleshooting. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Assay optimization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Consideration for the Use of ARF . . . . . . . . . . . . . . . . . . . Lipid/Detergent and Nucleotide Requirements . . . . . . . . Development of other Assay Conditions . . . . . . . . . . . . .

CHAPTER 3. Pertussis Toxin C . LOCHTand R . ANTOINE . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 3.2 3.3 3.4 3.5 3.6

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genetic Regulation of Pertussis Toxin Production . . . . . . . Biogenesis of Pertussis Toxin . . . . . . . . . . . . . . . . . . . . . . . Receptor-binding and Translocation . . . . . . . . . . . . . . . . . ADP-ribosyltransferase Activity and Enzyme Mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biological Activities and Role of Pertussis Toxin in Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

CHAPTER 4 . Pertussis Toxin as a Cell Biology Tool B.NURNBERG. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1 4.2 4.2.1 4.2.2 4.2.3

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pertussis Toxin as a Tool to Modify Cellular Functions . . . Cell Culture of Bordetella pertussis . . . . . . . . . . . . . . . . . . Source of Pertussis Toxin and Preparation of Solution . . . Treatment of Mammalian Cell Cultures with Pertussis Toxin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3 Pertussis Toxin as a Tool to Study Cellular Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.1 Activation of Pertussis Toxin for in in vitro ADPribosylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.2 Preparation of Cell Homogenates and Fractions . . . . . . 4.3.3 ADP-ribosylation of Membrane Proteins by Pertussis Toxin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.4 ADP-ribosylation of Proteins by Pertussis Toxin . . . . . . . . . 4.3.5 Preparation of Samples for SDS-PAGE . . . . . . . . . . . . . . 4.3.6 Cleavage of ADP-ribose from Ga Subunits . . . . . . . . . . . SDS-Gel Electrophoresis. . . . . . . . . . . . . . . . . . . . . . . . . . 4.4 Reagents and Chemicals . . . . . . . . . . . . . . . . . . . . . . . . . 4.5 Contents

25 25 25 26 26 26 26 26 26 27 27 28 28 29

33 33 34 35 36 38 41

47 47 48 48 48 49 50

50 51 51 54

55 56 57 57

CHAPTER 5. Clostridium botulinum ADP-ribosyltransferase C3 K.AKTORlES and G. KOCH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

5.1 5.2 5.3 5.4 5.5

5.6

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Family of C3-like Transferases . . . . . . . . . . . . . . . . . . Modification of Rho Proteins by C3-like Transferases . . . Characterization of the C. botulinum C3 ADP-Ribosyltransferase Reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Factors Affecting ADP-ribosylation by C3 . . . . . . . . . . . . C3-Transferases in Cell Biology and Pharmacology . . . .

61 61 62 64 65 65

.

CHAPTER 6 Clostridium botulinum C3 Exoenzyme and Studies on Rho Proteins C. D. NOBES and A . HALL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Purification of Recombinant C3 Transferase from ............................... 6.2.1 Purification of Recombinant GST-C3 . . . . . . . . . . . . . . . . 6.2.2 Recovery of Cleaved C3 Transferase . . . . . . . . . . . . . . . . 6.2.3 Dialysis and Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2.4 Determination of Protein Concentration and Assay of Activity . . ........................ Preparation of Swiss 3T3 Cells . . . . . . . . . . . . . . . . . . . . . 6.3 6.4 Microinjection of C3 Transferase . . . . . . . . . . . . . . . . . . . Fixation and Staining of Cells . . . . . . . . . . . . . . . . . . . . . . 6.5 6.6 C3 Protein Inhibits LPA-stimulated Stress Fibre Assembly and Focal Adhesion Clustering . . . . . . . . . . . . . . . . . . . . . Timecourse of Focal Adhesion Breakdown . . . . . . . . . . . 6.7 6.8 Alternative Delivery of C3 Transferase . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.9 Reagents and Chemicals . . . . . . . . . . . . . . . . . . . . . . . . . 610

61 6.2

71 71 72 73 74 74 74 75 76 77 80 81 81 81

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CHAPTER 7 Preparation of Clostridium botulinum C3 Exoenzyme and Application of ADP-ribosylation of Rho Proteins in Biological Systems Y.SAlTO and S . NARUMIYA . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

7.1 7.2 7.3

7.4

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Expression and Purification of Recombinant C3 Exoenzyme . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ADP-ribosylation Reaction . . . . . . . . . . . . . . . . . . . . . . . . Application of C3 Exoenzyme in Biological Systems . . . .

85 85 86 87 89

Contents

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CHAPTER 8 Actin-ADP-ribosylating Toxins: Cytotoxic Mechanisms of Clostridium botulinum C2 Toxin and Clostridium perfringens Iota Toxin K.AKTORlES, f? SEHR and I . JUST . . . . . . . . . . . . . . . . . . . . . . . .

8.1 8.2 8.3 8.4 8.5

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Clostridium botulinum C2 Toxin . . . . . . . . . . . . . . . . . . . . . Other actin-ADP-ribosylatingToxins . . . . . . . . . . . . . . . . . Actin as the Substrate for ADP-ribosylation . . . . . . . . . . . Model for the Cytopathic Effects of ActinADP-ribosylatingToxins . . . . . . . . . . . . . . . . . . . . . . . . . . .

93 93 93 94 95 98

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CHAPTER 9 Purification. Activation and Endocytosis of Botulinum C2 Toxin

I.OHlSHl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

103

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Assay Method for the Toxin . . . . . . . . . . . . . . . . . . . . . . . Purification Procedures for the Two Components of C2 Toxin . . . . . . . . . . . . . . . . . . . 9.3.1 Preparation of Culture Medium . . . . . . . . . . . . . . . . . . . . 9.3.2 Preparation of Inoculum and Incubation . . . . . . . . . . . . . 9.3.3 Ammonium Sulfate Fractionation . . . . . . . . . . . . . . . . . . . 9.3.4 Adsorption and Elution on Calcium Phosphate Gel . . . . 9.3.5 CM-Sephadex Column Chromatography; Separation of Components II from I . . . . . . . . . . . . . . . . . 9.3.6 Gel Filtration of Component II on Sephacryl S-300 . . . . . 9.3.7 DEAE-Sephadex Chromatography of Component I . . . . 9.3.8 Hydroxyapatite Chromatography of Component I . . . . . 9.3.9 Gel Filtration on Sephacryl S-300 . . . . . . . . . . . . . . . . . . . 9.3.10 Storage of the Two Purified Two Components of C2T . . . 9.4 Activation of Component II . . . . . . . . . . . . . . . . . . . . . . . Endocytosis of Two Nonlinked Protein Components in 9.5 Cultured Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.5.1 Preparation of Polyclonal Antibodies for Two Components of C2 Toxin . . . . . . . . . . . . . . . . . . . . . . 9.5.2 Purification of IgG Specific for Each of the Components I and II by Affinity Chromatography . . . . . . . . . . . . . . . . . . 9.5.3 Visualization of the Two Components Bound to Cultured Cells by Indirect lmmunoflurescence Labeling . 9.5.4 Cell Culture and Indirect lmmunofluorescence Labeling of Cell-Bound Component I I . . . . . . . . . . . . . . . Reagents and Chemicals . . . . . . . . . . . . . . . . . . . . . . . . . 9.6

103 103

9.1 9.2 9.3

Contents

105 106 106 106 106 107 107 107 108 108 108 109 110 111

112 112

113 115

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CHAPTER 10 The Role of the Clostridium botulinum C2 Toxin as a Research Tool to Study Eucaryotic Cell Biology I. L. SIMPSON . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

10.1 10.2 10.3 10.4 10.4.1 10.4.2 10.4.3

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Identifying Potential Uses . . . . . . . . . . . . . . . . . . . . . . . . Isolation and Purification . . . . . . . . . . . . . . . . . . . . . . . . C2 Toxin as a Research Tool . . . . . . . . . . . . . . . . . . . . . . Binding Step . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Internalization Step . . . . . . . . . . . . . . . . . . . . . . . . . . . . . lntracellular Step . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

117 117 118 120 121 121 122 123

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CHAPTER 11 Probing the Actin Cytoskeleton by Clostridium botulinum C2 Toxin and Clostridium perfringes Iota Toxin K . AKTORIES. U. PREPENS. P. SEHR. and I . JUST . . . . . . . . . . . . . . 129 11.1 11.2

11.2.1 11.2.2 11.2.2.1 11.2.2.2 11.2.3 11.2.3.1 11.2.3.2 11.2.4 11.3 11.3.1 11.3.2 11.3.3 11.3.4 11.4

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

129

Effects of Clostridium botulinum C2 Toxin

in Intact Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Treatment of Intact Cells with Clostridium botulinum C2Toxin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effects of C2 on the Microfilament System . . . . . . . . . . . Visualization of the Microfilament System with FITC-phalloidin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Quantitative Determination of F-actin Depolymerization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Detection of C2-catalyzed ADP-ribosylation in Intact Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Direct Method by Metabolical Labeling. . . . . . . . . . . . . Quantification of ADP-ribosylated Actin . . . . . . . . . . . . . Controls of the in vivo Effects of C2 Toxin . . . . . . . . . . . . ADP-ribosylation of Actin . . . . . . . . . . . . . . . . . . . . . . . . ADP-ribosylation of Actin in Cell Lysates . . . . . . . . . . . . ADP-ribosylation of Skeletal Muscle a-Actin to Study Actin Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . ADP-ribosylation of Skeletal Muscle a-Actin for Microinjection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Troubleshooting. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Reagents and Chemicals . . . . . . . . . . . . . . . . . . . . . . . .

130 130 131 131 131 133 133 133 134 134 134 135 136 138 138

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CHAPTER 12 Clostridium difficile Toxins M. THELESTAM. I . FLORIN and E . CHAVES-OLARTE 12.1 12.2 12.3 12.3.1 12.3.2 12.3.3

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141 Molecular Characteristics of the Toxins . . . . . . . . . . . . . 142 Cellular Effects of the Toxins . . . . . . . . . . . . . . . . . . . . . . 143 Actin Cytoskeleton is the Toxin Target . . . . . . . . . . . . . . . 144 The Receptor for ToxA Contains Galactose . . . . . . . . . . .144 The Toxins Act lntracellularly after Endocytosis . . . . . . . 144 Contents

12.4 12.4.1 12.4.2 12.5 12.5.1 12.5.2 12.6 12.7

Molecular Mode of Action of the Toxins . . . . . . . . . . . . . C. difficile Toxins Modify Rho Proteins . . . . . . . . . . . . . . . C. difficile Toxins are Glucosyltransferases . . . . . . . . . . . Biological Consequences of Toxin Action on Cells . . . . In Cultured Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . In Animal Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Relationship of the C. difficile Toxins to Other Large Clostridial Cytotoxins . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions and Perspectives for the Future . . . . . . . . .

145 145 146 147 147 148 150 151

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CHAPTER 13 Clostridium difficile Toxin B as a Probe for Rho GTPases I. JUST. J . SELZER. F. HOFMANN and K.AKTORlES . . . . . . . . . . . 159 13.1 13.2 13.3 13.3.1 13.3.2

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Purification of Toxin A and Toxin B . . . . . . . . . . . . . . . . . GlucosyltransferaseActivity of Toxin B . . . . . . . . . . . . . . Glucosylation in Cell Lysates . . . . . . . . . . . . . . . . . . . . . Glucosylation of Recombinant Rho Proteins or Membranous Fractions . . . . . . . . . . . . . . . . . . . . . . . . . . 13.3.3 Glucosylation of Recombinant GTPases for Microinjection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Toxin B Action on Intact Cells . . . . . . . . . . . . . . . . . . . . . 13.4 13.4.1 Treatment of Intact Cells with Toxin B . . . . . . . . . . . . . . . 13.4.2 Assessment of the Extent of Glucosylation . . . . . . . . . . 13.4.2.1 Differential Glucosylation by Toxin B . . . . . . . . . . . . . . . 13.4.2.2 ADP-Ribosylation of Rho by Clostridiurn botulinurn Exoenzyme C3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.5 Toxin B a Tool in Cell Biology . . . . . . . . . . . . . . . . . . . . . 13.6 Reagents and Chemicals . . . . . . . . . . . . . . . . . . . . . . . .

159 160 161 162 163 163 164 164 164 165 165 166 167

.

CHAPTER 14 Clostridial Neurotoxins G.SCHlAV0 and C.MONTECUCC0. . . . . . . . . . . . . . . . . . . . . 14.1 14.2 14.3 14.3.1 14.3.2 14.3.3 14.3.3.1 14.3.3.2 14.3.3.3 14.3.3.4 14.4 14.5 14.5.1 Contents

The Origin of Clostridial Neurotoxins . . . . . . . . . . . . . . . Structure of Clostridial Neurotoxins . . . . . . . . . . . . . . . . Mechanism of Action of Clostridial Neurotoxins . . . . . . Cell Binding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Internalization and Membrane Translocation. . . . . . . . . lntracellular Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VAMPISynaptobrevin . . . . . . . . . . . . . . . . . . . . . . . . . . . SNAP-25 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Syntaxin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Target Recognition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Clostridial Neurotoxins and the Blockade of Neurotransmitter Release . . . . . . . . . . . . . . . . . . . . . . . . Biomedical Applications of Clostridial Neurotoxins . . . . Safety Warnings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

169 169 170 174 174 175 176 178 179 179 180 181 182 182

14.5.2 14.5.3 14.5.3.1 14.5.3.2 14.6 14.7 14.8

Purification of Clostridial Neurotoxins . . . . . . . . . . . . . . . 182 Functional Assay of Clostridial Neurotoxins . . . . . . . . . . . 183 In vitro Assay of Clostridial Neurotoxins . . . . . . . . . . . . . . 184 Use in Cell Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184 Therapeutic Uses of Botulinum Neurotoxins. . . . . . . . . . . 185 Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185 Chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186

.

CHAPTER 15 Isolated Nerve Terminals as a Model System for the Study of Botulinum and Tetanus Toxins J . BLASI. E. LINK and R . JAHN . . . . . . . . . . . . . . . . . .:. . . . . . . . . 193 15.1 15.2 15.3 15.3.1 15.3.2 15.3.3 15.3.4 15.3.5 15.3.6 15.3.7 15.4 15.4.1 15.4.2 15.4.3 15.4.4 15.4.5 15.4.6 15.5

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193 Clostridial Neurotoxins: How Do They Work? . . . . . . . . 193 Model Systems for the Study of Toxin Action . . . . . . . . . 196 Bioassay . . . . . . . . . . . . . . .................... 197 Neuromuscular Junction Preparations . . . . . . . . . . . . . . 197 Synaptic Preparations Derived from the Autonomic Nervous System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198 Invertebrate Neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . 198 Primary Cultures of CNS Neurons . . . . . . . . . . . . . . . . . 198 Endocrine Cells in Culture and Endocrine Cell Lines . . . 199 Synaptosomes from Mammalian Brain . . . . . . . . . . . . . 199 Practical Considerations . . . . . . . . . . . . . . . . . . . . . . . . . 201 Sources for Clostridial Neurotoxins . . . . . . . . . . . . . . . . 201 Precautions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201 Preparation of Synaptosomes . . . . . . . . . . . . . . . . . . . . 202 Monitoring Release of Neurotransmitter . . . . . . . . . . . . 204 Poisoning of Synaptosomes . . . . . . . . . . . . . . . . . . . . . . 207 Subfractionation of Synaptosomes. . . . . . . . . . . . . . . . . 210 Relevant ChemicaIs . . . . . . . . . . . . . . . . . . . . . . . . . . .211

.

CHAPTER 16 Clostridial Toxins and Endocrine Secretion: Their Use in Insulin-Secreting Cells J . LANG. R. REGAZZI and C. .B. WOLLHEIM . . . . . . . . . . . . . . . . 217

Exocytosis of Insulin from.the Pancreatic P-cell . . . . . . . 217 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217 Regulated Exocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . 218 The SNARE Hypothesis of Exocytosis. . . . . . . . . . . . . . . 219 Application of the SNARE Hypothesis to Insulin Exocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220 Role of Ca2+and Rab3A in Insulin Exocytosis. . . . . . . . . 221 16.1.5 Permeabilization of Insulin-secreting Cells . . . . . . . . . . . 222 16.2 General Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222 16.2.1 Material Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224 16.2.2. . . . . . . 224 16.2.3 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225 16.2.4 Final Working Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . .

16.1 16.1.1 16.1.2 16.1.3 16.1.4

Contents

16.2.5 16.2.5.1 16.2.5.2 16.2.5.3 16.2.6 16.2.7 16.2.7.1 16.2.7.2 16.3 16.3.1 16.3.2 16.3.3 16.3.4 16.3.5 16.3.5.1 16.3.5.2 16.4 16.4.1 16.4.2 16.4.3 16.4.4 16.4.4.1 16.4.4.2 16.4.4.3 16.4.4.4 16.4.5 16.4.5.1 16.4.5.2 16.5 16.6

Experimental Outline. . . . . . . . . . . . . . . . . . . . . . . . . . . . 225 General Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225 Determination of SLO Concentrations . . . . . . . . . . . . . . 226 Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226 Cell Permeabilization and Exocytosis Assay . . . . . . . . . 226 Troubleshooting. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228 Cell Detachment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228 Insufficient or No Response to Ca2+. . . . . . . . . . . . . . . . 228 Transient Cotransfection Assay for Exocytosis . . . . . . . . 228 General Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228 Material Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230 Troubleshooting. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231 Insufficient Gene Expression. . . . . . . . . . . . . . . . . . . . . . 231 Considerable Variability in the Expression Levels between the same of Different Plasmids . . . . . . . . . . . . 232 Tetanus Toxin as a Tool for Studying the Role of VAMPs in Exocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232 General Considerations . . . . . . . . . . . . . . . . . . . . . . . . . 232 Specific Considerations for the Study of Insulin-Secreting Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . 233 Practical Approach .................... 233 Material Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233 Solutions Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234 Preparation of Neurotoxins. . . . . . . . . . . . . . . . . . . . . . . 234 Detailed Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235 Troubleshooting. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236 TeTx Does not Inhibit Exocytosis. . . . . . . . . . . . . . . . . . . 236 No Cleavage of VAMPs . . . . . . . . . . . . . . . . . . . . . . . . . 237 The Use of Other Clostridial Neurotoxins for the Study of Exocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . 237 237 Reagents and Chemicals . . . . . . . . . . . . . . . . . . . . . . . .

.

CHAPTER 17 Pore-forming Toxins

S. BHAKDI. A.VALEVA, I. WALEV, U. WELLER and M. PALMER . . . 241 17.1 1z2 1Z3 17.4 17.4.1 17.4.2 17.4.3 17.5 17.5.1 17.5.1.1 17.5.1.2 Contents

Origin of Pore-formingToxins (PFTs) . . . . . . . . . . . . . . . . 241 Evolution of the PFT Field . . . . . . . . . . . . . . . . . . . . . . . . 241 Relationship to Other Toxins . . . . . . . . . . . . . . . . . . . . . . 242 Molecular Mechanism of Action . . . . . . . . . . . . . . . . . . 243 243 Binding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 01igomerizat ion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244 Pore formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245 Cell-biological Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . 246 Cell-biological Effects Resulting from Ion Fluxes . . . . . . 246 Reactions Governed by Flux of Monovalent Ions . . . . . 246 Reactions Triggered by Ca2+Influx . . . . . . . . . . . . . . . . . 247

Reactions not Recognizably Governed by Flux of Ions through Toxin Pores . . . . . . . . . . . . . . . . . . . . . . . 17.5.2.1 Short Circuiting of G- protein- de pend e nt Sig na IIing

17.5.2

17.5.2.2 17.6 17.7 17.7.1 17.72 17.8 17.9

247

247 Pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Proteolytic Shedding of Membrane Proteins . . . . . . . . . 248 Pathogenic Aspects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248 Purification Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249 Purification of Staphylococcal Alpha-Toxin . . . . . . . . . . . 249 Purification of Streptolysin 0 . . . . . . . . . . . . . . . . . . . . . 251 Application of PFTs as Tools in Research . . . . . . . . . . . . 252 Reagents and Chemicals . . . . . . . . . . . . . . . . . . . . . . . . 254

CHAPTER 18. Application of Alpha-toxin and Streptolysin 0 as Tools in Cell Biological G.AHNERT-HILGER and U. WELLER . . . . . . . . . . . . . . . . . . . . . . .

259

Permeabilized Cells: an Approach to Study Intracellular Processes . . . . . . . . . . . . . . . . . . . . . . . . . . 259 Alpha-Toxin and Streptolysin 0 as Tools in the Study 18.2 of Secretory Processes . . . . . . . . . . . . . . . . . . . . . . . . . . 260 260 18.21 Biological Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18.2.1.1 Assay to Compare Biological Activity of Various Pore-Forming Toxins Using Rabbit 261 Erythrocytes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Assay for Exocytosis in Permeabilized Cells . . . . . . . . . . 263 18.2.2 Regulation of Vesicular Transmitter Transporters 18.3 in Permeabilized Cells and Synaptosomes . . . . . . . . . . 266 18.3.1 Catecholamine Uptake into SLO-Permeabilized PC 12 Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 266 18.3.2 Acidification of Small Synaptic Vesicles in SLO-Permeabilized Synaptosomes. . . . . . . . . . . . . . . 267 Reagents and Chemicals . . . . . . . . . . . . . . . . . . . . . . . . 270 18.4

18.1

CHAPTER 19. Toxins as Transporting Tools S . OLSNES. 0. KLINGENBERG. R . MUNOZ. f? 0. FALNES and A . WIEDLOCHA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.1 19.2 19.2.1 19.2.2 19.2.3 19.2.4 19.2.5 19.3 19.4 19.5

273

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273 Diphtheria Toxin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273 Toxin-resistant Cell Mutants . . . . . . . . . . . . . . . . . . . . . . 274 Receptor for Diphtheria Toxin . . . . . . . . . . . . . . . . . . . . . 275 276 Translocation of the Toxin . . . . . . . . . . . . . . . . . . . . . . . . Further Requirementsfor Translocation . . . . . . . . . . . . . 276 Formation of Cation Channels . . . . . . . . . . . . . . . . . . . . 277 Pseudomonas Exotoxin A . . . . . . . . . . . . . . . . . . . . . . . . 278 Non-cytocidal Toxins with lntracellular Sites of Action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279 Methods to Assess Translocation . . . . . . . . . . . . . . . . . . 280 Contents

19.6 19.7 19.8 19.8.1 19.8.2 19.8.3 19.8.4 19.8.5 19.8.6 19.8.7 19.8.8

283 285 286 286 S DS-PAGE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287 Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288 Cell Binding and Translocation Assay . . . . . . . . . . . . . . 288 Example of Translocated Peptides and Proteins . . . . . . . Limitations and Possibilities of the System . . . . . . . . . . . Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . In vitro Transcription and Translation. . . . . . . . . . . . . . . .

Expression in Bacteria and Purification of Recombinant Proteins . . . . . . . . . . . . . . . . . . . . . . . . . 288 In vitro Farnesylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289 Analysis of in vivo Farnesylation . . . . . . . . . . . . . . . . . . . 289 Triton X-114 Partitioning of Farnesylated Proteins . . . . . . 290

CHAPTER 20. A Brief Guide to the Safe Handling of Biological Toxin A . B.MAKSYMOWYCH and L . L.SlMPSON . . . . . . . . . . . . . . . . . 295 20.1 20.2 20.3 20.3.1 20.3.2 20.3.3 20.4 20.5 20.6 20.7 20.8

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Preventive Measures . . . . . . . . . . . . . . . . . . . . . . . . . . . . Containment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Containment Procedures . . . . . . . . . . . . . . . . . . . . . . . . Containment Facilities. . . . . . . . . . . . . . . . . . . . . . . . . . . Containment within the Laboratory and beyond the Laboratory. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Disposal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Decontamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Emergency Intervention . . . . . . . . . . . . . . . . . . . . . . . . . Resources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Contents

295 295 296 296 296 296 298 298 299 300 300 301

Abbreviations The following is a list of the most common abbreviations used throughout the book.

5- HT

5- hyd roxytryptamine

ACh

acetylcholine

ACTSK

actin cytoskeleton

ADP

adenosine diphosphate

ARF

ADP-ribosylation factor

ATP

adenosine triphosphate

BFA

brefeldin A

BHI

brain-heart infusion

BME

2-mercaptoethanol

BoNT

botulinum neurotoxin

BSA

bovine serum albumin

C2T CB

botulinum C2 toxin

CNT

clostridial neurotoxin

CPE CROP

cytopathogenic effect

CT

cholera toxin

CTL DEPC

cytotoxic T lymphocyte diethyl pyrocarbonate

DMPC

dimyristoylphosphatidylcholine

DT

diphtheria toxin

DTT

dithiothreitol

EDlN

epidermal differentiation inhibitor

E DTA

ethylenediaminetetraacetic acid

EF-2

elongation factor

ER

endoplasmic reticulum

cellubrevin

combined repetitive oligopeptides

2 Abbreviations

Abbreviations

ETA

exotoxin A from Pseudomonas aeruginosa

EXAFS

X-ray absorption fine structure

FCS

fine calf serum

FGF

fibroblast growth factor

GABA

y-aminobutyric acid

GDI

guanine nucleotide dissociation inhibitor

GDP

guanosine diphosphate

GDS

guanine nucleotide dissociation stimulator

GERL

Golgi-endoplasmic recticulum-lysosome

GPI

glycophosphoinositol

GST

glutathione S-transferase

GTP

guanosine triphosphate

GTPase

guanosine triphosphatase

HIC

hydrophobic interaction chromatography

HlyA

E. coli hemolysin

HT

hemorrhagic toxin

ICE IL

interleukin converting enzyme

IMAC

immo bilized-metal-ion -affinity chromatography

I PTG

isopropyl-fi-D-thiogalactopyranoside

LDCV

large dense core vesicles

LF

lethal factor

LPA

lysophosphatidic acid

LT

E. coli heat-labile enterotoxin

MHC NAD

maior histocompatibility class

NMJ

neuromuscula r iunction

NSF

N-ethylmaleimide-sensitive factor

PA

protective antigen

PAGE

polyacrylamide gel electrophoresis

PBS

phosphate-buffered saline

PDGF

platelet-derived growth factor

PFT

pore-forming toxins

PGE2

prostaglandin E2

interleukin

nicotinamide adenine dinucleotide

PLD

phospholipase D

PMSF

phenylmethylsulfonyl fluoride

PT

pertussis toxin

RBL

rat basophilic leukemia

RTX

repeats in toxin

SDS- PAGE

sodium dodecyl sulfate-polyacrylamide gel electrophoresis

SDS SLMV

sodium dodecyl sulfate

SLO SNAP-25 SNAPS

streptolysin 0

SNARE

SNAP receptor

ssv

small synaptic vesicles

t-SNARE

target SNAP receptor

TCA

trichloroacetic acid

TeNT

tetanus neurotoxin

TeTx

tetanus toxin

Tox A

toxin A

Tox B

toxin B

UDP-Glc

uridine disphosphate glucose

synaptic-like microvesicles synaptosomal-associated protein of 25 kDa soluble NSF attachment proteins

UDP-GIcNAc uridine diphosphate N-acetylglucosamine vesicle SNAP receptor V-SNARE VAMP

vesicle-associated membrane protein (= synaptobrevin)

Abbreviations

List of Contributors

Ahnert-Hilger, G., lnstitut fur Anatomie, Universitatsklinikum Charite, Humboldt-Universitatzu Berlin, Philippstr. 12, 10115 Berlin, Germany Aktories, K., lnstitut fur Pharmakologie und Toxikologie, AlbertLudwigs-Universitat Freiburg, Hermann-Herder-Str. 5, 79104 Freiburg, Germany Antoine, R., Laboratoire de Microbiologie Genetique et Moleculaire, INSERM CJF9109, lnstitut Pasteur de Lille, 1, rue du Professeur Calmette, 59019 Lille Cedex, France Bhakdi, S., lnstitut fur Medizinische Mikrobiologie und Hygiene, Johannes-Gutenberg-Universitat Mainz, Hochhaus am Augustusplatz, 55101 Mainz, Germany Blasi, J., Departement de Biologia Cellular i Anatomia Patologica, Facultat d'Odontologia, Universitat de Barcelona, c/Feixa Llarga s/n, 08907 L'Hospitalet del Llobregat, Spain Falnes, i? 0.,Institute of Cancer Research, The Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway Florin, I., Microbiology and Tumorbiology Center (MTC), Karolinska Institute, Box 280, 17177 Stockholm, Sweden Hall, A., CRC Oncogene and Signal Transduction Group, MRC Laboratory for Molecular Cell Biology, Department of Biochemistry, University College London, Gower Street, London WC1 E6BT, England Hofmann, F., lnstitut fur Pharmakologie und Toxikologie, AlbertLudwigs-Universitat Freiburg, Hermann-Herder-Str. 5, 79104 Freiburg, Germany Jahn, R., Departments of Pharmacology and Cell Biology, Howard Hughes Medical Institute, Yale University, School of Medicine, 295 Congress Avenue, New Haven, CT 06510, USA Just, I., lnstitut fur Pharmakologie und Toxikologie, Albert-LudwigsUniversitat Freiburg, Hermann-Herder-Str. 5, 79104 Freiburg, Germany Klingenberg, O., Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway

List of Contributors

Koch, G., lnstitut fur Pharmakologie und Toxikologie, Albert-LudwigsUniversitat Freiburg, Hermann-Herder-Str. 5, 79104 Freiburg, Germany Lang, J., Division de Biochimie Clinique et Diabetologie Experimentale, Departement de Medicine Interne, Centre Medical Universitaire, Universite de Geneve, Rue Michel-Servet 1, 1211 Geneve 4, Switzerland Link, E., Departments of Pharmacology and Cell Biology, Howard Hughes Medical Institute, Yale University, School of Medicine, 295 Congress Avenue, New Haven, CT 06510, USA Locht, C., Laboratoire de Microbiologie Genetique et Moleculaire, INSERM CJF9109, lnstitut Pasteur de Lille, 1, rue du Professeur Calmette, 59019 Lille Cedex, France Maksymowych, A. B., Departments of Medicine and Pharmacology, Jefferson Medical College, Thomas Jefferson University, 1020 Locust Street, Philadelphia, PA 19107, USA Montecucco, C., Centro CNR Biomembrane, Dipartimento di Scienze Biomediche, Universita di Padova, Via Trieste, 75, 35121 Padova, Italy Moss, J., Pulmonary-Critical Care Medicine Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, 10 Center Drive, MSC 1434, 9000 Rockville Pike, Bethesda, Maryland 20892-

1434, USA Mufioz, R., Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway Narumiya, S., Department of Pharmacology, Kyoto University, Faculty of Medicine, Yoshida, Sakyo-ku, Kyoto 606, Japan Nobes, C. D., CRC Oncogene and Signal Transduction Group, MRC Laboratory for Molecular Cell Biology, Department of Biochemistry, University College London, Gower Street, London WC1 E 6BT, England Nurnberg, B., lnstitut fur Pharmakologie, Universitatsklinikum Benjamin Franklin, Freie Universitat Berlin, Thielallee 67-73, 14195 BerlinDahlem, Germany Ohishi, I., Nipping Veterinary and Animal Science University, 1-7-1, Musashino, Tokyo 180, Japan Olarte, E. C., Microbiology and Tumorbiology Center (MTC), Karolinska Institute, Box 280, 17177 Stockholm, Sweden Olsnes, S., Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway Palmer, M., lnstitut fur Medizinische Mikrobiologie und Hygiene, Johannes Gutenberg Universitat Mainz, Hochhaus am Augustusplatz, 55101 Mainz, Germany

List of Contributors

Patton, W. A., Pulmonary-Critical Care Medicine Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, 10 Center Drive, MSC 1434, 9000 Rockville Pike, Bethesda, Maryland

20892-1434, USA Prepens, U., lnstitut fur Pharmakologie und Toxikologie, AlbertLudwigs-Universitat Freiburg, Hermann-Herder-Str. 5, 79104 Freiburg, Germany Rapak, A., Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway Regazzi, R., Division de Biochimie Clinique et Diabetologie Experimentale, Departement de Medicine Interne, Centre Medical Universitaire, Universite de Geneve, Rue Michel-Servet 1, 1211 Geneve 4, Switzerland Saito, Y., Department of Pharmacology, Kyoto University, Faculty of Medicine, Yoshida, Sakyo-ku, Kyoto 606, Japan Schiavo, G., Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY

10021, USA Sehr, I?, lnstitut fur Pharmakologie und Toxikologie, Albert-LudwigsUniversitat Freiburg, Hermann-Herder-Str. 5, 79104 Freiburg, Germany Selzer, J., lnstitut fur Pharmakologie und Toxikologie, Albert-LudwigsUniversitat Freiburg, Hermann-Herder-Str. 5, 79104 Freiburg, Germany Simpson, L. L., Departments of Medicine and Pharmacology, Jefferson Medical College, Thomas Jefferson University, 1020 Locust Street, Philadelphia, PA 19107, USA Thelestam, M., Microbiology and Tumorbiology Center (MTC),Karolinska Institute, Box 280, 17177 Stockholm, Sweden Valeva, A., lnstitut fur Medizinische Mikrobiologie und Hygiene, Johannes-Gutenberg-Universitat Mainz, Hochhaus am Augustusplatz, 55101 Mainz, Germany Vaughan, M., Pulmonary-Critical Care Medicine Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, 10 Center Drive, MSC 1434, 9000 Rockville Pike, Bethesda, Maryland 20892 1434, USA Walev, I., lnstitut fur Medizinische Mikrobiologie und Hygiene, Johannes-Gutenberg-Universitat Mainz, Hochhaus am Augustusplatz, 55101 Mainz, Germany Weller, U., lnstitut fur Medizinische Mikrobiologie und Hygiene, Johannes-Gutenberg-Universitat Mainz, Hochhaus am Augustusplatz, 55101 Mainz, Germany Wiedocha, A., Institutefor Cancer Research, The Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway List of Contributors

Wollheim, C. B., Division de Biochimie Clinique et Diabetologie Experimentale, Departement de Medecine Interne, Centre Medical Universitaire, Universite de Geneve, Rue Michel-Servet 1, 1211 Geneve 4, Switzerland Zhang, G.-F., Pulmonary-Critical Care Medicine Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, 10 Center Drive, MSC 1434, 9000 Rockville Pike, Bethesda, Maryland

20892-1434, USA

List of Contributors

Bacterial Toxins

Klaus Aktories Copyright 0 2002 WILEY-VCH Verlag GmbH & Co. KGaA

CHAPTER 1

Cholera Toxin: Mechanism of Action and Potential Use in Vaccine Development G.-F. ZHANG, W. A. PATTON, J. MOSS and M. VAUGHAN

1.1 Introduction Cholera results from an intestinal infection with the pathogenic bacterium Vibrio cholerae that causes a debilitating, and even deadly, diarrhea. Successful treatment of cholera requires effective rehydration with solutions of glucose and salts (Kaper et al., 1995). Administration of antibiotics decreases the duration of disease (Kaper et a/., 1995); vaccines are only partially effective. Koch, who first described Vibrio cholerae as the causative agent of cholera, suggested that it was a toxin-mediated disease (Koch, 1884). Over a half-century later, the existence of cholera toxin (CT) was demonstrated in cell-free culture filtrates (De, 1959; Dutta et a/., 1959); a decade later, purification of the protein toxin was achieved (Finkelstein and LoSpalluto, 1969). Since then, it has been found that CT is just one member of a family of bacterial toxins that are ADP-ribosyltransferases, which catalyze transfer of the ADP-ribose portion of nicotinamide adenine dinucleotide (NAD) to a nucleophilic acceptor (Moss and Vaughan, 1988a,b; Burnette, 1994; Merritt and Hol, 1995). CT specifically modifies arginine, either free or as part of a protein. In all cells, including intestinal epithelial cells, the maior substrate is the guanine nucleotide-binding subunit of the stimulatory regulator of adenylyl cyclase, G, (Moss and Vaughan, 1988b).ADP-ribosylation of arginine-201 in G, (Robishaw et a/., 1986) leads to persistently high intracellular levels of CAMP (Moss and Vaughan, 1988b), perhaps other effects on arachidonate metabolites (Peterson et al., 1994; Reitmeyer and Peterson, 1990), and the massive fluid and electrolyte flux that are characteristic of cholera (Kaper et a/., 1995). Cholera toxin and the very similar (in structure and mechanism of action) heat-labile enterotoxin from E. coli (LT-1 or LT, which is responsible for the syndrome of traveler's diarrhea) have been widely applied as molecular tools to facilitate understanding of signalling systems. Examples of molecules besides G, that were identified because of work on CT are the ADP-ribosylation factors (ARFs),now known to play a critical role in intracellular vesicular transport (Moss and Vaughan, 1995), and the mammalian ADP-ribosyltransferases (Zolkiewska et al., 1994).As more is learned about CT structure and biochemistry, modified or mutant CT and LT molecules are being gen-

action of cholera toxin

K. Aktories (Ed.),Bacterial Toxins. 0Chapman & Hall, Weinheim, 1997. ISBN 3-8261-0080-8

erated for use as targeting molecules to direct covalently attached ligands to specific cells, or as adiuvants for use in vaccine development (Holmgren et al., 1994).This chapter is a review of recent studies on CT and LT structure, mode of cell entry, mechanism of action, and their use as tools in biochemical studies.

1.2 Molecular Aspects of Cholera Toxin Action 1.2.1 Structure and Relationship to Other Toxins related toxins

structure of cholera toxin

GUI-FENGZHANG et al

Cholera toxin and E. coli heat-labile enterotoxins, members of the AB5 family of toxins (Burnette, 1994; Merritt and Hol, 1995) consist of a single catalytic A subunit and five B subunits (Spangler, 1992).Another oligomeric toxin is pertussis toxin (PT), a secretory product of Bordetella pertussis (for review and primary references, see Moss and Vaughan, 198813; Burnette, 1994; Merritt and Hol, 1995).CT, LT, and PT are ADP-ribosyltransferases that use NAD as an ADP-ribose donor and a subunits of the heterotrimeric guanine nucleotide-binding (G) proteins as an acceptor (Moss and Vaughan, 198813). CT and LTADPribosylate an arginine (Robishaw et al., 1986) in G, causing its activation (Gill and Meren, 1978; Johnson et al., 1978; Northup et al., 1980), whereas PT ADP-ribosylates a cysteine in G,,, resulting in uncoupling from its receptors (Bokoch et al., 1983, 1984; West et al., 1985). Diphtheria toxin (DT) from Corynebacterium diphtheriae and the closely related exotoxin A (ETA) from Pseudomonas aeruginosa are also ADP-ribosylating toxins, but are monomeric proteins that ADP-ribosylate elongation factor 2 (EF-2), leading to inhibition of protein synthesis and cell death (Wilson and Collier, 1992). CT is an oligomeric protein of 84 kDa composed of one A subunit (CTA, approx. 29 kDa) and five B (CTB, approx. 11.6 kDa) subunits (Ohtomo et al., 1976). CTA is synthesized as a single protein that is later proteolytically nicked to produce two polypeptides (Mekalanos et al., 1979), CTA, and CTAp, which remain covalently linked by a single disulfide bond near the carboxyl end of CTA,. Both the proteolytic cleavage, in a short sequence between the two cysteines, and reduction of the disulfide bond are required for generation of active CTAl (Mekalanos et al., 1979, Tomasi et al., 1979). Both CT and LT holotoxins have a "doughnut-shaped" pentameric B subunit into which amino acids of the A2 subunit are anchored (Spangler, 1992). One face of the B subunit contains five binding sites for ganglioside G M 1 (Sixma et al., 1991)) the cell-surface binding site for CT and LT. Each binding site is composed (for the most part) of residues from a single individual monomer, but binding to G M 1 occurs only when CTB is in the pentameric form (Fishman, 1982). CTB anchors the holotoxin to the membrane in an orientation such that the GM1 binding sites face and interact with the membrane, whereas the portion of CTA that does not penetrate the center of the B pentamer is above the opposite face of the pentamer (Sixma et al., 1991, 1993).

Considerable information on the three-dimensional structure of both toxins has come from X-ray crystallographic analysis of LT (Merritt et al., 1994a, 1994b, 1995; Sixma et al., 1991, 1993) and of CT (Merritt et al., 1994c; Zhang et al., 1995a, 1995b). Crystals of LT holotoxin exhibit an AB5 toxin structure in which the five identical B subunits are assembled in a ring-like manner creating a highly charged central pore lined with five long amphiphilic helices (Sixma et al., 1993).The carboxy-terminal portion of LTA2serves as a link between the A and B subunits by extending into the pore of B pentamer. The N-terminus of the LTA2 subunit forms a long a-helix that is in close contact with the surface of the LTAl protein; the region close to the trypsinization site was not visualized in the structure, most likely due to its flexibility (Sixma et al., 1993).The recently published CT holotoxin structure indicated that the most striking difference between the LT and CT structures is in the A2 subunit (Zhang et al., 1995a); the portion of LTA2that extends through the B pentamer is mostly an extended chain (Sixma et al., 1993), whereas that of CTA? appears to be a continuous a-helix throughout its length (Zhang et al., 1995a).

1.2.2 Toxin Entry into Cells and Events leading to Pathogenesis To enter a cell, CT must first bind to ganglioside GM1 (Moss and Vaughan, 198813).Through an unknown mechanism, binding results in endocytosis of the holotoxin (Lencer et al., 1995).When CTB binds to GM1 in a model system, at least two events are known to occur. One is an apparent change in membrane lipid packing (Picking, 1993; Picking et al., 1995) that may be due to GM1 being forced further into or out of the plane of the membrane as well as possibly becoming distributed in the membrane less randomly. Another effect of CTB binding to cells is an increase in calcium influx (Knoop and Thomas, 1984; Maenz and Forsyth, 1986; Dixon et al., 1987). Recently, CTB-induced calcium influx in Swiss 3T3 fibroblasts was reported to stimulate DNA binding by transcription factor AP-1 (Buckley et al., 1995). It has been known for some time that CT is endocytosed into Golgi-endoplasmic reticulum-lysosome (GERL) structures (Joseph et al., 1978). More recent data from several groups are consistent with endocytosed CT being transported within the cell via a brefeldin A (BFA)-sensitivepathway (Lencer et al., 1993; Nambiar et al., 1993; Orlandi et al., 1993); one of the recognized actions of BFA, a fungal metabolite, is to inhibit vesicle formation in the Golgi apparatus and the ER (Lippincott-Schwartzet al., 1989, 1990).After endocytosis at the apical surface of the cell, and the BFA-sensitive transport step, CT is transferred to a compartment where it is reduced to generate the enzymatically active CTA, subunit (Lencer et al., 1993).Temperaturesensitive transcytosis of CTA, or ADP-ribosylated G, to the basolatera1 membrane for the activation of adenylyl cyclase then follows (Lencer et al., 1993). The primary cellular substrate for cholera toxin ADP-ribosyltransferase is G, (Gill and Meren, 1978; Johnson et al., 1978; Kaslow Cholera Toxin: Mechanism of Action and Potential Use in Vaccine Development

fluid and electrolyte loss

et al., 1980; Northup et al., 1980).G,, composed of a, P, and y subunits, is responsible for coupling cell-surface receptors to intracellular effectors, which include adenylyl cyclases and certain Ca2+channels (Casey and Gilman, 1988). G, in the GTP-bound form, activates adenylyl cyclase and Ca2+channels. The intrinsic GTPase activity of G, then hydrolyzes bound GTP to produce an inactive, GDP-bound G, (Casey and Gilman, 1988). After ADP-ribosylation by CT, GsaGTP, with its GTPase activity inhibited (Cassel and Selinger, 1977), dissociates from the Py complex (Kahn and Gilman, 1984a), and is persistently activated. In turn, it persistently activates adenylyl cyclase, leading to increased intracellular CAMP (Taussig and Gilman, 1995). Fluid and electrolyte loss in cholera patients is closely related to elevated concentrations of CAMP,which can stimulate chloride secretion in intestinal epithelial cells (Field, 1971; Moss and Vaughan, 1988a).There is also evidence for the importance of other substances, such as prostaglandin E2 (PGE2)and 5-hydroxytryptamine (5-HT), in CT-induced secretion (Kaper et al., 1995). Nilsson et al. (1983) reported that 5-HT was released from enterochromaffin cells in the cat small intestine upon CT treatment. 5-HT could then stimulate PGE2 synthesis (Beubler et al., 1989) and activate the enteric nervous system (Eklund et al., 1984). Cholera toxin also caused release of PGE2into the lumen of intestinal loops in vitro (Peterson and Ochoa, 1989), via an effect on arachidonic acid formation (Peterson et al., 1990; Reitmeyer and Peterson, 1990).The contribution of these, and perhaps other, CT effects to the pathogenesis of cholera remains to be elucidated (Peterson et al., 1994).

1.2.3 Enzymology of Cholera Toxin In addition to catalyzing ADP-ribosylation of G, (ADP-ribosyltransferase activity), CT can also ADP-ribosylate: (i) simple guanidino compounds such as arginine (Moss and Vaughan, 1977a) and agmatine (Moss and Stanley, 198l), (ii) proteins unrelated to the adenylyl cyclase system, that contain a suitable arginine (Moss and Vaughan, 1978; Abood et al., 1982; Van Dop et al., 1984), and (iii) its own CTA, subunit, in an auto-ADP-ribosylation reaction (Trepel et al., 1977; Moss etal., 1980). CTcan also catalyze the hydrolysis of NAD to ADPribose and nicotinamide (NAD-glycohydrolase activity) (Moss et al., 1976). CT and LT, like other mono-ADP-ribosyltransferases (Moss et al., 1979a), catalyze a stereospecific SN2-likereaction (Oppenheimer, 1978; Moss et al., 1979b). Using agmatine, an arginine analogue, as an ADP-ribose acceptor, the CT-catalyzed reaction proceeds by a random sequential rapid-equilibrium mechanism (Osborne et al., 1985; Larew et al., 1991).

GUI-FENG ZHANG et al

1.2.4 In Vitro Stimulation of Cholera Toxin Activity by ARF Cholera toxin-catalyzed ADP-ribosylation and/or activation of adenyIyI cyclase can be enhanced by membrane-bound and soluble protein factors from a variety of species and tissues (Enomoto and Gill, 1980; LeVine and Cuatrecasas, 1981; Pinkett and Anderson, 1982; Schleifer etal., 1982; Kahn and Gilman, 198413; Gill and Coburn, 1987; Tsai et al., 1987, 1988).GTP or GTP analogues are also required for CT activation (Moss and Vaughan, 197713; Lin et al., 1978; Enomoto and Gill, 1979, 1980; Nakaya et al., 1980). One of the cofactors, ADPribosylation factor or ARF, initially purified from rabbit liver membranes, migrated as a doublet of approx. 21.5 kDa on denaturing polyacrylamide gel electrophoresis (Kahn and Gilman, 1984b). ARF was later shown to bind, with high affinity in the presence of 3 mM dimyristoylphosphatidylcholine (DMPC) and 2 mM MgCI2, guanine nucleotides (GTP,GDP, and GTPyS), but not adenine nucleotides (Kahn and Gilman, 1986). GTP, but not GDP, supported the ARF-stimulated, CTcatalyzed ADP-ribosylation of G, (Kahn and Gilman, 1986). Two soluble ARFs, sARFl and sARFll (Tsai et al., 1988), as well as a membrane-bound ARF (Tsai et al., 1987), were purified from bovine brain. sARFl and sARFll were later identified as ARFl and ARF3, respectively (Tsai et al., 1992). In the presence of cholate, ARFstimulated ADP-ribosylation of G, by cholera toxin was enhanced by DMPC (Tsai et al., 1988). However, DMPUcholate was not required for ARF-stimulated auto-ADP-ribosylation of CTA (Tsai et a/., 1988). sARFII-stimulated NAD:agmatine ADP-ribosyltransferase activity was enhanced by SDS in a concentration-dependent manner; 0.003 % produced maximal activity (Noda et al., 1990). ARFs are a family of highly conserved proteins, found in all eukaryotic species from Giardia to mammals (Moss and Vaughan, 1993). Their primary function in cells appears to be the regulation of vesicular trafficking in both secretory and endocytic pathways (Moss and Vaughan, 1995). Relatively recently, ARF was identified as an activator of a membrane-associated phospholipase D (PLD) (Brown et al., 1993; Cockcroft et al., 1994),which was subsequently demonstrated in Golgi preparations (Ktistakis et al., 1995).Taken together with the evidence for CT trafficking through the Golgi apparatus following cell entry, en route to activation of G, it is possible that CT might encounter ARF at several places within the cell. It remains to be determined, however, whether CT is activated by ARF in cells.

1.3 Practical Aspects of Cholera Toxin Use 1.3.1 Vaccine and Vaccine Development Cholera continues to be a maior problem in developing countries where poor sanitation and limited amounts of clean water cause difficulties in both treatment and prevention of the disease. An effective Cholera Toxin: Mechanism of Action and Potential Use in Vaccine Development

immunogenicity

GUI-FENG ZHANG et 01.

cholera vaccine would be an important contribution to the control of this potentially lethal diarrheal disease. Experiments in animals and humans have shown that both CT and the nontoxic CTB can induce strong intestinal IgA immune responses, when administered orally (Svennerholm, et al., 1978, 1984; Elson and Ealding, 1984; Quiding et al., 1991).The marked immunogenicity of CT and CTB may be due in part to their ability to bind to GMlganglioside on the surface of intestinal mucosal cells (Holmgren et al., 1994). It was recently suggested that the mucosal effects of CT in vivo could result from inhibition of certain mucosal T cell functions and alteration of the regulatory T cell environment in gut-associated lymphoid tissue (Elson et al., 1995). Two different types of oral vaccines are being developed (Mekalanos and Sadoff, 1994). One is a mixture of CTB and killed whole L! cholerae cells containing CT (Clemens et al., 1986).A large-scale field trial in Bangladesh proved the efficacy, immunogenicity, and safety of this combined CTB/killed whole-cell vaccine (Clemens et al., 1986, 1990). Some of its weaknesses, however, include lesser protection against strains (such as El Tor) other than that used for the vaccine, and apparently limited protection of individuals of blood group type 0 (Mekalanos and Sadoff, 1994). Another vaccine type is the group of live attenuated L! cholerae vaccines (for review see Davis and Spencer, 1995).An attenuated L! cholerae 01 vaccine strain was genetically engineered by deleting, from wild-type L! cholerae 01 Classical lnaba strain 5698, 94 % of the gene encoding the enzymatically active A subunit toxin (Levine et al., 1988; Kaper and Levine, 1990).The recent emergence of a new non-01 strain of L! cholerae, 0139 strain, which caused a massive cholera epidemic throughout and beyond the Indian subcontinent (Albert et al., 1993; Chongsa-nguan et al., 1993; Ramamurthy et al., 1993), has increased the need for new effective vaccines. Progress has been made in the development of a new live vaccine strain using recombinant DNA techniques (Waldor and Mekalanos, 1994).One maior concern with live vaccines is safety, as live attenuated strains can potentially revert to the fully virulent wildtype infectious agent (Mekalanos, 1994). Recombinant proteins, with point mutations that inactivate CT or LT ADP-ribosyltransferase activity, have been made, and have potential for use in vaccine development (e.g., Burnette et al., 1991). Nontoxic derivatives of LT, with Ser63Lys (Pizza et al., 1994) or Arg7Lys (Douce et al., 1995) mutations, were reported to be effective in inducing significant titers of anti-toxin antibodies. Recently, several nontoxic mutants of CTA were constructed by site-directed mutagenesis (Fontana et a/., 1995).The CT-K63 (Ser63Lys) mutant induced neutralizing antibodies against both the A and B subunits, suggesting its potential use for development of an effective vaccine.

1.3.2 Cholera Toxin as a Molecular Tool Some bacterial toxins have therapeutic applications, for example, as novel antigen delivery systems (Aitken and Hirst, 1995). CT, LT, and their non-toxic B subunit moieties have attracted much attention among immunologists. Of particular interest is the use of CTB as an effective carrier for oral delivery of foreign antigens (Lebens and Holmgren, 1994). Covalent coupling of horseradish peroxidase to CTB significantly enhanced gut immune responses to the enzyme after oral administration (McKenzie and Halsey, 1984). Studies by Czerkinsky et al.(1989)demonstrated that oral administration of small amounts of a CTB-linked streptococcal protein antigen stimulated both mucosal IgA and extramucosal IgG antibody responses in mice. More recently, CTB was recognized as a valuable transmucosal carrier for enhancing induction of peripheral immunological tolerance (Sun et al.,1994).CT has also been used successfully as an adjuvant when mixed with unrelated antigens to stimulate mucosal IgA immune responses (Elson and Ealding, 1984; Dertzbaugh and Elson, 1991). It seems that this adjuvant activity is closely related to the ADPribosyltransferase activity of CTA and increased formation of cyclic AMP in the affected cells (Holmgren et al., 1994). CT and CTB can also be used as targeting molecules. In one example, covalent attachment of CTB to liposomes, via the reaction of free sulfhydryl groups in CTB with maleimide on the surface of phosphatidylethanolamine liposomes, enhanced its ability (200 times compared with native CTB) to compete with RCA12,, agglutinin for binding to brush border surfaces (Uwiera et al.,1992). It was suggested that such a modification could be useful for directing liposomes to cell surfaces that contain GM, ganglioside. Recent studies confirmed and extended this finding by demonstrating that covalent coupling of CT or CTB to small unilamellar liposomes via a thioether bond could be used to target vesicles to M cells in Peyer’s patches (Harokopakis et al., 1995). In these studies, liposome-coupled CT or CTB retained its ability to interact with GMl ganglioside and its immunogenicity. The gene fusion technique provides an alternative to chemical conjugation for preparing CTB-containing vaccines (Sanchez et al., 1990). Several plasmid vectors have been developed to generate translational fusions of a protein of interest to the ctxB gene product (Dertzbaugh and Macrina, 1990; Sanchez et al., 1990). A synthetic peptide of 15 amino acids from glucosyltransferase B was attached to the amino terminus of CTB without changing the structure or the biological activity of CTB (Dertzbaugh et al., 1990). By preparing fusion proteins of CTA2 with bacterial alkaline phosphatase, maltosebinding protein, or P-lactamase, Jobling and Holmes (1992) investigated the interaction of CTA2 with CTB, as well as CTB-GMl interactions. These fusion proteins bound to membranes containing GMl, although only the alkaline phosphatase and P-lactamase constructs retained enzymatic activity (Jobling and Holmes, 1992). Cholera Toxin: Mechanism of Action and Potential Use in Vaccine Development

1.4 Summary Extensive studies on cholera toxin have led to a better understanding of cholera and have facilitated the initial development of effective treatments and therapies. Despite improvement in the vaccines for cholera, a successful vaccine has yet to be developed. CT has been applied broadly in studies of a variety of biological systems and has provided valuable insight into mechanisms of cellular signal transduction. It has been employed to probe key components of various signalling pathways such as the elucidation of functional domains of transducin and G, by labeling or modifying the activity of a specific G protein a subunit in vitro (Navon and Fung 1984; Ho et al., 1989), determination of the presence of G, and ganglioside G,,in cellular fractions, and elucidation of the effects of increased cyclic AMP on cellular events (Moss and Vaughan, 198813). Knowledge and understanding of ARF, another important component of cell signaling pathways, has also been facilitated by using CT-catalyzed ADPribosylation as a general method to define and assay ARF activity (Moss and Vaughan, 1995).The assays for assessing CT and LT activity in vitro are described in Chapter 2.

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Fishrnan PH (1982): Role of membrane gangliosides in the binding and action of bacterial toxins. In J. Membr. Biol. 69:85-97. Fontana MR, Manetti R, Giannelli V, et a/. (1995): Construction of nontoxic derivatives of cholera toxin and characterization of the immunological response against the A subunit. In Infect. Imrnun. 63:2356-2360. Gill DM, Coburn J (1987): ADP-ribosylation by cholera toxin: Functional analysis of a cellular system that stimulates the enzymatic activity of cholera toxin fragment A,. In Biochemistry 26:6364-6371. Gill DM, Meren R (1978): ADP-ribosylation of membrane proteins catalyzed by cholera toxin: Basis of the activation of adenylate cyclase. In Proc. Natl. Acad. Sci. USA 753050-3054. Cholera Toxin: Mechanism of Action and Potential Use in Vaccine Developmeni

Harokopakis E, Cholders NK, Michalek SM, et a/. (1995):Conjugation of cholera toxin or its B subunit to liposomes for targeted delivery of antigens. In J. Immunolog. Meth. 18531-42. Ho Y-K, Hingorani VN, Navon SE, et a/. (1989): Transducin: A signalling switch regulated by guanine nucleotides. In Curr. Top. Cell. Regul. 30:171-202. Holmgren J, Czerkinsky C, Lycke N, et a/. (1994): Strategies for the induction of immune responses at mucosal surfaces making use of cholera toxin B subunit as immunogen, carrier, and adiuvant. In Am. J. Fop. Med. Hyg. 50:42-54. Jobling MG, Holmes RK (1992):Fusion proteins containing the A2 domain of cholera toxin assemble with B polypeptides of cholera toxin to form immunoreactive and functional holotoxin-like chimeras. In Infect. Immun. 60:4915-4924. Johnson GL, Kaslow HR, Bourne HR (1978):Genetic evidence that cholera toxin substrates are regulatory components of adenylate cyclase. In J. Biol. Chem. 253:7120- 7123. Joseph KC, Kim SU, Strieber A, et al. (1978): Endocytosis of cholera toxin into neuronal GERL. In Proc. Natl. Acad. Sci. USA 75:2815-2819. Kahn RA, Gilman AG (1984~): ADP-ribosylation of Gs promotes the dissociation of its a and p subunits. In J. Biol. Chem. 259:6235-6240. Kahn RA, Gilman AG (198413): Purification of a protein cofactor required for ADPribosylation of the stimulatory regulatory component of adenylate cyclase by cholera toxin. In J. Biol. Chem. 259:6228-6234. Kahn RA, Gilman AG (1986):The protein cofactor necessary for ADP-ribosylation of Gs by cholera toxin is itself a GTP-binding protein. In J. Biol. Chem. 261 :7906-7911. Kaper JB, Levine MM (1990):Recombinant attenuated Vibrio cholerae strains used as live oral vaccines. In Res. Microbiol. 141:901-906. Kaper JB, Morris JG, Levine MM (1995):Cholera. In Clin. Microbiol. Rev. 8:48-86. Kaslow HR, Johnson GL, Brothers VM, et a/. (1980):A regulatory component of adenylate cyclase from human erythrocyte membranes. In J. Biol. Chem. 25513736-3741. Knoop FC, Thomas DD (1984):Effect of cholera enterotoxin on calcium uptake and cyclic AMP accumulation in rat basophilic leukemia cells. In Int. J. Biochem. 16:275-280. Koch R (1884): An address on cholera and its bacillus. In Brit. Med. J August 30:403-407;453-459. Ktistakis NT, Brown HA, Sternweis PC, etal. (1995):Phospholipase D is present on Golgi-enriched membranes and its activation by ADP-ribosylation factor is sensitive to brefeldin A. In J. Biol. Chem. 92:4952-4956. Larew JS-A, Peterson JE, Graves DJ (1991):Determination of the kinetic mechanism of arginine-specific ADP-ribosyltransferases using a high performance liquid chromatographic assay. In J. 6/01. Chem. 266:52-57. Lebens M, Holmgren J (1994):Mucosal vaccines based on the use of cholera toxin B subunit as immunogen and antigen carrier. In Dev. Biol. Stand. 82:215-227. Lencer WI, de Almeida JB, Moe S, etal. (1993):Entry of cholera toxin into polarized human intestinal epithelial cells: Identification of an early brefeldin A-sensitive event required for A,-peptide generation. In J. Clin. Invest. 92:2941-2951. Lencer WI, Moe S, Rufo PA, et a/. (1995):Transcytosis of cholera toxin subunits across model human intestinal epithelia. In Proc. Natl. Acad. Sci. USA 92: 10094- 10098. LeVine H, Cuatrecasas P (1981): Activation of pigeon erythrocyte adenylate cyclase by cholera toxin. In Biochim. Biophys. Acta 672:248-261. Levine MM, Kaper JB, Herrington D, et a/. (1988):Volunteer studies of deletion mutants of Vibrio cholerae 01 prepared by recombinant techniques. In Infect. Immun. 56: 161- 167. Lin MC, Welton AF, Berman MF (1978): Essential role of GTP in the expression of adenylate cyclase activity after cholera toxin treatment. In J. Cycl. Nuc. Res. 4: 159- 168. Lippincott-Schwartz J, Donaldson JG, Schweizer A, et al. (1990): Microtubuledependent retrograde transport of proteins into the ER in the presence of brefeldin A suggests an ER recycling pathway. In Cell 60:821-836. GUI-FENGZHANG et al.

Lippincott-SchwartzJ, Yuan LC, Bonifacino JS, et al. (1989):Rapid redistribution of Golgi proteins into the ER in cells treated with brefeldin A: Evidence for membrane cycling from the Golgi to the ER. In Cell 56:801-813. Maenz DD, Forsyth GW (1986):Cholera toxin facilitates calcium transport in jeiunal brush border vesicles. In Can. J. Physiol. Pharmacol. 64568-574. McKenzie SJ, Halsey JF (1984):Cholera toxin B subunit as a carrier protein to stimulate a mucosal immune response. In J. Immunol. 133:1818-1824. Mekalanos JJ (1994):Live bacterial vaccines: Environmental aspects. In Curr. Opin. Biotech. 5:312-319. Mekalanos JJ, Collier RJ, Romig WR (1979):Enzymatic activity of cholera toxin: II. Relationships to proteolytic processing, disulfide bond reduction, and subunit composition. In J. Biol. Chem. 2545855-5861. Mekalanos JJ, Sadoff JC (1994):Cholera vaccines: Fighting an ancient scourge. In Science 265: 1387- 1389. Merritt EA, Hol WG (1995):AB5 toxins. In Curr. Opin. Struct. Biol. 5:165-171. Merritt EA, Pronk SE, Sixma TK, et a/. 1994~): Structure of partially-activated E. coli heat-labile enterotoxin (LT) at 2.6 resolution. In FEBS let. 337:88-92. Merritt EA, Sarfaty S, Pizza M, et a/. (1995): Mutation of a buried residue causes loss of activity, but no conformational change in the heat-labile enterotoxin of Escherichia coli. In Struct. Biol. 2:269-272. Merritt EA, Sarfaty S, Van Den Akker F, et al. (1994~): Crystal structure of cholera toxin B-pentamer bound to receptor G ,, pentasaccharide. In Prot. Sci. 3:166-175. Merritt EA, Sixma TK, Kalk KH, et al. (199413):Galactose-binding site in Escherichia coli heat-labile enterotoxin (LT) and cholera toxin (CT). In Mol. Microbiol. 13:745-753. Moss J, Garrison S, Oppenheimer NJ, et a/. (197913): NAD-dependent ADPribosylation of arginine and proteins by Escherichia coli heat-labile enterotoxin. In J. Biol. Chem. 254:6270-6272. Moss J, Manganiello VC, Vaughan M (1976): Hydrolysis of nicotinamide adenine dinucleotide by choleragen and its A protomer: Possible role in the activation of adenylate cyclase. In Proc. Natl. Acad. Sci. USA 73:4424-4427. Moss J, Stanley, SJ (1981): Histone-dependent and histone-independentforms of an ADP-ribosyltransferase from human and turkey erythrocytes. In Proc. Natl. Acad. Sci. USA 78x4809-4812. Moss J, Stanley SJ, Oppenheimer NJ (1979~): Substrate specificity and partial purification of a stereospecific NAD- and guanidine-dependent ADPribosyltransferasefrom avian erythrocytes . In J. Biol. Chem. 255:7835-783Z Moss J, Stanley SJ, Watkins PA, et al. (1980): ADP-ribosyltransferase activity of mono- and multi-(ADP-ribosylated) choleragen. In J. Biol. Chem. 255:7835- 7837. Moss J, Vaughan M (1977~): Mechanism of action of choleragen: Evidence for ADP-ribosyltransferase activity with arginine as an acceptor. In J. Biol. Chem. 252 :2455-2457. Moss J, Vaughan M (1977b):Choleragen activation of solubilized adenylate cyclase: Requirement for GTP and protein activator for demonstration of enzymatic activity. In Proc. Natl. Acad. Sci. USA 74:4396-4400. Moss J, Vaughan M (1978): Isolation of an avian erythrocyte protein possessing ADP-ribosyltransferase activity and capable of activating adenylate cyclase. In Proc. Natl. Acad. Sci. USA 753621 -3624. Moss J, Vaughan M, (1988~): Mechanism of action of choleragen (cholera toxin) and E. coli heat-labile enterotoxins. In BacterialToxins (Handbook of Natural Toxins), 4, (Tu AT, Hardegree MC) Dekker, New York. Moss J, Vaughan M (198813):ADP-ribosylation of guanyl nucleotide-binding regulatory proteins by bacterial toxins. In Adv. Enzym. 61 :303-379. Moss J, Vaughan M (1993): ADP-ribosylation factors, 20,000 Mr guanine nucleotide-binding protein activators of cholera toxin and components of intracellular vesicular transport systems. In Cell. Signalling 5367-379. Moss J, Vaughan M (1995): Structure and function of ARF proteins: Activators of cholera toxin and critical components of intracellular vesicular transport processes. In J. Biol. Chem. 270: 12327- 12330.

8,

Cholera Toxin: Mechanism of Action and Potential Use in Vaccine Development

Nakaya S, Moss J, Vaughan M (1980): Effects of nucleoside triphosphates on choleragen-activated brain adenylate cyclase. In Biochemistry 19:4871-4874. Nambiar MP, Oda T, Chen C, etal. (1993): Involvement of the Golgi region in the intracellular trafficking of cholera toxin. In J. Cell. Physiol. 154:222-228. Navon SE, Fung BK (1984):Characterization of transducin from bovine retinal rod outer segments. Mechanism and effects of cholera toxin-catalyzed ADPribosylation. In J. Biol. Chem. 259:6686-6693. Nilsson 0, Cassuto J, Larsson L-I, et a/. (1983): 5-hydroxytryptamine and cholera secretion: A histochemical and physiological study in cats. In Gut 24:543-548. Noda M, Tsai S-C, Adamik R, et a/. (1990):Mechanism of cholera toxin activation by a guanine nucleotide-dependent 19-kDa protein. In Biochim. Biophys. Acta 1034:195- 199. Northup JK, Sternweis PC, Smigel MD, et al. (1980): Purification of the regulatory component of adenylate cyclase. In Proc. Natl. Acad. Sci. USA 77:6516-6520. Ohtomo N, Muraoka T, Tashiro A, et a/. (1976):Size and structure of the cholera toxin molecule and its subunits. In J. Infect. Dis. 133:S31-S40. Oppenheimer NJ (1978): Structural determination and sterospecificity of the choleragen-catalyzed reaction of NAD' with guanidines. In J. Biol. Chem. 253:4907-4910. Orlandi PA, Curran PK, Fishman PH (1993):Brefeldin A blocks the response of cultured cells to cholera toxin. In J. Biol. Chem. 268: 12010- 12016. Osborne JC Jr, Stanley SJ, Moss J (1985):Kinetic mechanisms of two NAD:arginine ADP-ribosyltransferases: The soluble, salt-stimulated transferase from turkey erythrocytes and choleragen, a toxin from Vibrio cholerae. In Biochemistry 24x523555240. Peterson JW, Jackson CA, Reitmeyer JC (1990):Synthesis of prostaglandins in cholera toxin-treated Chinese hamster ovary cells. In Microb. Pathogen. 9:345-353. Peterson JW, Lu Y, Duncan S, etal. (1994):Interactions of intestinal mediators in the mode of action of cholera toxin. In J. Med. Microbiol. 41 :3-9. Peterson JW, Ochoa LG (1989):Role of prostaglandins and CAMPin the secretory effects of cholera toxin. In Science 245:857-859. Picking WD (1993): Interaction of pyrene-labeled monosialoganglioside GM1 micelles with cholera toxin. In Biochem. Biophys. Res. Comm. 195:1153-1158. Picking WL, Moon H, Wu H, et al. (1995): Fluorescence analysis of the interaction between ganglioside GMl-containing phospholipid vesicles and the B subunit of cholera toxin. In Biochim. Biophys. Acta 1247:65-73. Pinkett MO, Anderson WB (1982): Plasma membrane-associated component(s) that confer(s) cholera toxin sensitivity to adenylate cyclase. In Biochim. Biophys. Acta. 714:337-343. Pizza M, Domenighini M, Hol W, etal. (1994):Probing the structure-activity relationship of Escherichia coli LT-A by site-directed mutagenesis. In Mol. Microbiol. 14:51-60. Quiding M, Nordstrom I, Kilander A, et a/. (1991): Intestinal immune responses in humans. Oral cholera vaccination induces strong intestinal antibody responses and interferon-y production and evokes local immunological memory. In J. Clin. Invest. 88: 143- 148. Ramamurthy T, Garg S, Sharma R, et a/. (1993):Emergence of novel strain of Vibrio cholerae with epidemic potential in southern and eastern India. In lancet 341:703-704. Reitmeyer JC, Peterson JW (1990): Stimulatory effects of cholera toxin on arachidonic acid metabolism in Chinese hamster ovary cells (43022). In Proc. SOC. Exper. Biol. Med. 193:181- 184. Robishaw JD, Russell DW, Harris BA, etal. (1986):Deduced primary structure of the a subunit of the GTP-binding stimulatory protein of adenylate cyclase. In Proc. Natl. Acad. Sci. USA 83: 1251- 1255. Sanchez J, Johansson S, Lowenadler B, et a/. (1990):Recombinant cholera toxin B subunit and gene fusion proteins for oral vaccination. In Res. Microbiol. 141:971-979. GUI-FENG ZHANG et al.

Schleifer LS, Kahn RA, Hanski E, et a/. (1982): Requirements for cholera toxindependent ADP-ribosylation of the purified regulatory component of adenylate cyclase. In J. Biol. Chem. 257:20-23. Sixma TK, Kalk KH, van Zanten BAM, et a/. (1993):Refined structure of Escherichia coli heat-labile enterotoxin, a close relative of cholera toxin. In J. Mol. Biol.

230:890-918. Sixma TK, Pronk SE, Kalk KH, et al. (1991):Crystal structure of a cholera toxinrelated heat-labile enterotoxin from €. coli. In Nature 351 :371-377. Spangler BD (1992):Structure and function of cholera toxin and the related Escherichia coli heat-labile enterotoxin. In Microbiol. Rev. 56:622-647. Sun J-B, Holmgren J, Czerkinsky C (1994):Cholera toxin B subunit: An efficient transmucosal carrier-delivery system for induction of peripheral immunological tolerance. In Proc. Natl. Acad. Sci. USA 91 :10795- 10799. Svennerholm A-M, Jertborn M, Gothefors L, et a/. (1984):Mucosal antitoxic and antibacterial immunity after cholera disease and after immunization with a combined B subunit-whole cell vaccine. In J. Infect. Dis. 149:884-893. Svennerholm A-M, Lunge S, Holmgren J (1978):Correlation between intestinal synthesis of specific immunoglobulin A and protection against experimental cholera in mice. In Infect. Immun. 21 :1 -6. Taussig R, Gilman AG (1995):Mammalian membrane-bound adenylyl cyclases. In J. Biol. Chem. 27O:l-4. Tomasi M, Battistini A, Araco A, et al. (1979):The role of the reactive disulfide bond in the interaction of cholera-toxin functional regions. In Eur. J. Biochem.

93:621-627. Trepel JB, Chuang D-M, Neff NH (1977):Transfer of ADP-ribose from NAD to choleragen: A subunit acts as catalyst and acceptor protein. In Proc. Natl. Acad. Sci.

USA 74:5440-5442. Tsai S-C, Adamik R, Haun RS, et a/. (1992): Differential interaction of ADPribosylation factors 1, 3, and 5 with rat brain Golgi membranes. In Proc. Natl. Acad. Sci. USA 89:9272-9276. Tsai S-C, Noda M, Adamik R, et al. (1987): Enhancement of choleragen ADPribosyltransferase activities by guanyl nucleotides and a 19-kDa membrane protein. In Proc. Natl. Acad. Sci. USA 8 4 5 1 3 9 4 4 2 , Tsai S-C, Noda M, Adamik R, et al. (1988):Stimulation of choleragen enzymatic activities by GTP and two soluble proteins purified from bovine brain. In J. Biol. Chem. 263: 1768- 1772. Uwiera RE, Romancyia DA, Wong JP, etal. (1992):Effect of covalent modification on the binding of cholera toxin B subunit to ileal brush border surfaces. In Anal. Biochem. 204:244-249. Van Dop C,Tsubokawa M, Bourne HR, etal. (1984):Amino acid sequence of retinal transducin at the site ADP-ribosylated by cholera toxin. In J. Biol. Chem.

259:696-698. Waldor MK, Mekalanos JJ (1994):Emergence of a new cholera pandemic: Molecular analysis of virulence determinants in Vibrio cholerae and 0139 and development of a live vaccine prototype. In J. Infect. Dis. 170:278-283. West RE Jr, Moss J, Vaughan M et al. (1985): Pertussis toxin-catalyzed ADPribosylation of transducin. Cysteine 347 is the ADP-ribose acceptor site. In J. Biol. Chem. 260: 14428- 14430. Wilson BA, Collier RJ (1992):Diphtheria toxin and Pseudomonas aeruginosa exotoxin A: Active-site structure and enzymatic mechanism. In Curr. Top. Microbiol. & Immunol. 175:27-41. Zhang R-G, Scott DL, Westbrook ML, et a/. (1995~): The three-dimensional crystal structure of cholera toxin. In J. Mol. Biol. 251 563-573. Zhang R-G, Westbrook ML, Westbrook EM, et a/. (1995b):The 2.4 8, crystal structure of cholera toxin B subunit pentamer: Choleragenoid. In J. Mol. Biol.

251 550-562. Zolkiewska A, Okasaki IJ, Moss J (1994): Vertebrate mono-ADP-ribosyltransferases. In Mol. Cell. Biochem. 138:107-112.

Cholera Toxin: Mechanism of Action and Potential Use in Vaccine Developmeni

Bacterial Toxins

Klaus Aktories Copyright 0 2002 WILEY-VCH Verlag GmbH & Co. KGaA

Cholera Toxin and Escherichia coli Heat Ia biIe Ente rot oxin: Biochemica I Methods for Assessing Enzymatic Activities

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W. A. PATTON, G.-F. ZHANG, J. MOSS and M. VAUGHAN

2.1 Introduction Cholera toxin (CT) and E. coli heat-labile enterotoxin (LT-1 or LT) are members of the AB5family of ADP-ribosylating toxins (Merritt and Hol, 1995).The B subunits of CT and LT (CTB and LTB, respectively) form a homo-pentameric structure that is critical for toxin-cell interaction, whereas the A subunits of both CT and LT (CTA and LTA, respectively), after cleavage near their carboxy termini with trypsin or similar proteases, followed by reduction of a single disulfide bond, yield the enzymatically active Al peptide and a smaller A2 peptide (Spangler, 1992). CTAl and LTAl catalyze the cleavage of the glycosidic bond linking ADP-ribose to nicotinamide in NAD, with transfer of ADPribose to an acceptor amino acid or water (Moss and Vaughan, 1992). The products formed in the SN2-like reaction depend on the presence of specific nucleophilic acceptors. This fact is the basis of the four biochemical assays described here that measure the amounts of ADP-ribose transferred to simple guanidino compounds (e.g., arginine, agmatine) or arginine-containing proteins (ADP-ribosyltransferase activity), the toxin itself (auto-ADP-ribosyltransferase activity), or water (NAD glycohydrolase activity). These assays, originally developed to assess the activity of native CT or LT, are used also to determine the activity of specific toxin constructs alone or in the presence of GTP-dependent activating proteins known as ADPribosylation factors or ARFs. ARFs were originally identified as activators of CT-catalyzed ADPribosylation of a heterotrimeric G-protein subunit, G, that is the stimulatory regulator of adenylyl cyclase (Kahn and Gilman, 1984), and were later recognized as allosteric activators of CT (Noda et al., 1990).They are now known to be a family of monomeric approx. 20kDa guanine nucleotide-binding proteins belonging to the Ras superfamily, found in eukaryotic organisms from Giardia (Murtagh et al., 1992) to humans (Tsuchiya et al., 1991), that stimulate toxin activity when they are in the GTP-bound (activated) state (Moss and Vaughan, 1995; Welsh et al., 1994a). Initial studies showed that in yeast, ARF was localized to the Golgi apparatus (Stearns et a/., 1990a),and ARFs have since been shown to be essential regulators of membrane vesicle dynamics (Stearns et al., 1990b) in the ER and

basis of assays for toxins

K. Aktories (Ed.),Bacterial Toxins. 0 Chapman & Hall, Weinheim, 1997. ISBN 3-8261-0080-8

Golgi apparatus, the nucleus, and the plasma membrane (Donaldson and Klausner, 1994).The identification of ARFs as activators of an isoform of phospholipase D (Brown et al., 1993; Cockcroft et a/., 1994; Massenburg et al., 1994; Hammond et al., 1995), which appears to be present (at least in part) in the Golgi apparatus (Ktistakis et al., 1995), has helped to define further the function of ARF in cells. Investigations into the role of ARFs in these processes have employed recombinant wild-type and mutant ARF constructs. Thus, the assays discussed here have become commonly used to measure native ARFs, and to assess the integrity of recombinant proteins and the effects of mutations on their activity.

2.2 General Information on CT, LT, ARF and Reagents 2.2.1 Sources, Purification, and Activation of CTA and LTA Cholera toxin, first purified by Finkelstein and LoSpalluto (1969), can be purified by one of several more recently published methods including phosphocellulose chromatography (Mekalanos et a/., 1978), or affinity chromatography (based on the interaction of the B subunits with the carbohydrate moiety of ganglioside GM1) on IYSOGM~ bound alternative methods for purification to a Spherosil-DEAE-dextran support (Tayot and Tardy, 1980; Tayot et al., 1981). Pure CT, as iudged by isoelectric focusing, can also be obtained using Mono Q FPLC (Spangler and Westbrook, 1989). Purified CT can be purchased from several sources (see Section 2.2.3). Among the early descriptions of the purification of LT are those that utilize ammonium sulfate fractionation, gel filtration, and chromatography on hydrophobic or ion exchange matrices (e.g., Kunkel and Robertson, 1979 and references therein). Procedures have been developed (similar to that for CT) that make use of agarose A-5m (Clementsand Finkelstein, 1979) or immobilized D-galactose (Uesaka et al., 1994) as affinity resins; purified LT can be eluted from either matrix with galactose. Purified CTA or LTA is required for the assays described herein and can be generated and purified from CT or LT holotoxin using methods originally described for CT. Gel filtration in 0.1 M glycine buffer (pH 3.2) containing 6 M urea (Ohtomo etal., 1976) or 5 % formic acid (Lai etal., 1976) results in separation of the A subunit from B subunit monomers. More recently, a reverse-phase HPLC procedure has been described for cholera toxin subunit purification (Pearson et a/., 1986). Like CT, purified CTA can be purchased from several sources (see Section 2.2.3). Pure CTA and LTA are activated for use in these assays by incubation with thiol and proteolytic cleavage to produce the A, and A2 peptides. After secretion, CTA is proteolytically nicked by bacterial proteases (Mekalanos etal., 1979) and thus requires only reduction to be W. A. PATTON et al.

active. CT is reduced (activated) by incubation for 30 min at 30°C in 50 mM Tris-buffered saline (pH 8.0) containing 20 m M DTT, similar to the original description by Ohtomo et al. (1976). Purified LT is usually not nicked and must be proteolyzed with trypsin (Rappaport et al., 1976) (6pg per 30yg of LT (Moss et al., 1993)) as well as being reduced; proteolysis is terminated by the addition of 30 pg of trypsin inhibitor. Although reduced and alkylated toxin A, subunit can be separated from the A2 peptide (as described by Ohtomo et al. (1976) and Lai et al. (1976)))it is not necessary to do so; preparations of A, that contain A2 are routinely used and have enzymatic activity. Activated toxin is stored in portions sufficient for one experiment to avoid repeated freezing and thawing. Although CTA is a relatively stable protein, when reduced it may aggregate and precipitate in an inactive form.

activation of toxins

2.2.2 Sources and Purification of ARF Both native and recombinant ARFs (synthesized in E. coli or Sf9 cells) can be used to stimulate toxin activity. One advantage of using native ARFs or those expressed in Sf9 cells is that they are posttranslationally modified by the addition of an amino-terminal myristoy1 group (Kahn et al., 1988; Kunz et al., 1993), whereas recombinant ARFs synthesized in E. coli are not, unless coexpressed with a eukaryotic N-myristoyltransferase (Randazzo et al., 1992; Haun et al., 1993). Soluble, native ARFs are commonly prepared from bovine brain (Tsai et al., 1988). Briefly, proteins, precipitated between 25 % and 75 % saturation with ammonium sulfate from the supernatant fraction of bovine brain homogenized in buffer A (20mM potassium phosphate, pH 8.0, 1 mM EDTA, 1 mM DTT, 1 m M sodium azide, 0.5mM PMSF, and 10% sucrose), are suspended in and dialyzed against buffer B (20m M potassium phosphate, pH 7.0, 1 m M EDTA, 1 m M DTT, 1 mM sodium azide, 1 m M benzamidine, and 250mM sucrose) before chromatography on CM-Sepharose in the same buffer. The flow-through and wash are combined, adjusted to pH 5.35 with cold acetic acid, and chromatographed again on CMSepharose in buffer C (buffer B adjusted to pH 5.35). Elution with a linear gradient of 25 to 200 m M NaCl in buffer C yields two separate peaks of ARF protein and ARF activity as determined by SDSPAGE and stimulation of the NAD:agmatine ADP-ribosyltransferase activity of CTA (described below), respectively. Peak I (sARFI or the ARFl gene product) and peak II (sARFII or the ARF3 gene product) are further purified by hydroxylapatite chromatography in buffer D (20 mM Tris, pH 8.0, 1 m M EDTA, 1 m M DTT, 1 m M sodium azide, 5 mM magnesium chloride, and 250 m M sucrose) with a linear gradient of 0 to 5 0 m M potassium phosphate in the same buffer. Chromatography on Ultrogel AcA 54 (Section 2.2.3) in buffer D with the addition of 100mM NaCl (buffer E) can

preparation of native ARFs

Cholera Toxin and Escherichio coli Heat-labile Enterotoxin

preparation of recombinant ARFs

W. A. PATTON et al.

be used to purify each protein further. The purity of ARFl or ARF3 after gel filtration chromatography is greater than 90 %. ARF proteins should be stored in portions sufficient for a single experiment as they lose activity with repeated freezing and thawing, especially at concentrations below 1 mg/ml. Recombinant ARFs, e.g., recombinant human ARFl (rARFl), are also commonly used in these assays, as it is generally faster to obtain greater quantities of high-purity protein by over-expression in E. coli (e.g. Weiss et al., 1989; Price et al., 1992; Hong et a/., 1994) than from mammalian tissue. Sources of reagents are given in Section 2.2.3. For the preparation of recombinant, myristoylated human ARFl (mARFl), using the method of Hong etal. (1994), competent BL21(DE3) E. coli are cotransformed (by the heat-shock method) with an ampicillin-resistant PET-derived plasmid containing the hARFl gene and a kanamycin-resistant plasmid containing the yeast N-myristoyltransferase gene. Transformants from ampicillin and kanamycin plates are grown in LB broth (typically 500 or 1000 ml) containing ampicillin or carbenicillin, 100 yg/ml, and kanamycin, 100 yglml, to an OD600of approx. 0.6. As protein production is often variable, several transformants are picked and screened using 6-well dishes to select a colony that has the best protein production. Myristic acid and fatty acid-free BSA are then added to 0.5 and 0.06pM, respectively, as described by Franco et al. (1995). Ten minutes later, recombinant protein production is induced with the addition of IPTG to a final concentration of 0.5 mM. Franco et al. (1995) recommended reducing the temperature from 37°C to 27°C during induction to increase the efficiency of myristoylation. After further incubation for 2 h, cells are harvested and lysed in 15 ml of TE buffer (10m M Tris, p H 8.0, 1 m M EDTA) containing lysozyme, 5 mg/ml, by sonification or nitrogen decompression (Hong et al., 1994). The supernatant (100,000 g, 30min) is applied to a column ( 2 . 5 100cm) ~ of Ultrogel AcA 54 equilibrated and eluted, with buffer E. Fractions representing an ARF peak, identified by stimulation of CT ADP-ribosyltransferase activity in the agmatine assay (Assay 2, described below) and SDS-PAGE (in Tris-Glycine 14 % gels), are pooled, concentrated to approx. 1 mg/ml, divided into portions as described for native ARFs, and stored at -20°C. Protein obtained by this method is generally more than 90 % pure (as iudged by Coomassie staining of SDS gels), and can be purified futher by chromatography on a DEAE resin using buffer D, with a linear gradient of 0 to 200 m M NaCI. To obtain recombinant ARF protein preparations that contain a high percentage of myristoyluted protein, Franco et al. (1995) utilized both DEAE and Mono S chromatography. Such enrichment is especially important when the activation of toxin by nonmyristoylated and myristoylated proteins is to be compared.

2.2.3 Reagents and Chemicals Materials AG 1-X2 resin; chloride form, 200-400 mesh Agmatine p-Nicotinamide adenine dinucleotide (NAD’) Carbenicillin Cardiolipin Cholera toxin

Supplier

Cat-No.

BioRad (Hercules, CA)

140- 1251

Sigma (St. Louis, MO) A7127 Boehringer Mannheim (Indianapolis, 127 965 IN) Calbiochem (Sun Diego, CA) 205805 Avanti (Alabaster, AL) 840012 100 List Biologicals (Campbell, CA); or Sigma (St. Louis, MO) 68052 102 Cholera toxin A subunit List Biologicals (Campbell, CA); or 68180 Sigma (St. Louis, MO) Cibacron blue Fluka (Ronkonkoma, NY) 27319 69450-2 Competent BL21 (DE3) E.coli Novagen (Madison, WI) Fattty acid-free BSA Fluka (Ronkonkoma, NY) 05468 Guanosine-5’-0-(2-thioBoehringer Mannheim (Indianapolis, 528 536 diphosphate) (GDPBS) IN) Guanosine-5’-0-(3-thioBoehringer Mannheim (Indianapolis, 220 647 triphosphate) (GTPyS) IN) I PTG Gold Biotechnology (St. Louis, MO) 12481C 15502 or Sigma (St. Louis, MO) 16-8200-19 Microtube strips PGC (Gaithersburg, MD) Myristic Acid Fluka (Ronkonkoma, NY) 70140 [”PI NAD New England Nuclear (Boston, MA) NEG-023 CFA 497 [Adenine-U-14C]NAD Amersham (Arlington Heights, IL) [Carb~nyl-’~C]NAD Amersham (Arlington Heights, IL) CFA-372 Ready Gel Beckman (Fullerton, CA) 158728 230191 Biosepra (Malboraugh, MA) Ultrogel AcA 54 X-OMAT film Kodak (Rochester, NY) 165 1579

2.2.4 Stock Solutions Solutions are prepared in water using the reagents of the highest quality available, unless otherwise stated. They are divided into portions sufficient for 50 assays and stored at -20°C until use. Precipitates in agmatine, DMPC/cholate, and cardiolipin (formed with freezing) can be dissolved by heating to 50°C and vortex mixing.

Activated CTA or LTA: 0.1 mg/ml Potassium phosphate: 0.5 M, pH 7.5 DTT: 1.0M Magnesium chloride: 1.0M SDS: 0.045 % Ovalbumin: 10 mg/ml Agmatine: 0.1 M NAD: 20 m M Guanine nucleotide: 50 mM. Although natural nucleotides can be used, non-hydrolyzable analogues are often preferable, especially when working with samples that may contain nucleotidases. Guanosine-5‘-0-(3-thiotriphosphate) (GTPyS), but not guanosine-5’0-(2-thiodiphosphate) (GDPPS),stimulates ARF activity. Cholera Toxin and Escherichia coli Heat-labileEnterotoxin

DMPCKholate: 30mM DMPC in 2 % sodium cholate. Two percent sodium cholate is prepared in water or 10 m M potassium phosphate buffer, pH 7.5, and DMPC is added to a final concentration of 30 mM, followed by vortex mixing and warming to 37°C until all lipid is dissolved. Cardiolipin: Cardiolipin, Z5 mg/ml, in 10 m M potassium phosphate, pH Z5. Cardiolipin, 7.5mg in a methanol or chloroform solution, is transferred to a glass tube ( 1 2 ~ 7 5 m mand ) the solvent evaporated using a vacuum centrifuge or a stream of N2,followed by the addition of 1 ml of 10 m M potassium phosphate (pH Z5) and vortex mixing until the lipid is dispersed; sonification using a tip probe is required to obtain a uniform suspension without discernible particulates. (Usually 10 to 20seconds at a medium power setting is sufficient.) The solution is stored at -20°C and requires vortex mixing after thawing to produce a uniform suspension.

2.3 Assay 1 :The G,, Assay This assay measures toxin-catalyzed transfer of ADP-ribose from NAD to a specific arginine(s) in G, the heterotrimeric GTP-binding protein subunit that is the stimulatory regulator of adenylyl cyclase. In the assay, the toxin catalyzes the transfer of [32P]ADP-ribosefrom NAD to G, or to other arginine-containing proteins. The reaction is stopped with ice-cold Z5 % TCA, and the precipitated proteins are recovered for SDS-PAGE and autoradiography. In one version of this assay (Weiss etal., 1989), labeled proteins are precipitated and collected on nitrocellulose filters for direct quantification of incorporated ADPribose. Other G,,-based assays (e.g., Schleifer et al., 1982)) which exploit the activation of adenylyl cyclase by ADP-ribosylated G, utilize the reconstitution methods described by Ross and Gilman (1977), in which the ability of ADP-ribosyl-G,, to stimulate adenylyl cyclase activity in cyc-S49 lymphoma cell membrane preparations is assessed.

2.3.1 Additional Reagents and Materials Required ARF protein [32P]NAD(30Ci/mmol; 2 mCi/ml)

G, (or other arginine-containing proteins, see Section 2.7.2) Since G, is a poor substrate for ADP-ribosylation (Graziano etal., 1987, 1989; Toyoshige et al., 1994), the heterotrimeric form ( a P y ) of G, should be used. Crude, detergent-extracted G, , or purified native G,

can be prepared using the methods of Kaslow et al. (Kaslow et al.,

W. A. PATrON et al.

1980) and Northup et al. (Northup etal., 1980), respectively. Recombinant G, (Graziano et al., 1987, 1989) may also be used, but only with the addition of purified Gpy.

7.5 % TCA SDS-BME sample buffer 120 m M Tris, pH 6.8 20 % Glycerol 1 % Sodium dodecyl sulfate (SDS) 5 % 2-mercaptoethanol (BME) 0.05 % Bromphenol blue

2.3.2 Protocol (100pl assay volume) 1. Assays are performed in glass 12x75mm tubes; reagents are added in the order shown in Table 1. Incubate for 60min. at 30°C.

2. After incubation, tubes are placed on ice and reactions terminated by addition of 1 ml of ice-cold 7.5 % TCA and 5 pg of BSA.

the G,, assay

3. Samples are mixed by vortexing and kept on ice for 60 minutes before centrifugation (2300 g, 30 min).

4. The supernatant is aspirated and pellets are rinsed with 250 pI of ice-cold 100 % ethanol to remove residual TCA (optional). Table 1. Components of Assay 1 : NAD:G,, ADP-ribosyltransferaseActivity Component’

Volume

Assay Concentration

Phosphate buffer DMPC/cholate Water

lop1 10pl To volume 0.3 pI 0.5 pl

50 mM 3 mM/0.2 %

DTT Magnesium chloride Guanine nucleotide NAD [32P]NAD G, source ARF protein Activated toxin2 Total Volume

0.4 pI 0.1 pI 0.5 pI

x PI x

10 pl 100 pI

3 mM 5 mM 200 pM 20 pM 2 pCi

1 1 Pg 1 PS

’

Rather than adding individual components to each tube, phosphate buffer and lipid can be added as a mixture, followed by a mixture (of intermediate volume) that contains all components except G, ARF, and activated toxin, and then by successive addition of each of the other components. Addition of components in the order described prevents the precipitation of phosphate and magnesium. The reaction is started by the addition of toxin, followed by vortex mixing.

*

Cholera Toxin and Escherichia coli Heat-labile Enterotoxin

5. Proteins are dissolved in 100 PI of SDS-BME sample buffer with heating at approx. 80°C for 10 min before electrophoresis in 12 % gels. (Samples may turn yellow during heating, especially if pellets were not rinsed, and can be made blue (basic) by the addition of several microliters of 1.0 M Tris.)

6. After electrophoresis, gels are stained with Coomassie blue, destained and equilibrated with 30 % methanol/ 2 % glycerol (to avoid cracking during drying). restoring pH

7. Dried gels are exposed to X-OMAT film (Section2.2.3) at -70°C with intensifying screens.

2.4 Assay 2: The Agmatine Assay This assay measures toxin-catalyzed transfer of ADP-ribose to agmatine, an analogue of arginine lacking the carboxyl moiety, using [14Cadenine]NAD. The reaction product, ['4C]ADP-ribosylagmatine,has a net charge of zero and is eluted from an AG 1-X2 anion-exchange column with water, while the substrate, [14C]NAD, is retained. ['4C]ADP-ribosylagmatineis quantified by liquid scintillation counting. Other simple guanidino compounds such as arginine methyl ester can also be utilized in this assay (Moss and Vaughan, 1977), provided a suitable method is used to isolate the ADP-ribosylated product.

2.4.1 Additional Reagents and Materials Required [Adenine-U-14C]NAD(281mCi/mmole; 25 vCi/ml)

ARF protein AG 1-X2 resin The resin is equilibrated three times with two volumes of distilled/ deionized water. A 1 :1 resin:water slurry is used to prepare columns.

Plexiglass column rack with disposable columns We use a rack designed (in house) for 100 5Y4-inch Pasteur pipettes that fits over a l o x 10 tray of 20-ml scintillation vials such that eluate from each column is collected in a vial. Each pipette (containing a glass wool plug) is filled with AG 1-X2 slurry up to the constriction in the glass using a syringe fitted with a blunted wide-bore spinal needle or a piece of stiff tubing. The needle is used to compact the glass wool at the bottom of the pipette and the column is filled as the needle is withdrawn. Before use, each column is washed with 1 ml of distilled/ deionized water using a repeating pipet. Each column contains 1-1.5 ml of packed resin that quantitatively retains NAD while allowing greater than 99 % recovery of ADP-ribosylagmatine. W. A. PATrON et al

Gel-type scintillation mixture (e.g., Ready Gel)

A gel-type fluid that allows counting of samples with a high aqueous content is required, as 5 ml of water are used to elute each column.

2.4.2 Protocol (150 PI assay) 1. Assays are set-up in triplicate using either 12x75mm tubes (glass and polystyrene work equally well) or 1.1-ml Microtube strips (see Section 2.2.3). Components are added to each tube in the order shown in Table 2. Incubate for 60 min. at 30°C.

the agmatine assay

2. After incubation, tubes are placed in an ice-water bath to slow the reaction. Duplicate 70-pI samples from each tube are transferred to separate columns to yield duplicate measurements for each of the triplicate assays.

3. Each column is eluted five times with 1 ml of water using a repeating dispenser, followed by the addition of sufficient scintillation fluid to generate a sample suitable for counting. Table 2. Components of Assay 2: NAD:agmatine ADP-ribosyltransferase Activity Component’

Volume

Assay Concentration

15yl

50 m M 0.5 mg/ml

~~

Phosphate buffer Cardiolipin Water Agmatine Ovalbumin

10yl To volume 15yl 1.5 yI

DTT

3 CII

Magnesium chloride Guanine nucleotide NAD [adenine-I4C]NAD ARF protein Activated toxin’ Total Volume

1.5 yI

0.3 yl 0.75 yl 1 CII

x

lop1

lOmM

0.1 mg/ml 20 m M 5 mM 100 p M 100 p M 0.05 pCi 1 CLg 1 yg

150 pI

’

Rather than adding individual components to each tube, phosphate buffer and lipid can be added as a mixture, followed by a mixture (of intermediate volume) that contains all components except ARF and activated toxin, and then by successive addition of each of the other components. Addition of components in the order described prevents the precipitation of phosphate and magnesium. The reaction is started by the addition of toxin, followed by vortex mixing. When an assay contains a large number of tubes, it is prudent to stagger starting times by approx. 30sec, consistent with equal incubation times for all tubes, to allow the time needed to transfer samples to columns.

’

Cholera Toxin and Escherichio coli Heat-labile Enterotoxin

2.5 Assay 3: Auto-ADP-ribosylation Assay This assay, which does not require the addition of lipids or detergent, measures the ability of CT or LT to ADP-ribosylate itself in the absence of another ADP-ribose acceptor (except water). ARF added to this assay will also be ADP-ribosylated, but the ADP-ribosylation of it or CT under these conditions does not appear to decrease CT activity or the ability of ARF to stimulate CT (Tsai et al., 1991). Reaction products are separated by SDS-PAGE and analyzed by autoradiography as described for Assay 1 (Section2.3).This assay is the most sensitive of all those listed here and can be used to verify toxin activity observed in other assays.

2.5.1 Additional Reagents and Materials Required [32P]NAD(30Ci/mmol; 2 mCi/ml) ARF protein

7.5 % TCA SDS-BME sample buffer

2.5.2 Protocol (100 pl assay) 1. This assay (like Assay 1) is carried out in 12x75 mm glass tubes; reagents are added in the order shown in Table 3. Incubate for

60 min. at 30°C. auto-ADP-ribosylation assay

2. Electrophoresis and autoradiography, are carried out as described for Assay 1. Table 3. Components of Assay 3: Toxin Auto-ADP-ribosylationActivity Component’

Phosphate buffer Water

DTT Magnesium chloride Guanine nucleotide

NAD [32P]NAD ARF protein Activated toxin’ Total Volume

’

Volume

Assay Concentration

lop1

50 mM

To volume 0.3 pI 0.5 p1 0.4 pI

0.1 pI 0.5 pI

x PI

10pI 100 pl

3 mM 5 mM 200 pM 20 pM 2 pCi

1 1 Pg

Rather than adding individual components to each tube, a mixture that contains all components except ARF and activated toxin can be added, followed by the successive addition of each of the remaining components. Addition of components in the order described prevents the precipitation of phosphate and magnesium. Reaction is started by the addition of toxin, followed by vortex mixing.

’ W. A. PATTON et 01.

2.6 Assay 4: NAD Glycohydrolase Assay This assay measures the ability of CTA or LTA to act as an NADase (first described by Moss et al. (1976)),catalyzing cleavage of the glycosidic bond between the ADP-ribose and the nicotinamide moieties of NAD. Products of the reaction are separated on AG 1-X2 columns and quantified as described for Assay 2 (Section 2.4).

2.6.1 Additional Reagents and Materials Required [~arbonyI-’~C]NAD (53 mCi/mmol; 50 pCi/ml) AG 1-X2 resin, columns, and column rack ARF protein 2.6.2 Protocol (150 pl assay) 1. Assays are performed in triplicate using either 12x75 mm tubes (glass and polystyrene work equally well) or 1.1-ml Microtube strips (Section 2.2.3). Components are added to each tube in the order shown in Table 4. Incubate for 60 min. at 30°C.

NAD glycohydrolase

2. After the incubation, duplicate samples from each tube are transferred to AG 1-X2 columns and handled further as described in Assay 2. Table 4. Components of Assay 4: NAD glycohydrolase Activity Component’

Volume

Assay Concentration

Phosphate buffer Cardiolipin Water Ovalbumin DTT Magnesium chloride Guanine nucleotide NAD [~arbonyl-’~C]NAD ARF protein Activated toxin2 Total Volume

15pl 10 pl To volume 1.5 pI 3 PI 1.5 pI 0.3 pI 0.3 pl

50 m M 0.5 mg/ml

1 PI

x PI

10 pI 150 pl

0.1 mg/ml 20 m M 5 mM 100 p M 40 p M 0.025 pCi 1 Yg 1 Pg

’

Rather than adding individual components to each tube, phosphate buffer and lipid can be added as a mixture, followed by a mixture (of intermediate volume) that contains all components except ARF and activated toxin, and then by successive addition of each of the other components. Addition of components in the order described prevents the precipitation of phosphate and magnesium. The reaction is started by the addition of toxin, followed by vortex mixing. When an assay contains a large number of tubes, it is prudent to stagger starting times by approx. 30 sec, consistent with equal incubation times for all tubes, to allow the time needed to transfer samples to columns. Cholera Toxin and Escherichia coli Heat-labile Enterotoxin

2.7 Comments and Considerations 2.7.l Appropriate Controls and Analysis of Data controls

use of different

concentrations of ARF

2.2 7.7 Controls All assays described here should include controls for each sample tested (i.e.,+/- toxin, +/- ARF, +/- buffer (for added ARF or other component), + GTPyS, and + GDPPS) to ensure that any differences are due to the presence of a specific component. When using these assays to test for ARF activity, it is important to use several concentrations of ARF to establish that the effect is concentration-dependent, as well as GTP (or GTPyS)-dependent. In the NAD glycohydrolase assay, it is especially important when testing impure samples of toxin or ARF to run a control without toxin as the sample itself may contain NADase, which is ubiquitous in animal tissues.

run control without t

2.2 7.2 Data analysis

quantification

Radiolabeled bands on autoradiographs from Assays 1 and 3 can be quantified by densitometry and the activity of each sample graphed in arbitrary units. For Assays 2 and 4, counts in ADP-ribosylagmatine or nicotinamide are quantified directly and are converted to nanomoles of AD P- rib0 sy Ia g matine or nicot ina mide formed/ ho ur/ mg protein, respectively, based on the specific activity of NAD in the assay.

2.7.2 Interfering Substances, Troubleshooting, and Assay Optimization 2.22.7 Interfering substances

minimize effects of contaminating enzymes

W. A. PAnON et al.

The presence of two groups of contaminating enzymes in these assays, due to a need to use impure toxin, G, or ARF proteins, may complicate the interpretation of data. The effects of the first group, NADases and pyrophosphatases, which decrease substrate concentrations, can be minimized by the addition of 0.5mM Cibacron blue and 0.5 m M ATP, respectively. NADases can also be inhibited by isonicotinic acid hydrazide or DTT (Gilland Coburn, 1988); other analogs of NAD (Slama and Simmons, 1989)will also inhibit contaminating NADase activity, but may also interfere with the activities of the toxin. The second group of enzymes, ADP-ribosylarginine hydrolases, may reduce the amount of ADP-ribosyl product. ADP-ribosylarginine hydrolase activity can be inhibited by the addition of ADP-ribose to the assay.

2.22.2 Troubleshooting Problems in these assays that cannot be attributed to interfering NADases or pyrophosphatases are usually attributable to a single component (i.e. inactive toxin or ARF) and can be identified using data from the controls. When wild-type CT or LT display reduced (or zero) basal activity, one of several conditions may be the cause. It is possible that the toxin was not properly activated, and a new sample of toxin should be activated, using freshly prepared DTT. It is possible that the concentration of NAD in the assay was too low and was hydrolyzed. Because NAD in solution is susceptible to hydrolysis, it should be stored in small portions at -20°C and the stock supply replaced regularly. Mutant toxins may, in fact, be inactive and it is suggested that Assays 3 and 4 be used to confirm or negate this possibility. CT activity may also be affected by salt and/or protein concentrations in the assay. Excessive salt can stimulate toxin activity; buffer controls should, therefore, be run in each assay. High protein concentrations may result in decreased or variable toxin activity; in Assay 2, the presence of ovalbumin minimizes nonspecific protein effects. ARF stimulation of toxin activity is also subject to several types of interference. When an ARF does not stimulate toxin activity in these assays, Assay 2 is used to decide whether that ARF preparation is active. It is suggested that nonhydrolyzable nucleotides be used to minimize problems due to the presence of nucleotidases or pyrophosphatases in the assay. If Assay 2 confirms inactivity, be certain that agmatine and lipid stocks were completely dispersed before use. The GTP-binding ability of the ARF can be verified using a nitrocellulose filter-binding assay (e.g., Northup et al., 1982) that measures [35S]GTPySbinding to ARF under assay conditions. It is possible that an ARF or ARF mutant may be active in Assay 2 and not Assay 1. It has been suggested that this may be due to the ability of that ARF to stimulate modification of that particular substrate (Randazzo et al., 1994). The use of alternative substrate sources or substrates for ADPribosylation in Assays 1 and 2 may result in apparently lower activity. For Assay 1, arginine-containing proteins (other than Gsa)or membrane preparations that contain G, and ARF can be substituted for G, but the amount of labeling may be greatly reduced, depending on protein concentration or the susceptibility of a particular protein to modification. It should be noted that not all arginine residues in native proteins are modified, as they must be accessible to the ADP-ribosylating enzyme (e.g., Moss et al., 1990).Thus, the arginine content of a protein may not necessarily reflect its capacity to be ADP-ribosylated.

troubleshooting

activate new toxin sample

replace NAD stock regularly

effect of concentration on activity

assay for activity of ARF

2.22.3 Assay optimization The need to modify a particular assay may arise when CT is not satisfactorily stimulated by a particular ARF protein, although the ARF is active in Assay 2 and binds nucleotide as effectively as a control ARF (e.g., rARF1). Problems center most commonly around the nucleotide Cholera Toxin and Escherichia coli Heat-labile Enterotoxin

concentration

concentration or the use of a particular lipid. For any of the mammalian ARFs, 200 pM nucleotide is suitable. Its use should circumvent the possibility that a particular ARF is not capable of high affinity guanine nucleotide binding (e.g., Bobak et al., 1990). The lipid of choice for optimal activity in Assay 1 is DMPCkholate, and for Assays 2 and 4 is cardiolipin. SDS (0.003 %) or 3 mM DMPC in 0.2 % cholate can also be used in Assays 2 and 4. As demonstrated by Price et al. (1992),optimal lipid and nucleotide conditions differ for different ARFs. With no way to predict the optimal choice for either component, it best to determine it empirically.

choice of lipid

2.7.3 Considerations for the Use of ARF

2.X3.1

Lipid/Detergent and Nucleotide Requirements

ARF activity in any of these assays is strongly dependent on the presence of lipid and nucleotide in the assay, with the exception of mammalian ARF6 and several mutant ARF molecules with an altered or deleted amino terminus. Lipids and detergents at non-denaturing concentrations influence ARF directly by enhancing the exchange and binding of guanine nucleotides to ARF, as well as toxin and ARF, by influencing the formation of ARF-toxin complexes (Tsai et al., 1991).The addition of ARF to CT decreases the apparent K, for NAD substantially, without affecting;,V ,, the addition of 0.003 % SDS also results in a further decrease in Km and an increase in,V ,, (Noda et al., 1990). Lipids may also play a role in the action of toxin in vivo as the addition of lipid shifts the optimal temperature for the CT-catalyzed NAD:agmatine ADP-ribosyltransferase reaction towards physiological temperatures (Murayama et al., 1993). Lipids are postulated to exert their effects by facilitating the exchange of GDP for GTP (by effects on the ARF amino terminus (Randazzo et al., 1995)),which in turn, causes ARF, especially when myristoylated (Franco et al., 1995) to interact with the lipid membrane (Walker et al., 1992). The effects of certain lipids on nucleotide exchange may be due, in part, to their acidic nature (e.g., Terui et al., 1994) that favors interaction with a positively charged patch on the surface of ARF, of which residues from the amino terminus are a part (Amor et al., 1994). There have been several situations, however, in which lipid effects on ARF activity were minimal, or the addition of a standard lipid concentration significantly decreased the nucleotide requirement for that ARF. In all of these instances, there were modifications of the amino terminus, either addition of sequence (Welsh et al., 1994b), absence of sequence present in other ARFs (Price et al., 1992), replacement with sequence that was predicted to form a similar structure (Hong et al., 1994), or deletion (Hong et al., 1994, 1995). These ARFs had a lower requirement for added GTP than did most other ARFs. They were isolated in an active GTP-bound state (Welsh et al., 199413; Hong et al., 1995) or were active without bound nucleotide (Hong etal., 1994,1995). W. A. PATTON et a1

2.Z3.2 Development of other Assay Conditions It is sometimes necessary to modify the conditions of the assays described here. In one specific example, conditions of the agmatine assay were changed to allow the assay of a second enzyme, phospholipase D (PLD), under identical conditions (Massenburg et a/., 1994).With the identification of ARF as an activator of PLD (Brown et a/., 1993; Cockcroft et a/., 1994)) it was desired to determine which mammalian ARFs activated PLD, and to identify ARF domains that were involved in the activation of CT and PLD. It was first determined that each ARF sample was active in Assay 2 in the presence of cardiolipin; each ARF was then assayed for its ability to stimulate PLD in a separate assay (Massenburg et a/., 1994).To compare the activities of ARF proteins in the CT and PLD assays, we used conditions under which both enzymatic activities could be measured. With the buffer conditions and sonified lipid vesicles of the composition required for the [3H-choline]-release assay used to measure PLD activity, all reagents required for Assay 2 were added to create a dual assay system to determine the formation of ADP-ribosylagmatine determined as described above. To perform the PLD assay, radiolabeled vesicles of the same composition replaced the non-radiolabeled vesicles and choline release was determined. Although these conditions resulted in a lower ARF stimulation of CT activity than observed in Assay 2, they allowed direct comparison of ARF activation of CT and PLD. In other experiments utilizing these conditions, we took advantage of a human ARF-like protein 1 (human ARLl or ARL1) that is relatively inactive towards CT and is only slightly active towards PLD, to identify domains in human ARFl that are involved in the activation of CT and PLD. ARFl and ARLl each contain 181 amino acids and are 53% identical in sequence. Chimeric proteins were synthesized, in which the amino and carboxyl termini (amino acids 1 to 73 and 74 to 181) were switched (Zhang et a/., 1995). Using the combination assay, a domain responsible for PLD activation was demonstrated in amino acids 1 to 73 of ARF, whereas amino acids 73 to 181 of ARF were responsible for CTA activation.

assay in presence of phospholipase D

References Amor J, Harrison D, Kahn R, et al. (1994):Structure of the human ADP-ribosylation factor 1 complexed with GDP In Nature 372:704-708. Bobak DA, Bliziotes MM, Noda M, et al. (1990):Mechanism of activation of cholera toxin by ADP-ribosylation factor (ARF): Both low- and high-affinity interactions of ARF with guanine nucleotides promote toxin activation. In Biochemistry 29:855 -861. Brown HA, Gutowski S, Moomaw CR, etal. (1993):ADP-ribosylation factor, a small GTP-dependent regulatory protein, stimulates phospholipase D activity. In Cell 75: 1137- 1144. Clements JD, Finkelstein RA (1979): Isolation and characterization of homogeneous heat-labile enterotoxins with high specific activity from Escherichia coli cultures. In Infect. Immun. 24:760-769. Cholera Toxin and Escherichia coli Heat-labile Enterotoxin

Cockcroft S, Thomas GMH, Fensome A, et al. (1994): Phospholipase D: A downstream effector of ARF in granulocytes. In Science 263523-526. Donaldson JG, Klausner RD (1994):ARF: A key regulatory switch in membrane traffic and organelle structure. In Curr. Op. Cell Biol. 6527-532. Finkelstein RA, LoSpalluto JJ (1969):Pathogenesis of experimental cholera: Preparation and isolation of choleragen and choleragenoid. In J. Exp. Med. 130:185-202. Franco M, Chardin P, Chabre M, et al. (1995):Myristoylation of ADP-ribosylation factor 1 facilitates nucleotide exchange at physiological Mg” levels. In J. Biol. Chern. 270: 1337- 1341. Gill DM, Coburn J (1988): ADP-ribosylation of membrane proteins by bacterial toxins in the presence of NAD glycohydrolase. In Biochim. Biophys. Acta 954165-72. Graziano MP, Cosey PJ, Gilman AG (1987):Expression of cDNAs for G proteins in Escherichia coli: Two forms of G, stimulate adenylate cyclase. In J. 6iol. Chem. 262~11375-11381. Graziano MP, Freissmuth M, Gilman AG (1989): Expression of G, in Escherichia coli. In J. Biol. Chern. 264:409-418. Hammond SM, Altshuller YM, Sung T-C, et al. (1995): Human ADP-ribosylation factor-activated phosphatidylcholine-specific phospholipase D defines a new and highly conserved gene family. In J. 6i0l. Chem. 270:29640-29643. Haun R, Tsai S-C, Adamik R, et al. (1993): Effect of myristoylation on GTPdependent binding of ADP-ribosylation factor to Golgi. In J. Biol. Chern. 268:7064-7068. Hong J-X, Haun RS, Tsai S-C, et al. (1994):Effect of ADP-ribosylation factor aminoterminal deletions on its GTP-dependent stimulation of cholera toxin activity. In J. Biol. Chern. 269:9743-9745. Hong J-X, Zhang X, Moss J, et al. (1995): Isolation of an amino-terminal deleted recombinant ADP-ribosylation factor 1 in an activated nucleotide-free state. In Proc. Natl. Acad. Sci. USA 92:3056-3059. Kahn RA, Gilman AG (1984): Purification of a protein cofactor required for ADPribosylation of the stimulatory regulatory component of adenylate cyclase by cholera toxin. In J. 6iol. Chern. 259:6228-6234. Kahn RA, Goddard C, Newkirk M (1988):Chemical and immunological characterization of the 21-kDa ADP-ribosylation factor of adenylate cyclase. In J. 6i0l. Chem. 263:8282-8287. Kaslow HR, Johnson GL, Brothers VM, et al. (1980):A regulatory component of adenylate cyclase from human erythrocyte membranes. In J. Biol. Chern. 25513736-3741. Ktistakis NT, Brown HA, Sternweis PC, et al. (1995):Phospholipase D is present on Golgi-enriched membranes and its activation by ADP-ribosylation factor is sensitive to brefeldin A. In J. 6i0l. Chern. 92:4952-4956. Kunkel SL, Robertson DC (1979):Purification and chemical characterization of the heat-labile enterotoxin produced by enterotoxigenic Escherichia coli. In Infect. Irnrn un. 25 :586 -596. Kunz BC, Muczynski KA, Welsh CF, et al. (1993):Characterization of recombinant and endogenous ADP-ribosylation factors synthesized in Sf9 insect cells. In 6iochemistry 32:6643-6648. Lai CY, Mendez E, Chang D (1976):Chemistry of cholera toxin: The subunit structure. In J. Infect. Dis. 133:S23-S30. Massenburg D, Hun J-S, Liyanage M, etal. (1994):Activation of rat brain phospholipase D by ADP-ribosylation factors 1,5, and 6: Separation of ADP-ribosylation factor-dependent and oleate-dependent enzymes. In Proc. Natl. Acad. Sci. USA 91 :11718-11722. Mekalanos JJ, Collier RJ, Romig WR (1978): Purificationof cholera toxin and its subunits: New methods of preparation and the use of hypertoxinogenic mutants. In Infect. Irnmun. 20:552-558. Mekalanos JJ, Collier RJ, Romig WR (1979): Enzymatic activity of cholera toxin: II. Relationships to proteolytic processing, disulfide bond reduction, and subunit composition. In J. Biol. Chem. 2545855-5861. W. A. PATTON et al.

Merritt EA, Hol WG (1995):AB5toxins. In Curr. Opin. Struct. Biol. 5:165-171. Moss J, Manganiello VC, Vaughan M (1976): Hydrolysis of nicotinamide adenine dinucleotide by chaleragen and its A protomer: Possible role in the activation of adenylate cyclase. In Proc. Natl. Acad. Sci. USA 73:4424-4427. Moss J, Stanley SJ, Levine RL (1990):Inactivation of bacterial glutamine synthetase by ADP-ribosylation. In J. Biol. Chem. 265:21056-21060. Moss J, Stanley SJ, Vaughan M, etal. (1993):Interaction of ADP-ribosylation factor with Escherichia coli enterotoxin that contains an inactivating lysine-112 substitution. In J. Biol. Chem. 268:6383-6387. Moss J, Vaughan M (1977):Mechanism of action of choleragen: Evidence for ADPribosyltransferase activity with arginine as an acceptor. In J. EGO/. Chem. 252:2455-2457. Moss J, Vaughan M, (1992)Activation of cholera toxin by ADP-ribosylation factors. In Natural Toxins: Toxicology, Chemistry and Safety (Keeler RF, Mandava NB, Tu AT eds) pp 266-282, Fort Collins, CO: Alaken, Inc. Moss J, Vaughan M (1995):Structure and function of ARF proteins: Activators of cholera toxin and critical components of intracellular vesicular transport processes. In J. Biol. Chem. 270: 12327-12330. Murayama T, Tsai S-C, Adamik R, et a/. (1993): Effects of temperature on ADPribosylation factor stimulation of cholera toxin activity. In Biochemistry 32561-566. Murtagh J, Mowatt M, Lee C-M, etal. (1992):Guanine nucleotide-binding proteins in the intestinal parasite Giardia lamblia. In J. Biol. Chem. 267:9654-9662. Noda M, Tsai S-C, Adamik R, et a/. (1990):Mechanism of cholera toxin activation by a guanine nucleotide-dependent 19-kDa protein. In Biochim. Biophys. Acta 1034:195- 199. Northup JK, Smigel MD, Gilman AG (1982):The guanine nucleotide activating site of the regulatory component of adenylate cyclase: Identification by ligand binding. In J. Biol. Chem. 257:11416-11423. Northup JK, Sternweis PC, Smigel MD, et al. (1980):Purification of the regulatory component of adenylate cyclase. In Proc. Natl. Acad. Sci. USA 77:6516-6520. Ohtomo N, Muraoka T, Tashiro A, et a/. (1976):Size and structure of the cholera toxin molecule and its subunits. In J. Infect. Dis. 133:S31-S40. Pearson SD, Dixon JD, Nothwehr SF, et al. (1986):Isolation of high-specific activity subunits of cholera toxin by reversed-phase high-performance liquid chromatography. In J. Chrom. 359:413-421. Price SR, Welsh CF, Haun RS, et al. (1992): Effects of phospholipid and GTP on recombinant ADP-ribosylation factors (ARFs): Molecular basis for differences in requirements for activity of mammalian ARFs. In J. Biol. Chem. 267: 17766-17772. Randazzo PA, Terui T, Sturch S, et al. (1994):The amino terminus of ADP-ribosylation factor (ARF) 1 is essential for the interaction of G, and ARF GTPase-activating protein. In J. Biol. Chem. 269:29490-29494. Randazzo PA, Terui T, Sturch S, et a/. (1995):The myristoylated amino terminus of ADP-ribosylation factor 1 is a phospholipid- and GTP-sensitive switch. In J. Biol. Chem. 270: 14809- 14815. Randazzo PA, Weiss 0, Kahn RA (1992): Preparation of recombinant ADPribosylation factor. In Meth. Enzym. 219:362-369. Rappaport RS, Sagin J F, Pierzchala WA, et al. (1976): Activation of heat-labile Escherichia coli enterotoxin by trypsin. In J. Infect. Dis. 133:S41-S54. Ross EM, Gilman AG (1977): Reconstitution of catecholamine-sensitive adenylate cyclase activity: interaction of solubilized components with receptor replete membranes. In Proc. Natl. Acad. Sci. USA 74:3715-3719. Schleifer LS, Kahn RA, Hanski E, et al. (1982): Requirements for cholera toxindependent ADP-ribosylation of the purified regulatory component of adenylate cyclase. In J. Biol. Chem. 257:20-23. Slama JT, Simmons AM (1989):Inhibition of NAD glycohydrolase and ADP-ribosyl transferases by carbocyclic analogues of oxidized nicotinamide adenine dinucleotide. In Biochemistry 28:7688-7693. Spangler BD (1992):Structure and function of cholera toxin and the related Escherichia coli heat-labile enterotoxin. In Microbiol. Rev. 56:622-647. Cholera Toxin and Escherichia

coli Heat-labile Enterotoxin

Spangler BD, Westbrook EM (1989):Crystallizationof isoelectrically homogeneous cholera toxin. In Biochemistry 28:1333-1340. Stearns T, Willingham MC, Botstein D, et al. (1990~):ADP-ribosylation factor is functionally and physically associated with the Golgi complex. In Proc. Natl. Acad. Sci. USA 87: 1238- 1242. Stearns T, Kahn RA, Botstein D, et al. (1990b):ADP-ribosylation factor is an essential protein in Saccharomyces cerevisiae and is encoded by two genes. In Mol. Cell. Biol. 10:6690-6699. Tayot J-L, Holmgren J, Svennerholm L, et al. (1981): Receptor-specific large-scale gangliopurification of cholera toxin on silica beads derivatized with IYSOGM, side. In Eur. J. Biochem. 113:249-258. Tayot J-L, Tardy M (1980): Isolation of cholera toxin by affinity chromatography on porous silica beads with covalently coupled ganglioside GM1. In Adv. Exp. Med. Biol. 125~471-478. Terui T, Kahn R, Randazzo P (1994): Effects of acid phospholipids on nucleotide exchange properties of ADP-ribosylation factor 1. In J. Biol. Chem. 269:28130-28135. Toyoshige M, Okuya S, Rebois RV (1994):Choleragen catalyzes ADP-ribosylation of the stimulatory G protein heterotrimer, but not its free a-subunit. In Biochemistry 33:4865-4871. Tsai S-C, Adamik R, Moss J, et al. (1991):Guanine nucleotide-dependent formation of a complex between choleragen (cholera toxin) A subunit and bovine brain ADP-ribosylation factor. In Biochemistry 30:3697-3703. Tsai S-C, Noda M, Adamik R, et al. (1988):Stimulation of choleragen enzymatic activities by GTP and two soluble proteins purified from bovine brain. In J. Biol. Chem. 263: 1768- 1772. Tsuchiya M, Price SR, Tsai S-C, et al. (1991): Molecular identification of ADPribosylation factor mRNAs and their expression in mammalian cells. In J. Biol. Chem. 266:2772-2777. Uesaka V, Otsuka Y, Lin Z, et al. (1994):Simple method of purification of Escherichia coli heat-labile enterotoxin and cholera toxin using immobilized galactose. In Microb. Pathogen. 16:71-76. Walker MW, Bobak DA, Tsai S-C, et al. (1992):GTP but not GDP analogues promote association of ADP-ribosylation factors, 20-kDa protein activators of cholera toxin, with phospholipids and PC-12 cell membranes. In J. Biol. Chem. 267:3230- 3235. Weiss 0, Holden J, Rulka C, et al. (1989): Nucleotide binding and cofactor activities of purified bovine brain and bacterially expressed ADP-ribosylation factor. In J. Biol. Chem. 264:21066-21072. Welsh CF, Moss J, Vaughan M (1994~):ADP-ribosylation factors: a family of approx. 20-kDa guanine nucleotide-binding proteins that activate cholera toxin. In Mol. Cell. Biochem. 138:157- 166. Welsh CF, Moss J, Vaughan M (1994b): Isolation of recombinant ADP-ribosylation factor 6, an approx. 20-kDa guanine nucleotide-binding protein, in an activated GTP-bound state. In J. Biol. Chem. 269:15583-15587. Zhang G-F, Patton WA, Lee F-JS, et al. (1995):Different ARF domains are required for the activation of cholera toxin and phospholipase D. In J. Biol. Chem. 270:21-24.

W. A. PATTON et a1

Bacterial Toxins

Klaus Aktories Copyright 0 2002 WILEY-VCH Verlag GmbH & Co. KGaA

Pertussis Toxin C. LOCHT and R. ANTOINE

3.1 Introduction Among the various bacterial toxins, pertussis toxin (PT) has the most complex structure known so far. It is composed of five dissimilar subunits, named S 1 - S . PT is a member of the A-B toxins, in which the A moiety, composed here of the S1 subunit, is an enzyme, and the B moiety, composed of subunits S2-S5, constitutes the target-cell receptor-binding portion. The B oligomer of PT can in turn be divided into two dimers, named D1 and D2, composed of subunits S2-S4 and subunit S3-S4, respectively (Tamura et a/., 1982).The molecular steps of PT action include: (i) binding of the toxin to the target-cell receptors via the B oligomer, with specific involvement of D1 or D2; (ii) membrane translocation of the enzymatically active S1 subunit; and (iii) expression of the ADP-ribosyltransferase activity catalyzed by the internalized S1 subunit. All three steps are required for the full expression of most - but not all - biological activities of PT. Depending on the target cell, the physiological effects of PT may vary tremendously. These effects are responsible for most of the systemic features of whooping cough, the disease caused by Bordetella pertussis, the micro-organism that produces PT (Pittman, 1984). Accordingly, the existence of PT was first predicted by the expression of its numerous biological activities detected after infection with 6. pertussis or administration of whole cell pertussis vaccines. These activities include histamine sensitization, islet activation, induction of leukocytosis, immunopotentiation and many others (Munoz, 1985). Some of the pharmacological effects of whole cell pertussis vaccines were recognized as early as the 1940s (Parfentiev and Goodline, 1948). However, considering the wide diversity of biological activities of pertussis vaccines, they were initially believed to be caused by distinct 6. pertussis products. Many workers therefore attempted to separate the different activities, but without success (Munoz and Bergman, 1977). In the late 1970s several independent groups managed to purify to a high degree what is now known as pertussis toxin. Today the most widely used purification procedure is that developed by Sekura et al. (1983).The purification of this substance in a crystalline form allowed one to conclude that the diverse biological activities are all caused by a single protein (Munoz et al., 1981).This

structure of pertussis toxin

biological activities of pertussis toxin

K. Aktories (Ed.),Bacterial Toxins. 0 Chapman & Hall, Weinheim, 1997. ISBN 3-8261-0080-8

toxin is distinct, however, from other toxic molecules produced by B. pertussis, such as endotoxin, dermonecrotic toxin, adenylate cyclase toxin and tracheal cytotoxin. The oligomeric nature of PT was then soon recognized, and the toxin was included in the growing family of A-B toxins (Tamura et al., 1982). It was also found that the A moiety of PT expresses an ADP-ribosyltransferase activity, using NAD' and the a subunits of a class of G proteins as donor and acceptor substrates, respectively (Katada and Ui, 1982). The cloning (Locht et al., 1986, Nicosia et al., 1986) and sequencing (Locht and Keith, 1986; Nicosia et al., 1986) of the genes encoding the PT subunits, and especially the recent determination of the crystal structure of the toxin (Stein et al., 1994a), confirmed and firmly established the quaternary structure of the protein. The homology to other toxins, such as cholera toxin, also confirmed that PT may be included in the family of ADP-ribosylating toxins.

3.2 Genetic Regulation of Pertussis Toxin Production

transactivating factors

C. LOCHT and R. ANTOINE

The genes encoding all five PT subunits are arranged in a polycistronic operon on the Bordetella chromosome (Locht and Keith, 1986; Nicosia et al., 1986). Interestingly, other members of the Bordetella genus, such as Bordetella parapertussis and Bordetella bronchiseptica, also contain the full set of ptx genes (Marchitto et al., 1987). However, only 6. pertussis expresses these genes. The other two species have accumulated several inactivating mutations in the ptx promoter region (Arico et al., 1987))raising the questions of a possible negative selective pressure of PT, and of the origin of the ptx genes. The expression of the ptx genes requires a transactivating factor, named BvgA. This transactivator is part of a two-component system composed of the sensor BvgS and the activator BvgA (for review, see Uhl and Miller, 1995). PTX is coordinately produced together with other 6. pertussis virulence factors. All known virulence factors are under the control of the same BvgA/S two-component system. BvgS is an inner-membrane protein with two transmembrane domains, a large periplasmic domain and a large cytoplasmic domain. Genetic studies have suggested that BvgS functions as a dimer in the active configuration. Although the signals that are sensed by BvgS in vivo are not known, it can be reversibly inactivated by low temperature, or by the addition of modulators to the culture medium, a phenomenon known as phenotypic modulation (Lacey, 1960).In the active form, the cytoplasmic portion of BvgS autophosphorylates itself, and then serves as a phosphate donor for the phosphorylation of BvgA. Frameshift mutations in BvgS result in PT- B. pertussis strains. Such mutations may occur at relatively high frequencies by insertion or deletion of a C in a run of six C residues (Stibitz et al., 1989), and are known as phase variations. BvgA is a cytoplasmic protein with an N-terminal receiver domain and a C-terminal DNA-binding domain. In the active form, this pro-

tein also functions as a dimer (Scarlato et al., 1990; Boucher et al., 1994).Phosphorylation of BvgA by phosphorylated BvgS or by acetylphosphate increases the affinity of the protein for target DNA in the promoter regions of the Bordetella virulence genes (Boucher et al., 1994).Although so far all the identified virulence genes are regulated by BvgA, the requirement of BvgA phosphorylation.for DNA binding is stronger for some virulence genes than for others (Boucher and Stibitz, 1995). This initially led people to believe that expression of some virulence genes, such as the ptx genes, might require accessory factors in addition to BvgA. However, very recent evidence indicated that the BvgAS activity is sufficient for activation of the pfx genes (Uhl and Miller, 1995)and that, unlike for certain other virulence genes, phosphorylation of BvgA is an absolute requirement for binding of the activator synergistically with RNA polymerase to the ptx promoter (Boucher and Stibitz, 1995).However, this does not exclude possible additional help by other factors for optimal transactivation of the ptx genes (DeShazer et al., 1995).Finally, the DNA topology may also play an enhancing role in ptx gene expression (Scarlato et al., 1993).

3.3 Biogenesis of Pertussis Toxin The individual PT subunits are produced as pre-proteins containing typical signal peptides at their N-terminal extremities (Locht and Keith, 1986; Nicosia et al., 1986).Although the B. pertussis sec genes have not yet been identified, it is likely, considering the similarities of the PT signal peptides to standard signal peptides, that the individual subunits are translocated through the inner membrane by a Sec-dependent mechanism. The different subunits are assembled into the holotoxin molecule in the periplasm of the organism. This assembly produces a B oligomer composed of two copies of S4 and one copy each of S2,S3 and S5, arranged as a ring in the following order : S2-S4-S3-S4-S5 (Stein et al., 1994~). Although S2 and S3 share about 70 % amino acid identity, the wild type holotoxin always appears to incorporate the S2 and S3 subunits at their correct positions. The association of the S1 subunit with the B oligomer requires the intramolecular disulfide bond of S1 and its C-terminal domain (Antoine and Locht, 1990).However, the secretion of the toxin does not require the presence of S1. S1 alone cannot be secreted by B. pertussis, but the assembled B oligomer is secreted even in the absence of S1, indicating that the secretion determinants are located in the B oligomer. Nevertheless, even in the unsecreted form, the assembled toxin in the periplasm is fully active. The expression of maximal virulence of B. pertussis in animal models, however, requires not only the production, but also the secretion of PT (Weiss and Goodwin, 1989). PT secretion through the outer membrane depends on the expression of accessory genes located directly downstream from the five structural genes (Weiss et al., 1993).These genes, named ptl genes

assembly of the holotoxin

secretion of pertussis toxin

Pertussis Toxin

(for pertussis toxin liberation) are under the control of the ptx promoter and probably constitute, with the ptx genes, a single polycistronic operon, composed of the five ptx genes followed by eight ptl genes (Kotob et al., 1995; Baker et al., 1995; Antoine et al., 1995). Interestingly, replacement of the non-functional ptx promoter in B. bronchiseptica by the functional 6. pertussis ptx promoter results not only in the production of PT, but also in its proper secretion by this organism. This observation indicates that B. bronchiseptica also contains the full array of functional ptx and ptl genes (Kotob et al., 1995), which might provide a useful application. Indeed, 5. bronchiseptica may be an interesting alternative source for PT, since this organism grows significantly faster than 6. pertussis and is biologically less hazardous (Lee et al., 1989). The ptl gene products are homologous to some of the gene products of the Agrobacterium tumefaciens virB operon. The VirB proteins of this organism are responsible for the transport of its virulence plasmid Ti into the plant host cell (Ward et al., 1991). Some of the Ptl proteins have been detected by immunoblot analysis, and their subcellular location has been determined (Johnson and Burns, 1994). Like its homologue in A. tumefaciens, PtlE is most likely an inner-membrane protein that may be associated with outer-membrane components. PtlF may be an outer-membrane protein or may, like its VirB9 homologue, be associated with both the outer and the inner membranes. PtlG cofractionates with the membranes and is insoluble in Triton X100, suggesting an inner-membrane location. However, like VirB10, it may also interact tightly with components of the outer membrane. As has been proposed for the VirB proteins of A. tumefaciens, the Ptl proteins might form a gate to transport PT through the membranes, in particular through the outer membrane. The precise architecture of this structure relevant to its function, however, is not yet known. Interestingly, two of the Ptl proteins, PtlC and PtlH, contain putative ATPbinding sites in the predicted cytoplasmic locations, and might therefore perhaps provide the energy necessary for membrane translocation. The ptlC gene has also been suggested to be involved in the translation of ptx mRNA in B. pertussis (Baker et a/., 1995).

3.4 Receptor-binding and Translocation The first step in PT action is its binding, via the B oligomer, to the receptor on the target cells. Virtually all cells that have been studied so far contain PT receptors on their surface. PT receptors on red blood cells can conveniently be used to follow the presence of PT by hemagglutination. However, none of the receptors has as yet been characterized, although it is believed that they are all sialoglycoproteins (Armstrong et al., 1988). Unlike most of the other bacterial toxins, it appears that different cell types contain different PT receptors which may vary in size from 70 kDa to more than 165 kDa (Clark and Armstrong, 1990; Brennan et al., 1988). Several glycoproteins have been C. LOCHT and R. ANTOINE

used as model systems to study the PT-receptor interactions. These proteins include fetuin, haptoglobin, and transferrin. The complex structure of the PT B oligomer may reflect its ability to interact with such a wide variety of receptors.The B oligomer contains at least two binding domains with distinct specificities. For example, Dimer 1 (S2 and S4) is able to bind to haptoglobin, whereas dimer 2 (S3 and S4) binds to PT receptors on the surface of Chinese hamster ovary (CHO) cells. The recently determined crystal structure of PT complexed with an undecasaccharide from transferrin showed that the carbohydrate-binding domain includes residues 101 to 105 in subunits S2 and S3 (Stein et al., 199413). Interestingly, deletion of Asn-105 of S2 abolishes haptoglobin binding of PT, and deletion of the analogous Lys-105 in S3 abolishes interaction of PT with CHO cells (Lobet et al., 1993). However, both subunits bind to the sugar moieties in essentially the same manner, and hydrogen-bond contacts with the sialic acid involve amino acid residues that are common to S2 and S3 (Stein et al., 199413). This suggests that the specificities involve determinants that are outside the sialic acid sugar ring-binding site. Indeed, other regions, in particular the N-terminal domain, may also be involved in receptor recognition (Saukkonen et al., 1992). The receptor-binding site, determined by site-directed mutagenesis and the crystal structure of the PT-carbohydrate complex, is located close to the S1 subunit. This suggests that PT may bind to the cell surface in such a manner that S1 is positioned in close proximity to the target cell membrane, which would be ideal for its translocation. This is in contrast to cholera toxin, where the receptor-binding portion of the B oligomer is on the opposite side from the enzymatically active A subunit (Merritt et al., 1994). It remains to be determined, however, whether the two toxins translocate their enzymatic subunits by different mechanisms. Unlike diphtheria toxin, little is known about the structures required for the translocation of the enzymatic subunit of PT. In diphtheria toxin and Pseudornonas aeruginosa exotoxin A, the B moiety can be clearly subdivided into two distinct domains, one responsible for receptor binding, composed essentially of fi sheets, and one responsible for translocation of the A subunits, essentially composed of a helices (Allured et al., 1986; Choe et al., 1992).There is no clear translocation domain in PT, and much less is known about the internalization step of PT, compared to diphtheria toxin and exotoxin A. Recently, Xu and Barbieri (1995) showed that NH4CItransiently inhibits in vivo ADP-ribosylation of target proteins in CHO cells. It is therefore likely that intoxication of C H O cells by PT involves acidification of endosomes. This is in contrast to earlier studies that showed that NH4CIdid not inhibit PT-mediatedCHO cell clustering (Hausman et al., 1992).Together these observations suggest that acidic pH may help to translocate S1 but is not absolutely essential, unlike the situation with diphtheria toxin. Brefeldin A also blocked in vivo ADPribosylation (Xu and Barbieri, 1995), suggesting the involvement of Golgi-mediated intracellular trafficking beyond the endosome. This is again unlike diphtheria toxin, but it is similar to cholera toxin and exoPertussis Toxin

toxin A, as well as to a number of other A-B toxins. No endoplasmic reticulum retention sequence is apparent in PT, suggesting that PT intoxication may perhaps not involve the endoplasmic reticulum. In vivo, PT ADP-ribosylates membrane-bound G proteins at the cytoplasmic site of the membrane, including G proteins that are associated with the Golgi apparatus (Stow et al., 1991). However, cytosolic proteins are not ADP-ribosylated in vivo, although they may constitute PT substrates in vitro (Xu and Barbieri, 1995), suggesting that S1 probably remains membrane-associated after its translocation.

3.5 AD P-ribosyIt ra nsferase Activity and Enzyme Mechanism

G proteins as substrates for pertussis toxin

NAD+-glycohydrolysis

C.LOCHT and R. ANTOINE

The expression of S1-catalyzed ADP-ribosyltransferase activity requires the dissociation of S1 from the B oligomer and the reduction of the intramolecular disulfide bond (Moss et al., 1983). It is not known whether this dissociation takes place within the endosome, in the membrane, or at the cytoplasmic side of the membrane. In the presence of ATP, the concentration of glutathione in the cell may be sufficient for reduction of the disulfide bond and dissociation of S1 (Kaslow et al., 1987), suggesting that this activation step may occur at the cytoplasmic side of the membrane. After intracellular activation of PT, the dissociated S1 subunit catalyses ADP-ribosylation of signaltransducing G proteins of the Gi/o class. The subunit of these heterotrimeric G proteins that serves as the PT-acceptor substrate is the a subunit, in particular the cysteine residue located near the C-terminal end of this subunit. Cholera toxin ADP-ribosylates an arginine residue of the a subunit of the Gs type of G proteins. The G proteins are efficient PT substrates only in their trimeric forms, whereas isolated Ga monomers are much less well ADPribosylated (Katada et al., 1986). However, peptides composed of only the C-terminal residues of Ga may also be efficiently ADPribosylated by PT (Graf et al., 1992), suggesting that the C-terminal region of the full-length Ga may be relatively unavailable in the monomeric form. One of the regions of the S1 subunit found to be involved in the interaction with the G protein acceptor substrates is the C-terminal portion of the enzyme. Deletion of this region results in a dramatic decrease of ADP-ribosylation efficiency, whereas binding to the donor substrate NAD' and the catalytic rate are not affected (Locht et al., 1990; Cortina et al., 1991). In the absence of the G protein, PT is able to catalyze the transfer of the ADP-ribose moiety of NAD' to a water molecule in a reaction known as NAD+-glycohydrolysis (for review, see Locht and Antoine, 1995). This property is also shared with cholera toxin. In contrast, the NAD+-glycohydrolase activities of diphtheria toxin and exotoxin A are much less efficient. The affinity of PT for NAD' is in the micromolar range, as evidenced by fluorescent quenching and K, determina-

tions. In contrast, the affinity of cholera toxin for NAD' is in the millimolar range. The higher affinity of PT for NAD' may be in part attributed to the presence of a tryptophan residue located in the NAD+binding site (Trp-26).This residue is not present in cholera toxin, and site-directed alteration of the PT Trp-26 results in a significant decrease in NAD+-affinity of S1. So far, only two catalytic residues have been identified in S1: Glu129 (Antoine et al., 1993) and His-35 (Antoine and Locht, 1994).The first residue is conserved in all known ADP-ribosylating toxins, whereas His-35 is only conserved in cholera toxin and a recently identified mosquitocidal toxin produced by Bacillus sphaericus. This residue is not present in diphtheria toxin and exotoxin A (Fig. 1). The absence of this catalytic residue in the latter two toxins may explain their inefficiency in carrying out the NAD+-glycohydrolysis reaction, compared to PT and cholera toxin. The acceptor substrate of diphtheria toxin and exotoxin A is diphthamide, a modified histidine residue in EF2. Perhaps this residue may take on some of the functions of the catalytic His residue in the other toxins. We have recently proposed a model for the PT-catalyzed ADPribosyltransferase activity (Locht and Antoine, 1995). This model is based on the catalytic role of Glu-129 in the NAD+-glycohydrolase reaction, the difference in catalytic rates between NAD+-

DTX

LTX H21

f j

132

Fig. 1. The active sites of PT S1 and of the A subunits of diphtheria toxin and Escherichia coli heat labile toxin. The thin lines represent the carbon backbones. Only those strands and a helices that are relevant for the active-site geometry are shown. The side chains of residues involved in the enzyme activity, especially those of the catalytic Glu and His residues, are represented by the thick lines. The a2 helix present in PT (top) and E. coli heat labile toxin (LT, right) is completely missing in diphtheria toxin (DT, left) Pertussis Toxin

glycohydrolysis and ADP-ribosylation, the differences in catalytic NAD+-glycohydrolase properties of PT and cholera toxin compared to diphtheria toxin and exotoxin A, the complete conservation of the catalytic Glu residues among ADP-ribosylating toxins and active site geometry (Fig. l), the less well conserved catalytic His residues, the differences in acceptor substrate specificities of the various toxins, the stereochemistry of the products of some of the reactions, and the proposed mechanism of hydroxide-catalyzed NAD+-glycohydrolysis.The model predicts that Glu-129 weakens the N-glycosidic bond linking ADP-ribose to the nicotinamide ring by stabilizing or promoting the formation of an oxocarbonium-like intermediate. This may occur through ionization of the nicotinamide ribose diol, which would result in intramolecular electrostatic stabilization of the intermediate (see Fig. 2). However, this transition-state intermediate is probably not a stable oxocarbonium, since the existence of such an intermediate would result in a SN1-type reaction, whereas ADP-ribosylating toxins probably catalyze their reaction by a SN2-type mechanism. This part of the reaction may be conserved among all ADP-ribosylating toxins, as well as possibly among eukaryotic ADP-ribosyltransferases.

oc-NH 0

II

ry"

His 35

w

ADP

0- 6+;

H

N

s".,,S / H..,'. 6- 0,".~ \ CH2 H., \ cys -Cia "O\;?/,

0.. '.__ ...

lL

-------...,-, '.....

--------

__/

C

I Glu 129 Fig. 2. The proposed transition-state intermediate of the ADP-ribosyltransferase reaction catalyzed by PT. The carboxylate group of Glu-129 is proposed to interact with the hydrogen of the ribose 2'-hydroxyl group, thereby promoting the ionization of the diol which weakens the N-glycosidic band by intramolecular stabilization of an oxocarbonium-like intermediate. The His-35-activated cysteine of the Gia protein may simultaneously exert its nucleophilic attack on the weakened Nglycosidic bond to cleave completely the pyridine-ribosyl bond C. LOCHT and R. ANTOINE

His-35 is proposed to act on the acceptor substrate by increasing its nucleophilicity via hydrogen-bonding (Fig. 2). The activated nucleophile can then attack the weakened N-glycosidic bond, which would result in the transfer of the ADP-ribose moiety of NAD' to the acceptor substrate cysteine of the Gia or water through a SN2-type mechanism. This part of the reaction may differ among the various ADP-ribosyltransferases. Given the conservation of the catalytic His residue in PT, cholera toxin and mosquitocidal toxin, its role in increasing the nucleophilicity of the acceptor substrate may be conserved in this subgroup of toxins. However, given the absence of this residue in diphtheria toxin and exotoxin A, this part of the reaction may perhaps be fulfilled directly by their acceptor substrate diphthamide.

3.6 Biological Activities and Role of Pertussis Toxin in Pathogenesis PT-mediated ADP-ribosylation of the Ga subunits of the Gi/o class of proteins results in uncoupling of the G proteins from their cognate receptors. The initial observations were made by Ui and coworkers who studied the effect of PT on insulin secretion by pancreatic B cells. Secretion of insulin by these cells was inhibited via a2-adrenergic receptors, and this inhibition was lost upon ADP-ribosylation of Gia by PT (Murayama and Ui, 1983). In the resting state, the receptor is linked to its corresponding G protein in the trimeric inactive form containing GDP bound to the a subunit. Recognition of the receptor by the cognate extracellular signal results in the dissociation of the trimeric G protein from the signalreceptor complex, and in the displacement of GDP by GTP This activates the G protein which can dissociate into G a G T p and GPy. G a G T p may then interact productively with the effector protein. Depending on the type of G protein, this may result in activation or inhibition of the effector. The intrinsic GTP-hydrolase activity of the G protein cleaves GTP to yield GDP and phosphate, thereby inactivating the Ga protein again, promoting its dissociation from the effector and its reassociation with the P and y subunits (Fig. 3). The first effector recognized in this signal transduction cascade was the membrane-bound adenylate cyclase. However, other effectors have now been identified, and include ion channels, phosphodiesterases, and enzymes that produce diacylglycerol and inositol phosphates (Ui, 1988). The extracellular signals may also be very diverse and range from neurotransmitters and hormones to light. The vast diversity of the biological activities of PT can thus easily be accounted for by its enzymatic ADP-ribosyltransferase activity. However, some biological activities of PT, such as its mitogenicity and its ability to agglutinate erythrocytes, are independent of the enzymatic activity. Biological effects that have been related to the action of PT in tissue cultures include suppression of cell proliferation, morphological Pertussis Toxin

S -R

S -R

GBTp+ Gpy+ 1"

+K

11

Fig. 3. The G-protein dependent signal transduction cycle. The signal transduction unit is represented by R (receptor), G (G protein), and E (effector). G is composed of Ga with bound GTP or GDP and of Gpy. The active and inactive complexes or proteins are shown in black and grey, respectively. S represents the stimulus that triggers the cycle. Proteins interacting in a complex are linked by hyphens

changes, exocrine secretion, inhibition of histamine secretion, stimulation of lipolysis and many others. In vivo, the biological effects are recognized as islet activation, lymphocyte promoting activity, histamine sensitization, and increase of vascular permeability (Munoz, 1985), some of which are the hallmarks of systemic pertussis in patients (Pittman, 1984). It is therefore not surprising that PTis a valuable tool for dissecting the involvement of G-proteins in signal transduction (Ui, 1990) on the one hand, and as a potent protective antigen in vaccines against whooping cough (Ad hoc group, 1988) on the other hand.

Acknowledgements We gratefully acknowledge all the workers in the PT field for the many exciting experiments and the excellent work that has been done over the years. The work in our laboratory was supported by the lnstitut Pasteur de Lille, Region Nord-Pas-deCalais, INSERM, Ministere de la Recherche.

References Ad hoc group for the study of pertussis vaccines (1988): Placebo-controlled trial of two acellular pertussis vaccines in Sweden - protective efficacy and adverse events. In Lancet ii: 955-960. Allured VS, Collier RJ, Carroll SF, et al. (1986):Structure of exotoxin A of Pseudomonas aeruginosa at 3.0-Angstrom resolution. In Proc. Natl. Acad. Sci. USA 83: 1320-1324. C. LOCHT and R. ANTOINE

Antoine R, Locht C (1990): Roles of the disulfide bond and the carboxy-terminal region of the S1 subunit in the assembly and biosynthesis of pertussis toxin. In Infect. Irnmun. 58: 1518- 1526. Antoine R, Locht C (1994):The NAD-glycohydrolase activity of the pertussis toxin S1 subunit : involvement of the catalytic His-35 residue. In J. Biol. Chem. 269: 6450-6457. Antoine R, Raze D, Locht C. (1995): Genetic organization of the pertussis toxin locus. In Zbl. Bakteriol. Suppl. 28: 44-45. Antoine R, Tallett A, van Heyningen S, et al. (1993):Evidence for a catalytic role of glutamic acid 129 in the NAD-glycohydrolase activity of the pertussis toxin S1 subunit. In J. Biol. Chern. 268: 24149-24155. Arico B, Rappuoli R (1987):Bordetella paraperfussis and Bordetella bronchiseptica contain transcriptionnally silent pertussis toxin genes. In J. Bacteriol. 169: 2847-2853. Armstrong GD, Howard LA, Peppler MS (1988): Use of glycosyltransferases to restore pertussis toxin receptor activity to asialogalactofetuin. In J. Biol. Chern. 263: 8677-8684. Baker SM, Masi A, Liu D-F, et al. (1995): Pertussis toxin export genes are regulated by the ptx promoter and may be required for efficient translation of ptx mRNA in Bordetella pertussis. In Infect. Immun. 63: 3920-3926. Boucher PE, Menozzi FD, Locht C (1994):The modular architecture of bacterial response regulators : insights into the activation mechanism of the BvgA transactivator of Bordetella pertussis. In J. Mol. Biol. 241 : 363-377. Boucher PE, Stibitz S (1995): Synergistic binding of RNA polymerase and BvgA phosphate to the pertussis toxin promoter of Bordetella pertussis. In J. Bacteriol. 177: 6486-6491. Brennan MJ, David JL, Kenimer JG, etal. (1988):Binding of pertussis toxin to a 165kilodalton Chinese hamster ovary cell glycoprotein. In J. Biol. Chem. 263: 4895-4899. Choe S, Bennett MJ, Fujii G, etal. (1992):The crystal structure of diphtheria toxin. In Nature 357: 216-222. Clark CG, Armstrong GD (1990):Lymphocyte receptors for pertussis toxin. In Infect Irnrnun. 58: 3840-3846. Cortina G, Krueger KM, Barbieri JT (1991):The carboxyl terminus of the S1 subunit of pertussis toxin confers high affinity binding to transducin. In J. Biol. Chem. 266: 23810-23814. DeShazer D, Wood GE, Friedman RL (1995):Identification of a Bordetella pertussis regulatory factor required for transcription of the pertussis toxin operon in Escherichia coli. In J. Bacteriol. 177: 3801 -3807. Graf R, Codina J, Birnbaurner L (1992): Peptide inhibitors of ADP-ribosylation by pertussis toxin are substrates with affinities comparable to those of the trimeric GTP-binding proteins. In Mol. Pharmacol. 42: 760-764. Hausman SZ, Burns DL (1992): Interaction of pertussis toxin with cells and model membranes. In J. Biol. Chern. 267: 13735-13739. Johnson FD, Burns DL (1994): Detection and subcellular localization of three Ptl proteins involved in the secretion of pertussis toxin from Bordetellapertussis. In J. Bacteriol. 176: 5350-5356. Kaslow Hi?, Lirn LK, Moss J, et al. (1987):Structure-activity analysis of the activation of pertussis toxin. In Biochemistry 26: 123-127. Katada T, Oinuma M, Ui M (1986):Two guanine nucleotide-binding proteins in rat brain serving as the specific substrate of islet-activating protein, pertussis toxin. Interaction of the a-subunits with &-subunits in development of their biological activities. In J. Biol. Chem. 261 : 8182-8191. Katada T, Ui M (1982):Direct modification of the membrane adenylate cyclase system by islet-activating protein due to ADP-ribosylation of a membrane protein. In Proc. Natl. Acad. Sci. USA 79: 3129-3133. Kotob SI, Hausman SZ, Burns DL (1995): Localization of the promoter for the ptl genes in Bordetella pertussis, which encode proteins essential for secretion of pertussis toxin. In Infect. Irnrnun. 63: 3227-3230. Lacey BW (1960): Antigenic modulation of Bordetella pertussis. In J. Hyg. 58: 57-93. Pertussis Toxin

Lee CK, Roberts A, Perrin S (1989):Expression of pertussis toxin in Bordetella bronchiseptica and Bordetella parapertussis carrying recombinant plasmids. In Infect. Immun. 57: 1413- 1418. Lobet V, Feron C, Dequesne G, et al. (1993): Site-specific alterations in the Boligomer that affect receptor-binding activities and mitogenicity of pertussis toxin. In J. Exp. Med. 177: 79-87. Locht C, Antoine R (1995):A proposed mechanism of ADP-ribosylation catalyzed by the pertussis toxin S1 subunit. In Biochimie 77: 333-340. Locht C, Barstad PA, Coligan JE, et al. (1986):Molecular cloning of pertussis toxin genes. In Nucl. Acids Res. 14: 3251 -3261. Locht C, Keith JM (1986): Pertussis toxin gene : nucleotide sequence and genetic organization. In Science 232: 1258- 1264. Locht C, Lobet Y, Feron C, etal. (1990):The role of cysteine 41 in the enzymatic activities of the pertussis toxin S1 subunit as investigated by site-directed mutagenesis. In J. Biol. Chem. 265: 4552-4559. Marchitto KS, Smith SG, Locht C, et al. (1987):Nucleotide sequence homology to pertussis toxin gene in 6. bronchiseptica and 6. parapertussis. In Infect. Immun. 55: 497-501. Merritt EA, Sarfaty S, van den Akker F., et al. (1994):Crystal structure of cholera toxin B-pentamer bound to receptor G ,, pentasaccharide. In Prot. Sci. 3: 166-175. Moss J, Stanley SJ, Burns DL, et al. (1983):Activation by thiol of the latent NAD glycohydrolase and ADP-ribosyltransferase activities of Bordetella pertussis toxin (Islet activating protein). In J. Biol. Chem. 258: 11879-11882. Munoz JJ (1985): Biological activities of pertussigen (pertussis toxin). In Pertussis toxin (Sekura RD, Moss J, Vaughn M, eds) pp 1-18, Orlando, FL: Academic Press. Munoz JJ, Arai H, Bergman RK, et al. (1981): Biological activities of crystalline pertussigen from Bordetella pertussis. In Infect. Immun. 33: 820-826. Munoz JJ, Bergman RK (1977): Bordetella pertussis : immunological and other biological activities. Immunological Series, Vol. 4, pp 1-235, New York: Marcel Dekker. Murayama T, Ui M (1983): Loss of the inhibitory function of the guanine nucleotide regulatory component of adenylate cyclase due to its ADP-ribosylation by isletactivating protein, pertussis toxin, in adipocyte membrane. In J. Biol. Chem. 258: 381 -390. Nicosia A, Perugini M, Franzini C, et al. (1986):Cloning and sequencing of the pertussis toxin gene : operon structure and gene duplication. In Proc. Natl. Acad. Sci. USA 83: 4631 -4635. Parfentjev IA, Goodline M A (1948):Histamine shock in mice sensitized with Hemophilus pertussis vaccine. In J. Pharmacol. Exp. Ther. 92: 411 -413. Pittman M (1984):The concept of pertussis as a toxin mediated disease. In Pediatr. Infect. Dis. 3: 467-486. Saukkonen K, Burnette WN, Mar VL, etal. (1992):Pertussis toxin has eukaryotic-like carbohydrate recognition domains. In Proc. Natl. Acad. Sci. USA 89: 118-122. Scarlato V, Arico B, Rappuoli R (1993):DNA topology affects transcriptional regulation of the pertussis toxin gene of Bordetellapertussis in Escherichia coli in vitro. In J. Bacteriol. 175: 4764-4771. Scarlato V, Prugnola A, Arico B., et al. (1990): Positive transcriptional feedback at the bvg locus controls expression of virulence factors in Bordetella pertussis. In Proc. Natl. Acad. Sci. USA 87: 6753-6757. Sekura RD, Fish F, Manclark CR, et al. (1983):Pertussis toxin, affinity purification of a new ADP-ribosyltransferase.In J. Biol. Chem. 258: 14647- 14651. Stein PE, Boodhoo A, Armstrong GD, et al. (1994~): The crystal structure of pertussis toxin. In Structure 2: 45-57. Stein PE, Boodhoo A, Armstrong GD, et al. (199413):Structure of a pertussis toxin sugar complex as a model for receptor binding. In Structural Biology 1 : 591 -596. Stibitz S, Aaronson W, Monack D, et al. (1989):Phase variation in Bordetella pertussis by frameshift mutation in a gene for a novel two-component system. In Nature 338: 266-269. C. LOCHT and R. ANTOINE

Stow JL, de Almeida JB, Narula N, etal. (1991):A heterotrimericG protein GQ, on Golgi membranes regulates the secretion of a heparan sulfate proteoglycan in LLC-PK, epithelial cells. In J. Cell Biol. 114: 1113-1124. Tamura M, Nogimori K, Murai S, et al. (1982):Subunit structure of islet-activating protein, pertussis toxin, in conformity with the A-B model. In Biochemistry 21 : 5516-5522. Uhl MA, Miller JF (1995): Bordetella pertussis BvgAS virulence control system. In two-component signal transduction. (Hoch J, Silhavy T, eds) pp 333-349, Washington DC: American Society for Microbiology. Uhl MA, Miller JF (1995): BvgAS is sufficient for activation of the Bordetellapertussis pfx locus in Escherichia coli. In J. Bacteriol. 177: 6477-6485. Ui M (1988):The multiple biological activities of pertussis toxin. In Pathogenesis and Immunity in Pertussis (Wardlaw AC, Parton R, eds) pp 121-145, Chichester: John Wiley and Sons. Ui M (1990):Pertussis toxin as a valuable probe for G-protein involvement in signal transduction. In ADP-ribosylating toxins and G proteins. Insights into signal transduction (Moss J, Vaughan M, eds) pp 45-77. Ward JE, Dale EM, Binns AN (1991):Activity of the Agrobacteriurn T-DNA transfer machinery is affected by virB gene products. In Proc. Natl. Acad. Sci. USA 88: 9350-9354. Weiss AA, Goodwin M (1989):Lethal infection by Bordetella pertussis mutants in the infant mouse model. In Infect. Immun. 57: 3757-3764. Weiss AA, Johnson FD, Burns DL (1993):Molecular characterization of an operon required for pertussis toxin secretion. In Proc. Natl. Acad. Sci. USA 90: 2970-2974. Xu V, Barbieri JT (1995):Pertussis toxin-mediated ADP-ribosylation of target proteins in Chinese hamster ovary cells involves a vesicle trafficking mechanism. In Infect. Immun. 63:825-832.

Pertussis Toxin

Bacterial Toxins

Klaus Aktories Copyright 0 2002 WILEY-VCH Verlag GmbH & Co. KGaA

CHAPTER 4

Pertussis Toxin as a Cell Biology Tool B. NURNBERG

4.1 Introduction Use of bacterial toxins such as pertussis toxin (PT) and cholera toxin (CT) permitted crucial studies allowing one to detect, identify and understand the structure and function of heterotrimeric regulatory guanine nucleotide binding proteins (Gproteins) (Ui, 1984; Gilman, 1995; Nurnberg etal., 1995).Today, both toxins are still appreciated as important tools for elucidating the physiological roles of G proteins and for identifying G-protein coupled pathways (for review see Chapter 5 and references Milligan, 1988; Gierschik and Jakobs, 1993). Pertussis toxin (or islet-activating protein) is a 105 kDa hexameric enzyme catalyzing ADP-ribosylation of most Ga isoforms belonging to the Gi subfamily, but not members of the G,, G, and GI2 subfamilies. PT catalyzes both hydrolysis of NAD' and the transfer of the resulting ADP moiety to a cysteine residue near the carboxy terminus of the a subunit of Gi, Goand transducin,, in the presence of Gpy. PT-resistantGi proteins lacking a carboxy terminal cysteine include G, and a splice variant of Gi2(Gi2(L1). The amino acid motif required for PT-mediated ADP-ribosylation is similar to the consensus site for isoprenylation of Gy subunits or some monomeric GTPases, except for the C-terminal aromatic amino acid, which obviously determines the enzyme's specificity. Since the modified cysteine is located four residues upstream from the carboxy terminus, PT-catalyzed ADPribosylation prevents interaction of the transmembrane receptor with the G protein, which is anchored to the cytoplasmic surface of the membrane. This results in a functional receptor-G-proteinuncoupling. Nevertheless, PT-modified G proteins are capable of interacting with effectors, although the time required to observe the maximal effect produced by GTPyS can be markedly increased (Jakobs et a/., 1984; Ahnert-Hilger et a/., submitted). Pertussis toxin consists of two components, the enzymatically active A protomer consisting of a single polypeptide, and the pentameric B oligomer (Tamura et al., 1982).The B component is involved in binding to the surface of eukaryotic target cells, and presumably in translocation of the toxin across the plasma membrane. Once inside the cell, the enzymatically active A component needs to undergo activation that depends on reduced gluthathione; this effect can be

action of pertussis toxin

structure of pertussis toxin

K. Aktories (Ed.), Bacterial Toxins. 0 Chapman & Hall, Weinheim, 1997. ISBN 3-8261-0080-8

mimicked in a cell-free system with the sulfhydryl reducing agents dithiothreitol (DTT)or 2-mercaptoethanol (6-ME). Use of holoenzyme is therefore mandatory for treating whole cells, whereas the preactivated A protomer is able to modify cellular proteins when injected or infused into cells, or in cell homogenates, or in protein preparations that have been purified further. This overview of methods will first concentrate on sources of pertussis toxin and its application to whole cells (section 4.2). Section 4.3 describes preactivation of the holotoxin and treatment of cellular components, including purified proteins, while high resolution SDS-PAGE techniques to separate modified proteins are described in Section 4.4.

4.2 Pertussis Toxin as a Tool to Modify Cellular Functions 4.2.1 Cell Culture of Bordetella pertussis Pertussis toxin is produced by certain Bordetella pertussis strains, which are pathogenic organisms (Yaiima et al., 1978a,b). Since the toxin is commercially available in adequate quality we do not purify PT from cultured B. pertussis. Those who are interested in culturing B. pertussis and purifying PT are referred to detailed laboratory protocols published previously (Sekura, 1985).

4.2.2 Source of Pertussis Toxin and Preparation of Solutions We obtain pertussis toxin from List Biological Laboratories (Campbell, CA, USA; European distributor: Quadratech, Epsom, Surrey, UK); it is

A

avoid inhalation or contamination

B. NURNBERG

supplied in 50pg quantities as a lyophilized powder in glass bottles. The preparation also contains sodium chloride (25 pmol) and phosphate buffer, pH 7.0 (5pmol).Since we usually order at least 10 ampoules we keep them at +4"C until use. For adding pertussis toxin to mammalian cell cultures we reconstitute 50pg of the toxin (one bottle) in 500 to 1OOOpl of a solution containing 2 M urea and 100mM K,HPO,/ KH2P04buffer, pH 7.0, in the glass bottle, as originally suggested by Dr. M. Yajima. Make sure that inhalation of the toxin or contamination of open wounds is avoided. The final concentration is 50 to 100 pg/ml of pertussis toxin. The solution is also stored at +4 "C; no significant loss of function is observed for at least 2 months. For studies employing preactivated toxin (see section 4.31) we prepare pertussis toxin stock solutions in essentially the same way, but at a final concentration of 1 mg toxin per mI (50pg/50pI). In contrast to our practice List Laboratories recommends reconstitution of the toxin in sterile water. Storage of aliquots should be well tolerated at -70°C as reported elsewhere (Carty, 1994). Pertussis toxin is also available from Sigma Chemical Co. (St. Louis, MO, USA), supplied as lyophilized powder or solution.

4.2.3 Treatment of Mammalian Cell Cultures with Pertussis Toxin For ADP-ribosylation of G proteins in cultured mammalian cells, we supplement the medium with pertussis toxin to a final concentration of at least 25 ng/ml, and not more than 200 ng/ml. The optimal concentration has to be found empirically. For pertussis toxin to be fully effective, we incubate at least overnight (24 h) although a few hours of treatment have been reported to be sufficient in most cases; higher PT concentrations are required with a shorter time. It appeared to be very helpful to aliquot the batch of cells and treat one portion with the vehicle alone, omitting toxin, to allow comparison and quantification of the effect of pertussis toxin on cells. Under these conditions the toxin works well, e.g. G, proteins of HL-60 cells are more than 9 8 % modified (25 ng/ml, 24 h) (Hageluken et al., 1995). However, other cells may respond differently to the toxin. In particular, platelets are able to resist the entry of pertussis toxin, either because they lack surface binding sites for the toxin B oligomer or because they are unable to internalize the bound toxin (Brass et al., 1990). PT-sensitive G proteins expressed in insect Sf9 cells (ovary cells from the insect Spodoptera frugiperda; Sf9 cells are employed as an overexpression system for G proteins and other signal transduction components by infection with recombinant baculovirus) are also not modified by the toxin (Mulheron et al., 1994). Therefore, for functional studies, it is mandatory to ensure quantitative modification of G, proteins after pertussis toxin treatment. This can be done by functional tests, i.e., recording the degree of inhibition of a cellular signal after stimulation by a certain receptor agonist. Cellular responses of a known signal transduction pathway that is exclusively pertussis toxin-sensitive in a particular cell type should be selected. Different possibilities for signal read-outs are possible. For instance, in HL-60 cells, rise of intracellular free calcium or GTPase activity can be measured after exposure of cells to the chemotactic formyl peptide fMLP (Hageluken et al., 1995).As an alternative, measurement of ADP-ribosylation of cellular G proteins by radiolabeled NAD' may be performed (see below and Figure 1).Whereas the former assay strategy is usually more rapid than the latter, it has to be kept in mind that it is indirect and gives less precise measurement of ADP-ribosylation due to variable endpoints, whereas the latter is more time consuming but allows exact and direct measurement of the degree of ADP-ribosylation of proteins. Nevertheless, either measurement should be done in comparison with controls treated only with the vehicle (see above).

cells resistant to pertussis toxin

Pertussis Toxin as a Cell Biology Tool

Fig. 1. Pertussis toxin-mediated ADP ribosylation of membrane G proteins. Isolated cell membranes (50 pg of protein) from N1E 115 cells (mouse neuroblastoma cell line), N2A cells (mouse neuroblastoma cell line), S49-1 cyc' cells (S49(-);mutated mouse lymphoma cell line deficient in Ga,), S49 wt cells (wild-type mouse lymphoma cell line), RBL (RBL 2H3; rat basophilic leukemia cell line), GH3 cells (GH,; rat hypophyseal tumor cell line), PC-12 (rat pheochromocytoma cell line), HIT-T15 cells (hamster insulinoma cell line), Y-1 cells (mouse adrenal cortex tumor cell line), 108cc 15 cells (mouse/rat neuroblastoma x glioma hybrid cell line), HL-60 cells (DMSO-differentiated human leukemia cell line), HL-60 (+PT) cells (HL-60cells pretreated with 25 ng/ml of pertussis toxin for 24 h prior to preparation of membranes), RINm5F cells (rat insulinoma cell line), and C6-2 cells (rat glioma cell line) were subjected to 32P-ADP-ribosylation as described in section 4.3.3. Samples were precipitated as outlined in section 4.3.5 and subjected to SDS-PAGE with separating gels containing 8 % acrylamide (w/v). An autoradiogram of the dried gel is shown. Molecular masses of marker proteins ore indicated (kDa). Modified Ga proteins migrate at approximately 40 kDa. Radioactivity running in front of the 30 kDa marker protein comigrates with the dye front

4.3 Pertussis Toxin as a Tool to Study Cellular Components 4.3.1 Activation of Pertussis Toxin for in vitro ADP-ribosylation Dissociation of A protomer and B oligomer, reduction, and activation of the enzymatically active A component prior to incubation with the substrate are not absolutely required but generally result in more extensive ADP-ribosylation of G proteins when studying cell-free systems.

6. NURNBERG

We preactivate the enzymes routinely by incubating 5pI of PT holoenzyme (1 mg/ml PT; see section 4.2.2) together with 5 pI of 10 mM ATP, 25 pI of 40 mM DTT, and 15 pI of water in an Eppendorf tube at 37 "C for 30 min. Thereafter, the mixture is diluted with 450pl of a buffer made of 125 pI of 1 % of bovine serum albumin (BSA) in water (w/v), 50 yl of 0.5 M DTT, 31 pI of 40mM EDTA, 31 yl of 1 M Tris-CI, pH 7.5, and 1013 pl of water.

@

enzyme activation

Alternative protocols are described elsewhere (Ribeiro-Net0 et al.,

1985; Kopf and Woolkalis, 1991; Carty, 1994).ATP, phospholipids and small amounts of certain ionic and nonionic detergents (i.e. SDS, CHAPS, Lubrol PX) promote dissociation of the A and B protomers (Moss et al., 1986).We do not use SDS since it denatures solubilized G proteins very easily. DTT or b-ME are necessary to break the disulfide bonds of the A protomer. Supplementation with BSA (final concentration: approx. 0.9 mg/ml) helps prevent loss of enzyme through adsorption to the walls of the tube, and ensures recovery of proteins following precipitation with sodium chloride/acetone, trichloroacetic acid (TCA), or chloroform/methanol. Furthermore, when samples are subjected to SDS-PAGE, intensities of the stained 67 kDa BSA protein bands allow rough estimation of incomplete recovery of the precipitated sample (see section 4.5). Preactivated PT should be used immediately, and enzyme left over from an experiment should be discarded, since reduced toxin has been shown to lose activity rapidly (Kaslow et al., 1989).

alternative procedures

4.3.2 Preparation of Cell Homogenates and Fractions G proteins are located in different compartments within the cell (Nurnberg and Ahnert-Hilger, 1996). Although most G proteins are found attached to the plasma membrane and intracellular membranes, some are also located within the cytoplasm (Rudolph et al., 1989). Therefore, G proteins in preparations of disrupted cells, or in cell and tissue extracts are also subject to pertussis toxin-mediated ADP-ribosylation. In this case, precautions have to be taken to prevent proteolysis, and protease inhibitors should be included in the buffer (aprotonin, p-aminobenzamidine, leupeptin, phenylmethylsulfonyl fluoride, or soybean or lima bean trypsin inhibitors) (Carty,

1994).

use protease inhibitors

4.3.3 ADP-ribosylation of Membrane Proteins by Pertussis Toxin Membranes exhibit the highest concentration of G proteins in the cell although there is great variation among different cells (see Fig. 1). Usually, brain cell membranes contain the largest amount of subPertussis Toxin as a Cell Biology Tool

strates for pertussis toxin. We routinely use cell membranes which are stored in aliquots of 250 and 500 pg/tube at -70 "C. Membranes are thawed on ice. Just before the experiment a washing step is performed using a microcentrifuge (13 OOOg, 4"C, 10min). The membrane pellet is resuspended in a buffer containing 25 mM Tris-CI, pH 7.5, and 1 mM EDTA. The volume of the membrane suspension is adjusted to 20 pl per sample and kept at 0 "C to 4 "C. The amount of membrane proteins per sample should be estimated from preliminary experiments. In general, a range of 10 to 1OOpg of total protein per sample is employed. The reaction buffer is freshly made from stock solutions stored at -20°C. As a standard procedure we prepare the reaction buffer as detailed below (volume per assay tube); all steps are carried out at 0 "C to 4 "C: Reaction buffer: 1.5 pI of 40 mM of EDTA, 3 pl of 100 mM ATP, 0.6 pl of 100 mM GTP, 1.8 pI of 10 % Lubrol PX, 6 PI of 100 mM thymidine, 0.6 PI of DNAase (2 mg/ml), 5 PI of 1 M Tris-CI, pH 7.5, and 0.3 pI of 200 p M unlabeled NAD'; 32P-NAD+is added to give 500 000 to 1 000 000 c.p.m per tube (approx. 0.25 to 0.5pCi). The buffer is diluted with water to give a final volume of 30 pl per tube. Note that any NAD' containing solutions must be protected from light, which is easily done by wrapping containers with aluminum foil. Radiolabelled 32P-NAD+is either synthesized from [a-32P]ATPby the method of Cassel and Pfeuffer (1978)or purchased and stored frozen. Although the half-life of the radioactive phosphate is approximately two weeks, we do not use 32P-NAD+longer than three to four weeks after preparation because of radiolysis. For detection of ADP-ribosylated G proteins we have also used an affinity-purified polyclonal rabbit antibody (T2B3) directed against + adenosine 5'-[methylene]diphosphate (ADP[CH2]) which was developed by Meyer and Hilz (1986) (Fig. 2). This antibody detects ADP-ribosylated G proteins, and it may be employed to avoid using radioactivity [Nurnberg et al., 19941. The non-ionic detergent Lubrol PX at a final concentration of 0.3 % significantly increases the extent of ADP-ribosylation. Since Lubrol PX is no longer commercially available, other non-ionic detergents have to be used. Polyoxyethylene 10 lauryl ether (Sigma, Cat. No. P9769), which is chemically related to Lubrol PX, may be an effective substitute (Pang et al., 1994). GTP is included to protect G proteins from degradation. Since considerable amounts of GTP are hydrolyzed, GDP shifts the equilibrium of dissociated G protein a and by subunits towards the heterotrimeric state, thereby facilitating the enzymatic reaction. In order to reduce ADP-ribosylation by glycohydrolases other than pertussis toxin, thymidine is included in the reaction mixture as an inhibitor of poly(ADP-ribose)synthase (NAD' ADP-ribosyltransferase, EC 2.4.2.30) (Moss et al., 1980). Ten mM or inhibition of unwanted less of isonicotinic acid hydrazide (INN: isoniazid) may be added to enzyme activities inhibit NAD hydrolases present in membranes (Sternweis and Pang, 1990).The optimal concentration has to be found empirically, since isoniazid inhibits pertussis toxin at high concentrations. 6. NURNBERG

Fig. 2. Detection of ADP-ribosylated Ga by specific antibodies. Purified bovine brain Gaol (500 ng) was treated with pertussis toxin in the presence of 32P-NADand brain GPy complexes (1250ng) as outlined in section 4.3.4. Precipitated proteins (see section 4.3.5) were subsequently resolved on gels (6 M urea, 9 % acrylamide, 13cm length, 4 mm width of slots). After blotting, nitrocellulosefilters were exposed to X-ray films, or detected by incubating nitrocellulose filters with antibody AS 6 (anti-a OCOmmOn serum diluted 1 :300 (Spicher et al., 1992) or antibody T2B3 (antiADP-ribose antibody, affinity purified). The molecular mass (kDa) of a marker protein is indicated. An enhanced chemoluminescence (ECL) system was used to detect filter-bound antibodies. Please note an apparent higher molecular weight (gel-mobility shift) of the modified Gaol (for details see section 4.5)

ADP-ribosylation of the samples is carried out in glass tubes (approximate volume 5 ml) in case the reaction has to be followed by precipitation with organic solvents. Therefore, the markings on the tubes should withstand solvents. Each tube contains 10 PI of preactivated toxin (100ng PT/tube) and 20 pI of membrane suspension. The reaction is readily started by addition of 30pl of the 32P-NAD+containing buffer, resulting in a total volume of 60pl per tube. ADPribosylation of the thoroughly vortexed sample is conducted for 30 min at 30°C or for 20 min at 37°C while the tubes are agitated contino usly. The assay should be validated. Appropriate controls for specificity and sensitivity are at least one sample known to contain PT-sensitive G proteins, e.g. mammalian brain membranes, and samples treated without the toxin as negative controls. Addition of stable guaninetrinucleotides, e.g. GTPyS and GppNHp, or ligand-bound G-proteincoupled-receptors, activate G proteins, resulting in decreased labeling of PT-substrates. Therefore, the latter experimental approach can be used to identify receptors activating PT-sensitive G proteins (Watkins et a/., 1985; Korner et a/., 1995). We stop the reaction after not more than 45 min. If desired, isoform identification of labeled proteins may be done by immunoprecipitation employing specific antisera. First, the membranes are pelleted in

ADP-ribosylation procedure

validation of assay

Pertussis Toxin as a Cell Biology Tool

Fig. 3. lmmunoprecipitation of ADP-ribosylated Ga,-proteins. Bovine brain membranes were ADP-ribosylated in the presence of pertussis toxin and 32P-NADas detailed in section 4.3.3. Proteins were resolved on 8 YO acrylamide gels supplemented with 4.3 M urea, length 9 cm, width of slots 4 mm. An autoradiogram is shown (lanes 1 -4), after 32P-ADP-ribosylation of bovine brain membrane proteins (lane 1) and after immunoprecipitation with antiserum AS6 (anti-ao,,,,,,; lane 2), separating Gaol (upper band) and Ga02(lower band). For immunoprecipitation sedimented (10min at 13 OOOg, 4°C) brain membranes (200pg of protein) were solubilized in 40 pl of a solution containing SDS (4 YO)and phenylmethylsulfonylfluoride (PMSF; 0.2 pM) at room temperature. After 30 min, 280 pl of precipitation buffer was added (150mM NaCI, 10 mM Tris-HCI, pH 7.4, 1 mM EDTA, 1 mM dithiotreitol, 1 YO (w/v) sodium deoxycholate, 0.3 pM aprotinine, 0.2 pM PMSF, and 1 % (v/v) Nonidet P-40) followed by addition of 1Opl with 1.25yg of Protein A bound to Sepharose beads. Following incubation (30min at room temperature) and centrifugation (30min at 13 OOOg, 4"C), the clear supernatant was removed and supplemented with 30 yl of AS 6 serum or non-immune serum and 50 pI of 10 % (w/v) Protein A Sepharose beads. The mixture was gently shaken for 16 h at 4°C. Protein A Sepharose beads were pelleted and washed twice with precipitation buffer and once with precipitation buffer containing 300 mM NaCI. Bound proteins were eluted by adding Laemmli's sample buffer, and loaded onto polyacrylamide gels. Lane 3: Autoradiography of 32PADP-ribosylated purified bovine brain Gaol supplemented with purified By-dimers. Lane 4: Autoradiography of 32P-NADand pertussis toxin treated purified brain Gpy-dimers. Lane 5: lmmunostaining of bovine brain Gaol (lower band) and another as yet unknown Ga,-isoform (upper band); proteins were detected by incubating nitrocellulose-filterswith antiserum AS 248 (antiaOl serum diluted 1:300 (Spicher et al., 1992)),and the ECL system was used for detection of filter-bound antibodies

a microcentrifuge (13 OOOg) for 10min at 4°C. This removes the vast majority of non-incorporated radioactivity. Afterwards membranes are subjected to routine immunoprecipitation procedures (Fig. 3) (Laugwitz etal., 1994; Nurnberg etal., 1994).At this stage, it has to be borne in mind that recognition of ADP-ribosylated proteins by antibodies directed against the carboxy terminus of Ga may be impaired.

4.3.4 ADP-ribosylation of Proteins by Pertussis Toxin Extraction of G proteins from membranes usually enhances ADPribosylation. In addition, small amounts of detergents like SDS, CHAPS or Lubrol PX facilitate the toxin's enzymatic activity (see above). For detection and identification of G proteins during G protein purification procedures, aliquots are also subjected to PTcatalyzed ADP-ribosylation for detection and identification (Rosenthal 6. NURNBERG

et al., 1986). The protocol for preparation of the reaction buffer outlined in this section is slightly different from the one used for membrane treatment (see section 4.3.3). Again, volumes are given per assay tube, and all steps are carried out at 0°C to 4°C.

Reaction buffer: 0.75 pl of 40 mM of EDTA, 1.5 pI of 100 mM ATP, 0.3 pI of 100 mM GTP, 0.9 p1 of 10 % Lubrol PX, 0.75 pI of 1 M Tris-CI, pH 7.5, and 0.15 pI 200 pM of unlabeled NAD'; 32P-NAD+ is added to reach a activity of 1 000 000 c.p.m per tube. The buffer is diluted with water to give a final volume of 10 pI per tube. Ten pl of G-protein-containing samples is mixed with 10 pl of preactivated toxin (see section 4.3.1; 100 ng PT/tube) and started by addition of another 10 pl of reaction buffer (total volume: 30pl). The assay is conducted as outlined in section 4.3.3. After termination of the reaction samples may be quantified by filtration assay as described by Sternweis and Pang (1990). For efficient PT-mediated ADP-ribosylation of G proteins precautions are required (Moss et al., 1986). Final concentrations of detergents should be low. For instance, the concentration of cholate (a commonly used detergent for purification of G proteins) should be less than 0.1 % under optimal conditions. To enhance ADPribosylation, addition of dimyristoylphosphatidylcholine (1 mg/ml final concentration) to purified preparations may be useful. The stock sohtion contains 8 mg/ml of dimyristoylphosphatidylcholine, which is sonicated to clarity. Since only the Gapy holoprotein is sensitive to PT, G proteins activators such as GTPyS, GppNHp or AIF4-/Mg2+ decrease ADP-ribosylation and should be removed. AIF4- may be inactivated by addition of at least 1 mM EDTA in excess of the concentration of Mg2+contributed by the sample. Samples containing large amounts of non-hydrolyzable GTP-analogues may be dialyzed against non-activating buffers. Purified Ga proteins must be supplemented with Gpy prior to PT-treatment. GBy increases ADPribosylation in a concentration-dependent manner. In fact, PTmediated 32P-ADP-ribosylationof a known amount of purified Ga, in the presence of unkown concentrations of Gpy can be used to quantitate the amount of functional active GBy (Iniguez-Lluhi et al., 1992).

optimization of ADPribosylation

4.3.5 Preparation of Samples for SDS-PAGE Samples subjected to analysis of labeled proteins by SDS-PAGE should be devoid of detergents. In addition, non-incorporated radioactivity should be extracted before loading gels, for safety reasons. Therefore, the reaction is terminated by adding 10 pI of 1.5 M NaCl (4°C) and 600 pI of ice cold acetone, followed by centrifugation at 3000g for 30 min at 4°C. Additionally, samples may be stored overnight at -20°C after precipitation. This may produce higher recovery of proteins. Following sedimentation, supernatants are carefully and completely removed by aspiration. Pellets are carefully dissolved in 1 ml of ice cold trichloroacetic acid (TCA, 20 % (v/v))with gentle shak-

A

remove non-incorporated radioactivity

Pertussis Toxin as a Cell Biology Tool

ing of the tube (not vortexing!). This step removes the NaCl which otherwise affects SDS-PAGE. Exposure of the sample to TCA should be short. Therefore, centrifugation (20 to 25 min, 3000g, 4°C) and aspiration of the supernatant is completed as quickly as possible to avoid degradation of proteins. Afterwards, TCA and lipids are removed by adding 1 ml of ice cold chloroform/methanol (l:l,v/v) to the pellet. Alternatively, diethylether may be used, but has the disadvantage of being explosive due to generation of peroxides when stored incorrectly. After centrifugation and careful aspiration as before, the samples are completely dried. Pellets are dissolved in sample buffer (25 to 75~1, room temperature), thoroughly vortexed and subjected to SDS-PAGE (see section 4.5.). Samples should not be boiled before loading onto gels because G proteins sometimes tend to aggregate under these conditions and will not enter separating gels (Codina et a/., 1991).

4.3.6 Cleavage of ADP-ribose from G a Subunits

A

disposal of mercury compounds

ADP-ribosylation of the carboxyterminal cysteine by thioether-linkage is irreversible and chemically stable (Krantz and Lee, 1976). However, PT-catalyzed modifications are susceptible to treatment with mercury (11) ions, allowing removal of ADP-ribose without degrading the remaining Ga subunit (Fig. 4) (Meyer et a/., 1988; Nurnberg et a/., 1994). Solutions containing biological material, e.g. cell membranes, crude or purified proteins supplemented with 1 mg/ml of BSA, should be buffered at pH 7.5. Samples are kept in a glass tubes and treated in parallel with 1 mM HgCI2(final concentration) or water for 60 min at 37°C. The reaction is followed by precipitation with acetone/NaCl, TCA and chloroform/methanol (see section 4.3.5). Analysis is usually done by SDS-PAGE. 32P-ADP-ribosylatedmembranes or other G protein preparations should serve as positive controls. Please note legal rules for disposal of mercury-containing material.

Fig. 4. Cleavage of ADP-ribose from Ga subunits. A mixture of purified heterotrimeric G,/G, proteins (2.2 pg/tube) were 32P-ADP-ribosylatedby pertussis toxin (PT; for details see section 4.3.4). Thereafter, the sample was split and incubated with buffer alone or with buffer supplemented with mercury (11) ions (Hg2+)(for details see section 4.4). The reaction was stopped by triple precipitation as described in section 4.3.5. Proteins dissolved in sample buffer (Laemmli, 1970) were resolved on gels (6 M urea, 9 YO acrylamide, 13 cm length, 4 mm slot width), blotted on nitrocellulose-filters and exposed to X-ray films (32P). Subsequently, identical filters were incubated with antibodies, affinityantiserum AS 6 (anti-aocommon purified) for detection of Ga, proteins. The ECL system was used to visualize filter-bound antibodies. AS 6-stained unmodified Ga, proteins served as controls (left lane) B. NURNBERG

4.4 SDS-Gel Electrophoresis ADP-ribosylated proteins are visualized by SDS-PAGE according to the method of Laemmli (Laemmli, 1970) followed by autoradiography. Usually, discontinuous SDS-polyacrylamide gels are used. For separating gels (9 cm length), 8 to 12 % (w/v) polyacrylamide is employed. The ratio of bisacrylamide to acrylamide is 1 :37.5. For better resolution we add to separating gels either 4.3M urea in the presence of 8 % acrylamide, or 6 M urea in the presence of 9 % acrylamide (Schmidtetal., 1991; Spicher etal., 1992).In the latter case, separating gels should be 13 cm in length. The advantage is better resolution of mixtures of ADP-ribosylated proteins. Using urea-supplemented gels a higher apparent molecular weight, (i.e. a reduced electrophoretic mobility) of the modified proteins in comparison to the untreated counterpart is visible (see Figs 2-4). This phenomenon is the result of a shift in electrophoretic mobility of the modified protein. This can be exploited to check and quantify ADP-ribosylation by immunoblot analysis, comparing the intensities of the faster migrating unmodified protein band with the PT-modified protein band. The BSA band should be inspected to ensure uniform recovery of the proteins after precipitations of samples. Alternatively, resolution is improved by alkylating proteins with N-ethylmaleimide prior to electrophoresis. For details see Sternweis and Pang (1990).

improving resolution

Acknowledgements The continous support of Gunter Schultz is greatly appreciated. Many thanks to my colleagues for valuable discussions. The author’s own research reported here was supported by Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 366, and Fonds der Chemischen Industrie.

4.5 Reagents and Chemicals Material

Supplier

Cat-No.

!-ME

Sigma

M-6250 NEG-023X 236624

2

~

aprotinin ATP BSA CHAPS DNAase DTT

EDTA GDP

-

~

~

~

NEN Boehringer Mannheim Boehringer Mannheim Serva Sigma Boehringer Mannheim Biomol Serva Boehringer Mann heim

519 979 11926 C-3023 104159

04010 11278 106208 Pertussis Toxin as a Cell Biology Tool

Material

Supplier

Cat-No.

GPPNHP

Boehringer Mannheim Boehringer Mannheim Boehringer Mannheim E. Merck E. Merck Serva Boehringer Mannheim Sigma Quadratech, Epsom, Surrey (UK) Sigma Sigma Sigma E. Merck Serva Sigma Gibco

106402

GTP GTPyS K2HP04 KH2P04 Leupeptin NAD p-aminobenzamidine pertussis toxin phenylmethylsulfonylfluoride polyoxyethylene 10 lauryl ether SDS thymidine tris trypsin inhibitor urea

106364 220647 5104 4873 51867 127981 8-6506

180 P-7626 P-9769 L-4509 8206 37190 T-9003 540-5505UV

References Ahnert-Hilger G, Nurnberg B, Exner T et al.: The heterotrimeric G-protein Ga,? inhibits catecholamine uptake into secretory granules. In revision Brass LF, Manning DR, Shattil DR (1990):GTP-binding proteins and platelet activation. In Progr. Hemostasis Thrornb. 10: 127-174 Carty DJ (1994): Pertussis toxin-catalyzed ADP-ribosylation of G-proteins. In Methods Enzymol. 237: 63-70 Cassel D, Pfeuffer T (1978): Mechanism of cholera toxin action: covalent modification of the guanyl nucleotide-binding protein of the adenylate cyclase system. In Proc. Natl. Acad. Sci. (USA)75: 2669-2673 Codina J, Carty DJ, Birnbaumer L et al. (1991):Purification of G proteins. In: Methods Enzymol. 195: 177-188 Gierschik P, Jakobs KH (1993): ADP-ribosylation of signal-transducing guanine nucleotide binding proteins by cholera and pertussis toxin. In Selective neurotoxicity (Herken H, Hucho F, eds), Handbook of Experimental Pharmacology, Vol. 102 pp0.807-839, Springer Verlag: Heidelberg Gilman, A.G. (1995) G proteins and regulation of adenylyl cyclase. In Bioscience Reports 15,65-97 Hageluken A, Nurnberg B, Harhammer R et al. (1995):The class Ill antiarrhythmic drug amiodarone directly activates pertussis toxin-sensitive G proteins. In Mol. Pharmacol. 47: 234-240 Iniguez-LluhiJA, Simon MI, Robishaw JD et al. (1992):G protein By subunits synthesized in Sf9 cells. In J. Biol. Chem. 267: 23409-23417 Jakobs KH, Aktories K, Schultz G (1984): Mechanism of pertussis toxin action on the adenylate system: inhibition of the turn-on reaction of the inhibitory regulatory site. In Eur. J. Biochem. 140: 177-181 Kaslow HR, Schlotterbeck JD, MarVL etal. (1989):Alkylation of cysteine 41, but not cysteine 200, decreases the ADP-ribosyltransferaseactivity of the S1 subunit of pertussis toxin. In J . Biol. Chem. 264: 6386-6390 Kopf GS, Woolkalis MJ (1991): ADP-ribosylation of G proteins with pertussis toxin. In Methods Enzymol. 195: 257-266 Korner C, Nurnberg B, Uhde M et al. (1995):Mannose 6-phosphate/insulin-like growth factor II receptor fails to interact with G-proteins. In J. Biol. Chem. 270: 287-295 B. NURNBERG

Krantz MJ, Lee YC (1976): Quantitative hydrolysis of thioglycosides. In Anal. Biochemistry 71 : 318-321 Laemmli UK (1970): Cleavage of structural proteins during the assembly of the head of bacteriophage T4. In Nature (London) 340: 680-685 Laugwitz KL, Spicher K, Schultz G et a/. (1994): Identification of receptor-activated G proteins: selective immunoprecipitation of photolabeled G-protein a subunits. In Methods Enzymol. 237: 283-294 Meyer T, Hilz H (1986): Production of anti-(ADP-ribose)antibodies with the aid of a dinucleotide-pyrophosphatase-resistent hapten and their application for the detection of mono(ADP-ribosyl)ated polypeptides. In Eur. J. Biochem. 155: 157- 165 Meyer T, Koch R, Fanick W et al. (1988):ADP-ribosyl proteins formed by pertussis toxin are specifically cleaved by mercury ions. In Biol. Chem. Hoppe Seyler 369: 579-583 Milligan G (1988):Techniques used in the identification and analysis of function of pertussis toxin-sensitive guanine nucleotide binding proteins. In Biochem. J. 255: 1-13 Moss J, Stanley SJ, Watkins PA (1980): Isolation and properties of an NAD- and guanidine-dependentADP-ribosyltransferasefrom turkey erythrocytes. In J. Biol. Chem. 255: 5838-5840 Moss J, Stanley SJ, Watkins PA et a/. (1986): Stimulation of the thiol-dependent ADP-ribasyltransferase and NAD glycohydrolase activities of Bordetella pertussis toxin by adenine nucleotides, phospholipids, and detergents. In Biochemistry 25: 2720-2725 Mulheron JG, Casanas SJ, Arthur JM et a/. (1994): Human 5-HTIA receptor expressed in insect cells activates endogenous Go-like G protein(s). In J. Biol. Chem. 269: 12954- 12962 Nurnberg B, Ahnert-Hilger G (1996):Trimeric G proteins of the endomembrane system. In FEBS Lett 389: 61 -65 Nurnberg B, Gudermann T, Schultz G (1995):Receptors and G proteins as primary components of transmembrane signal transduction. Part 2. G proteins: structure and function. In J. Mol. Med. 73: 123-132; corrections: 73: 379 Nurnberg B, Spicher K, Harhammer R etal. (1994):Purification of a novel G-protein a,-subtype from mammalian brain. In Biochem. J. 300: 387-394 Pang IH, Smrcka AV, Sternweis PC (1994):Synthesis and application of affinity matrix containing immobilized By subunits of G proteins. In Methods Enzymol. 237: 164- 174 Ribeiro-Net0 FAP, Mattera R, Hildebrandt JD et al. (1985): ADP-ribosylation of membrane components by pertussis and cholera toxin. In Methods Enzymol. 109: 566-572 Rosenthal W, Koesling D, Rudolph U et al. (1986): Identification and characterization of the 35-kDa fisubunit of guanine nucleotide-binding proteins by an antiserum raised against transducin. In Eur. J. Biochem. 158: 255-263 Rudolph U, Koesling D, Hinsch KD et al. (1989):G-protein a-subunits in cytosolic and membranous fractions of human neutrophils. In Mol. Cell. Endocrinol. 63: 143- 153 Schmidt A, Hescheler J, Offermanns S et a/. (1991) Involvement of pertussis toxinsensitive Ca2' currents in an insulin-secretingcell line (RINmSF).In J. €301. Chem. 266: 18025-18033 Sekura RD (1985): Pertussis toxin: a tool for studying the regulation of adenylate cyclase. In Methods Enzymol. 109: 558-566 Spicher K, Nurnberg B, Jager B et a/. (1992):Heterogeneity of three electrophoretically distinct Goa-subunits in mammalian brain. In FEBS Lett. 307: 215-218 Sternweis PC, Pang IH (1990): Preparation of G-proteins and their subunits. In Receptor-effector coupling. A practical approach (Hulme EJ, ed) pp 1-30, Oxford: University Press Tamura M, Nogimori K, Murai S et a/. (1982):Subunit structure of islet-activating protein, pertussis toxin, in conformity with the A-B model. In Biochemistry 21: 5516-5522

Pertussis Toxin as a Cell Biology Tool

Ui, M (1984): Islet-activating protein, pertussis toxin: a probe for functions of the inhibitory guanine nucleotide regulatory component of adenylate cyclase. In Trends Pharrnacol Sci 5: 277-279 Watkins PA, Burns DL, Kanaho Y et a/. (1985): ADP-ribosylation of transducin by pertussis toxin. In J. Biol. Chern. 260: 13478-13482 Yaiima M, Hosoda K, Kanbayashi Yet a/. (1978~): Islets-activating protein (IAP) in Bordetella pertussis that potentiates insulin secretory responses of rats. In J Biochern (Tokyo) 83: 295-303 Yaiima M, Hosoda K, Kanbayashi Y et a/. (1978b): Biological properties of isletsactivating protein (IAP) purified from the culture medium of Bordetella pertussis. In J Biochem (Tokyo) 83: 305-312

B. NURNBERG

Bacterial Toxins

Klaus Aktories Copyright 0 2002 WILEY-VCH Verlag GmbH & Co. KGaA

Clostridium botulinum AD P-ribosyltransferase C3 K. AKTORIES and G. KOCH

5.1 Introduction ADP-ribosyltransferase C3 is an exoenzyme produced by several strains of Clostridium botulinum. It was serendipitously detected during screening for high producer strains of C. botulinum C2 toxin (Aktories et al., 1987; Aktories et al., 1988b). The novel ADPribosyltransferase was termed C. botulinum ADP-ribosyltransferase C3, because it proved to be distinct from C. botulinum neurotoxin C1 and the actin ADP-ribosylating C. botulinum C2 toxin. C3 ADPribosylates members of the Rho protein family at asparagine-41 thereby inactivating the GTP-binding protein. Therefore, C3 ADPribosyltransferase has become a molecular tool to study the function of Rho proteins.

5.2 The Family of C3-like Transferases The C3 ADP-ribosyltransferase gene was shown to be located on a DNA bacteriophage together with neurotoxin C1 and D (Popoff et al., 1991; Hauser et al., 1993).Various isoforms of the C3 transferase have been described (Popoff et al., 1990; Nemoto et al., 1991; Popoff et al., 1991; Moriishi et al., 1991; Moriishi et al., 1993).Whereas the gene for C3 from C. botulinum strain C468 encodes a protein of 211 amino acids (without signal peptide) with a molecular mass of 23546 Da (Popoff et al., 1990; Popoff et al., 1991), C3 from C. botulinum strain C 003-9 encodes a protein of 204 amino acids with a molecular mass of 23119 Da (Nemoto etal., 1991) showing about 65 % identity with the other C3 isoform. Moreover, C3-related transferases are produced by Clostridium limosum (C. limosum exoenzyme) (Just et al., 1992a), Staphylococcus aureus (EDIN: epidermal differentiation inhibitor) (Inoue et al., 1991), and Bacillus cereus (B. cereus exoenzyme) (Just et al., 199213; Just etal., 1995).Amino acid sequences of the transferases from C. limosum and S. aureus (EDIN)are about 63 % and 30 % identical with those from C. botulinum strains, respectively. So far, the transferase from B. cereus has not been cloned. Partial amino acid analysis indicates that this transferase is rather distantly related to

C3 isoforms

K. Aktories (Ed.), Bacterial Toxins. 0 Chapman & Hall, Weinheim, 1977. ISBN 3-8261-0080-8

other C3-like toxins. All C3-like tranferases are 23-28 kDa proteins with a high content of basic amino acids (pl 9-10). C3 transferase is rather heat stable (1 min, 95°C) and is resistant to short-term trypsin treatment (Aktories et al., 1987). Recently, the active site of C3-like transferases has been identified. It was shown that Glu174 of C. botulium C3 transferase (strain C468; Glu173 in strain 0003-9) is a catalytic important glutamic acid residue, which is strictly conserved in all bacterial ADP-ribosyltransferases studied so far (Jung et al., 1993; Aktories et al., 1995; Bohmer et al., 1996; Saito et al., 1995). Interestingly, it was found that C3 like transferases show a significant degree of sequence similarity (especially in the region around the catalytic site) with various eukaryotic ADPribosyltransferases like rabbit muscle AD P-ribosyltransferase (Okazaki etal., 1994) and the family of RT6-like transferases (Koch-Nolte et al., 1996).

5.3 Modification of Rho Proteins by C3-like Transferases

functions of

Rho proteins

K. AKTORIES and G. KOCH

Rho proteins belong to a subfamily of low molecular mass GTPases that include RhoA,B,C, Racl,2, Cdc42 (G25K), RhoG and TClO (for review see references: Nobes and Hall, 1994; Hall, 1994; Takai et al., 1995; Machesky and Hall, 1996). At the amino acid level, Rho subfamily proteins are about 55% identical to each other and about 30 % identical to Ras proteins. Like other regulatory GTPases (Bourne et al., 1990; Bourne et al., 1991), Rho subfamily proteins are inactive in the GDP bound form and active with GTP bound (Fig. 1). Activation is induced by nucleotide exchange factors (GEF),also called guanine nucleotide dissociation stimulators (GDS) (Feig, 1994; Quilliam et al., 1995; Overbeck et al., 1995; Quilliam et al., 1995). In the active state, Rho subfamily proteins interact with their specific effectors (more than ten potential effectors have been identified and their number is still growing) which appear to be involved in various regulatory processes (see below). The active state of Rho proteins is terminated by hydrolysis of GTP catalyzed by their endogenous GTPase activity. The basal GTPase activity is very low, but is dramatically increased by GAP proteins (GTPase-stimulatingproteins) (Lamarcheand Hall, 1994). Finally, Rho subfamily proteins are regulated by guanine nucleotide dissociation inhibitors (GDI), which inhibit nucleotide exchange. GDI proteins are able to prevent binding of Rho subfamily proteins to membranes where, it is suggested, they act (Isomura et al., 1991). Rho proteins are involved in regulation of the actin cytoskeleton and induce formation of stress fibers and focal adhesions (Paterson et al., 1990; Ridley and Hall, 1992; Tominaga et al., 1993; Laudanna et al., 1996). Most probably independently of their role in actin regulation, Rho proteins have been proposed to participate as molecular switches in the control of phosphoinositide-3-kinase (Zhang et al., 1993)) phosphatidylinositol-4-phosphate-5-kinase (Chong et al.,

Fig. 1. ADP-ribosylation of Rho by C3 transferases. Rho proteins are regulated by a GTPose cycle. The GTP-binding proteins are inactive with GDP bound, and active in the GTP-bound form. GDP/GTP exchange is facilitated by guanine nucleotide dissociation stimulator(s) (GDS) and inhibited by guanine nucleotide dissociation inhibitor(s) (GDI).In the active form, Rho protein interacts with its effector(s) and induces several cellular responses, one of which is polymerization of actin. Rho is ADP-ribosylatedby C3 transferases at asparagine-41. Most likely, the modification inhibits the interaction of Rho with its effector(s) which results in inhibition of Rho dependent processes (e.g. F-actin depolymerization)

1994))phospholipase D (Malcolm et al., 1994), myosin phosphatase (Kimura et al., 1996) and smooth muscle contraction (Hirata et al., 1992), cell-cell contact (Tominaga et al., 1993) and endocytosis (Schmalzing et al., 1995).Furthermore, Rho subtype proteins may play a role in transcriptional activation (Olson et al., 1995; Hill et al., 1995)) and in the transformation of cells induced by the oncogene product Ras (Khosravi-Faret al., 1995; Qiu et al., 1995). Rac proteins (Racl,2), which are also members of the Rho subfamily, are involved in membrane ruff ling and lamellipodia formation induced by growth factors (Ridley et al., 1992; Hall, 1994),and it has also been suggested that they are important for Ras-induced transformation (Qiu et al., 1995). Moreover, Rac proteins control NADPH oxidase (Abo et al., 1991; Bokoch, 1994), and may have a role in phospholipase A2 regulation (Peppelenbosch et al., 1995). Cdc42, another member of the Rho family, which occurs in at least two iso-

functions of Rac and Cdc42 proteins

Clostridium botulinum ADP-ribosyltransferase C3

substrate specificity of C3

forms, participates in receptor-induced formation of microspikes and filopodia (Nobes and Hall, 1995; Kozma et al., 1995). C3 shows a remarkable substrate specificity and modifies RhoA, B, C (Hall, 1994; Paterson et al., 1990; Chardin et al., 1989; Wiegers et al., 1991;Aktories et al., 1989; Braun et al., 1989), while other members of the Rho subfamily are poorly modified (e.g. 5-10% ADPribosylation of Rac in the presence of 0.01 % SDS), or not at all (Cdc42) (Just et al., 1992~). Other low molecular mass GTP-binding proteins or heterotrimeric G-proteins are not substrates for C3. The other C3-like transferases appear to share the same substrate specificity. C3-like transferases ADP-ribosylate Rho proteins at asparagine41 (Sekine et al., 1989).Asparagine-41 is located in the effector region of the small GTP-binding proteins and attachment of the bulky ADPribose group by ADP-ribosylation may inhibit the interaction of Rho with its effectors. Alternatively, ADP-ribosylation may affect the activation of the Rho protein by guanine nucleotide exchange factors.

5.4 Characterization of the C. botulinum C3 ADP-Ribosyltransferase Reaction Like other bacterial ADP-ribosylating toxins (e.g. diphtheria toxin, Pseudornonas aeruginosa exotoxin A, cholera toxin, pertussis toxin, and C. botulinurn C2 toxin (Aktories and Just, 1993)),C3 is a monoADP-ribosyltransferase (Aktories et al., 198813). Treatment of ADPribosylated Rho with phosphodiesterase releases 5'-AMP and not phosphoribosyl-AMP, a cleavage product of poly(ADP-ribose) (Aktories et al., 198813; Rubin et al., 1988).Accordingly, thymidine, an inhibitor of poly(ADP-ribose)polymerase, does not block C3-like ADPribosyltransferases, and can be included in C3 ADP-ribosylation assays to block poly-ADP-ribosylation reactions. Asparagine is unique as an acceptor amino acid for ADPribosylation by C3-like transferases (Sekine et al., 1989). In contrast, pertussis toxin modifies cysteine residues, and cholera toxin and C. botulinurn C2 toxin specifically ADP-ribosylate arginine residues (for details see the relevant chapters). The ADP-ribose-asparagine bond formed by C3-like transferases is stable towards neutral hydroxylamine (OSM, 2h) and mercury ions (2mM, 1 h), whereas cysteine and arginine-specific ADP-ribosylation, respectively, are sensitive towards these agents (Aktories et al., 1988~). ADP-ribosylation by C3 and its isoenzymes is a reversible reaction, i.e. in the presence of high concentrations of nicotinamide (10mM) and at low pH (pH <7) it can be reversed (Habermann et al., 1991). The de-ADP-ribosylation reaction has been exploited to identify the acceptor amino acid of ADP-ribosylation by C3-like isoforms. Like other AD P- ribosyItra nsferases, C3-Iike exoenzymes exhibit NAD g lycohydrolase activity (Aktories et al., 1988b). However, this enzyme activity is at least 100-fold lower than the transferase activity and most likely has no physiological significance. K. AKTORIES and G. KOCH

5.5 Factors Affecting ADP-Ribosylation by C3 In principle, ADP-ribosylation of Rho by C3 needs no other factors in addition to Rho and NAD (for review see also (Aktories and Just, 1995)).Rho is ADP-ribosylated in intact cells, in cell lysate (membranes and cytosol), and as a recombinant protein. Even Rho-glutathione S-transferase fusion proteins are substrates for ADPribosylation by C3. ADP-ribosylation by C3 is affected by guanine nucleotides, divalent cations, detergents and temperature. Purified endogenous Rho, recombinant Rho proteins, and the membranous Rho protein are better substrates for ADP-ribosylation when bound to GDP rather than GTP (Habermann et al., 1991). In contrast, ADPribosylation of cytosolic Rho is enhanced by addition of GTPyS or GTP (Williamson etal., 1990).It is suggested that the nucleotides facilitate dissociation of RhoGDI, because affinity of GTP-bound RhoA for RhoGDl is lower than of GDP-bound RhoA (Kikuchi et al., 1992; Regazzi et al., 1992; Bourmeyster et al., 1992). Free Rho protein is a better substrate for C3 than Rho complexed with RhoGDI. Similarly, C3 ADP-ribosylation of cytosolic or partially purified Rho protein is increased in the presence of detergents (sodium cholate, deoxycholate) or phospholipids (phosphatidylinositol bisphosphate). This suggests that detergents and phospholipids dissociate the complex between RhoGDl and Rho protein and provide access for C3-like exoenzymes to ADP-ribosylate Rho (Bourmeyster et al., 1992; Just et al., 1993). In the absence of GDI, C3-catalyzed ADP-ribosylation is also affected by various detergents. Sodium cholate (0.2 %), deoxycholate, dimyristoylphosphatidylcholine (3mM), and SDS (0.01 %) increase C3-catalyzed ADP-ribosylation. In contrast, CHAPS, LubrolPX, and SDS (>0.03 %) impair ADP-ribosylation (Just et al., 1992a; Just et al., 1993; Maehama et al., 1990).

5.6 C3-Transferases in Cell Biology and Pharmacology In intact animals, toxicity of C3 is low compared with other toxins that affect Rho proteins. lntraperitoneal injection of 100 pg of C3 into mice is apparently without obvious consequences. (Note that the minimum lethal dose of C. difficile toxins A and B, which also modify Rho proteins, is about 50 ng per mouse). C3-like transferases appear to lack any specific cell-binding or transport subunits, a fact that calls into question the designation of these exoenzymes as toxins. However, when these bacterial exoenzymes reach the eukaryotic cytosol (e.g. by unspecific uptake or microinjection, see below) they are potent cytotoxins (Wiegers et al., 1991; Didsbury et al., 1989; Chardin et al., 1989; Paterson et al., 1990). Thus, the use of C3 toxin as a cell biological tool is hampered by the fact that C3 is not able to enter cells readily. The problem of cell accessibility was first by-passed by using

low toxicity of C3

Clostridium botulinum ADP-ribosyltransferase C3

osmotic shock to introduce C3 into cells (Rubin et al., 1988). Many excellent studies from the laboratory of Alan Hall have been performed using the microinjection technique (Paterson et al., 1990; Ridley and Hall, 1992) (see Chapter 6). Other approaches to study the effects of C3 in eukaryotic cells used expression of transfected C3 DNA (Hill et al., 1995), a C3 fusion toxin consisting of C3 and the receptor and translocation domain of diphtheria toxin (Aullo et al., 1993), and permeabilization of cells before C3 treatment (Koch et al., 1994). However, most studies exploited the property of C3 of entering most cells when applied at rather high concentrations (30-150 pg/ml) (Wiegers et al., 1991; Nishiki et al., 1990).Additionally, it appears that some C3 isoforms are somewhat more capable of entering culture cells. In some studies, effects of C3 were even observed at low concentrations (0.5 to 1 pg/ml) of the transferase (Sugai et al., 1992).So far, it is not clear whether these effects are caused by different C3 isoforms or by specific conditions of cell culture (serum-free treatment).

References Abo A, Pick E, Hall A et al. (1991):Activation of the NADPH oxidase involves the small GTP-binding protein p21rac. In Nature 353: 668-70 Aktories K, Weller U, Chhatwal GS (1987):Clostridium botulinum type C produces a novel ADP-ribosyltransferasedistinct from botulinum C2 toxin. In FEBS Lett. 212: 109- 13 Aktories K, Just I, Rosenthal W (1988a):Different types of ADP-ribose protein bonds formed by botulinum C2 toxin, botulinum ADP-ribosyltransferaseC3 and pertussis toxin. In Biochem. Biophys. Res. Commun. 156: 361 -7 Aktories K, Rosener S, Blaschke U et a/. (198813):Botulinum ADP-ribosyltransferase C3. Purification of the enzyme and characterization of the ADP-ribosylation reaction in platelet membranes. In Eur. J. Biochem. 172: 445-50 Aktories K, Braun U, Rosener S et a/. (1989):The rho gene product expressed in E. coli is a substrate of botulinum ADP-ribosyltransferaseC3. In Biochem. Biophys. Res. Commun. 158: 209-13 Aktories K, Jung M, Bohmer J et al. (1995):Studies on the active site structure of C3-like exoenzymes: Involvement of glutamic acid in catalysis of ADPribosylation. In Biochimie 77: 326-32 Aktories K, Just I (1993): GTPases and actin as targets for bacterial toxins. In GTPases in biology I, (Dickey BF, Birnbaumer L., eds) pp. 87-112 BerlinHeidelberg: Springer-Verlag Aktories K, Just I (1995): In vitro ADP-ribosylation of Rho by bacterial ADPribosyltransferases. In Methods Enzymol. 256: 184-95 Aullo P, Giry M, Olsnes S et al. (1993): A chimeric toxin to study the role of the 21 kDa GTP binding protein rho in the control of actin microfilament assembly. In EMBO J. 12: 921 -31 Bokoch G M (1994): Regulation of the human neutrophil NADPH oxidase by the Rac GTP-binding proteins. In Curr. Opin. Cell Biol. 6: 212-8 Bourmeyster N, Stasia M-J, Garin J et al. (1992):Copurification of rho protein and the rho-GDP dissociation inhibitor from bovine neutrophil cytosol. Effect of phosphoinositides on rho ADP-ribosylation by the C3 exoenzyme of Clostridium botulinum. In Biochemistry 31 : 12863-9 Bourne HR, Sanders DA, McCormick F (1990):The GTPase superfamily: a conserved switch for diverse cell functions. In Nature 348: 125-32 Bourne HR, Sanders DA, McCormick F (1991):The GTPase superfamily: conserved structure and molecular mechanism. In Nature 349: 117-27 K. AKTORIES and G. KOCH

Bohmer J, Jung M, Sehr P et a/. (1996): Active site mutation of the C3-like ADPribosyltransferase from Clostridium limosum - Analysis of glutamic acid 174. In Biochemistry35: 282-9 Braun U, Habermann B, Just I et a/. (1989): Purification of the 22 kDa protein substrate of botulinum ADP-ribosyltransferase C3 from porcine brain cytosol and its characterization as a GTP-binding protein highly homologous to the rho gene product. In FEBS Lett. 243: 70-6 Chardin P, Boquet P, Madaule P et al. (1989): The mammalian G protein rhoC is ADP-ribosylated by Clostridium botulinum exoenzyme C3 and affects actin microfilaments in Vero cells. In EM60 J. 8: 1087-92 Chong LD,Traynor-Kaplan A, Bokoch GM etal. (1994):The small GTP-binding protein Rho regulates a phosphatidylinositol 4-phosphate 5-kinase in mammalian cells. In Cell 79: 507-13 Didsbury J, Weber RF, Bokoch GM et al. (1989): Rac, a novel ras-related family of proteins that are botulinum toxin substrates. In J. Biol. Chem. 264: 16378-82 Feig LA (1994): Guanine-nucleotide exchange factors: A family of positive regulators of Ras and related GTPases. In Curr. Opin. Cell Biol. 6 : 204-11 Habermann B, Mohr C, Just I et a/. (1991): ADP-ribosylation and de-ADPribosylation of the rho protein by Clostridium botulinum exoenzyme C3. Regulation by EDTA, guanine nucleotides and pH. In Biochim. Biophys. Acta 1077: 253-8 Hall A (1994):Small GTP-binding proteins and the regulation of the actin cytoskeleton. In Annu. Rev. Cell Biol. 10: 31 -54 Hauser D, Gibert M, Eklund M W et a/. (1993): Comparative analysis of C3 and botulinal neurotoxin genes and their environment in Clostridium botulinum types C and D. In J. Bacteriol. 175: 7260-8 Hill CS, Wynne J, Treisman R (1995): The Rho family GTPases RhoA, Racl, and CDC42Hs regulate transcriptional activation by SRF. In Cell 81: 1159-70 Hirata K, Kikuchi A, Sasaki T et al. (1992): Involvement of rho p21 in the GTPenhanced calcium ion sensitivity of smooth muscle contraction. In J. Biol. Chem. 267: 8719-22 lnoue S, Sugai M, Murooka Y etal. (1991):Molecular cloning and sequencing of the epidermal cell differentiation inhibitor gene from Staphylococcus aureus. In Biochem. Biophys. Res. Cornmun. 174: 459-64 lsomura M, Kikuchi A, Ohga N et a/. (1991): Regulation of binding of rhoB p20 to membranes by its specific regulatory protein GDP dissociation inhibitor. In Oncogene 6: 119-24 Jung M, Just I, van Damme J et al. (1993): NAD-binding site of the C3-like ADPribosyltransferase from Clostridium limosum. In J. Biol. Chem. 268: 23215-8 Just I, Mohr C, Schallehn G et a/. (1992~): Purification and characterization of an ADP-ribosyltransferase produced by Clostridium limosum. In J. Biol. Chem. 267: 10274-80 Just I, Schallehn G, Aktories K (1992b):ADP-ribosylation of small GTP-binding proteins by Bacillus cereus. In Biochem. Biophys. Res. Commun. 183: 931 -6 Just I, Mohr C, Habermann B etal. (1993): Enhancement of Clostridium botulinum C3-catalyzed ADP-ribosylation of recombinant rhoA by sodium dodecyl sulfate. In Biochem. Pharmacol. 45: 1409- 16 Just I, Selzer J, Jung M et a/. (1995):Rho-ADP-ribosylating exoenzyme from Bacillus cereus -purification, characterization and identification of the NAD-binding site. In Biochemistry34: 334-40 Khosravi-Far R, Solski PA, Clark GJ et a/. (1995): Activation of Racl, RhoA, and mitogen-activated protein kinases is required for Ras transformation. In Mol. Cell. Biol. 15: 6443-53 Kikuchi A, Kuroda S, Sasaki T et a/. (1992): Functional interactions of stimulatory and inhibitory GDP/GTP exchange proteins and their common substrate small GTP-binding protein. In J. Biol. Chem. 267: 14611-5 Kimura K, Ito M, Amano M etal. (1996):Regulation of myosin phosphatase by Rho and Rho-associated kinase (Rho-kinase). In Science 273: 245-8 Koch G, Norgauer J, Aktories K (1994): ADP-ribosylation of Rho by Clostridium limosum exoenzyme affects basal but not N-formyl-peptide-stimulated actin Clostridium botulinum ADP-ribosyltransferase C3

polymerization in human myeloid leukaemic (HL60) cells. In Biochem. J. 299: 775-9 Koch-Nolte F, Petersen D, Balasubramanian S etal. (1996):MouseTcell membrane proteins Rt6- 1 and Rt6-2 are arginine protein mono(ADPribosy1)transferases and share secondary structure motifs with ADP-ribosylating bacterial toxins. In J. Biol. Chem. 271 : 7686-93 Kozma R, Ahmed S, Best A et al. (1995):The Ras-relatedprotein Cdc42Hs and bradykinin promote formation of peripheral actin microspikes and filopodia in Swiss 3T3 fibroblasts. In Mol. Cell. Biol. 15: 1942-52 Lamarche N, Hall A (1994): GAPS for rho-related GTPases. In Trends Genet. 10: 436-40 Laudanna C, Campbell JJ, Butcher EC (1996): Role of Rho in chemoattractantactivated leukocyte adhesion through integrins. In Science 271 : 981 -3 Machesky LM, Hall A (1996): Rho: a connection between membrane receptor signalling and the cytoskeleton. In Trends Cell Biol. 6: 304-10 Maehama T, Ohoka Y,Ohtsuka T et al. (1990): Botulinum ADP-ribosyltransferase activity as affected by detergents and phospholipids. In FEBS Lett. 263: 376-80 Malcolm KC, Ross AH, Qiu R-G et al. (1994):Activation of rat liver phospholipase D by the small GTP-binding protein RhoA. In J. Biol. Chem. 269: 25951 -4 Moriishi K, Syuto B, Yokosawa N et al. (1991): Purification and characterization of ADP-ribosyltransferases (ExoenzymeC3) of Clostridium botulinum type C and D strains. In J. Bacteriol. 173: 6025-9 Moriishi K, Syuto B, Saito M et al. (1993): Two different types of ADPribosyltransferaseC3 from Clostridium botulinum type D lysogenized organisms. In Infect. Immun. 61 : 5309- 14 Nemoto Y, Namba T, Kozaki S et al. (1991): Clostridium botulinum C3 ADPribosyltransferase gene. In J. Biol. Chem. 266: 19312-9 NishikiT, Narumiya S, Morii N etal. (1990):ADP-ribosylation of the rho/rac proteins induces growth inhibition, neurite outgrowth and acetylcholine esterase in cultured PC-12 cells. In Biochem. Biophys. Res. Commun. 167: 265-72 Nobes C, Hall A (1994): Regulation and function of the Rho subfamily of small GTPases. In Curr. Opin. Genet. Dev. 4: 77-81 Nobes CD, Hall A (1995):Rho, Rac, and Cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. In Cell 81 : 53-62 Okazaki IJ, Zolkiewska A, Nightingale MS et al. (1994): Immunological and structural conservation of mammalian skeletal muscle glycosylphosphatidylinositollinked ADP-ribosyltransferases. In Biochemistry 33: 12828-36 Olson MF, Ashworth A, Hall A (1995):An essential role for Rho, Rac, and Cdc42 GTPases in cell cycle progression through GI. In Science 269: 1270-2 Overbeck AF, Brtva TR, Cox AD et al. (1995):Guanine nucleotide exchange factors: Activators of Ras superfamily proteins. In Mol. Reprod. Dev. 42: 468-76 Paterson HF, Self AJ, Garrett M D etal. (1990):Microinjection of recombinant p21rh0 induces rapid changes in cell morphology. In J. Cell Biol. 111: 1001-7 Peppelenbosch MP, Qiu R-G, De Vries-Smits AMM et al. (1995): Rac mediates growth factor-induced arachidonic acid release. In Cell 81 : 849-56 Popoff MR, Boquet P, Gill D M et al. (1990): DNA sequence of exoenzyme C3, an ADP-ribosyltransferase encoded by Clostridium botulinum C and D phages. In Nucl. Acids Res. 18: 1291 Popoff MR, Hauser D, Boquet P et al. (1991): Characterization of the C3 gene of Clostridium botulinum types C and D and its expression in Escherichia coli. In Infect. Immun. 59: 3673-9 Qiu R-G, Chen J, Kirn D et al. (1995): An essential role for Rac in Ras transformation. In Nature 374: 457-9 Qiu RG, Chen J, McCormick F et al. (1995):A role for Rho in Ras transformation. In Proc. Natl. Acad. Sci. USA 92: 11781-5 Quilliam LA, Khosravi-Far R, Huff SY et al. (1995):Guanine nucleotide exchange factors: Activators of the Ras superfamily of proteins. In Bioessays 17: 395-404

K. AKTORIES and G. KOCH

Regazzi R, Kikuchi A, Takai Y et al. (1992):The small GTP-binding proteins in the cytosol of insulin-secreting cells are complexed to GDP dissociation inhibitor proteins. In J. Biol. Chem. 267: 17512-9 Ridley AJ, Paterson HF, Johnston CL etal. (1992):The small GTP-binding protein rac regulates growth factor-induced membrane ruffling. In Cell 70: 401 -10 Ridley AJ, Hall A (1992):The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. In Cell 70: 389-99 Rubin EJ, Gill DM, Boquet P et al. (1988):Functional modification of a 21-kilodalton G protein when ADP-ribosylated by exoenzyme C3 of Clostridium botulinum. In Mol. Cell. Biol. 8: 418-26 Saito Y, Nemoto Y, lshizaki T etal. (1995):Identificationof GIu”~ as the critical amino acid residue for the ADP-ribosyltransferase activity of Clostridium botulinum C3 exoenzyme. In FEBS Lett. 371 : 105-9 Schmalzing G, Richter HP, Hansen A etal. (1995):Involvement of the GTP binding protein Rho in constitutive endocytosis in Xenopus laevis oocytes. In J. Cell Biol. 130: 1319-32 Sekine A, Fujiwara M, Narumiya S (1989): Asparagine residue in the rho gene product is the modification site for botulinum ADP-ribosyltransferase.In J. Biol. Chem. 264: 8602-5 Sugai M, Hashimoto K, Kikuchi A etal. (1992):Epidermal cell differentiation inhibitor ADP-ribosylates small GTP-binding proteins and induces hyperplasia of epidermis. In J. Biol. Chem. 267: 2600-4 Takai V, Sasaki T, Tanaka K et a/. (1995):Rho as a regulator of the cytoskeleton. In Trends Biochem. Sci. 20: 227-31 Tominaga T, Sugie K, Hirata M et a/. (1993): Inhibition of PMA-induced, LFA-1dependent lymphocyte aggregation by ADP-ribosylation of the small molecular weight GTP binding protein, rho. In J. Cell Biol. 120: 1529-37 Wiegers W, Just I, Muller H etal. (1991):Alteration of the cytoskeleton of mammalian cells cultured in vitro by Clostridium botulinum C2 toxin and C3 ADPribosyltransferase.In Eur. J. Cell Biol. 54: 237-45 Williamson KC, Smith LA, Moss J et al. (1990): Guanine nucleotide-dependent ADP-ribosylation of soluble rho catalyzed by Clostridium botulinum C3 ADPribosyltransferase.In J. Biol. Chem. 265: 20807- 12 Zhang J, King WG, Dillon S et al. (1993):Activation of platelet phosphatidylinositide 3-kinase requires the small GTP-binding protein Rho. In J. Biol. Chem. 268: 22251-4

Clostridiurnbotulinurn ADP-ribosyltransferose C3

Bacterial Toxins

Klaus Aktories Copyright 0 2002 WILEY-VCH Verlag GmbH & Co. KGaA

Clostridium botulinum C3 Exoenzyme and Studies on Rho Proteins C. D. NOBES and A. HALL

6.1 Introduction The Rho family of small GTP-binding proteins functions to regulate the assembly of distinct actin structures in cells; Rho regulates stress fiber assembly, Rac regulates lamellipodia protrusion and Cdc42 stimulates protrusion of the plasma membrane to form filopodia (Ridley and Hall, 1992; Ridley et al., 1992; Nobes and Hall, 1995; Kozma et al., 1995). All three regulate attachment of cells to the extracellular matrix via adhesive integrin structures (Nobes and Hall, 1995). We and others have used the bacterial exoenzyme C3 ADPribosyltransferase from Clostridium botulinum to analyze the signaling pathways controlled by the Rho GTPase. The introduction of C3 transferase into a variety of cell types causes them to lose their actin stress fibers, round up, and eventually detach from the underlying substrate (Rubin et al., 1988; Chardin et al., 1989; Paterson et al., 1990).The targets of C3 in cells are the three isoforms of the Rho protein, RhoA, RhoB and RhoC (Narumiya et al., 1988); the enzyme catalyzes the transfer of an ADP-ribose group from NAD' to an asparagine residue at codon 41 of Rho (Aktories et al., 1989; Sekine et al., 1989) and renders the protein inactive (Paterson et al., 1990). Other Rho family members such as Rac and Cdc42 are essentially not substrates for C3 in vitro (Ridley et al., 1992; Just et al., 1992), and microinjection of C3 into cells does not affect the activity of either Rac or Cdc42 (Ridley et al., 1992; Nobes and Hall, 1995).The introduction of C3 transferase into cells has, therefore, been a valuable tool for assessing the specific cellular roles of the Rho GTPase.

6.2 Purification of Recombinant C3 Transferase from Escherichia coli High levels of recombinant C3 transferase have been expressed in E. coli using the glutathione S-transferase (GST) gene fusion vector, pGEX-2T (Pharmacia LKB Biotechnology, Inc).The GST-C3 expression vector was constructed in the laboratory of Dr. Larry Feig (Tufts UniverK. Aktories (Ed.),Bacterial Toxins. 0 Chapman & Hall, Weinheim, 1997. ISBN 3-8261-0080-8

sity School of Medicine, Boston, USA) as described in (Dillon and Feig, 1995), and introduced into the E. coli strain JM101 and stored as a glycerol stock at -70 "C. During cloning, additional amino acids were introduced at the amino terminus of the protein; consequently, after cleavage of the GSTfusion protein, the recombinant C3 is 26 amino acids longer than the enzyme purified from Clostridia. The N-terminal extension comprises 19 amino acids from the pUC19 polylinker and the last 7 amino acids of the signal peptide sequence of C3 in C. botulinurn (Dillon and Feig, 1995).We have never compared directly, in the same microinjection experiments, the relative activities of recombinant C3 and that purified from C. botulinurn. However, both preparations have been microinjected separately in this laboratory and we are unaware of any significant differences between them.

6.2.1 Purification of Recombinant GST-C3 We routinely grow 1 litre of E. coli containing the GST-C3 protein expression plasmid, and this yields approximately 2-3 mg of recombinant C3 protein. 1. L-broth (100ml) containing 50 pg/ml ampicillin is inoculated with bacteria containing the GST-C3 construct and incubated overnight at 37 "C in a bacterial shaker. 2. The following morning the culture is diluted 1 :10 into fresh Lbroth/ampicillin (room temperature) and incubated further with shaking in two 2-litre flasks (500ml culture/flask) at 37 "C for 1 h.

3. GST-C3 fusion protein expression is induced by adding isopropyl-(3-D-thiogalactopyranoside (IPTG; Calbiochem) to 0.2 m M (1 ml of 0.1 M stock made in water and stored at -20 "C), and the culture is incubated with shaking for a further 3 h. 4. The cells are pelleted by centrifugation (4000 r.p.m for 10 min at 4 "C) and resuspended (on ice) in 3 ml cold lysis buffer (50 mM Tris-HCI, pH Z6, 50 m M NaCI, 5 m M MgC12, 1 m M dithiothreitol [DTT], and 1 m M phenylmethylsulfonyl fluoride [PMSF]).

5. Resuspended bacteria are lysed by sonication on ice using a small probe on an MSE Soniprep 150 sonicator at an amplitude of 12pm (six bursts for 10s each), and the bacterial debris removed by centrifugation (10 000 r.p.m for 10 min at 4 "C). 6. Glutathione-agarose beads (1 ml of a 1 : l suspension, Sigma G4510) are first washed with three changes of lysis buffer (5 ml), and the bacterial supernatant (about 4 ml) is added to the beads. The slurry is incubated for 30 min on a rotating wheel at 4 "C.

7. The beads, with bound GST-C3 fusion protein, are pelleted in a bench top centrifuge at 4000 r.p.m for 1 min and the supernatant C. D. NOBES and A. HALL

removed and discarded. The beads are washed with 10ml of cold lysis buffer (with 1 m M DTT but without PMSF) three times to remove unbound proteins.

6.2.2 Recovery of Cleaved C3 Transferase 1. The washed beads are transferred to a 1.5ml microcentrifuge tube, resuspended in 0.5 ml of thrombin digestion buffer (50 m M Tris-HCI, pH 8.0, 150mM NaCI, 2.5mM CaCI2, 2 m M MgCI2, 1 mM DTT) containing 5 units of bovine thrombin (SigmaT6634), and incubated on a rotating wheel overnight at 4 "C. 2. After thrombin digestion, the beads are pelleted in a microcentrifuge (1 min at 4000 r.p.m) and the supernatant is removed. 3. Any remaining protein associated with the beads is recovered with 0.5 ml of high salt buffer (50m M Tris-HCI, pH Z6, 150 m M NaCI, 5 mM MgCI2, 1 mM DTT) for a further 2 min at 4 "C, and the two supernatants combined. The efficiency of thrombin cleavage of GST-C3 approaches 90 % (Fig. 1).

4. Thrombin is removed by adding 20 pl of a 1 : 1 suspension of paminobenzamidine-agarose beads (Sigma A7155) to the supernatant and incubating for a further 30 min at 4 "C on a rotating wheel.

5. Finally, the beads with bound thrombin are pelleted in a microcentrifuge for 1 min at 4000 r.p.m.

Fig. 1. Purification of thrombin-cleaved C3 transferase. Samples loaded: GST-C3 protein bound to beads before thrombin cleavage (lane 1); protein remaining on beads after thrombin cleavage (mainly GST) (lane 2); C3 protein eluted from beads (lane 3)

Clostridiurn botulinurn C3 Exoenzyme and Studies on Rho Proteins

6.2.3 Dialysis and Storage 1. The supernatant (1 ml) containing C3 protein is first dialyzed against two changes of buffer (15 mM Tris-HCI, pH 7.5, 150 mM NaCI, 5 mM MgC12and 0.1 m M DTT) at 4 "C for 1 h each. The protein is concentrated to approximately 5OOp.I in an Amicon Centricon filter unit by centrifugation in a fixed angle rotor at 7000 r.p.m.

do not over-concentrate C3

It is important not to over-concentrate C3, since it tends to precipitate at high concentration (>5mg/ml). Solutions of recombinant C3 prepared in this way generally contain 3-5 mg/ml, and are suitable for microinjection. Small aliquots (201.11) of the C3 transferase are stored at -20 "C. A working stock, of approximately 0.7-1 mg/ml in dialysis buffer, can be stored at 4 "C and dilutions made from this working stock at the time of microinjection.

6.2.4 Determination of Protein Concentration and Assay of Activity The concentration of each C3 protein preparation is determined with a Biorad protein assay kit, using BSA as a standard. The relative activities of C3 transferase preparations can be determined by ADPribosylation of recombinant Rho protein or by microinjection. For the ribosylation assay, 10 ng of recombinant RhoA protein are incubated for 1 h at 37 "C in 50 pl of ADP-ribosylation buffer (50 m M Tris-HCI, pH 7.4, 50mM NaCI, 5 m M MgCI2, 0.3mM GTP, 1 m M DTT, 1 pCi [32P]NAD)with varying amounts of C3 protein, ranging from 1 to 200 ng. Gel electrophoresis sample buffer is added, the samples are boiled, and proteins resolved by SDS-polyacrylamide gel electrophoresis (13.5 70). Protein is first visualized with Coomassie blue, and then the gel is destained with many washes to remove any unincorporated [32P]NAD.ADP-ribosylated rho is visualized by autoradiography. Each new preparation of C3 protein is also assayed by microinjection into quiescent serum-starved Swiss 3T3 cells, followed by treatment with lysophosphatidic acid, LPA (100ng/ml for 15 or 30min; Sigma L7260) to stimulate Rho and stress fibre assembly (see Section 6.6). The lowest concentration of C3 that still blocks the LPA effect provides a measure of the relative activity of different batches of C3.

6.3 Preparation of Swiss 3T3 Cells Swiss 3T3 cells are maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS), 0.11 g/l sodium pyruvate, 4.5 g/l glucose, 100 U/ml penicillin and 100 pg/ml streptomycin at 37 "C with 10 % C 0 2 . Cells are grown to 90 % confluence in 80cm2 flasks and passaged (1 :7) twice a week. We culture Swiss 3T3 cells up to passage 13 (our stock cells are described as C. D. NOBES and A. HALL

passage 5)after which we discard and thaw a new stock of low passage (passage 5)cells. We find that when Swiss 3T3 cells are cultured for longer than passage 13 they show an increasing tendency not to become contact inhibited and quiescent; such populations retain polymerized actin structures (mainly stress fibres) on serum starvation (see Section 6.6). We inject Swiss 3T3 cells in three different states: confluent, quiescent; subconfluent with serum; and subconfluent without serum.

use low passage cells

Confluent, quiescent: The cells are prepared by seeding at high density (5xlo4 cells) onto 13mm acid washed, baked glass coverslips in 15 mm diameter tissue culture wells (Nunc) containing 1 ml DMEM/10% FCS. Prior to washing, glass coverslips are marked at their approximate midpoint with a cross, using a diamond pen, to facilitate localization of injected cells after staining. After 6-10 days, when the cells are quiescent, the medium is removed and replaced (without washing) with 1 ml DMEM (from powder stock; Gibco cat. no. 52100-021) supplemented with 2 g/l N a H C 0 3for 16 h. 2. Subconfluent, with serum: Cells are plated onto coverslips at a density of 6 x lo3 cells in DMEM/10 % FCS and allowed to attach for 2-3 h before microinjection.

3. Serum-starved, subconfluent cells: Cells are seeded at a density of 3 x lo5 cells into 80 cm2 flasks and grown to confluence. The cells are left without medium change to become quiescent (normally 7-10 days after seeding) before serum starving overnight in DMEM with 2g/l NaHC03. After washing with PBS, cells are trypsinized briefly, resuspended in serum-free medium containing 0.5mg/ml soybean trypsin inhibitor (SigmaT9003) pelleted, and resuspended in serum-free medium. Cells are plated on coverslips coated with fibronectin (50pg/ml, [Bio Products Laboratory]) in serum-free medium. These cells contain few polymerized actin structures, and few if any clusters of vinculin in focal adhesions. Coverslips for microinjection are transferred to 60 mm dishes containing 4 ml DMEM (with or without 10 % FCS as appropriate) and are replaced in their original tissue culture wells after microinjection.

6.4 Microinjection of C3 Transferase Routinely, 10 x stock solutions of recombinant C3 protein (0.7-1 mg/ ml) are stored at 4 "C. Long term storage of more concentrated stocks (4-5mg/ml) is at -20 "C. Appropriate dilutions of C3 transferase for microinjection are made up fresh on the day of injection in 150mM NaCI, 50mM Tris pH 7.5,5 m M MgCI2, on ice. So as to be able to identify injected cells later, C3 protein is co-iniected with either rat immunoglobulin (final concentration 0.5mg/ml [Pierce cat. no. 318851) or FITC- or Texas Red-lysinated dextrans (final concentration 2 mg/ml [Molecular Probes D1820 and D18631).This mixture is centriClostridium botulinum C3 Exoenzyme and Studies on Rho Proteins

fuged for 5 min at 15, 000 g at 4 "C to spin down any large particles which may block the microinjection needle. Micropipettes for injection are pulled from glass capillaries (Clarke Electromedical Instruments GC120F-10) with a programmable pipette puller (Campden Instruments Model no. 773). The coil temperature, pull force and the time and distance of pull are optimized to obtain optimization of micropipettesmicropipettes of approximately 0.5 pm tip diameter. We microinject cells using a Zeiss/Eppendorf microinjection workstation. With this set-up, the cells are maintained at 37 "C with an atmosphere of 10 % C 0 2 during microinjection. Cells are injected in the manual mode using an Eppendorf micromanipulator (model 5170) and an Eppendorf microinjector (model 5242), and the injection pressure is adjusted to give a constant flow rate of injection material. C3 protein is injected into the cytoplasm of cells. It has been estimated that between 1 and 2 x lo-'' ml is injected per fibroblast cell (Graessmann and Graessmann, 1983). With practice it is possible to inject routinely between 100 and 200 cells per coverslip in a 10-15 minute period. The cells on the coverslips are then returned to the incubator for varying lengths of time as appropriate.

6.5 Fixation and Staining of Cells 1. Cells injected with C3 transferase are rinsed in PBS before being fixed for 10 min at room temperature in a freshly prepared solution of 4 % paraformaldehyde in PBS.

A

use in fume cupboard

2. Cells are permeabilized with 0.2% Triton X-lOO/PBS at room temperature for 5 min.

3. After reducing free aldehyde groups by treatment with sodium borohydride (1 mg/ml in PBS for 10 min), cells are stained for filamentous actin by incubating for 30 min with 0.1 pg/ml tetramethylrhodamine isothiocyanate (TRITC)-phalloidin (Sigma P1951) in PBS. 4. In the case of co-injection with rat IgG, the cells are incubated at the same time with a 1 :300 dilution of fluorescein isothiocyanate (FITC)-conjugated goat anti-rat immunoglobulin antibody (Sigma F7631).

5. Cells can also be labeled for components of focal adhesions using antibodies against, for example, vinculin (Sigma V4505), paxillin (Zymed cat. no. 03-6100), FAK (2A7 clone, a kind gift of Tom Parsons) or talin (a kind gift of Keith Burridge). Incubation in the presence of appropriate primary antibodies is for 60 min at room temperature. Antibody incubations are carried out by placing coverslips face up on inverted tube caps attached to 10cm petri dishes by double sided adhesive tape. C. D. NOBES and A. HALL

6. The coverslips are rinsed by sequential dipping into beakers containing PBS. 7. The coverslips are transferred to the appropriate second antibody mixture containing FITC-conjugated goat anti-mouse (in the case of vinculin, paxillin and FAK staining [Pierce cat. no. 31544]), or goat anti-rabbit (for talin staining [Pierce cat. no. 316701) and rhodamine-labeled rabbit anti-rat IgG (for detection of injected cells [Sigma T57781).To visualise both actin and vinculin, along with the injection marker, three color immunofluorescence is used. In this case a Cascade Blue-conjugated anti-rat IgG (Molecular Probes C2794) is used to detect the injected cells.

8. After the final wash, the coverslips are drained of excess liquid and are mounted by inverting onto 5 p1 Mowiol mountant (Calbiochem) containing p-phenylenediamine (1 mg/ml) as an antifading agent.

9. The coverslips are observed with a Zeiss Axiophot microscope using Zeiss x 40 (NA 1.3), x 63 (NA 1.4) and x 100 (NA 1.3) oil immersion objectives. Fluorescence images are recorded on Kodak T-MAX 400 ASA film.

6.6 C3 Protein Inhibits LPA-stimulated Stress Fibre Assembly and Focal Adhesion Clustering Serum-starved confluent quiescent Swiss 3T3 cells display few polymerized actin structures. TRITC-phalloidin staining reveals only a thin ring of polymerized actin at the periphery of the cells (Fig. 2). In addition, unlike growing cells, vinculin and other focal adhesion components are not clustered into focal adhesion structures. The addition to these cells of serum (the major active constituent of which is lysophosphatidic acid, LPA) induces the rapid assembly of actin stress fibres and the clustering of vinculin and talin to form new focal adhesions (Fig. 2; Ridley and Hall, 1992). This response to LPA is blocked completely if cells are first microinjected with C3 transferase (Fig. 3; Ridley and Hall, 1992). Normally we microinject C3 protein at a concentration between 75 and 125 pg/ml 10-20 min prior to stimulation of the cells with LPA (100ng/ml [Sigma L72601) or 1 % FCS. Each new preparation of C3 protein is titrated by microinjecting serially diluted samples of the C3 protein, for example 200, 100, 75, 50 and 20pglml before stimulating cells with LPA for 15-30 min. The cells are then fixed and stained for filamentous actin with TRITC-labeled phalloidin, or for focal adhesion components as described above (Fig. 3). Since focal adhesions are generally located at the periphery of the cell, it is often difficult to determine whether or not a C3 injected cell has focal adhesions in a confluent monolayer where cells are in close apposition to each other. In this case we sometimes stain for vinculin in serumstarved subconfluent cells prepared as described earlier.

test each new c3 preparation

Clostridium botulinum C3 Exoenzyme and Studies on Rho Proteins

Fig. 2. LPA-induced actin reorganization. Filamentous actin in untreated control Swiss 3T3 cells serum-starved for 16h (a)or stimulated with LPA (lOOng/ml) for 30min (b)

It should be noted that microinjection of C3 into quiescent serumstarved Swiss 3T3 cells is not without effect. Despite the cells having few if any actin stress fibres, or visible vinculin staining in focal adhesions, C3 causes cells to round up and to become detached from the underlying substrate. This indicates that there is, even in serum-free conditions, a low basal activity of Rho, perhaps maintained by a tyrosine kinase/phosphotyrosine phosphatase cycle upstream of Rho (Nobes et a/., 1995). Interestingly, the cell rounding and detachment induced by injection of C3 into quiescent cells is inhibited by coinjection of activated Rac or by addition of PDGF (5ng/ml), which C. D.NOBES and A. HALL

Fig. 3. C3 transferase inhibits LPA-induced stress fiber assembly. C3 was injected into confluent serum-starved Swiss 3T3 cells at concentrations of 200yglml (a,b), 100yg/ml (c, d) and 20yglml (e, f). Cells were stimulated after 15min with LPA (100ng/ml), fixed 30min later and stained for filamentous actin with TRITCconjugated phalloidin (a,c, e). FITC-conjugated dextran was co-injected with C3 transferase as a marker of injected cells (b, d, f). Note injection of C3 transferase at 200 yg/ml caused rapid rounding and detachment of the cells, and stress fibre assembly was not completely inhibited in cells injected with 20 yg/ml C3

activates endogenous Rac, to the culture medium. This is due to the formation of focal complexes induced by Rac (Nobes and Hall, 1995; Hotchin and Hall, 1995).

Clostn'dium botulinurn C3 Exoenzyme and Studies on Rho Proteins

Fig. 4. Active Rho is required for the maintanence of focal adhesion plaques. C3

(100pg/ml) was injected into Swiss 3T3 cells growing in the presence of 10 % FCS. Cells were fixed after 5 min (b),10 min (c) and 20 min (d) and labeled to show vinculin distribution. (a)Vinculin labeling in a control uninjected cell

6.7 Timecourse of Focal Adhesion Breakdown Rho Is Also Required for Maintenance of Focal Adhesions In addition to being required for the formation of focal adhesions, Rho is required for their maintenance. Swiss 3T3 cells newly attached and spread on glass coverslips in the presence of serum contain large focal adhesions (Fig. 4). Injection of C3 protein into such cells results in loss of staining for vinculin and other focal adhesion components. If cells are fixed at different times after microinjection of C3, and then stained for vinculin, it is possible to estimate how rapidly focal adhesions are broken down after inhibition of Rho. Within 15-20 min of C3 injection, vinculin is no longer clustered to focal adhesions suggesting a half life of around 10 min in the absence of rho (Fig. 4). This indicates that focal adhesions are dynamic structures and are likely to be turning over rapidly under normal conditions.

C. D. NOBES and A. HALL

6.8 Alternative Delivery of C3 Transferase C3 transferase can also be introduced into cells simply by adding it to the external culture medium, and it appears to enter cells by pinocytosis (Weigers et al., 1991; Morii and Narumiya, 1995).A range of C3 concentrations are used depending on cell type, but generally pg/ml concentrations of C3 are required. It is recommended that cells should be incubated with 1, 3, 10, 30, and 100 pg/ml for 24 to 48 h (Morii and Narumiya, 1995). Once Rho in cells has been ADPribosylated, it is no longer a substrate for C3, and the extent of ADPribosylation can be estimated by incubation of cell homogenates with C3 and [32P]NADin vitro (Morii and Narumiya, 1995). More effective uptake of C3 has been achieved by making a fusion protein of C3 transferase with the subunit of diphtheria toxin involved in binding and transport across membranes (Aullo eta/., 1993).Unfortunately this delivery system does not work for all cell types since mouse and rat cells do not bind diphtheria toxin. Vero cells appear to be the cells most sensitive to this construct (Aullo et a/., 1993; Boquet et al.I 1995). lntracellular C3 transferase expression has also been used to test whether endogenous Rho is required for regulation of transcriptional activation by SRF (Hill et a/.! 1995). Cells can be transfected with expression vectors containing C3.

6.9 Conclusion C3 transferase is a specific inhibitor of the Rho GTPase and has been used in lymphocytes, neutrophils, neuronal cells, epithelial cells and platelets as well as fibroblasts. Although all members of the Rho family of GTPases contain an asparagine residue at codon 41, only Rho seems to be a substrate for C3 transferase. However, many members of the Rho family have yet to be characterized, and it is possible therefore that other substrates for C3 transferase will be identified.

6.10 Reagents and Chemicals Materials

Supplier

Cat-No.

FlTC goat anti-rabbit IgG ampicillin anti-paxillin anti-vinculin Biorad protein assay kit BSA Cascade blue anti-rat IgG

Pierce Sigma Zymed Sigma Biorad Sigma Molecular probes Gibco

31670 A9518 03-6100

DMEM (powder)

V4505 500-0006 A2153 C2794 52100-021

Clostridiurn botulinurn C3 Exoenzyme and Studies on Rho Proteins

Materials

Supplier

Cat-No.

DMEM DTT fetal calf serum (FCS) FlTC goat anti-mouse IgG FlTC goat anti-rabbit IgG FlTC goat anti-rat IgG FlTC lysinated dextran Glutathione-agarose beads IPTG lysophosphatidic acid Mowiol mountant p-aminobenzamidine agarose beads p-phenylenediamine (anti-fade) paraformaldehyde penicillin/streptomycin PMSF rat IgG Rhodamine rabbit anti-rat IgG sodium borohydride sodium pyruvate Soybean trypsin inhibitor Texas red lysinated dextran thrombin (bovine) TRITC-phalloidin Triton X-100 [32P]NAD

Gibco Sigma Sigma Pierce Pierce Sigma Molecular probes Sigma Calbiochem Sigma Calbiochem Sigma Sigma Sigma Gibco Sigma Pierce Sigma Sigma Gibco Sigma Molecular probes Sigma Sigma Sigma NEN/Dupont

41966-029 D9779 F7524 31544 31670 F7631 D 1820 G4510 420322 L7260 475804 A7155 P6001 P6148 15140-106 P7626 31885 T5778 S9125 11360-039 T9003 D 1863 T6634 P1951 x-100 023x10055

References Aktories, K, Braun, S, Rosener, S et al. (1989):The rho gene product expressed in E. coli is a substrate for botulinum ADP-ribosyl transferase C3. In Biochem. Biophys. Res. Commun. 158:209-13. Aullo, P, Giry, M, Olsnes, S et al. (1993):A chimeric toxin to study the role of the 21 kDa GTP-binding protein rho in the control of actin microfilament assembly. In EM60 J. 12:921-31. Boquet, P, Popoff, MR, Giry, M etal. (1995):Inhibition of p21 rho in intact cells by C3 diptheria toxin chimera proteins. In Methods in Enzymology (Balch WE, Der CJ and Hall A eds) Vol 256, pp297-306, Sun Diego, Academic Press. Chardin, P, Boquet, P, Maduale, P et a/. (1989):The mammalian protein rhoC is ADP-ribosylated by Clostridium botulinum exoenzyme C3 and affects actin microfilaments in vero cells. In EM60 J. 8:1087-92. Dillon, ST and Feig, LA (1995): Purification and assay of recombinant C3 transferase. In Methods in Enzymology (Balch, WE, Der, CJ, Hall, A eds) Vol 256, pp174-84, Sun Diego, Academic Press. Graessmann, M and Graessmann, A (1983):Microinjection of tissue culture cells. In Methods in Enzymology (R. Wu, 1. Grossman and K. Moldave eds) Vol 101, pp482- 92, Sun Diego, Academic Press. Hill, CS, Wynne, J and Treisman, R (1995):The rho family GTPases, rhoA, racl and cdc42Hs regulate transcriptional activation by SRF. In Cell 8:1159-70. Hotchin, NA and Hall, A (1995): The assembly of integrin adhesion complexes requires both extracellular matrix and intracellular rho/rac GTPases. In J. Cell Biol. 131:1857-65. Just, I. Mohr, C. Schallehn, G. et a/. (1992):Purification and characterisation of an ADP-ribosyltransferase produced by Clostridium limosum. In J. 6i0l. Chem. 267: 10274-80.

C. D. NOBES and A. HALL

Kozma, R, Ahmed, S, Best, A etal. (1995):The ras-related protein cdc42Hs and bradykinin promote formation of peripheral active microspikes and filopodia in Swiss 3T3 fibroblasts. In Mol. Cell. Biol. 151942-52. Morii, N and Narumiya, S (1995): Preparation of native and recombinant Clostridium botulinum C3 ADP-ribosyltransferase and identification of rho proteins by ADP-ribosylation. In Methods in Enzymology (Balch WE, Der CJ, Hall A eds) Vol 256, pp196-206, Sun Diego, Academic Press. Narumiya, S, Sekine, A and Fuiiwara, M (1988): Substrate for botulinum ADPribosyltransferase, Gb, has an amino acid sequence that is homologous to a putative rho gene product. In J. Biol. Chem. 263:17255-57. Nobes, CD and Hall, A (1995):Rho, rac and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with stress fibres, lamellipodia and filopodia. In Cell 8153-62. Nobes, CD, Hawkins, P, Stephens, L et al. (1995): Activation of the small GTPbinding proteins rho and rac by growth factor receptors. In J. Cell Sci. 108:225 -33. Paterson, HF, Self, AJ, Garrett, M D et a/. (1990): Microinjection of recombinant p21rho induces rapid changes in cell morphology. In J. Cell Biol. 111: 1001- 1007. Ridley, AJ and Hall, A (1992): The small GTP-binding protein rho regulates the assembly of focal adhesions and stress fibres in response to growth factors. In Cell 70:389-99. Ridley, AJ, Paterson, HF, Johnston, CL et a/. (1992):The small GTP-binding protein roc regulates growth factor-induced membrane ruffling. In Cell 70:401-10. Rubin, EJ, Gill, MD, Boquet, P et a/. (1988):Functional modification of a 21 kilodalton G protein when ADP-ribosylated by exoenzyme C3 of Clostridium botulinum. In Mol. Cell. Biol. 8:418-26. Sekine, A, Fujiwara, M and Narumiya, S (1989):Asparagine residue in the rho gene product is the modification site for botulinum ADP-ribosyltransferase.In J. Biol. Chem. 264:8602-5. Weigers, W, Just, I, Muller, H etal. (1991):Alteration of the cytoskeleton of mammalian cells cultured in vitro by Clostridium botulinum C2 toxin and C3 ADPribosyltransferase.In Eur. J. Cell Biol. 54: 237-45.

Clostridium botulinum C3 Exoenzyme and Studies on Rho Proteins

Bacterial Toxins

Klaus Aktories Copyright 0 2002 WILEY-VCH Verlag GmbH & Co. KGaA

CHAPTER 7

Preparation of Clostridium botulinum C3 Exoenzyme and Application of ADP-ribosylation of Rho Proteins in Biological Systems Y. SAITO and S. NARUMIYA

7.1 Introduction Clostridium botulinum C3 ADP-ribosyltransferase (botulinum C3 exoenzyme) is an exoenzyme which is produced as a single polypeptide of about 23 kDa by Clostridium botulinum type C and D (Aktories et al., 1987; Morii et al., 1990). Its isolated cDNA encodes a protein consisting of 244 amino acid residues with a calculated molecular weight of 27362 and pl of 9.18, in which the N-terminal 40 amino acid peptide serves as a signal peptide and is removed to yield a mature form of the enzyme of 204 amino acids with a molecular weight of 23119 (Nemoto et al., 1991). It specifically ADP-ribosylates rho gene products (Rho) at an asparagine residue at the 41st position from the N-terminus (Narumiya et al., 1988; Sekine et al., 1989).The reaction catalyzed by C3 exoenzyme is thus: Rho + NAD + ADP-ribose-Asn41 Rho + nicotinamide + H +. Mammalian Rho is a low-molecular weight GTP binding protein and is composed of Rho A, Rho B, and Rho C (Madaule and Axel, 1985; Chardin et al., 1988; Yeramian et al., 1987).All of these serve as substrates for C3 exoenzyme (Hoshiiima et al., 1990; Chardin et al., 1989; Narumiya et al., 1988). On the other hand, other members of the Rho subfamily of low-molecular weight GTP binding proteins, Rac 1, Rac 2, and Cdc 42 Hs, are not ADP-ribosylated by C3 exoenzyme unless they are denatured in the presence of detergents (Just et al., 1992).Since Am4’ of Rho is located in a putative effector domain, ADP-ribosylation of this residue was presumed to interfere with the interaction of Rho with its putative effector(s) in cells, and block its signal transduction. This assumption has been corroborated by a number of studies. C3 exoenzyme can, therefore, be used to examine the role of Rho in various cellular responses. Here we describe our methods of expression and purification of recombinant C3 exoenzyme, an ADP-ribosylation reaction, and the application of this exoenzyme in biological systems. Using site-directed mutagenesis, we recently found that G I u ’ of ~ ~this enzyme is critical in binding NAD and, hence, for the enzyme activity (Saito et al., 1995). The use of a recombinant enzyme with a mutation at this residue as a negative control is also shown. K. Aktories (Ed.), Bacterial Toxins. 0 Chapman & Hall, Weinheim, 1997. ISBN 3-8261-0080-8

7.2 Expression and Purification of Recombinant C3 Exoenzyme The recombinant C3 exoenzyme that we used is a modified enzyme that does not contain the signal peptide, but has Met-Ala attached to Ser’ of the mature enzyme (Kumagai et al., 1993).The modified gene was cloned into PET 3a vector, resulting in PET 3a C3, which allows the enzyme to be expressed under control of the bacteriophage T7 promoter. €. coli strain BL21 (DE3) cells are transformed with PET 3a C3.

1.

A single colony is picked up and inoculated into 10ml of LB medium containing 100 pg/ml ampicillin and allowed to grow. Ten ml of the overnight culture of cells is diluted 1 :lo0 into M9 medium containing 1 m M MgS04, 0.1 mM CaCI2, 0.5 % (w/v) casamino acids, 0.4 % (w/v) glucose, and 100 pg/ml ampicillin. The cells are incubated at 37°C with agitation until the absorbance at 600 nm reaches 0.5.

2.

Isopropyl-1-thio-p-D-galactopyranoside is added to a final concentration of 1 mM to induce C3 exoenzyme expression, followed by incubation at 37°C for another 3 h with agitation.

3.

The cells are harvested by centrifugation at 5000 g for 10 min, and frozen and thawed several times.

4.

The pellet is then suspended in 50ml of ice-cold 50mM Tris HCI, pH 7.5,2 mM EDTA and 2 mM phenyl methyl sulfonyl fluoride (PMSF) and is subjected to 4 sonication bursts of 30sec each on ice.

5. Three mg of lysozyme (0.06mg/ml, final concentration) is added to the homogenate, followed by incubation for 20 min at 37°C. 6.

DNase 1 (Sigma) and MgCI2 are then added to final concentrations of 0.2 pg/ml and 5 mM, respectively. Incubation is continued for another 20 min at 37°C.

7.

The homogenate is centrifuged at 10000 g for 30 min.

8. The supernatant is diluted with an equal volume of 20 mM HEPES NaOH, pH 7.5 (buffer A), and applied to a CM-Sepharose column (2.5cm i.d., x 10cm) equilibrated with buffer A. The column is washed with 100 ml of buffer A. Elution is performed by a linear gradient between 0 and 0.5 M NaCl in buffer A.

9.

Each fraction is subjected to SDS-PAGE on a 12 % polyacrylamide gel, followed by staining with Coomassie Brilliant Blue. The enzyme is typically eluted at 0.15 M to 0.2 M NaCI, and is detected as a maior 23 kDa protein on SDS-PAGE gel.

10. The fractions containing the enzyme are concentrated using Centriprep 10 (Amicon) and applied to gel filtration on a TSKgel 3000SW column (Toso, Japan). Elution is performed with buffer A containing 0.15 M NaCI. The fractions containing the enzyme can be determined using SDS-PAGE analysis. This Y. SAITO and S. NARUMIYA

C

.-0

8 1.0-

_.

h

In 0

0 L

a n a a .? 0.55 a r

00

15

30

Time ( min )

60

Fig. 1. Time course of ADP-ribosylation of Rho. 1 pg of recombinant GST-Rho (fusion protein) was incubated with 20pM of [32P]-NADand 3ng of the wild type ( 0 ) or the E173Q (0)mutant of C3 exoenzyme in a total volume of 50 pl of ADPribosylation buffer, as described in the text, at 30°C for times indicated. The reaction was terminated by the addition of trichloroacetic acid and Nu deoxycholate. After centrifugation, the pellets were subjected to SDS polyacrylamide gel electrohoresis, followed by staining with Coomassie Brilliant Blue and autoradiography. ‘P-ADP-ribosylation of Rho was quantified by cutting out the radiolabeled GSTRho bands and determining their radioactivities.The relative ADP-ribosylation was expressed as the ratio of the 32P incorporation into GST-Rho at each point to the maximal 32P incorporation by the wild type C3 exoenzyme

procedure yields about 3mg of the purified enzyme. A mutant enzyme with GIu”~substitution with Gln (E173Q)can be expressed and purified by the same method. Figure 1 shows a time course for ADP-ribosylation using 1 pg of recombinant Rho fused with glutathione S transferase and 3 ng of C3 exoenzyme. The reaction proceeds in a time-dependent manner and reaches a plateau at 30min. In contrast, no ADP-ribosylation is observed in the reaction using the E173Q mutant (Fig. 1).The recombinant wild-type enzyme has a catalytic activity of 1.3 pmol of ADPribose transferred/ng/h and the K, value for NAD is 0125 p M (Fig. 2).

7.3 ADP-ribosylation Reaction The ADP-ribosylation reaction is utilized to determine the amount of Rho in cells and tissues. When [32P]-NADwith a higher specific activity is used, the ADP-ribosylation reaction is an extremely sensitive method for detecting a small amount of Rho.

1. Cells or tissues are suspended in an appropriate volume of homogenization buffer composed of 20mM Tris HCI, pH Z5, 0.25 M sucrose, 5 mM MgCI2, 1 mM EDTA, 1 m M dithiothreitol, Preparation of Clostridium botulinum C3 Exoenzyme and Application of ADP-ribosylation of Rho Proteins in Biologicol Systems

Fig. 2. Determination of the affinity for NAD of C3 exoenzyme. 1 pg of GST-Rho was incubated with various concentrations of [32P]-NADand 3 ng of the wild type of C3 exoenzyme at 30°C for 5 min. ADP-ribosylation of Rho was determined as described in Fig. 1. The results were analyzed with a double-reciprocal plot

0.5mM PMSF, and 1 m M benzamidine, and homogenized on ice by 3 sonication bursts of 5 sec duration each, or by 3 strokes in a Potter- EIvehiem ho mogenizer.

2. The homogenate

is centrifuged at 700 g for 10min and the supernatant (1OOyg-2OOyg protein) is used for the ADPribosylation reaction. Alternatively, cells are suspended in the ADP-ribosylation buffer (see below), homogenized using sonication as described above and subjected to the in vitro ADP-ribosylaton reaction. In order to examine the activity of C3 exoenzyme, recombinant Rho is used as a substrate.

3. The reaction mixture consists of 100 m M Tris HCI, pH 8.0, 10 m M nicotinamide, 10 m M thymidine, 10 m M dithiothreitol, 5 m M MgCI2,10 y M [32P]-NAD(900cpm/pmol) (ADP-ribosylation buffer), purified C3 exoenzyme, and recombinant Rho or a crude homogenate in a total volume of 100 PI. The mixture is incubated at 30°C. To measure the amount of Rho, the ADP-ribosylation reaction should reach a plateau. When 50 ng of C3 exoenzyme is used, most of the substrate is ADP-ribosylated after 2 h incubation. The reaction is terminated by addition of 200yl of 24 % trichloroacetic acid and 400yl of 0.02 % N a deoxycholate.

4. The mixture is put on ice for 20 min, followed by centrifugation at 10000 g for 15 min at 4°C. 5. The pellet is suspended in Laemmli sample buffer containing 100 m M Tris HCI, p H 8.0 and is applied to 12 % SDS PAGE.

Y. SAITO and S. NARUMIYA

6. After staining, the gel is dried and subjected to autoradiography. ADP-ribosylated Rho is detected as a radiolabeled band with a molecular weight of 21-23kDa. The band is cut out and the radioactivity is determined by liquid scintillation counting. For an optimal reaction, it is important to keep Rho in an intact guanine nucleotide-binding conformation. The presence of free magnesium ion is essential for the reaction. The addition of guanine nucleotides is also helpful, but detergents such as Triton and Tween inhibit the reaction. For optimization, it is advisable to use a homogenization buffer without the detergent.

7.4 Application of C3 Exoenzyme in Biological Systems C3 exoenzyme is quite a valuable tool for elucidating the function of Rho in biological systems. Previous studies using C3 exoenzyme have revealed that Rho is involved in the regulation of the formation of stress fibers (Patersonet al., 1990; Ridley and Hall, 1992)and contractile rings during cytokinesis (Mabuchi et al., 1993; Kishi et al., 1993),cell adhesion (Tominaga et al., 1993),cell motility (Takaishi et al., 1993),smooth muscle contraction (Hirata et al., 1992),cell proliferation (Yamamotoet al., 1993)and the serum response factor-mediated transcriptional activation of serum response element (Hill et al., 1995). It has also been used to identify the biochemical pathways downstream of Rho (Kumagai et al., 1993; Kumagai et al., 1995). C3 exoenzyme can be introduced into cells by simple addition to culture medium, by permeabilization, or by microinjection. There is no specific receptor for C3 exoenzyme on cells, and the exoenzyme is presumably taken into cells by nonspecific endocytosis. Sensitivities to C3 exoenzyme differ among cell types. Therefore, a preliminary study is required for any type of cell to determine the optimal C3 exoenzyme concentration and incubation time. It is recommended that cells are incubated with 1, 3, 10, and 30 pg of C3 exoenzyme for 24 to 72 h. The amount of Rho ADPribosylated in situ in the cell can be determined by in vitro ADPribosylation of lysates of treated cells with [32P]-NAD,because once Rho is ADP-ribosylated in vivo in the cell, it can no longer serve as a substrate for the in vitro ADP-ribosylation reaction. C3 exoenzyme tends to stick to the cell surface during the incubation of cells, and Rho is often ADP-ribosylated with contaminated C3 exoenzyme and endogenous NAD during sample preparation, resulting in incorrect estimation of the amount of the ADP-ribosylated Rho in situ. To avoid this misinterpretation, the cells should be washed extensively with phosphate-buffered saline and homogenized in ADP-ribosylation buffer containing [32P]-NADas described, and the homogenate subjected to the reaction immediately. The biological effects of C3 exoenzyme correlate well with the extent of in situ ADP-ribosylation of Rho. However, the biological effect does not vary linearly with the extent of ADP-ribosylation, but is

functions of

sensitivity to

C3 exoenzyme

C3 varies

C3 sticks to cell surface

correlation between biological effects and ADP-ribosylation

Preparation of Clostridiumbotulinum C3 Exoenzyme and Application of ADP-ribosylation of Rho Proteins in Biological Systems

controls

inversely proportional to the logarithm of the amount of unmodified Rho remaining in the cell. For example, C3 exoenzyme reduces the growth of Swiss 3T3 cells (Yamamoto et al., 1993). Growth of the cells treated with the exoenzyme at 1, 3, 10, 30pg/ml was 95, 71, 65 and 19 % of the control cells, respectively. In situ ADP-ribosylation of Rho in these cells was 56, 75, 87 and 95 % of the control, i.e. the amount of unmodified Rho remaining was 44, 25, 13 and 5 %, respectively. The B lymphoblast cell line, JV, forms a Rho-mediated aggregation in response to phorbol myristate acetate (PMA) which is dependent on lymphocyte function-associated antigen 1 and intercellular adhesion molecule (Tominaga et al., 1993). When the cells were treated with 10pg/ml and 30yglml of C3 exoenzyme for 24 h, in situ ADPribosylation of Rho were 61.0 and 83.2 %, respectively. The cells treated with 10pg/ml of the exoenzyme showed loose aggregates in response to PMA. In the cells treated with 30 pglml of C3 exoenzyme, inhibition of the PMA-induced aggregation was clearly observed. These results suggest that more than 80 % of in situ ADP-ribosylation of Rho is required to observe the biological effects of ADP-ribosylation. Although C3 exoenzyme affects many biological responses caused by extracellular stimuli, presumably due to the ADP-ribosylation of Rho (Kumagai et al., 1993; Paterson et al., 1990; Ridley and Hall, 1992; Mabuchi et al., 1993; Kishi et al., 1993; Tominaga et al., 1993; Takaishi et al., 1993; Hirata et al., 1992; Yamamoto et al., 1993; Hill et al., 1995; Kumagai et al., 1995), there has been no proper con-

Fig. 3. Autoradiogram of 32P-ADP-ribosylationof Rho in Swiss 3T3 cell homogenate. Swiss 3T3 cells were treated with buffer alone (lane 1), 30pg/ml of the wild type (lane 2) or the E173Q mutant (lane 3) of C3 exoenzyme for 72 h in DMEM containing 10 % fetal bovine serum. The cells were washed twice in phosphatebuffered saline (PBS), and incubated with 0.05 % ( w h ) trypsin in PBS. The detached cells were collected and suspended in ADP-ribosylation buffer containing [32P]-NAD,followed by sonication. The homogenates were incubated with 100 ng of the wild type C3 enzyme at 30°C for 2 h. The reaction was terminated by the addition of trichloroacetic acid and Nu deoxycholate. The pellets were subjected to 12 % SDS polyacrylamide gel electrophoresis and autoradiography. The positions of molecular weight markers are indicated on the left. The position of ADP-ribosylated Rho is indicated by an arrow Y. SAITO and S. NARUMIYA

Fig. 4. Effects of the wild type and mutant C3 exoenzyme on the morphology of Swiss 3T3 cells. Swiss 3T3 cells were treated with buffer alone (a), the wild type (b) or the E173Q (c) mutant of C3 exoenzyme for 72 h. Morphology was examined using a phase-contrast microscope and photographed

trol in most experiments. The E173Q mutant of C3 exoenzyme, which lacks ADP-ribosyltransferase activity, is useful as a negative control for examining the effect of ADP-ribosylation of Rho (Saito et al., 1995). For example, when Swiss 3T3 cells were incubated with 30pg/ml of the wild type or of the E173Q mutant of C3 exoenzyme for 72 h, levels of 32P-ADP-ribosylatedRho in the cells were 19 and 107 % of those of the control cells, respectively (Fig. 3) which is consistent with the ADPribosyltransferase activities evaluated by the in vitro reactions using recombinant Rho as a substrate. Figure 4 shows the morphology of Swiss 3T3 cells treated with the wild-type and the E173Q mutant of C3 exoenzyme. The cells with the wild-type exoenzyme showed marked cell rounding with beaded dendritic processes. In contrast, the cells treated with the E173Q mutant do not show any phenotypic changes, but show the same morphology as that of the control cells.

References Aktories K, Weller U, Chhatwal GS (1987) Clostridium botulinum type C produces a novel ADP-ribosyltransferase distinct from botulinum C2 toxin. FEBS Lett. 212: 109-113. Chardin P, Madaule P, Tavitian A (1988) Coding sequence of human rho cDNAs clone 6 and clone 9. Nucleic Acids Res. 16: 2717-2722. Chardin P, Boquet P, Madaule P et al. (1989) The mammalian G protein rho C is ADP-ribosylated by Clostridium botulinum exoenzyme C3 and affects actin microfilaments in Vero cells. EM60 J. 8: 1087-1092. Preparation of Clostridium botulinum C3 Exoenzyme and Application of ADP-ribosylationof Rho Proteins in Biological Systems

Hill CS, Wynne J, and Treisman R (1995)The Rho family GTPase Rho A, Rac 1, and CdC 42Hs regulate transcriptional activation by SRF. Cell 81 : 1159-1170. Hirata K, Kikuchi A, Sasaki S et al. (1992) Involvement of Rho in the GTP-enhanced calcium ion sensitivity of smooth muscle contraction. J. Biol. Chem. 267: 8719-8722. Hoshijima M, Kondou J, Kikuchi A et a/. (1990) Purification and characterization from brain membrane of a GTP-binding protein with a Mr 21,000, ADPribosylated by ADP-ribosyltransferasecontaminated in botulinum toxin type C1. Mol. Brain Res. 7: 9-16. Just I, Mohr C, Schallehn G et a/. (1992) Purification and characterization of an ADP-ribosyltransferase produced by Clostridium limosum. J. Biol. Chem. 267: 10274- 10280. Kishi K, Sasaki T, Kuroda S et a/. (1993) Regulation of cytoplasmic division of Xenopus embryo by rho and its inhibitory GDP/GTP exchange protein (rho GDI). J. Cell. Biol. 120: 1187-1195. Kumagai N, Morii N, Fujisawa K et a/. (1993)ADP-ribosylation of rho inhibits lysophosphatidic acid-induced protein tyrosine phosphorylation and phosphatidylinositol 3-kinase activation in cultured Swiss 3T3 cells. J. 6iol. Chem. 268: 24535-24538 Kumagai N, Morii N, lshizaki T et a/. (1995) Lysophosphatidicacid-induced activation of protein Ser/Thr kinases in cultured rat 3Y1 fibroblasts: possible involvement in Rho mediated signalling. FEBS Lett. 366: 11 -16. Mabuchi I, Hamaguchi Y, Fujimoto H etal. (1993)A rho-like protein is involved in the organization of the contractile ring in dividing sand dollar eggs. Zygotes 1: 325-331. Madaule P, Axel R (1985) A novel ras-related gene family. Cell 41: 31 -40. Morii N, Ohasi Y, Nemoto Y et al. (1990) lmmunochemical identification of the ADPribosyltransferasein Botulinum C1 neurotoxin as C3 exoenzyme-likemolecole.J. Biochem. 107: 769-775. Narumiya S, Sekine A, Fujiwara M (1988) Substrate for botulinum ADPribosyltransferase, Gb, has an amino acid sequence homologous to a putative rho gene product. J. Biol. Chem. 263: 17255-17257. Nemoto Y, Namba T, Kozaki S et a/. (1991) Clostridium botulinum C3 ADPribosyltransferase gene: Cloning, sequencing and expression of a functional protein in Esherichia coli. J. Biol. Chem. 266: 19312-19319. Paterson HF, Self AJ, Garrett M D et a/. (1990) Microinjection of recombinant rho induces rapid changes in cell morphology. J. Cell. Biol. 111: 1001- 1007. Ridley AJ, Hall A (1992)The small GTP binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell 70: 389 -399. Saito Y, Nemoto Y, ishizaki T et a/. (1995) Identification of Glu173 as the critical amino acid residue for the ADP-ribosyltransferaseactivity of Clostridium botulinum C3 exoenzyme. FEBS Lett. 371 : 105- 109. Sekine A, Fujiwara M, Narumiya S (1989)Asparagine residue in rho gene products is the modification site for botulinum ADP-ribosyltransferase.J. Biol. Chem. 264: 8602-8605. Takaishi K, Kikuchi A, Kuroda S et a/. (1993) involvement of rho and its inhibitory GDP/GTP exchange protein (rho GDI) in cell motility. Mol. Cell. Biol. 13: 72-79. Tominaga T, Sugie K, Hirata M et a/. (1993) Inhibition of PMA-induced, LFA-1mediated lymphocyte aggregation by ADP-ribosylation of the small molecular weight GTP-binding protein, rho. J. Cell. Biol. 120: 1529-1537. Yamamoto M, Marui N, Sasaki T et a/. (1993) ADP-ribosylation of the rho A gene product by C3 exoenzyme causes Swiss 3T3 cells to accumulate in the G1 phase of cell cycle. Oncogene 8: 1449-1455. Yeramian P, Chardin P, Madaule P et al. (1987) Nucleotide sequence of human rho cDNA clone 12. NucleicAcids Res. 15: 1869.

Y. SAITO and S. NARUMIYA

Bacterial Toxins

Klaus Aktories Copyright 0 2002 WILEY-VCH Verlag GmbH & Co. KGaA

CHAPTER 8

Actin-ADP-ribosylating Toxins: Cytotoxic Mechanisms of Clostridium botulinum C2 Toxin and Clostridium perfringens Iota Toxin K. AKTORIES, P. SEHR and I. JUST

8.1 Int roduct ion Whereas most of the known bacterial ADP-ribosylating toxins modify GTP-binding proteins (Aktories and Just, 1993) (see chapter 1, 3 and 5), there is a family of clostridial toxins that ADP-ribosylates the ATPbinding protein actin (for review see (Aktories et al., 1992; Considine and Simpson, 1991; Aktories and Just, 1990; Aktories and Wegner, 1992; Aktories and Wegner, 1989; Ohishi and DasGupta, 1987)). These toxins have proved to be a valuable tool in cell biology because they are most effective agents to induce depoylmerization of actin in intact cells. Therefore, they are used to study the role of the microfilament protein actin in various cell functions. The family of actin ADP-ribosylating toxins comprises Clostridium botulinum C2 toxin, C. perfringens iota toxin, C. spiroforme toxin and a transferase produced by certain strains of C. difficile. Besides their common eukaryotic substrate actin, these toxins are characterized by their binary structure. The toxins are constructed according the A-B model and consist of a binding component and an enzyme component. However, in contrast to other toxins which are A-B toxins, like cholera toxin or pertussis toxin, the components of the actin ADP-ribosylating toxins are separate proteins and are not linked by either covalent or non-covalent bonds.

8.2 Clostridium botulinum C2 Toxin The binding component (C211)of C. botulinum C2 toxin has a molecular mass of about 100 kDa (Ohishi et al., 1980) and has to be activated by trypsin (Ohishi, 1987).Trypsin treatment cleaves the 100 kDa component and releases a fragment of about 20 kDa. The approx. 80 kDa activated component of C211 binds to the cell surface, thereby inducing a binding site for the enzyme component (C21).So far, the nature of the eukaryotic cell surface receptor is not known. Most probably the toxin complex (C21and C211) is internalized by receptormediated endocytosis, followed by translocation of the enzyme component (C21)into the cytosol (Simpson, 1989; Ohishi and Yanagimoto,

structure of toxin

K. Aktories (Ed.), Bacterial Toxins. 0 Chapman & Hall, Weinheim, 1997. ISBN 3-8261-0080-8

specificity of ADPribosylation

1992). Here, C21 catalyzes the ADP-ribosylation of actin, resulting in dramatic redistribution of F-actin. Recently, the gene of C21 has been reported encoding a protein of 431 amino acids (M,49400) with significant similarity to the enzyme component of iota toxin (see below) (Fujii et al., 1996). ADP-ribosylation of actin by C. botulinum C2 toxin is highly selective (see below). Neither G proteins, which are substrates for cholera or pertussis toxin nor other cytoskeletal proteins such as tubulin are ADP-ribosylated. As with other bacterial toxins, the enzyme-catalyzed modification is a mono-ADP-ribosylation. Accordingly, the actinbound ADP-ribose is cleaved by Crotalus durissus phosphodiesterase and releases 5'-AMP. The K, for NAD of the ADP-ribosylation reaction was determined to be about 4 pM. Using protein chemistry and mutagenesis, arginine-177 has been identified as the acceptor amino acid of ADP-ribose. Typically for an ADP-ribose-arginine bond, ADP-ribose incorporated at arginine-177 by C2 toxin is released by neutral hydroxylamine (OSM, 2 h) (Aktories et al., 1988). The ADP-ribosylation by C2 toxin is reversible in the presence of a high concentration of nicotinamide (30-50mM) and in the absence of free NAD (Just et al., 1990).

8.3 Other actin-ADP-ribosylating Toxins Closfridium perfringens iota toxin consists of an enzyme component with a molecular mass of about 52317 Da containing a signal sequence of 41 amino acids and a binding component of about 98467 Da with a signal sequence of 39 amino acids (Perelle et al., 1993; Perelle et al., 1995; Simpson et al., 1987; Stiles and Wilkens, 1986; Stiles and Wilkins, 1986). The enzyme component shows about 31 % identity and 43 % similarity with the sequence of C21 (Fujii et al., 1996). Interestingly, the binding component has about 55 % similarity to the protective antigen of the anthrax toxin (Perelle et al., 1993).N o effect of trypsin on the purified components was observed. However, it has been reported that the toxicity of iota toxin is increased by proteolysis, most probably indicating the action of a bacterial protease (Stiles and Wilkins, 1986). The enzyme component of C. spiroforme toxin appears to be heterogeneous with an M, ranging from 43 to 47 kDa and its binding component is also activated by proteolysis (Simpson et al., 1989). So far no binding component has been reported for the actin-ADP-ribosylatingtoxin from C. difficile (Popoff et al., 1988) (Note that this transferase is distinct from the C. difficile toxins A and B described in chapter 12).The binding components of iota toxin and C. spiroforme toxin (but not that of C2 toxin) are interchangeable and can even translocate C. difficile ADP-ribosyltransferase into the cell, suggesting that there is a subgroup of more closely related iota-like actin-ADP-ribosylating toxins (Popoff and Boquet, 1988; Simpson et al., 1987; Simpson et al., 1989). K. AKTORIES, P. SEHR and I. JUST

8.4 Actin as the Substrate for ADP-ribosylation Actin is found in all eukaryotic cells, and moreover, it is one of the most abundant proteins in many of them. Besides its role in skeletal muscle contraction, it is a maior component of the microfilament system of the cytoskeleton, and is involved in various cellular motile functions, such as migration, phagocytosis, secretion or intracellular transport. Thus, actin appears to be a crucial target for toxins, because of its essential role for a variety of cellular functions. Actin is a single-chain polypeptide of 375 amino acids that is structurally divided into four domains (IIV), with a cleft between domain 1/11 and III/IV. Here are localized the high affinity binding sites for ATP/ADP and the divalent cation. In cells, actin has bound Mg”, whereas actin purified according to the procedure by (Pardee and Spudich, 1982) contains Ca”. In addition to ATP/ADP-binding, actin possesses ATPase activity. Essential for the physiological role of actin is its ability to polymerize and to form filaments. Actin filaments are polar structures with two nonequivalent ends. Polymerization of actin occurs faster at the plus (barbed) end of filaments than at the minus (pointed) ends. Because barbed ends have a higher apparent affinity for actin monomers than the pointed ends, actin filaments tend to polymerize at the barbed ends and to depolymerize at the pointed ends. In non-muscle cells, about half of the cellular actin is monomeric and half polymeric. A large number of actin-binding proteins are involved in the dynamic regulation of the state of actin. At least six mammalian actin isoforms have been identified: skeletal muscle a-actin, cardiac muscle a-actin, smooth muscle a- and y-actin and cytoplasmic 6- and y-actin (Vandekerckhove and Weber, 1979; Vandekerckhove and Weber, 1978).All these isoforms are highly homologous, with a maximal difference in the primary structure of about 5 % (Vandekerckhove and Weber, 1978). Although arginine at position 177 is conserved in all actin isoforms, C. botulinum C2 toxin specifically ADP-ribosylates cytoplasmic actin and y-smooth muscle actin but not a-actin isoforms (Aktories et al., 1986b; Mauss et a/., 1990).In contrast to C2 toxin, C. perfringens iota toxin ADP-ribosylates all actin isoforms studied (Mauss et al., 1990). Additional actin substrates of C2 toxin are Physarum polycephalum actin (unpublished observation), Drosophila indirect flight muscle actin (and also the actin-ubiquitin conjugate) (Just et al., 1993a), and actin from Saccharomyces cerevisiae, Didyostelium and the green alga Chara (Grolig et al., 1996). ADP-ribosylation of actin depends on the native structure of the protein substrate. In the presence of EDTA, which chelates and removes the actin-bound magnesium ion, resulting in denaturation of actin, ADP-ribosylation is completely blocked (Just et al., 1990). C. botulinum C2 toxin or C. perfringens iota toxin ADP-ribosylate monomeric G-actin, but not polymerized F-actin (Aktories et al., 198613; Schering et al., 1988).This is due to the fact that the acceptor amino acid arginine-177, which is located in domain Ill of actin, is at or near

characteristics of actin

actin isoforms

Actin-ADP-ribosylating Toxins: Cytotoxic Mechanisms of Clostridium botulinurn C2 Toxin and Clostridium perfringens IotaToxin

Table 1. Consequences of ADP-ribosylation of actin 1. 2. 3. 4. 5. 6.

Inhibition of actin polymerization Capping protein function of ADP-ribosylated actin Increase in the critical actin concentration for polymerization Inhibition of actin ATPase activity Increase in ATP exchange rate of actin Inhibition of nucleation activity of the gelsolin-actin complex

an actin-actin contact site (Holmes et a/., 1990).Similarly, the position of arginine-177 explains why ADP-ribosylation of monomeric actin inhibits actin polymerization (Aktories et al., 198613) most probably by steric hindrance (Table 1). Actin polymerization is inhibited even in the presence of phalloidin, which markedly decreases the critical concentration for polymerization (Aktories et a/., 1986~).However, ADP ribosylated actin still interacts with unmodified actin and binds like a capping protein to the fast-polymerizing end (barbed or plus end) of F-actin (Wegner and Aktories, 1988; Weigt et a/., 1989).This interaction inhibits further association of monomeric actin at this end. In contrast, ADP-ribosylated actin does not affect polymerization and depolymerization of unmodified actin at the pointed end of filaments. The equilibrium constant for binding of ADP-ribosylated actin to the barbed end of F-actin was determined to be K, approx. lo8 M-’. By capping the barbed end of F-actin, the critical concentration of actin for polymerization increases to values that correspond to the critical actin concentration at the pointed (minus) end of actin filaments. ADP-ribosylation completely blocks the actin ATPase activity and increases the rate of ATP exchange by about twofold (Geipel et a/., 1990; Geipel et a/., 1989). This effect is not due to inhibition of polymerization, because the basal ATPase activity of G-actin is also inhibited. Moreover, the ATPase activity of actin is blocked even in the quasi-monomeric actin-DNAse I complex after stimulation with the mycotoxin cytochalasin (Geipel et a/., 1990).Thus, by analogy with the ADP-ribosylation of G-proteins by cholera toxin, which inhibits Gprotein-associated GTP hydrolysis, the ADP-ribosylation of actin inhibits its intrinsic ATPase activity. ADP-ribosylation not only blocks actin-actin interaction but also affects the interaction of actin with actin-binding proteins like gelsolin. Gelsolin (M,approx. 82000) is an actin-binding protein that (i),severs F-actin and increases the number of short filaments, (ii) acts like a barbed-end-capping protein thereby inhibiting fast polymerization of actin, and (iii) binds two actin monomers to form a 1 :2 complex with nucleation activity for actin polymerization (Pollard et a/., 1994). As demonstrated with isolated proteins, ADP-ribosylated actin still interacts with gelsolin, however, the nucleation activity of the gelsolin-actin complex was inhibited when ADP-ribosylated actin was bound to the Ca2+-sensitivebinding site of gelsolin (Wille et a/., 1992) (Fig. 1).

K. AKTORIES, P. SEHR and I. JUST

Fig. 1. Toxin-catalyzedADP-ribosylation inhibits nucleation activity of the gelsolinactin complex. In the presence of Ca2+,gelsolin forms a 1 :1 and a 1:2complex with

actin monomers at the so-called EGTA-resistant (a)and Ca2+-sensitive(b) binding site, respectively. Gelsolin-actin complexes act as nuclei for actin polymerization. Actin bound to both sites (a, b) can be ADP-ribosylated.Whereas ADP-ribosylation of actin bound to the EGTA-resistant site has no effect on nucleation, ADPribosylation of actin bound to the Ca2+-sensitivesite inhibits nucleation activity of the gelsolin-actin complex

Actin-ADP-ribosylatingToxins: Cytotoxic Mechanisms of Clostridium botufinum C2 Toxin and Clostridium perfringens IotaToxin

8.5 Model for the Cytopathic Effects of Actin-ADP-ribosylating Toxins effect of toxins on cell structure

It has been shown in a large variety of cell types and tissues that actin is the pathobiochemical substrate for the ADP-ribosylating toxins in intact cells. The typical effect of the toxins on cell culture is the redistribution of the microfilament network (Reuner et al., 1987; Wiegers et al., 1991) (Table2). In fact, the toxins induce a decrease of F-actin, with a concomitant increase in the G-actin content of cells. These effects can be explained by the inhibition of actin polymerization after ADPribosylation and by the capping function of ADP-ribosylated actin, which results in blockage of polymerization at the fast growing end of actin filaments, whereas depolymerization at the so-called pointed end of filaments remains possible (Fig. 2). It is worth noting that, even in the absence of toxin, microinjection of ADP-ribosylated skeletal muscle actin induces dramatic morphological changes and depolymerization of F-actin, a finding which underlines the functional importance of the capping property of ADP-ribosylated actin (Kiefer et al., 1996). Moreover, it is possible that the modification of actin in complexes with actin-binding proteins participates in the cytotoxic or cytopathic effects. Thus, it was shown that the gelsolin-actin complex is a

Fig. 2. Model of the cytopathic effects of actin ADP-ribosylating toxins. The activated binding component of C. botulinum C2 toxin binds to a receptor of the eukaryotic cell. This induces a binding site for the enzyme component (C21).Most likely, C21 enters the cell by endocytosis and subsequent translocation. In the cell, G-actin is ADP-ribosylated, which inhibits its polymerization and traps actin in the monomeric form. ADP-ribosylated actin binds in a capping protein-like manner to the barbed ends of filaments to inhibit further polymerization at the fast-growing end of F-actin. The toxin has no effects on the pointed end of filaments where actin depolymerization takes place. Additionally, ADP-ribosylation may affect functions of complexes of actin with binding proteins as examplified in Fig. 1 (From (Aktories, 1990) with permission) K. AKTORIES, P. SEHR and I. JUST

Table 2. Effects of octin ADP-ribosylating toxins on the cytoskeleton 1. Morphological changes (rounding-up of cells) 2. Redistribution of the microfiloment cytoskeleton 3. Depolymerizotion of F-octin 4. Increase in the amount of G-octin

substrate for C2 toxin in intact fibroblasts (Just et al., 199313).Thus, all these effects and properties of ADP-ribosylated actin may participate in the dramatic depolymerizing activity of this family of toxins.

References Aktories K, Ankenbauer T, Schering B et a/. (1986~): ADP-ribosylation of platelet actin by botulinum C2 toxin. In Eur. J. Biochem. 161: 155-62 Aktories K, Barmonn M, Ohishi I etal. (l986b): Botulinum C2 toxin ADP-ribosylates actin. In Nature 322: 390-2 Aktories K, Just I, Rosenthal W (1988):Different types of ADP-ribose protein bonds formed by botulinum C2 toxin, botulinum ADP-ribosyltransferaseC3 and pertussis toxin. In Biochem. Biophys. Res. Commun. 156: 361 -7 Aktories K (1990):Clostridial ADP-ribosyltransferases - modification of low molecular weight GTP-binding proteins and of actin by clostridial toxins. In Med. Microbiol. Immunol. 179: 123-36 Aktories K, Wille M, Just I (1992):Clostridial actin-ADP-ribosyloting toxins. In Curr. Top. Microbiol. Immunol. 175: 97- 113 Aktories K, Just I (1990): Botulinum C2 Toxin. In ADP-ribosyloting toxins and Gproteins, (Moss J, Vaughan M. 79-95 Washington,D.C. American Society for Microbiology. Aktories K, Just I (1993): GTPases and actin as targets for bacterial toxins. In GTPoses in biology I, (Dickey BF, Birnbaumer L. 87-112 Berlin-Heidelberg: Springer-Verlag. Aktories K, Wegner A (1989):ADP-ribosylation of actin by clostridial toxins. In J. Cell B i d . 109: 1385-7 Aktories K, Wegner A (1992):Mechanisms of the cytopathic action of actin-ADPribosyloting toxins. In Mol. Microbiol. 6: 2905-8 Considine RV, Simpson LL (1991):Cellular and molecular actions of binary toxins possessing ADP-ribosyltransferase activity. In Toxicon 29: 913-36 Fujii N, Kubota T, Shirakawa S etal. (1996):Characterization of component-l gene of botulinum C2 toxin and PCR detection of its gene in clostridial species. In Biochemical and Biophysical Research Communications 220: 353-9 Geipel U, Just I, Schering B etal. (1989):ADP-ribosylation of octin causes increase in the rate of ATP exchange and inhibition of ATP hydrolysis. In Eur. J. Biochem. 179: 229-32 Geipel U, Just I, Aktories K (1990):Inhibition of cytochalasin D-stimulated G-actin ATPase by ADP-ribosylationwith Clostridium perfringens iota toxin. In Biochem. J. 266: 335-9 Grolig F, Just I, Aktories K (1996):ADP-ribosylation of actin from the green alga Chara corallina by Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin. In Protoplasma in press Holmes KC, Popp D, Gebhard W et al. (1990):Atomic model of the actin filament. In Nature 347: 44-9 Just I, Geipel U, Wegner A etal. (1990):De-ADP-ribosylationof actin by Clostridium perfringensiota-toxin and Clostridium botulinum C2 toxin. In Eur. J. Biochern. 192: 723-7 Just I, Hennessey ES, Drummond DR etol. (1993~): ADP-ribosylation of Drosophila indirect flight muscle actin and arthrin by Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin. In Biochem. J. 291 : 409-12 Actin-ADP-ribosylating Toxins: Cytotoxic Mechanisms of Clostridium botulinum C2 Toxin and Clostridiurn perfringens IotaToxin

Just I, Wille M, Chaponnier C etal. (199313): Gelsolin-actin complex is target for ADP-ribosylation by Clostridium botulinum C2 toxin in intact human neutrophils. In Eur. J. Pharmacol. Mol. Pharmacol. 246: 293-7 Kiefer A, Lerner M, Sehr P et a/. (1996):Depolymerization of F-actin by microinjection of ADP-ribosylated skeletal muscle G-actin in PtK2 cells in the absence of the ADP-ribosylating toxin. In Med. Microbiol. Immunol. 184: 175-80 Mauss S, Chaponnier C, Just I et a/. (1990):ADP-ribosylation of actin isoforms by Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin. In Eur. J. Biochem. 194: 237-41 Ohishi I, lwasaki M, Sakaguchi G (1980): Purification and characterization of two components of botulinum C2 toxin. In Infect. Immun. 30: 668-73 Ohishi I (1987): Activation of botulinum C2 toxin by trypsin. In Infect. Immun. 55: 1461-5 Ohishi I, DasGupta BR (1987): Moleculare structure and biological activities of Clostridium botulinum C2 toxin. In Avian Botulism, (Eklund MW, Dowell VR., eds) 223-47 Springfield: Thomas. Ohishi I, Yanagimoto A (1992):Visualizations of binding and internalization of two nonlinked protein components of botulinum C2 toxin in tissue culture cells. In Infect. Immun. 60: 4648-55 Pardee JD, Spudich JA (1982):Purification of muscle actin. In Methods Enzymology 85: 164-81 Perelle S, Gibert M, Boquet P et a/. (1993):Characterization of Clostridium perfringens iota-toxin genes and expression in Escherichia coli. In Infect. Immun. 61: 5147-56 Perelle S, Gibert M, Boquet P etal. (1995):Characterization of Clostridium perfringens iota-toxin genes and expression in Escherichia coli. In Infect. Immun. 63: 4967 PollardTD, Almo S, Quirk S etal. (1994):Structure of actin binding proteins: Insights about function at atomic resolution. In Annu. Rev. Cell Biol. 10: 207-49 Popoff MR, Rubin EJ, Gill D M et a/. (1988): Actin-specific ADP-ribosyltransferase produced by a Clostridium difficile strain. In Infect. Immun. 56: 2299-306 Popoff MR, Boquet P (1988): Clostridium spiroforme toxin is a binary toxin which ADP-ribosylates cellular actin. In Biochem. Biophys. Res. Commun. 152: 1361-8 Reuner KH, Presek P, Boschek CB etal. (1987):Botulinum C2 toxin ADP-ribosylates actin and disorganizes the microfilament network in intact cells. In Eur. J. Cell B i d . 43: 134-40 Schering B, Barmann M, Chhatwal GS etal. (1988):ADP-ribosylation of skeletal muscle and non- muscle actin by Clostridium perfringens iota toxin. In Eur. J. Biochem. 171: 225-9 Simpson LL, Stiles BG, Zapeda HH et al. (1987): Molecular basis for the pathological actions of Clostridium perfringens Iota toxin. In Infect. Immun. 55: 118-22 Simpson LL (1989):The binary toxin produced by Clostridium botulinum enters cells by receptor-mediated endocytosis to exert its pharmacologic effects. In J. Pharmacol. Exp. Ther. 251 : 1223-8 Simpson LL, Stiles BG, Zepeda H etal. (1989):Production by Clostridium spiroforme of an iotalike toxin that possesses mono(ADP-ribosy1)transferaseactivity: Identification of a novel class of ADP- ribosyltransferases.In Infect. Immun. 57: 255-61 Stiles BG, Wilkens TD (1986): Purification and characterization of Clostridium perfringens iota toxin: dependence on two nonlinked proteins for biological activity. In Infect. Immun. 54: 683-8 Stiles BG, Wilkins TD (1986):Clostridium perfringens iota toxin: Synergism between two proteins. In Joxicon 24: 767-73 Vandekerckhove J, Weber K (1978):At least six different actins are expressed in a higher mammal: An analysis based on the amino acid sequence of the aminoterminal tryptic peptide. In J. Mol. Biol. 126: 783-802 Vandekerckhove J, Weber K (1979):The complete amino acid sequence of actins from bovine aorta, bovine heart, bovine fast skeletal muscle and rabbit slow skeletal muscle. In Differentiation 14: 123-33 Wegner A, Aktories K (1988):ADP-ribosylated actin caps the barbed ends of actin filaments. In J. 6iol. Chem. 263: 13739-42 K. AKTORIES, P. SEHR and I. JUST

Weigt C, Just I, Wegner A et al. (1989):Nonmuscle actin ADP-ribosylated by botulinum C2 toxin caps actin filaments. In FEBS Lett. 246: 181-4 Wiegers W, Just I, Muller H et al. (1991):Alteration of the cytoskeleton of mammalian cells cultured in vitro by Clostridium botulinum C2 toxin and C3 ADPribosyltransferase. In Eur. J. Cell 6iol. 54: 237-45 Wille M, Just I, Wegner A et a/. (1992):ADP-ribosylation of the gelsolin-actin complex by clostridial toxins. In J. Biol. Chem. 267: 50-5

Actin-ADP-ribosylating Toxins: Cytotoxic Mechanisms of Closfridium botulinum C2 Toxin and Closfridium perfringens IotaToxin

Bacterial Toxins

Klaus Aktories Copyright 0 2002 WILEY-VCH Verlag GmbH & Co. KGaA

CHAPTER 9

Purification, Activation and Endocytosis of Botulinum C2 Toxin I. OHlSHl

9.1 Introduction Clostridium botulinum type C was firstly isolated by Bengston in 1922 (Bengston, 1922). In 1935, Mason and Robinson reported that C. botulinum type C produced three different toxic factors, C1, C2 and D (Mason and Robinson, 1935), although it had been considered at that time that other types of C. botulinum, types A and B, produced only one antigenic type of the toxin. This was the first use of the term C2 toxin in the literature. Later on, Jansen applied this notion to the toxins produced by C. botulinum C,and C, strains, which had been classified by immunological cross-neutralization; c, produces C1, C2 and D toxins and C,only C2 toxin (Jansen, 1971).Thus, C2 toxin had been thought of as a botulinum neurotoxin until it was purified and characterized in 1980 (Ohishi et al., 1980). Botulinum C2 toxin is produced by certain strains of C. botulinum types C and D (Ohishi and Sakaguchi, 1982).The toxin is constructed with two unlinked protein components, neither covalently nor noncovalently linked, designated component I and II. These were named after the elution sequence from a column of cation exchanger, e.g., CM-Sephadex (Iwasaki et al., 1980, Ohishi et al., 1980). Because of this unique molecular construction, the purification of the toxin had encountered difficulties, especially in assaying the activity; when the toxin preparation was chromatographed on either ion exchanger or gel-filtration column, the two components of the toxin were eluted separately due to their different charges or their different molecular weights (Iwasaki etal., 1980; Ohishi etal., 1980).This was resolved by the finding that the activity of C2 toxin requires two separate protein components; only a mixture of these components manifests the toxicity (Ohishi et a/., 1980).

identification of C2 toxin

structure of C2 toxin

9.2 Assay Method for the Toxin One of the important points for purification of proteins is that an assay for biological activity can be done rapidly within a short period. After C2 toxin had been purified, it was found that the toxin, in addition to K. Aktories (Ed.), Bacterial Toxins. 0Chapman & Hall, Weinheim, 1997. ISBN 3-8261-0080-8

biological activities of C2 toxin

lethality, has various biological activities, i. e. increase in vascular permeability, fluid accumulation in ligated intestine, rounding of tissue-cultured cells (Miyake and Ohishi, 1987; Ohishi et al., 1980; Ohishi, 1983). Of these activities, however, the appropriate assay method for C2 toxin is the determination of "a time-to-death" after intravenous injection of toxin preparation into mice (Iwasaki et al., 1980; Ohishi et al., 1980). This is because the assay can be done within about 120min, if it contains 5-100 ipLDS0/ml, which roughly corresponds to 0.2-5 pg of C2/ml as a mixture of components I and II in a ratio of 1:2. The assay by the time-to-death method is as follows.

1. Prepare a culture of a C2 toxin-producing strain of C. botulinum: assay by %me-to-death"

it is preferable to use strains, that do not produce botulinum neurotoxin, because the neurotoxin interferes the determination of toxicity of C2 toxin. Otherwise, use anti-neurotoxin serum to neutralize the neurotoxic activity.

2. Concentrate the culture supernatant by adding solid ammonium sulfate at 65 % saturation. Collect the precipitate by centrifugation at 10000 g for 10min at 4 "C, suspend it in 10 mM K, Naphosphate buffer (PB), pH 7.3, containing 150mM NaCl (PBS) and dialyze the suspension against the buffer at 4 "C. 3. Before assaying the activity, C2 toxin must be trypsinized (Ohishi et al., 1980; Ohishi, 1987). To prepare the activated toxin, incubate the dialyzed preparation with trypsin at a final concentration of 200 pg/ml in 50 m M PB, pH 8.0, for 30 min at 37 "C. Terminate the trypsinization by adding twice the weight of soybean trypsin inhibitor into the reaction mixture.

activation of component II

To activate purified component II, a ratio of trypsin to purified component II of 1 :10 is required; trypsinization is required only for component II but not for component I (Ohishi et al., 1980; Ohishi, 1987) and the activity of the toxin can be assayed by mixing untrypsinized component I and trypsinized component II at a protein ratio of 1 :2.

4. Determine the intraperitoneal50 % lethal dose ( i ~ L D ~ ~ of ) / the ml trypsinized toxin sample in mice.

5. Dilute the toxin sample with PBS in an ice bath, inject intravenously into three or four mice, and determine the time-todeath (survival time) in min.

6. Plot the time-to-death (30 to 120min) against the toxin doses (5 to 100 ipLD,,/ml) both in logarithms; this results in a linear relationship between them. From this curve, the time-to-death in min after intravenous injection of a toxin sample can be converted into the lethal activity (ipLD50). Toxin preparations, whether activated or not activated, should be detoxified by boiling in a water bath for 3 to 5 min. If it might possibly I. OHlSHl

contain culture or culture supernatant of C2 toxin producing bacteria, all materials used for the experiments, e.g. toxin solution, injectors, test tubes, tips etc., should be autoclaved at 1 kg/cm2 for at least 15min or soaked in 1 N N a O H overnight, as they may be possibly contaminated with heat-resistant spores.

A

decontaminateall materials and equipment after use

9.3 Purification Procedures for the Two Components of C2 Toxin The flow sheet for purification steps is shown in Table 1. Table 1. Flow sheet for purification of the two components of C2 toxin Culture fluid of C. botulinum type C strain 92-13

1 Ammonium sulfate fractionation

1 Calcium phosphate gel (Batchwise fractionation)

1 CM-Sephadex chromatography

1

Pass-through fraction (component I)

1 DEAE-Sephadex chromatography

1 Hydroxyapatite chroma tog rap hy

I

Adsorbed and eluted fraction (Component II)

1 Gel filtration on Sephacryl S-300

1 Component II

I Gel filtration on Sephacryl S-300

1

Component I

Purification,Activation and Endocytosis of Botulinurn C2 Toxin

9.3.1 Preparation of Culture Medium

cool quickly

The culture medium for toxin production consists of 3 % nutrient broth (Difco Laboratories, Detroit, MI, USA), 1.5 % yeast extract (Difco Laboratories), 1 % ammonium sulfate, 1 % glucose, 0.4% calcium carbonate, 0.4 % soluble starch and 0.4 % cysteine hydrochloride. Adjust the pH of the medium to 7.5 with 4 N N a O H and autoclave at 1 kg/cm2for 15 min. After the temperature of the autoclave has fallen below 100 "C, cool the medium as soon as possible to 40-45 "C in tap water to avoid a decrease in reduction state of the medium, and then inoculate the culture of the toxin producing strain prepared as in Section 9.3.2.

9.3.2 Preparation of lnoculum and Incubation

use strain producing only C2 toxin

C. botulinum type C strain 92-13, which was kindly provided by Prof. S. Nakamura, Kanazawa University, Kanazawa, Japan, is suitable for an inoculum, because it produces only C2 toxin. Prepare the inoculum by incubating the strain overnight at 37 "C in cooked meat medium (Difco Laboratories) supplemented with 1 70ammonium sulfate, 1 % glucose, 1 % yeast extract and 0.2 % cysteine hydrochloride (pH 7.5). Transfer a 1-ml portion of the culture into 4 I of the toxin production medium in a flat-bottomed bottle (ca. 23 cm in diameter) and culture for 2 days at 37 "C.

9.3.3 Ammonium Sulfate Fractionation All of the following steps should be performed at 4 "C unless otherwise stated. Add solid ammonium sulfate to 4 I of whole culture to 60 % saturation and stand overnight. Collect the precipitate by centrifugation for 15min at 5000 g and suspend it in about 400ml of 50mM PB, pH Z5, by using a paint brush until it becomes homogeneous. Then, centrifuge the suspension for 15min at 8000 g and concentrate the supernatant by adding solid ammonium sulfate at 65 % saturation. Again, collect the precipitate by centrifugation and dissolve it in about 100ml of the buffer. Dialyze the suspension for 2 days against 2 I of 5 m M PB, p H 7.5, by exchanging the buffer 3 times.

9.3.4 Adsorption and Elution on Calcium Phosphate Gel Prepare the gel by mixing 500ml of 1 M calcium chloride and 500ml of 1M dibasic sodium phosphate. Wash the gel on a glass filter with 1000 ml of ice-cold water and then suspend in 500 ml of I. OHlSHl

water. Add the dialyzed fraction to the gel suspension, mix well and stand for 30 min in an ice bath. Wash the gel on the glass filter with 500 ml of 5 mM PB, pH 7.5, and then with 500 ml of the buffer containing 0.5 M ammonium sulfate to elute the toxin fraction. Concentrate the second filtrate by adding solid ammonium sulfate to 70 % saturation. Collect the precipitate by centrifugation, suspend it in the buffer and dialyze the suspension against 1 I of 10 m M PB, pH 6.0 for 2 days, changing the buffer 3 times.

9.3.5 CM-Sephadex Column Chromatography; Separation of Components II from I A column of CM-Sephadex (2.5 x 20 cm, Pharmacia Biotech, Uppsala, Sweden) is prepared according to the manufacturer’s instruction by using 10mM PB, pH 6.0, as an equilibration buffer. Apply the dialyzed fraction to the column, wash with 200 mI of 10 m M PB, pH 6.0 (this percolate contains component I), and elute component II with a gradient from 0 to 0.4 M sodium chloride in 1000 ml of 10 mM PB, pH 6.0. Collect the fractions containing component II and concentrate by ultrafiltration through a PO200 membrane (UHP-43K, Advantec Co., Tokyo, Japan).

9.3.6 Gel Filtration of Component II on Sephacryl S-300 Apply the concentrated component II to a column of Sephacryl S-300 (2.5 x 95 cm, Pharmacia Biotech), which is equilibrated with 50 m M PB, pH 7.5, and elute with the buffer. Collect the fractions containing component I I and concentrate by ultrafiltration. At this stage, check the purity of the component by sodium dodecylsulfatepolyacrylamide gel electrophoresis (SDS-PAGE). If the concentrated fraction still contains any impurities, repeat the gel filtration under the same conditions.

repeat if preparation contains impurities

9.3.7 DEAE-Sephadex Chromatography of Component I Dialyze the concentrated percolate fraction from Step 5 against 1OOOml of 25mM PB, pH 8.0, for 2 days, changing the buffer twice, and apply to a column of DEAE-Sephadex (1.5x 20 cm, Pharmacia Biotech) equilibrated with 25mM PB, pH 8.0. After washing the column with 50 ml of the buffer, elute component I with a gradient from 0 to 0.4 M NaCl in 600ml of 10mM PB, pH 8.0. Collect the fractions containing component I.

Purification, Activation and Endocytosis of Botulinurn C2 Toxin

9.3.8 Hydroxyapatite Chromatography of Component I Dilute the component I fraction from the DEAE-Sephadex column 2.5fold with water, adjust to pH 8.0 and apply to a column of hydroxyapatite (2.5 x 15 cm, Bio-Rad Laboratories, Hercules, CA, USA) equilibrated with 10 m M PB, pH 8.0. Wash the column with 50 ml of the buffer and elute with a gradient of 10 to 200mM PB, pH 8.0. Collect the fractions containing component I and concentrate by ultrafiltration through a PO200 membrane (UHP-43K, Advantec Co.).

9.3.9 Gel Filtration on Sephacryl S-300

repeat if preparation contains impurities

Apply the concentrated component I to a column of Sephacryl S-300 (2.5x95cm) equilibrated with 5 0 m M PB, pH 7.5, and elute with the buffer. Collect the fractions containing component I and concentrate by ultrafiltration as described above. Check the purity of the component using SDS-PAGE. If the preparation contains any impurities, run the gel filtration again on the column of Sephacryl S-300 under the same conditions as above. By these purification procedures, 1-3mg of component I and 20-40mg of component II are recovered from 4 I of culture of C. botulinum type C strain 92-13. The mixture of untrypsinized component I and trypsinized component II has high lethal activity in mice (specific activity is about 2 x lo4 mouse intraperitoneal 50 % lethal doses per mg of protein), although each of these components alone shows very low activity even after trypsinization. The purified components I and II each show one band in SDS-PAGE, and their molecular weights as determined by the electrophoresis are 45 kDa and 100 kDa, respectively.

9.3.10 Storage of the Two Purified Two Components of C2 Toxin The two purified components, component I and component II (the stability and storage of trypsinized component II will be described below), are relatively stable proteins and can be stored in the freezer (at -20 "C) for at least 3 months without loss of their activities, if freezing and thawing are not repeated too frequently. To store them for longer periods, however, it is preferable to freeze-dry and keep them in a deep freezer (-80°C) in a desiccator; both components are stable under the conditions for normal freeze-drying method without any additives.

I. OHlSHl

9.4 Activation of Component II The full biological activity of C2 toxin, a mixture of components I and II, is obtained by activation of the toxin with trypsin (Miyake and Ohishi, 1987; Ohishi et al., 1980; Ohishi et al., 1980; Ohishi, 1987). As described in Section 9.2 (Assay method for the toxin), the full activity of the toxin is produced by a mixture of untrypsinized component I and trypsinized component II. This indicates that activation of the toxin is brought about by the molecular cleavage of component 11, but not of component I, by trypsin. Therefore, to study the biological activity of botulinum C2 toxin and the effect of ADP-ribosylation of cytoplasmic actin by C2 toxin on whole cells, it is essential to prepare activated component II (trypsinizedcomponent 11). To prepare the activated component II, incubate 10 mg of component II with 1 mg of trypsin (Sigma Chemical Company, St. Louis, Mo., USA, type Ill-S) in 20ml of 50mM PB, pH 7.5, containing 200 mM NaCI. Apply the reaction mixture to a column of Sephacryl S-300 (2.5 x 95 cm), which is equilibrated with the buffer.

preparation of activated component II

Usually, not always, two protein peaks are eluted from the column

(Ohishi, 1987).The amount of protein in the first peak from the column is always larger than that in second peak. Both have toxicity when mixed with component I. They each show a single band in SDS-PAGE. Therefore, part of the trypsin-activated component II forms an oligomer. Collect the first peak, because, if the second peak is collected, it is occasionally contaminated with trypsin and/or trypsin-digested fragments, although it depends on the gel filtration conditions.

collect first peak only

Transfer the collected fractions to a dialysis bag and concentrate with Ficoll 400 (Pharmacia Biotech) at 4 "C. However, care should be taken to avoid the concentration of trypsinized component II exceeding 500 pg/ml, because the activated component II tends to aggregate at a higher concentration.

So far, it has not been possible to preserve the activated component II by freeze-drying. Therefore, it is desirable to prepare the activated component I1 at the time of use and to store it in the low temperature freezer (-80 "C) in vials. A gel-filtration pattern of trypsinized component II on Sephacryl S-300 and SDS-PAGE of the fractions are shown in Figure 1; in this case, one protein peak of activated component II was obtained.

prepare fresh of store at-80 "c

Purification, Activation and Endocytosis of Botulinum C2 Toxin

Fig. 1. Gel filtration of trypsinized component II on a column of Sephacryl S-300 (2.5 x95cm) equlibrated with 50mM PB, pH 7.5, containing 0.2 M NaCI. Component II (lOmg) was incubated with trypsin (1 mg) in 20ml of the buffer at 37 "C for 30 min. Fraction size; 3.8mVtube. Insert shows SDS-PAGE pattern of the representative fractions; figures indicates the fraction numbers of the gel filtration; (a) mixture of untrypsinized and trypsinized component II and (b)unfractionated reaction mixture

9.5 Endocytosis of Two Nonlinked Protein Components in Cultured Cells The two components of botulinum C2 toxin are functionally different proteins; component II is a binding molecule, whereas component I is an ADP-ribosyltransferase, of which substrate is cytoplasmic actin monomers (Ohishi and Tuyama, 1986; Ohishi, 1986; Ohishi et a/., 1990).This indicates that the toxin binds to the cell surface and enters the cytoplasm. These steps, the binding of the two nonlinked components of C2 toxin to the cells and the endocytotic vesicles containing the components, can be visualized either directly by incubating the cells with the two differently fluorescently labeled components (Ohishi, 1992), or indirectly by immunofluorescence labeling of the two proteins with their specific antibodies. This endocytotic incorporation of the protein is not a specific feature of this toxin, but is common to all the proteins that enter cells by receptor-mediated endocytosis. However, the internalization of the toxin may be of an interest to those readers, who would like use this characteristic toxin as a tool to analyze cellular responses, especially those who would like to compare the incorporation processes of the two non-linked components with I. OHlSHl

those of other proteins. In this section, I shall describe the visualization methods for binding and internalization of the two non-linked protein components of C2 toxin in tissue culture cells using their specific antibodies. In addition, I shall describe the immunization method for the two components of C2 toxin, which would be useful for those who wish to work with the toxin as a tool. The method for direct visualization for the cell-bound and the internalized components I and II of the toxin has already been presented elsewhere (Ohishi, 1992).

9.5.1 Preparation of Polyclonal Antibodies for Two Components of C2 Toxin Antisera to purified components I and II are prepared by injecting them separately into rabbits.

1. Before immunization in animals, prepare detoxified components by dialyzing them (200pg per ml) separately against 50 m M PB, pH Z5, containing 0.4 % formaldehyde at 30 "C for 24 h. 2. To induce a basal immunization, prepare an emulsion by mixing each of the formalinized components I and II with an equal vol-

@

immunization

ume of complete Freund's adjuvant (Difco Laboratories) and inject 100 pg of each subcutaneously into separate rabbits (it is better to use two animals for each component in case of an accident during the immunization). 3. Five to seven days after the first injection, repeat the same injection again. 4. 3 weeks after the first injection, boost the animals by subcutaneous injection of 50 pg each of unformalinized components I and II, emulsified with an equal volume of incomplete Freund's adjuvant.

5. Five to seven days after the first booster injection, repeat the same injection.

6. Five to seven days after the last injection, check the antibody production using the double diffusion technique in 1 % agar gel as described below. Dissolve agar (Agar Noble, Difco Laboratories) in 5 0 m M PB, pH 7.3, containing 150 m M NaCl in an electric oven. Spread the gel on a slide glass. Make a 3-mm diameter well in the center of the gel, and 6 wells around the center well at a distance of Z5 mm from center to center of the wells. Add 10 pI of antigen (100pg/ml) to the central well and 1OpI of 2-fold serially diluted serum to the outer wells. Incubate the gel at 4 "C for 1-2 days in a humid atmosphere in a Petri dish. Read the maximum dilution that results in an immunoprecipitin line. Usually, the antibody titer (the dilution factor) of rabbit serum immunized with component II is higher than that for component I.

check antibody production

Purification, Activation and Endocytosis of Botulinum C2 Toxin

7. Bleed the animals under anesthesia, prepare the serum and store it in a freezer at -20 "C or below. The sera can be stored in the freezer for two years or more with almost no loss of their titers.

9.5.2 Purification of I g G Specific for Each of the Components I and II by Affinity Chromatography IgG in antiserum prepared as described above is purified by affinity chromatography. To prepare the affinity column, conjugate the antigen (component I or II) to Affi-Gel 15 (Bio-Rad Laboratories) according to the manufacturer's Instruction Manual. The purification steps for IgG with the antigen-coupled affinity column are as follows.

1. Equilibrate the column with 5 0 m M PB, pH 7.3, containing antibody purification

150mM NaCI, and apply an appropriate volume of antiserum homologous to the coupled antigen. Wash the column with the same buffer to remove all unadsorbed proteins. 2. Elute the IgG from the column with 50 m M glycine-HCI, pH 2.5, containing 150 m M NaCI, and collect the fractions into test tubes containing 1 M Tris to neutralize the glycine-HCI buffer; the volume of 1M Tris to be added to the test tubes should be determined in advance, so as to neutralize the volume of the acid buffer eluted.

3. Concentrate the IgG with Ficoll 400 (Pharmacia Biotech) and dialyze it against 5 0 m M PB, p H 7.3, containing 150mM NaCI. Freeze-dry the purified IgG and store in a desiccator in a freezer at -80 "C.

9.5.3 Visualization of the Two Components Bound to Cultured Cells by Indirect lmmunofluorescence labeling

All tissue-cultured cells that I have examined so far show morphological changes when incubated with C2 toxin. These are FL (human amnion), HeLa (human uterus cancer), Intestine 407 (human embryonic intestine), L929 (mouse fibroblast), CHO (Chinese hamster ovary), BHK (baby hamster kidney), RK13 (rabbit kidney), CMK (cynomolgus monkey kidney) and Vero (green monkey kidney). The common response of the cells to C2 toxin is rounding up. Therefore, it seems that the binding and the endocytosis of the two components of C2 toxin can be visualized using any of these cells, although there must be some quantitative differences depending on the cell type. The morphological change in cultured cells in response to C2 toxin indicates that the cells have a binding site for the toxin on the cell membrane. In the following, the indirect immunofluorescence method for I. OHlSHl

demonstration of the cell-bound and the endocytosed component II in Vero cells is described. The same method is also applicable for the demonstration of the cell-bound component I, except that the cells must be exposed first to trypsinized component II and then to component I or exposed to a mixture of both components, because component I binds to the cells only in the presence of trypsin-activated component 11, or to cells that have already bound the component II.

9.5.4 Cell Culture and Indirect lmmunofluorescence labeling of Cell-Bound Component II Vero cells are maintained in minimal essential medium (MEM) containing 10 % fetal calf serum (MEM-FCS). 1. Before culturing the cells for immunolabeling, prepare glass coverslips cut in a size of about 3 x 3 mm, sterilize them by dryheating, and dispense aseptically into the wells of a 96-well plate; handle the cut coverslips with forceps, as used to handle for electron microscope grid. 2. Culture the cells in MEM-FCS on the coverslips in the wells of the plate. When the cells become confluent on the coverslip, wash the cells with 10 m M PB, pH Z3, containing 150 m M NaCl (PBS) and incubate them with component II, diluted with MEM containing 0.1 % bovine serum albumin (BSA); do not use FCS, because the binding of component II to the cells is inhibited by some factors in the serum. To observe the binding of the component to the cell surface, incubate the cells with component II at 37 "C for only 1-5 min, or at 4 "C for the same period. To demonstrate component II in endocytotic vesicles, incubate the cells with component II at 37 "C for 10-30 min. Remove unbound component II by washing the cells with PBSBSA. 3. Fix the cells in 10 % formaldehyde-PBS on ice for 30 min. Wash the cells again with PBS-BSA. To observe the endocytotic vesicles, incubate the cells with PBS containing 0.1 % Triton X-100 on ice for 1-2 min to increase the permeability of the cell membrane to IgG. Wash the cells thoroughly with PBS-BSA. Transfer the coverslips to the lid of a 96-well plate, which is turned upside down; the inside of the lid is cast so as to cover each of the wells of the plate, and these shallow wells are very useful for carrying out the immunofluorescence labeling.

handling coverslips

choice of serum

labelling of coverslips

4. Dispense 20pI of purified anti-component II IgG (100pg/ml) onto the fixed cells or onto the fixed and permealized cells on coverslips and incubate for 30min at room temperature (15-20 "C). Wash the cells with PBS-BSA. Purification, Activation and Endocytosis of Botulinurn C2 Toxin

Fig. 2. Indirect immunofluorescencelabeling of cell-bound component 11. Component I1 (0.5yg/well) was incubated with cultured Vero cells at 37 "C for 7.5 min; (a) not permeabilized with Triton X-100-PBS and (b) permeabilized with Triton X-100PBS. For details see text (Section 9.5.4)

5. Dispense 20 pl of appropriately diluted tetramethylrhodamine isothiocyate (TRITC)-labeled anti-rabbit IgG (Bio Source International, Inc., Tag0 Products, USA) onto the cells and incubate at room temperature for 30min. Wash the cells with PBS-BSA, and then transfer the coverslip to a glass slide by turning it over on a tiny amount (ca. 2-4 pI) of glycerol. Observe the cells in a microscope equipped for epifluorescence. Indirect fluorescence labeling of component II is shown in Figure 2.

I. OHlSHl

9.6 Reagents and Chemicals Materials

Supplier

Cat-No.

Affi-Gel 15 Agar Noble CM-Sepahdex A-50 Cooked meat medium DEAE-Sepahdex A-50 Ficoll 400 Hyd roxyapatite Nutrient broth Sephacryl S-300 TRITC-anti rabbit IgG

Bio-Rad Difco laboratoires Pharmacia Difco Laboratories Pharmacia Pharmacia Bio-Rad Difco Laboratories Pharmacia Biosource International (Tag0 Products) Sigma Sigma Difco Laboratories

153-6051 526-00054 17-0220-01 529-00804 17-0180-01 17-0400-01 157-0080 523-00483 17-0599-01 ALI-4406

Trypsin Trypsin inhibitor Yeast extract

T 7409 T 9003 527-00305

References Bengston IA (1922):Preliminary note on a toxin-producing anaerobe isolated from the larvae of lucilia caesar: Public Health Rep. 37: 164- 170. lwasaki M, Ohishi I, Sakaguchi G (1980): Evidence that botulinum C2 toxin has two dissimilar components. Infect. Immun. 29: 390-394. Jansen BC (1971):The toxic antigenic factors produced by Clostridium botulinum types C and D. Onderstepoort J. Vet. Res. 38: 93-98. Mason JH, Robinson EM (1935):The antigenic components of the toxins of C. botulinum types C and D. Onderstepoort J. Vet. Sci. Anim. Indus. 5: 65-75. Miyake M, Ohishi I (1987):Reponse of tissue-cultured cynomolgus monkey kidney cells to botulinum C2 toxin. Microbial Pathogenesis, 3: 279-286. Ohishi I, lwasaki M, Sakaguchi G (1980): Purification and characterization of two components of botulinum C2 toxin. Infect. Immun. 30: 668-673. Ohishi I, lwasaki M, Sakaguchi G (1980): Vascular permeability activity of botulinum C2 toxin elicited by cooperation of two dissimilar protein components. Infect. Immun. 31: 890-895. Ohishi I, Sakaguchi G (1982): Production of C2 toxin by Clostridium botulinum types C and D as determined by its vascular permeability activity. Infect. Immun. 35: 1-4. Ohishi I (1983): Response of mouse intestinal loop to botulinum C2 toxin: enterotoxic activity induced by cooperation of nonlinked protein components. Infect. immun. 40: 691 -695. Ohishi I, Tsuyama S (1986):ADP-ribosylation of nonmuscle actin with component I of C2 toxin. Biochem. Biophys. Res. Commun. 136: 802-806. Ohishi I (1986): NAD-glycohydrolase activity of botulinum C2 toxin: a possible role of component I in the mode of action of the toxin. J. Biochem. 100: 407-413. Ohishi I (1987): Activation of botulinum C2 toxin by trypsin. Infect. Immun. 55:

1461- 1465. Ohishi I, Morikawa Y, Baba T (1990):ADP-ribosylation of nonmuscle actin by component I of botulinum C2 toxin inactivates the ability to interact with unmodified actin. J. Biochem. 107: 420-425. Ohishi I (1992):Visualizations of binding and internalization of two nonlinked protein components of botulinum C2 toxin in tissue culture cells. 60: 4648-4655.

Purification, Activation and Endocytosis of Botulinum C2 Toxin

Bacterial Toxins

Klaus Aktories Copyright 0 2002 WILEY-VCH Verlag GmbH & Co. KGaA

The Role of the Clostridium botulinum C2 Toxin as a Research Tool to Study Eucaryotic Cell Biology 1. L. SIMPSON

10.1 Introduction Clostridial organisms are an unusually rich source of biological substances that act on eukaryotic cells (Simpson, 1989a). Many of these substances are noteworthy because of their abilities to cause diseases, the most important examples being clostridial toxins that cause gastrointestinal problems and those that cause neurological problems. In contrast, some of these substances have earned distinction because they can be used to treat human illness, one of the most exciting examples being the use of clostridial toxins to treat dystonias and related disorders. Yet another reason for focusing on clostridial toxins is that they have the potential to be research tools for analyzing the structure and function of eukaryotic cells. Clostridium botulinum type C2 toxin shares many properties with other clostridial toxins, but at the same time it has some unique features (for review, see Aktories and Wegner, 1989; Considine and Simpson, 1991; Aktories and Wegner, 1992).To begin with, this toxin has never been convincingly implicated in disease. Dose-response experiments on isolated cells and on whole organisms have shown that the toxin is quite potent - certainly potent enough to poison humans or other animals - but evidence of naturally occurring poisoning has not been reported. This may be a byproduct of the fact that C2 toxin is produced by organisms that also synthesize botulinum neurotoxin. The latter is orders of magnitude more potent than C2 toxin, and therefore dual exposure to neurotoxin and C2 toxin would probably result in a prevalence of signs and symptoms due to the neurotoxin. C2 toxin also differs from other clostridial toxins in that it has not been used as a therapeutic agent, although it does have desirable properties that may be exploited in the future (see below). Perhaps the most interesting feature of C2 toxin is that it is one of a relatively small group of biological agents known as binary toxins (Considine and Simpson, 1991). By definition, binary toxins are substances that possess two separate and independent components. The individual components express little or no toxicity when added to cells or administered to whole organisms, but when given in combination they cause serious adverse effects and even death. Relatively little is known about binary toxins, with the most puzzling question being

unique features of C2 toxin

C2 is a binary toxin

K. Aktories (Ed.),Bacterial Toxins. 0 Chapman & Hall, Weinheim, 1997. ISBN 3-8261-0080-8

that of why bacteria produce a toxin in two separate parts. Whatever the explanation, botulinum C2 toxin is a binary toxin capable of acting on a wide range of eukaryotic cells. In spite of these various differences, there is one respect in which C2 toxin is akin to other clostridial toxins, such as botulinum neurotoxin and tetanus toxin: its ability to act at low concentrations to produce a specific and irreversible effect on many types of cells can be translated into a benefit. This toxin has great potential as a research tool to study eukaryotic cell biology.

10.2 Identifying Potential Uses

internalization of toxin

L. L. SIMPSON

There are two aspects to the actual and potential uses of the binary toxin: (a) the mechanism of toxin action, and (b) the cells that are vulnerable to this action. The mechanism of C2 action was described in detail in Chapter 18, and will be discussed only briefly in this chapter. This mechanism can be envisioned as a sequence of three events, as follows: binding, productive internalization, and intracellular poisoning. To some extent, the structure-function relationships of the binary toxin have been determined. Thus, the heavy chain (M, approx. 100 000) plays an essential role in binding, and the light chain (Mr approx. 50 000) is an enzyme that possesses mono(ADP-ribosyl) transferase activity. These general concepts are easy to grasp when viewed in the context of a simple experiment. Addition of the light chain (viz., the enzymatic component) to intact cells does not cause poisoning, because this chain by itself does not associate with cells. Addition of the heavy chain (viz., the binding component) does not cause poisoning either, because this chain does not express toxicity. However, addition of both chains to intact cells leads to binding and eventual expression of toxicity (Ohishi, 1983a,b; Ohishi and Miyake, 1985). This outcome is intriguing, especially when one takes into account the fact that heavy chain and the light chain do not associate with one another in solution. This appears to indicate that: (a) the heavy chain changes conformation when bound to cell receptors, and the altered conformation leads to exposure of an occult binding site for the light chain, (b) the heavy chain modifies the membrane in such a way as to create a binding site for the light chain, or (c) the heavy chain and the membrane act cooperatively to form a binding site for the light chain. Because this point has not yet been resolved, the binding properties of C2 toxin have not been exploited as research tools. However, this is likely to change. There are experimental and possibly even clinical settings in which the unique properties of a binary toxin could be exploited. One such possibility is explored below. The second step in the sequence is productive internalization (Ohishi and Yanagimoto, 1992).Toxin that is associated with cell surface receptors is translocated into the cytosol to reach its substrate. The fact that the substrate is in the cell interior means that the toxin, or at

least the enzymatic component of the toxin, must be internalized. There is relatively little information on the underlying mechanism, although at least one study indicates that the toxin reaches the cell interior by receptor-mediated endocytosis (Simpson, 1989b). If this is correct, it would imply that the toxin must penetrate both the cell membrane and subsequently the endosome membrane. A sequence such as this is reminiscent of that utilized by other clostridial toxins, such as botulinum neurotoxin and tetanus toxin, as well as other microbial toxins (i.e., diphtheria toxin). As with the binding step, there is much that remains to be learned about the internalization step. Nevertheless, the fact that toxins possess the ability to penetrate biological membranes suggests that the mechanisms they use could have wide utility. To state the obvious, the ability to achieve efficient penetration of selected cell membranes would be highly advantageous in many areas of drug therapy. The final step in toxin action is enzymatic modification of certain forms of actin (Aktories et al., 1986; Ohishi and Tsuyama, 1986).The light chain component ADP-ribosylates non-polymerized actin at arginine residue 177 (Vandekerckhove ef a/., 1988).This has the combined effect of blocking further polymerization and promoting depolymerization (see Chapter 13), and as a result the cytoskeleton of cells is disrupted and may collapse. This action on the part of the toxin lends itself to the solution of many kinds of problems, but two have emerged as most prominent. First, the toxin has been widely used as a tool to determine the direct or indirect role of the actin-based cytoskeleton in various cell functions. Second, the toxin has been used as one in a battery of techniques to determine how the cell regulates synthesis and utilization of actin. The potential value of C2 toxin as a research tool depends on the vulnerability of cells. The experimental approach is straightforward when cells are susceptible to natural poisoning, because the two components of C2 toxin can be added to the exterior of cells and the enzymatic component will find its way to the cell interior (see for example Miyake and Ohishi, 1987; Reuner et al., 1987; Zepeda et al., 1988).The approach is more problematic when cells are resistant to natural poisoning. In theory, resistance could be due to absence of cell surface receptors, absence of a mechanism for productive internalization, or absence of an intracellular substrate, but thus far only an absence of receptors (Fritz et al., 1995) and an absence of substrate (Aktories et al., 1986) have been described. Cells without receptors can be rendered susceptible by using techniques that produce artificial internalization (e.g., permeabilizing the cell membrane or microinjection; see Muller et al., 1992). Cells that do not have substrate are permanently resistant to poisoning.

modification of actin

The Role of the Clostridium botulinum C2 Toxin as a Research Tool to Study Eucaryotic Cell Biology

10.3 Isolation and Purification

A

handle organisms with care

yse bacteria without phage

toxicity measured only by bioassay

L. L. SIMPSON

The components of C2 toxin can be isolated from culture supernatants of type C and type D Clostridium botulinum. It is important to remember that these organisms are capable of producing type C and type D botulinum neurotoxin. Although there are no documented cases of type C or type D poisoning in adults, recent work has shown that the isolated human neuromuscular junction can be poisoned by type C toxin (Coffield and Simpson, unpublished). Therefore, extreme caution should be used when handling organisms that produce neurotoxin. A simple alternative that greatly diminishes the hazard of isolating type C2 toxin is to use organisms from which bacteriophages have been eliminated. Both type C and type D toxin are encoded in phages that infect Clostridium botulinum (Hatheway, 1990).Therefore, eliminating the phages from the organisms removes the hazard associated with production of neurotoxin, but it does not alter growth and reproduction of the organisms, or their ability to make C2 toxin. All investigators isolate the two components of C2 toxin by techniques originally described by Ohishi, Sakaguchi and their colleagues (Iwasaki et d., 1980; Ohishi et al., 1980; Ohishi and Hama, 1992) (see Chapter 9). Although somewhat laborious, their method for isolating the toxin merely involves a series of chromatographic procedures. The heavy chain and light chain components d o not appear in the same fractions, so different fractions must be combined to achieve full toxicity. There are two techniques that can be used to confirm the identity of the isolated components: (a) cross-reactivity with chain-specific antibodies, and (b) bioassay. These techniques are equally easy to use, but the bioassay holds an enormous advantage. The immunologic technique confirms the presence of epitopes, but it reveals nothing about residual biological activity. Confirmation of toxicity can be achieved only by bioassay. Ideally, vulnerable cells should be exposed to toxin to evoke the characteristic rounding that results from loss of cytoskleleton, and substrate from poisoned cells should be isolated to confirm that cell rounding was indeed due to ADPribosylation. If one is interested only in enzymatic modification of isolated substrate, then only the light chain component needs to be purified. This material can be used in the form that it is obtained at the final fractionation step. However, if one wishes to study toxin action on intact cells, both components must be isolated. Furthermore, the heavy chain component must be treated with trypsin before it will attain full biological activity. This can be achieved by mixing trypsin with heavy chain (1 :10 on a protein basis) for 30min at 35°C at p H 75 (Ohishi, 1987). When run on polyacrylamide gels in the presence of sodium dodecyl sulfate, the native light chain migrates with an apparent molecular weight of approx. 50 000. The native heavy chain has an apparent molecular weight of approx. 100 000, whereas the activated

heavy chain migrates with an apparent molecular weight of 88 000. In experiments involving intact cells, activated heavy chain is normally added at a two-fold molar excess to light chain.

10.4 C2 Toxin as a Research Tool 10.4.1 Binding Step There are no published studies in which C2 toxin has been used explicitly as a research tool. However, there are two ideas that are clear extensions of the literature and which could lead to exciting applications of the toxin. One pertains to the novel expression of receptors and selective tissue-targeting of drugs, and the other relates to identification and characterization of an endogenous class of cell receptors. There is now a substantial literature on the synthesis and testing of chimeric molecules. These substances are each composed of at least two functional domains, such as a tissue-targeting domain and a pharmacologically active domain. One common strategy in constructing chimeric toxins is to isolate the binding domain of one toxin (e.g., diphtheria toxin) and attach this to the poisoning domain of another (e.g., ricin). This chimeric molecule attaches only to cells that have the diphtheria toxin receptor, and it expresses only the intracellular effects of ricin. Chimeric molecules are valuable both as research tools to study cell biology and as therapeutic agents for treating disease. For example, a host of chimeric toxins are currently being evaluated as potential anti-neoplastic drugs. The typical approach here is to attach an antibody directed against a cell surface antigen on a cancer cell to the enzymatic component of a lethal toxin. The resulting chimeric molecule is a potential anticancer drug. Chimeric molecules have been made both by the techniques of protein chemistry and by those of molecular biology. In the former, unrelated functional domains are linked by heterofunctional crosslinking reagents. In the latter, gene segments are ligated in the expectation that the expression product will have the correct structure and biological activity. The reason these linking techniques must be used is that heterologous components of unrelated toxins do not ordinarily form tight associations with one another. The existence of binary toxins raises the possibility that unique chimeric substances can be formed that have implicit within them "receptors-on-demand". The underlying concepts to support this premise are as follows. First, the light chain component of C2 toxin could be modified so that it no longer expressed enzymatic activity. When so modified, it could be envisioned as a tissue-targeting domain, as explained below. Next, a novel pharmacologically active domain could be attached to the light chain.

chimeric molecules

The Role of the Clostridium botulinum C2 Toxin as a Research Tool to Study Eucaryotic Cell Biology

cells resistant to C2 toxin

Ordinarily, toxicity of C2 toxin depends on exposing cells to the heavy chain component which leads to formation of a docking site for the light chain component. Therefore, one could treat cells with the heavy chain, subsequently add the modified light chain, and hopefully evoke a novel pharmacologic effect. This is entirely feasible, but it is hardly imaginative. A more intriguing possibility would be to create stable transformants that produce the heavy chain endogenously and under the control of an inducible element. If the gene encoding the heavy chain were altered to include an appropriate leader sequence, there is the possibility that the heavy chain would insert into the membrane, or be secreted and thus able to attach to its normal binding site on the cell surface. In either case, the expressed protein would serve as a "receptor-on-demand" for the native light chain or for a modified version of the light chain. Unlike natural receptors (e.g., neurotransmitter or hormone receptors), the unnatural heavy chain on the cell surface is already known to be part of a highly efficient mechanism for productive internalization of biologically active substances. And unlike natural receptors, the heavy chain would appear only when cells are exposed to the inducing reagent. The desirability of selecting for, selecting against, or specifically modifying only certain cells - especially when these cells are part of a mixed population - encourages efforts to meet the challenge of creating this novel brand of chimeric molecules. A less speculative use of the toxin to study receptors is an outgrowth of recent work by Aktories and his colleagues. Using standard techniques for mutagenesis, they were able to create cells that are resistant to C2 toxin (Fritz et al., 1995).Interestingly, the resistant cells had deficiencies suggesting that they had lost receptors for toxin as well as sensitivity to serum growth factors, which could mean that toxin receptors are also growth factor receptors. Given that C2 toxin binds almost ubiquitously to eukaryotic cells, isolation and characterization of its receptor could lead to identification of a universally important growth factor receptor.

10.4.2 Internalization Step As with the binding step, there are no studies in which the internalization of C2 toxin has been exploited to gain insight into cell biology. This may change though, given the results of a recent study by Schmid et al. (1994).These workers have shown that the activated heavy chain forms cation-selective and voltage-dependent channels in artificial channel formation in m ~ ~ b r a n e s lipid bilayers. It is a general property of internalized toxins and certain viruses that they form channels in membranes. The portion of the toxin molecule associated with channel formation is typically the same as that needed for internalization. This could mean that channel formation is the mechanism that underlies translocation, or that it is an epiphenomenon that occurs coincidentally with translocation. In either case, L. L. SIMPSON

procedures that block channel formation also block expression of toxicity. Regardless of the true role of channel formation in productive internalization, it is fascinating that microbial toxins, which presumably are ancient molecules, have this property. It is inevitable that investigators will compare the molecular biology and structure of toxin channels with corresponding properties of endogenous channels (e.9. sodium or potassium), and from this deduce something about the evolution of channels.

10.4.3 lntracellular Step (see Table 1) Table 1. Representative Studies Reflecting the Use of Clostridium botulinum C2 Toxin as a Research Tool Cell Line or Tissue Research Problem

3T3 Cells Adrenal Y-1 Cells Hepatocytes HIT-T15 Cells Ileum Lymphocytes Lymphocytes Lymphocytes Lymphocytes BW5147TLymphomaderived cell line Neuromuscular junction Mast Cells Macrophages Neutrophils Neutrophils Neutrophils Neutrophils PC-12 Cells Xenopus Oocytes

Reference

Autoregulation of actin polymerization Bershadsky et a/., 1995 Role of microfilaments in steroid release Considine et a/., 1992 Autoregulation of actin synthesis Reuner et a/., 1991 Role of actin filaments in insulin Li et al., 1994 secretion G/F-actin transition in smooth muscle Mauss et a/., 1989 contraction Receptor-mediated cell activation Melamed et a/., 1991 Proliferation of Epstein-Burr Virus Melamed etal., 1994 Role of cytoskeleton in nerve growth Melamed et a/., factor signaling 1995b Mitogen-activated protein kinases and Melamed et a/., signal transduction 1995a Role of microfilaments in motility of Verschueren etal., lymphoid cells 1995 Acetylcholine release

Simpson, 1982

Histamine release Cholesterol esterification Activation of oxidase

Bottinger etal., 1987 Tabas et a/., 1994 Al-Mohanna etal., 1987 Ligand-evoked lipid mediator Grimminger etal., generation 1991a Ligand-evoked signal transduction and Grimminger et al., secretion 1991b Nitric oxide stimulation of ADPClancy et a/., 1995 ribosylation Release of norepinephrine Matter et a/., 1989 Potassium channel clustering by the Honore et a/., 1992 cytoskeleton

The Role of the Clostridium botulinum C2 Toxin as a Research Tool to Study Eucaryotic Cell Biology

The first area of investigation in which C2 toxin was used as a research tool was in the analysis of storage and release of chemical mediators (Considine and Simpson, 1991). This was a natural outgrowth of the fact that other clostridial toxins, and particularly botulinum neurotoxin and tetanus toxin, have profound effects on transmitter release (Simpson, 1989a; Montecucco and Schiavo, 1994).It therefore seemed logical to determine whether clostridial binary toxins had similar effects. In addition, at the time that most of the original work on binary toxins and chemical mediators was done, there was a prevailing belief that the cytoskeleton played an integral role in governing the location and mobility of vesicles. Thus, it was reasonable to assume that any toxin that disrupted the cytoskeleton would have an impact on storage or release of mediators. The first report in this area compared the actions of botulinum neurotoxin and botulinum binary toxin on transmission in the phrenic nerve-hemidiaphragm preparation (Simpson, 1982). There was the expected finding that neurotoxin blocked transmission by blocking acetylcholine release from nerve terminals, but the binary toxin had no effect. Apart from showing that C2 toxin did not block exocytosis, this study showed that the toxin did not act on the diaphragm to block muscle twitch. This observation is in keeping with the fact that the predominant form of actin in striated muscle is not that which is ADPribosylated by C2 toxin. In related studies, norepinephrine action was studied on isolated strips of thoracic aorta (Simpson, 1982).It was found that the toxin neither altered agonist-induced responses nor the high affinity reuptake system. release of mediators

L. L. SIMPSON

The effect of C2 toxin on release of granular mediators such as norepinephrine (Matter et al., 1989) and histamine (Bottinger et al., 1987) has been examined, but there is no clear picture that emerges from this work. Studies published to date indicate that the toxin does not have any effect on basal release. However, there may be effects on stimulated release, but the nature and even the direction of effect hinges on experimental conditions. In contrast to granular mediators, the basal release of at least one non-granular mediator is affected by toxin. The constitutive release of steroids in Y-1 adrenal cells was markedly stimulated by C2 toxin (Considine et al., 1992). To the extent that these studies are revealing, they demonstrate that there is no simple and universal scheme that links integrity of the cytoskeleton with storage and release of mediators. This may not be a surprising finding. Even if the cytoskeleton did play a simple and direct role in mediator release, the indirect consequences of producing cellular collapse would probably cause many other outcomes that would complicate interpretation of results. This intuitively obvious matter may lead to what are the most important conclusions that can be drawn from studies with C2 toxin:

1. If an action of C2 toxin can be documented (i.e., ADP-ribosylation

of actin; disaggregation of the cytoskeleton), but there is no change in the cellular response under study, then this particular response is not immediately dependent on integrity of the cytoskeleton.

an action of C2 toxin can be documented, and if there is a change in the cellular response in question, then this response may be governed by the cytoskeleton. However, before a direct link between actin structure and cell response can be accepted, work must be done to demonstrate that the loss of response is not an indirect (and mundane) result of causing cell collapse. In relation to the second point, it is worth noting that most of the literature on C2 toxin as a research tool is flawed by the fact that few investigators have established whether changes in cell response are the direct or indirect consequence of modifying the cytoskeleton. There are, however, two areas of investigation for which this is not a serious problem. C2toxin has been used as a tool to analyze cell regulation of actin synthesis, and in separate studies it has been used to examine cell motility. It is safe to assume that changes in the actin based cystoskeleton would affect directly both of these phenomena.

2. If

The relationship between cytosolic levels of monomeric actin and the levels of mRNA associated with synthesis of actin has been examined by Reuner et al. (1991) and by Bershadsky et al. (1995). Although the two groups used different cell lines (hepatocytes and 3T3 cells), their experimental approaches and observations were similar. C2 toxin was used to promote depolymerization and thus an increase in cytosolic levels of monomeric actin. This treatment produced both a reduction in the rate of synthesis of actin and a decrease in the levels of mRNA. The latter finding was apparently due to a shortened half-life of mRNA (Bershadsky et al., 1995).The down regulation in synthesis of actin was a specific outcome, because synthesis of other proteins was largely unaffected. The down regulation could also be linked to increased levels of free actin rather than changes in the cytoskeleton, because the result was obtained both in monoculture and in cell suspension. It was interesting that procedures that promote polymerization had the opposite effect, causing both the levels of mRNA as well as the rates of actin synthesis to increase. This work demonstrates that there is autoregulation of actin synthesis. The relationship between actin polymerization and cell motility was examined by Verschueren et a/. (1995). Using a lymphoma-derived cell line, they showed that C2 toxin treatment greatly diminished pseudopodal protrusion as well as the ability to invade a monolayer of fibroblast-like cells. This is a predictable finding, and one that must be due at least in part to loss of actin filaments. In contrast to the studies reviewed above, in which a particular type of phenomenon was examined in various cell types (e.g., secretion, autoregulation of actin synthesis), there is other work in which several types of phenomena have been examined in a single cell type

regulation of actin synthesis

effect on cell motility

The Role of the Clostridiurn botulinurn C2 Toxin as a Research Tool to Study Eucaryotic Cell Biology

(see Table 1). Thus, Melamed and colleagues have analyzed the relationship between actin and various cell responses in lymphocytes, ranging from receptor-mediated cell activation to proliferation of Epstein-Barr virus (Melamed et a/., 1991; Melamed et a/., 1994; Melamed et a/., 1995~1,b). Other groups have investigated various phenomena in neutrophils (Al-Mohanna et a/., 1987; Grimminger et a/., 1991a, b; Clancy et a/., 1995).As discussed above, it is sometimes difficult to know whether an observed change in cell response is direct and can be related to specific changes in cytosolic actin or actin filaments, or indirect and merely due to a collapse in cell structure.

References Al-Mohanna FA, Ohishi I, Hallett MB (1987):Botulinum C2 toxin potentiates activation of the neutrophil oxidase. Further evidence of a role for actin polymerization. In FEBS letters. 219: 40-4 Aktories K, Barmann M, Ohishi I, et al. (1986):Botulinum C2 toxin ADP-ribosylates actin. In Nature. 322: 390-2 Aktories K, Wegner A (1989):ADP-ribosylation of actin by clostridial toxins. In J Cell Biol. 109: 1385-87 Aktories K, Wegner A (1992): Mechanism of the cytopathic action of actin-ADPribosylating toxins. In Mol Microbiol. 6: 2905-8 Bershadsky AD, Gluck U, Denisenko ON, et al. (1995):The state of actin assembly regulates actin and vinculin expression by a feedback loop. In J Cell Sci. 108:

1183-93 Bottinger H, Reuner KH, Aktories K (1987):Inhibition of histamine release from rat mast cells by botulinum C2 toxin. In lnternatl Archiv Allergy Appl Immun. 84:

380-4 Clancy R, Leszczynska J, Amin A, etal. (1995):Nitric oxide stimulates ADP ribosylation of actin in association with the inhibition of actin polymerization in human neutrophils. In Journal of Leukocyte Biology. 58: 196-202 Considine RV, Simpson LL (1991): Cellular and molecular actions of binary toxins possessing ADP-ribosyltransferaseactivity. In Toxicon. 29: 913-36 Considine RV, Simpson LL, Sherwin JR (1992): Botulinum C2 toxin and steroid production in adrenal Y-1 cells: The role of microfilaments in the toxin-induced increase in steroid release. In J Pharrnacol Exp The[ 260: 859-64 Fritz G, Schroeder P, Aktories K (1995): Isolation and characterization of a Clostridium botulinum C2 toxin-resistant cell line: Evidence for possible involvement of the cellular C211 receptor in growth regulation. In Infect Imrnun. 63: 2334-40 Grimminger F, Sibelius U, Aktories K, et al. (1991~):Inhibition of cytoskeletal rearrangement of botulinum C2 toxin amplifies ligand-evoked lipid mediator generation in human neutrophils. In Mol Pharmacol. 40: 563-71 Grimminger F, Sibelius U, Aktories K, et al. (1991b): Suppression of cytoskeletal rearranggement in activated human neutrophils by botulinum C2 toxin. In J Biol Chem. 266: 19276-82 Hatheway CL (1990): Bacterial Sources of Clostridial Neurotoxins. In Botulinum Neurotoxin and Tetanus Toxin. (Simpson, LL, ed) pp 3-24, San Diego: Academic Press Honore E, Attali B, Romey G, et al. (1992): Different types of K+ channel current are generated by different levels of a single mRNA. In EMBO Journal. 11: 2465-71 lwasaki M, Ohishi I, Sakaguchi G (1980):Evidence that botulinum C2 toxin has two dissimilar components. In Infect Irnrnun. 29: 390-4 Li G, Rungger-Brandle E, Just I, et al. (1994):Effect of disruption of actin filaments by Clostridium botulinum C2 toxin on insulin secretion in HIT-T15 cells and pancreatic islets. In Molecular Biology of the Cell. 5: 1199-213

L. L. SIMPSON

Matter K, Dreyer F, Aktories K (1989): Actin involvement in exocytosis from PC12 cells: studies on the influence of botulinum C2 toxin on stimulated noradrenaline release. In J Neurochern. 52: 370-6 Mauss S, Koch G, Kreye VA, et a/. (1989): Inhibition of the contraction of the isolated longitudinal muscle of the guinea-pig ileum by botulinum C2 toxin: evidence for a role of G/F-actin transition in smooth muscle contraction. In NaunSchrniedebergsArchiv Pharrnacol. 340: 345-51 Melamed I, Downey GP, Aktories K, et al. (1991): Microfilament assembly is required for antigen-receptor-mediated activation of human B lymphocytes. In J Irnrnunol. 147: 1139-46 Melamed I, Franklin RA, Gelfand EW (1995~): Microfilament assembly is required for'anti-lgM dependent MAPK and p90rsk activation in human B lymphocytes. In Biochern and Biophys Res Cornrn. 209: 1102-10 Melamed I, Stein L, Roifman C M (1994): Epstein-Burr virus induces actin polymerization in human B cells. In J Irnrnunol. 153: 1998-2003 Melamed I, Turner CE, Aktories K, et a/. (199513): Nerve growth factor triggers microfilament assembly and paxillin phosphorylation in human B lymphocytes. In J Exp Med. 181: 1071-9 Miyake M, Ohishi I (1987):Response of tissue-cultured cynomolgus monkey kidney cells to botulinum C2 toxin. In Microbial Pathogenesis. 3: 279-86 Montecucco C, Schiavo G (1994): Mechanism of action of tetanus and botulinurn neurotoxins. In Molecular Microbiology. 13: 1-8 Muller H, von Eichel-Streiber C, Habermann E (1992): Morphological changes of cultured endothelial cells after rnicroinjection of toxins that act on the cytoskeleton. In Infect Irnrnun. 60: 3007-10 Ohishi I (1983~):Lethal and vascular permeability activities of botulinum C2 toxin induced by separate injections of the two toxin components. In Infect Irnrnun. 40: 336-9 Ohishi I (1983b): Response of mouse intestinal loop to botulinum C2 toxin: enterotoxic activity induced by cooperation of nonlinked protein components. In Infect Irnrnun. 40: 691 -5 Ohishi I (1987): Activation of botulinum C2 toxin by trypsin. In Infect Irnrnun. 55: 1461-5 Ohishi I, Hama Y (1992): Purification and characterization of heterologous component IIs of botulinum C2 toxin. In Microbiol Irnrnunol. 36: 221 -9 Ohishi I, lwasaki M, Sakaguchi G. (1980): Purification and characterization of two components of botulinum C2 toxin. In Infect Irnrnun. 30: 668-73 Ohishi I, Miyake M (1985): Binding of the two components of C2 toxin to epithelial cells and brush borders of mouse intestine. In Infect Irnrnun. 48: 769-75 Ohishi I, Tsuyama S (1986):ADP-ribosylation of nonmuscle actin with component I of C2 toxin. In Biochern Biophys Res Cornrn. 136: 802-6 Ohishi I, Yanagimoto A (1992):Visualizations of binding and internalization of two nonlinked protein components of botulinum C2 toxin in tissue culture cells. In Infect Irnrnun. 60: 4648-55 Reuner KH, Presek P, Boschek CB, etal. (1987): Botulinum C2 toxin ADP-ribosylates actin and disorganizes the microfilament network in intact cells. In European Journal of Cell Biology. 43: 134-40 Reuner KH, Schlegel K, Just I, et al. (1991):Autoregulatory control of actin synthesis in cultured rat hepatocytes. In FEBS Letters. 286: 100-4 Schrnid A, Benz R, Just I, et al. (1994): Interaction of Clostridiurn botulinum C2 toxin with lipid bilayer membranes. Formation of cation-selective channels and inhibition of channel function by chloroquine. In J Biol Chem. 269: 16706-11 Simpson LL (1982):A comparison of the pharmacological properties of Clostridiurn botulinum type C1 and C2 toxins. In J Pharrnacol Exp Ther: 223: 695-701 Simpson LL (1989~): Botulinurn Neurotoxin and Tetanus Toxin. pp 1 -422, Sun Diego: Academic Press Simpson LL (198913):The binary toxin produced by Clostridiurn botulinurn enters cells by receptor-mediated endocytosis to exert its pharmacologic effects. In J Pharrnacol Exp Ther: 251 : 1223-8

The Role of the Clostridiumbotulinum C2 Toxin as a Research Tool to Study Eucaryotic Cell Biology

Tabas I, Xiaohui Z, Beatini N,et a/. (1994): The actin cytoskeleton is important for the stimulation of cholesterol esterification by atherogenic lipoproteins in macrophages. In J Biol Chem. 269: 22547-56 Vandekerckhove J, Schering B, Barmann M, et a/. (1988): Botulinum C, toxin ADPribosylates cytoplasmic G/F-actin in arginine 177. In J Biol Chem. 263: 696-700 Verschueren H, van derTaelen I, Dewit J, etal. (1995):Effects of Clostridium botulinum C2 toxin and cytochalasin D on in vitro invasiveness, motility and F-actin content of a murine T-lymphoma cell line. In EurJ Cell Biol. 66: 335-41 Zepeda H, Considine RV, Smith HL, et a/. (1988):Actions of the Clostridium botulinum binary toxin on the structure and function of Y-1 adrenal cells. In J Pharmacol Exp Ther: 246: 1183-9

L. L. SIMPSON

Bacterial Toxins

Klaus Aktories Copyright 0 2002 WILEY-VCH Verlag GmbH & Co. KGaA

CHAPTER 11

Probing the Actin Cytoskeleton by Clostridium botulinum C2 Toxin and Clostridium perfringens Iota Toxin K. AKTORIES, U. PREPENS, P. SEHR, and I. JUST

11.1 Introduction C. botulinum C2 toxin and C. perfringens iota toxin belong to the family of actin-ADP-ribosylating toxins that transfer ADP-ribose from NAD to arginine-177 of actin. This modification results in inhibition of actin polymerization, leading to depolymerization of the microfilament network. Origin, structure, molecular mechanisms and general aspects of the use of this family of toxins is described in chapter 8, 9 and 10. In recent years, C. botulinum C2 toxin has been widely used as a tool in cell biology to study the role of actin in various cell functions. The advantage of the use of C2 toxin is its clear molecular mechanism, its selective substrate specificity (only actin is ADP-ribosylated), and its high potency and efficacy. Treatment of intact cells with C2 toxin results in almost complete depolymerization of the actin cytoskeleton, an effect that is often not achievable with cytochalasins, mycotoxins that block polymerization and induce depolymerization of F-actin by capping actin filaments (Cooper, 1987). A further advantage of C2 toxin is based on its binary structure (the binding component and the biologically active component are separate proteins, C211 and C21, respectively). The isolated components are without toxic effects and are easily to handle. In principle, C. perfringens iota toxin shares the same properties. However, since iota toxin, especially the binding component, is more difficult to purify than C2 toxin, the latter toxin is more often used as a cell biological tool (but see below).

C2 toxin safe to components handle

K. Aktories (Ed.),Bacterial Toxins. 0Chapman & Hall, Weinheim, 1997. ISBN 3-8261-0080-8

11.2 Effects of Clostridium botulinum C2 Toxin in Intact Cells 11.2.1 Treatment of Intact Cells with Clostridium botulinum C2 Toxin

different cells differ in sensitivity to c* toxin

K. AKTORIES etal.

Because C. botulinum C2 toxin is a real binary toxin, studies of its effects on intact cells depend on the presence of both the binding (C211) and enzyme component (C21) (Reuner et al., 1987; Wiegers et al., 1991; Ohishi et al., 1984; Ohishi and Yanagimoto, 1992; Li et al., 1994; Prepens et al., 1996). The toxin components are usually applied at a C21:C211 ratio of 1 :2 (on a microgram basis) (Ohishi et al., 1980). C211 is only fully active in translocating C21 into intact cells when activated with trypsin (as described in Chapter 9). Both components are applied to the medium in a 1:lOO dilution step, to minimize salt effects on the cells. Predilution of C21 and C211 should be performed in PBS or another appropriate buffer supplemented with 100 yglml of serum albumin. Studies with C2 toxin revealed that cultured cells differ in their sensitivity to the toxin. While Vero (African green monkey kidney) cells are highly sensitive, with 100 % rounding up at 10 ng C2 toxin per ml after about 24 h of treatment, FL (human amnion) cells are much less sensitive and about 200-fold higher concentrations are needed for complete rounding up of cells (Ohishi et al., 1984). To some extent, less effective concentrations of the toxin can be compensated for by an increase in the incubation time. Furthermore, cells are usually more sensitive to the toxin under FCS-free conditions. Therefore, to study the toxin's effects with rather insensitive cells or to save toxin, the culture medium can be changed to FCS-free medium when tolerated by the cells being studied. Otherwise the toxin is added for 30 to 60 min in FCS-free medium and, thereafter, the appropriate medium is added again. Even at very high concentrations of the toxin, the earliest onset of cytotoxic effects is usually 15 to 30 min after intoxication (Reuner et al., 1987). This delay is probably due to the process of uptake and translocation of the toxin into the cytosol. In most cells, increase in toxin treatment time for 48h or longer results in detachment and degeneration of cells. In cell culture, individual cells appear to show different sensitivity towards the toxin. This is especially true at early phases of the intoxication process (1 to 2 h after toxin addition). Whether this effect depends on the phase of the cell cycle remains to be clarified.

11.2.2 Effects of C2 on the Microfilament System

7 7.2.2.7 Visualization of the Microfilament System with FITC-phalloidin

C2 toxin-induced depolymerization of actin filaments is visualized by fluorescence labeling of actin with FITC- or Rhodamine-phalloidin. 1. Cells grown on coverslips and treated with C2 toxin are rinsed with PBS.

2. The cells are then fixed with a solution containing 4 % (w/v) paraformaldehyde and 0.1 % (v/v) Triton X-100 in PBS for 60 min at room temperature, followed by rinsing three times with PBS.

W

actin labeling in cells

3. The cells are next incubated with 0.25 ELMFITC-phalloidin (Sigma) in PBS for 60 min at room temperature in the dark. 4. The coverslips are rinsed three times with PBS and mounted in pphenylenediamine (10pg/ml) in PBS/glycerol (1 :10 (v:v)).Pictures are taken with a fluorescence microscope at 490 nm (e.g. Zeiss Axiovert). In many cells, toxin-induced changes in the cytoskeleton are first observed in stress fibers, which become condensed, shortened and broken (Reuner et al., 1987). The cell morphology may still be quite normal, and the submembranous actin cytoskeleton appears to be intact. However, longer incubation with the toxin causes a complete loss of the microfilament network. At this stage more than 95 % of the cells are still viable (Reuner et al., 1987).The intermediate filaments are also redistributed after toxin treatment, whereas the microtubule system remains largely unaltered. However, it appears that the effects on the intermediate filaments are secondary and follow changes in the microfilament network (Wiegers et al., 1991).

7 7.2.2.2 Quantitative Determination of F-actin Depolymerization Although the toxin’s effects on the microfilament system are easily visualized by fluorescence microscopy, it may be important to determine the changes in the ratio of cellular F- and G-actin quantitatively. Because G-actin dissolves in Triton X-100, whereas F-actin (or at least a major fraction of F-actin) does not, fractionation by detergent solubility can be used to study changes in the G- and F-actin content of cells. A different approach is the determination of G-actin content by the DNAse inhibition assay according to Blikstad (Blikstad et al., 1978). The action of DNAse in cleaving DNA is inhibited by monomeric G-actin but not by F-actin. Thus, the extent of inhibition of DNAse is proportional to the concentration of G-actin in cell lysates. Probing the Actin Cytoskeleton by Clostridium botulinum C2 Toxin and Clostridiumperfringens Iota Toxin

Isolation of Triton X-110-soluble (G-actin) and Triton X-100-insoluble (F-actin) Cytoskeleton

1. Toxin-treated cells are rinsed with ice-cold PBS. Cold Triton solution ( 2 % (v/v) Triton X-100, 160mM KCI, 20mM EDTA, 8 m M sodium azide, 40 m M imidazole-HCI pH ZO; 300 pl per 50mm

culture dish) is added, the cells are mechanically removed, transferred into an Eppendorf vial, vortexed and incubated for 15 min on ice, followed by centrifugation for 15 min at 3000 g.

2. The pellets are washed once with Triton solution. 3. The pellets (containing F-actin) are dissolved in Laemmli sample buffer, whereas the proteins of the supernatants (containing Gactin) are concentrated by precipitation with trichloroacetic acid (20 %, w/v) and are then dissolved in Laemmli sample buffer.

4. The proteins are separated by 11 YO SOS gel electrophoresis followed by immunoblotting onto nitrocellulose.

5. Actin is probed with anti-actin (Boehringer or ICN) and the ratio of G-actin (Triton soluble fraction) and F-actin (Triton insoluble fraction) is analyzed using densitometric measurements of the immunoblots. DNAse Inhibition Assay

1. Toxin-treated cells are rinsed with ice-cold PBS. Lysis buffer ( 5 m M potassium phosphate pH 7.6, 150mM NaCI, 2 m M MgC12, 0.1 mM dithiothreitol, 0.2 mM ATP, 0.3 m M phenylmethylsulfonyl fluoride, 2 mM EGTA, 0.5% v/v Triton X-100, 15 % v/v glycerol; 300p1 per 50mm culture dish) is added, the cells are removed mechanically, placed on ice for 30min and are then homogenized with a Teflon homogenizer.

2. The supernatant from the ultracentrifugation 100,000 g ) is used for the DNAse inhibition assay.

(60 min at

3. In a quartz cuvette (at 25"C), 1 ml of a DNA solution (calf thymus, Sigma; 40 pg/ml in 100 m M Tris-HCI p H 7.5,4 m M MgC12, 1.8mM CaCI2) is added to 25pI DNAseI solution (Sigma, 0.1 mg/ml in 50m M Tris-HCI p H 7.5,0.2 m M CaCI2, 0.1 m M phenylmethylsulfonyl fluoride), mixed, and the hydrolysis of DNA is measured as increase in the extinction at 260 nm. Usually the linear phase of the absorbtion increase is about 30sec, and the slope is directly proportional to the enzyme activity. Addition of G-actin leads to an inhibition of DNAse activity. Applying defined concentrations of purified skeletal muscle a-actin gives a standard curve in which pg of actin is proportional to percentage inhibition. When the cell lysates are tested in this system, the determined % of inhibition gives via the standard curve pg of actin. 25 pI of cell extract is mixed with 25 pl DNAse I solution, and then 1 ml of DNA solution is added followed by measurement of the absorbtion. K. AKTORIES et al

11.2.3 Detection of C2-catalyzed ADP-ribosylation in Intact Cells 77.2.3.7 Direct Method by Metabolical Labeling More direct evidence that cellular actin is ADP-ribosylated in intact cells can be obtained, when cells are metabolically labeled with [32P]orthophosphoric acid or with [2-3H]adenine. Radioactively labeled phosphate/adenine is incorporated into cellular NAD which is the cosubstrate for C21-catalyzed ADP-ribosylation.

1. Depending on the cell line, the cells are incubated in phosphatefree medium in the presence of 1 mCi [32P]orthophosphoricacid per ml of medium for 2 to 24 h to allow equilibration of the different phosphate pools.

2. After treatment with C21VC21 (see 11.2.1) the cells are rinsed with ice-cold PBS.

3. Cell lysis is performed either directly by addition of Laemmli sample buffer, or in a lysis buffer containing 10 m M of unlabeled NAD. Alternatively the cells are metabolically labeled with 50 pCi [2-3H]adenine per ml of medium for 16 h (Staddon etal., 1991). Labeled actin can be identified by immunoprecipitation with anti-actin antibody (from Boehringer or ICN) or by immunoblot analysis of cell lysates electrophoretically resolved on 2 0 gels.

alternative labeling method

7 7.2.3.2 Quantification of ADP-ribosylated Actin To quantify the amount of toxin-induced ADP-ribosylation of actin in intact cells, the indirect method of differential ADP-ribosylation is appropriate. Assuming that in intact cells C2 modifies cellular actin, subsequent [32P]ADP-ribosylationof the lysates should result in a decreased incorporation of [32P]ADP-ribose. Because C21 shows transferase activity on ice, although less than at 37"C, the cells should be lysed in a buffer containing [32P]NADand C21.

1. Cultured cells are rinsed with ice-cold PBS and were then mechanically removed in the presence of 1 mM MgCI2,5 0 m M HEPES pH 7.5, 0.3 m M phenylmethylsulfonly fluoride, 30 pg/ml leupeptine, 1 mM dithiothreitol, 10 mM thymidine, 5 p M [32P]NAD, 1 pg/ml C21, sonicated five times on ice, and incubated for 30 min at 37°C. 2. The ADP-ribosylation reaction is stopped by addition of Laemmli sample buffer, or the proteins are precipitated with 1 ml of trichloroacetic acid (20%, w/v). Probing the Actin Cytoskeleton by Clostridium botulinum C2 Toxin and Clostridium perfringens Iota Toxin

3. The proteins are separated by 11 % SDS gel electrophoresis and labeled actin (42 kDa) is analyzed by autoradiography or phosphorimaging. The difference in ADP-ribosylation between control and toxin-treated cells gives the proportion of actin ADPribosylated in the intact cell.

11.2.4 Controls of the in vivo Effects of C2 Toxin

controls

In respect to the cell biological effects observed with the toxin, it is important to remember that the binding and the enzyme component itself should be without any biologic effect on intact cells. Thus, application of only one of the components is an excellent control to test whether the effect observed is specific for C2 toxin and depend on ADP-ribosylation of actin. The best control is to use both toxin components separately. In the case of a toxic effect observed in the presence of only one component (binding component or enzyme component) two explanations are possible. In most cases, the preparation of one component of the toxin applied is contaminated by the second component. This is especially true when high concentrations of either toxin component (e.g -1 pg/ml) is applied. To circumvent this effect, an antibody specific for one component can be used. Secondly, it is possible that the binding of C211 to the eukaryotic cell surface receptor induces effects on its own, as reported for the action of the binding component of pertussis toxin.

11.3 ADP-ribosylation of Actin 11.3.1 ADP-ribosylation of Actin in Cell Lysates ADP-ribosylation of actin in cell lysates is performed in the following way:

1. 40 pl of cell lysate plus 10 p M of [32P]NAD+(0.3pCi), plus 10 mM thymidine and 1 mM dithiothreitol is incubated with 0.1 pg/ml of iota toxin (total volume 50pI) for 30min at 37°C. Thymidine blocks the poly(ADP-ribose)polymerase (approx. 120 kDa), thereby preventing consumption of NAD', which is essential for quantitative ADP-ribosylation of actin. 2. The ADP-ribosylation reaction is terminated by addition of 10 p1 Laemmli sample buffer, or by precipitation with 1 ml of trichloroacetic acid (20 %, w/v).

3. The proteins are separated by 11 % SDS-gel electrophoresis, and labeled actin is analyzed by phosphorimaging or autoradiography.

K. AKTORIES et al.

Quantification of the amount of toxin-ADP-ribosylated actin in the intact cell can be done by the method of differential ADP-ribosylation. However, [32P]ADP-ribosylation of cell lysates prepared by standard procedures is critical because C21 can still work on ice. Thus, under the conditions of short term intoxication or analysis of the time course of intoxication, C21 continues to ADP-ribosylate actin during the preparation of the lysates. This results in an apparently higher amount of actin modified in the intact cell. To avoid this artefact, it is recommended that lysis of cells should be performed in the presence of [32P]NAD(as given in 11.2.3.2 ).

quantification

11.3.2 ADP-ribosylation of Skeletal Muscle a-Actin to Study Actin Functions Skeletal muscle a-actin is to purify easily in large amounts and is stable for several weeks when stored at 4°C. In contrast, non-muscle actin (p- and y-isoforms) is difficult to prepare and stable only for a few days. Therefore, skeletal muscle actin is the preferred isoform to study functions of G-actin. Skeletal muscle a-actin, however, is not a substrate for C2 toxin. In this case, C. perfringens iota toxin (enzyme component ia) has to be used, as this modifies all actin isoforms. To study the influence of ADP-ribosylation on the properties (e.g., ATPase activity, interactions with actin-binding proteins (Geipel et a/., 1989; Geipel et a/., 1990)) of isolated skeletal muscle actin, the following protocol is used. G-actin dissolved in G-buffer is ADP-ribosylated by addition of NAD (at least 10pM, but NAD concentration should exceed actin concentration by about fivefold), 1 mM dithiothreitol, and 1 yglml iota toxin (pH of NAD stock solution is to adjusted to Z5 otherwise the pH of the reaction decreases). Incubation time is 30 to 60 min at 37°C (depending on actin concentration) or overnight on ice. Enzyme activity of iota toxin (i,) is only slighly decreased on ice. Thus, iota toxin-catalyzed ADP-ribosylation has to be stopped by removing the cosubstrate NAD or the toxin itself. NAD can be removed by gel filtration (e.g. by passing through a PDlO column) or by dialysis against G-buffer. Iota toxin is removed by immunoprecipitation with anti-iota toxin as descibed in 11.3.3.

ADP-ribosylation of actin

The extent of ADP-ribosylation is checked by separating modified and unmodified actin using non-denaturing gel electrophoresis, whereas SDS gel electrophoresis is not efficient. ADP-ribosylation changes the isoelectric point of actin to more acid values, resulting in increased migration. This method is also appropriate to study double or triple ADP-ribosylation of actin, e.g., in the presence of iota toxin and/or turkey erythrocyte transferase (Just et a/., 1995). The latter transferase modifies actin even at two arginine residues.

Probing the Actin Cytoskeleton by Clostridium botulinum C2 Toxin and Clostridium perfringens Iota Toxin

1. Non-denaturing gel electrophoresis is performed in a Mini-Protean II gel system (Bio-Rad,Germany) according to Safer (Safer, 1989). Slab gel (degassed):25mM Tris-base, 194 mM glycine, 0.2 mM ATP, 0.5 Triton X-100, 7.5 % (w/v) acrylamide (acrylamide/bisacrylamide); Running buffer (degassed): 25mM Tris-base, 194mM glycine, 0.2mM ATP; Sample buffer: lOmM Tris-HCI pH 8.0, 0.5mM CaCI2, 1.0mM ATP, 0.5mM dithiothreitol, 50% glycerol (v/v) plus bromphenol blue. The gel is prerun for 1 h at 140Y

2. 4 pI of sample buffer is added to 20 pl of the ADP-ribosylation mixture followed by incubation for 10 min at room temperature. 3. The sample is centrifuged for 10min at 14,000 x g and 2-8pI (equivalent to 0.5 to 1.5 pg of actin) of the supernatant is loaded onto the gel and electrophoresis was performed for 50min at 140 V. 4. The gel is stained with Coomassie Blue, destained and dried. ADP-ribosylated actin shows increased migration.

5. The ratio of modified to unmodified actin can be calculated from densitometric measurements of the non-denaturing gel.

11.3.3 ADP-ribosylation of Skeletal Muscle a-Actin for Microinjection Skeletal muscle a-actin is ADP-ribosylated by iota toxin in the presence of unlabeled NAD. The reaction is terminated by immunoprecipitation of iota toxin. As a control, iota toxin and NAD are incubated but in absence of the actin.

1. Two ml of skeletal muscle a-actin (50pg/ml) are dialyzed against 2 x 2 I of a G-buffer containing 200 p M CaCI2, 250 p M ATP, 5 mM Tris-HCI pH 7.5 at 4°C to remove NaN3that is usually present in the G-buffer, and to depolymerize F-actin possibly present. 2. ADP-ribosylation of dialyzed G-actin is started by addition of 250 p M NAD and 2 pg/ml iota toxin. (The pH of the NAD stock solution has to be adjusted to 7.5).

3. After 90 min at 37"C, the reaction mixture is stored on ice.

4. Polyclonal anti-iota IgG coupled to Sepharose4B or, alternatively, bound to protein A-Sepharose is added to the control and to the ADP-ribosylation assay (100pI of a 50 % (v/v) suspension to 2 ml assay), and the mixture is agitated by repeated inversion for 90 min at 4°C.

5. The removal of iota toxin by anti-iota toxin antibody is repeated once.

6. The beads are removed by centrifugation twice for 3min at 14000g. K. AKTORIES eta/

7. ADP-ribosylated actin is concentrated to 200-350 p M by centrifugation in a Centricon (30 kDa cut-off, Amicon). 8. Concentration of actin is determined photometrically at 290 nm against G-buffer as blank: an extinction of 0.62 is equivalent to 23.8 p M actin.

To check complete removal of iota toxin, the following assay is performed.

1. The supernatant of toxin-freed ADP-ribosylated actin is diluted 1 : 10 with G-actin buffer, and ADP-ribosylation is started by addition of 50 p M MgClp, 5 p M [32P]NAD(0.5pCi), 2 p M G-actin. 2. After 60min at 37°C the reaction is terminated by addition of Laemmli sample buffer, heated 5min at 95"C, and the proteins are separated by 11 % SDS gel electrophoresis with subsequent

complete removal of toxin

analysis by autoradiography or phosphorimaging. Complete removal of iota toxin is indicated by the complete absence of any labeling of actin. The effects of microinjected ADP-ribosylated actin are monitored by detection of morphological changes or by visualization of the microfilament system with rhodamine- or FITC-phalloidin (Kiefer et al., 1996).

Fig. 1. Time dependency of ADP-ribosylation of actin by C2 toxin in rat basophilic leukemia (RBL) cells. Rbl cells were incubated with C2 holotoxin (lOOng/ml C21 + 200 ng/ml C211) for the times indicated. Controls were incubation with the enzyme component C21 (lOOng/ml) alone, or with the binding component C211 (200 ng/ml) alone, for 120 min. Another control (-) was not treated with toxin. After incubation, the medium was removed and cells were scraped off in the presence of lysis buffer (2 m M MgCI2, 0.1 m M phenylmethylsulfonyl fluoride, 10 pg/ml leupeptin, 25 m M triethanolamine, pH 7 3 , sonicated five times for 5 sec on ice and centrifuged for 10min at 1000 g. The supernatants were incubated with 1 pglml C21 and 5 p M [32P]NADfor 15min at 30°C. The proteins were separated by 12.5% SDSpolyacrylamide gel electrophoresis and analyzed by phosphorimaging

Probing the Actin Cytoskeletonby Clostridium botulinum C2 Toxin and Clostridium perfringens Iota Toxin

11.3.4 Troubleshooting

non-enzymatic modification

Cellular actin is also modified by non-enzymatic ADP-ribosylation. This modification occurs at cysteine residues and depends on the presence of free ADP-ribose. Thus, cleavage of NAD by endogenous NAD glycohydrolases or release of ADP-ribose from poly-ADPribosylated proteins can increase the amount of ADP-ribose and may induce non-enzymatic modification of actin. Non-enzymatic labeling of actin in the presence of ['4C]/[32P]NAD can be identified because this reaction is quenched by unlabelled ADP-ribose at rather high concentrations (1 mM), whereas enzymatically-catalyzed ['4Cc]/[32P]ADP-ribosylation is not influenced by ADP-ribose.

11.4 Reagents and Chemicals Materials

Supplier

Sigma-Aldrich Chemie GmbH Postfach, 82039 Deisenhofen, Germany Sigma Sigma Sigma Molecular Probes MoBiTec Wagenstieg 5, 37077 Gottingen, Germany anti-actin Boehringer Mannheim GmbH Sandhofer Str. 116, 68298 Mannheim, Germany [32P] o-phosphoric acid Du Pont de Nemours (Deutschland) GmbH Du Pont-Str.1, 61343 Bad Homburg, Germany Du Pont de Nemours (Deutschland) [3H]adenine GmbH [32P]NA D Du Pont de Nemours (Deutschland) GmbH Mini-Protean II gel elec- Bio-Rad Laboratories GmbH Heidemannstr.164, 80939 Munchen, Gertrophoresis cell many Centricon concentrator Amicon, Inc., Beverly, M A 01915, USA Deoxyribonucleic acid from calf thymus DNAse I solution FITC-phalloidin p-phenylendiamine rhodamine-phalloidin

Cat-No. D3664 D4263 P5282 P1519 R415

1378996 NEX-053

NET-063 NEG-023

References Blikstad I, Markey F, Carlsson L, etal. (1978):Selective assay of monomeric and filamentous actin in cell extracts, using inhibition of deoxyribonuclease I. In Cell 15: 935-43 Cooper JA (1987): Effects of cytochalasin and phalloidin on actin. In J. Cell Biol. 105: 1473-8 Geipel U, Just I, Schering B et al. (1989):ADP-ribosylation of actin causes increase in the rate of ATP exchange and inhibition of ATP hydrolysis. In Eur. J. Biochem. 179: 229-32 Geipel U, Just I, Aktories K (1990):Inhibition of cytochalasin D-stimulated G-actin ATPase by ADP-ribosylation with Clostridium perfringens iota toxin. In Biochem. J. 266: 335-9 K. AKTORIES et al.

Just I, Sehr P, Jung M et a/. (1995):ADP-ribosyltransferase type A from turkey erythrocytes modifies actin at Arg-95 and Arg-372. In Biochemistry34: 326-33 Kiefer A, Lerner M, Sehr P et al. (1996): Depolymerization of F-actin by microinjection of ADP-ribosylated skeletal muscle G-actin in PtK2 cells in the absence of the ADP-ribosylating toxin. In Med. Microbiol. Immunol. 184: 175-80 Li G, Rungger-Brandle E, Just I etal. (1994): Effect of disruption of actin filaments by Clostridium botulinum C2 toxin on insulin secretion in HIT-T15 cells and pancreatic islets. In Mol. Biol. Cell 5 : 1199-213 Ohishi I, lwasaki M, Sakaguchi G (1980): Purification and characterization of two components of botulinum C2 toxin. In Infect. Immun. 30: 668-73 Ohishi I, Miyake M, Ogura H et al. (1984): Cytopathic effect of botulinum C2 toxin on tissue-culture cells. In EMS Microbiol. Lett. 23: 281 -4 Ohishi I, Yanagimoto A (1992):Visualizations of binding and internalization of two nonlinked protein components of botulinum Cz toxin in tissue culture cells. In Infect. Immun. 60: 4648-55 Prepens U, Just I, Von Eichel-Streiber C et al. (1996): Inhibition of FceRI-mediated activation of rat basophilic leukemia cells by Clostridium difficile toxin B (monoglucosyltransferase). In J. Biol. Chem. 271 : 7324-9 Reuner KH, Presek P, Boschek CB et al. (1987):Botulinum C2 toxin ADP-ribosylates actin and disorganizes the microfilament network in intact cells. In Eur. J. Cell B i d . 43: 134-40 Safer D (1989):An electrophoretic procedure for detecting proteins that bind actin monomers. In Anal. Biochem. 178: 32-7 Staddon JM, Bouzyk MM, Rozengurt E (1991): A novel approach to detect toxincatalyzed ADP-ribosylation in intact cells: its use to study the action of Pasteurella multocida toxin. In J. Cell Biol. 115,No.4: 949-58 Wiegers W, Just I, Muller H et al. (1991):Alteration of the cytoskeleton of mammalian cells cultured in vitro by Clostridiurn botulinum C2 toxin and C3 ADPribosyltransferase. In Eur. J. Cell Biol. 54: 237-45

Probing the Actin Cytoskeleton by Clostridium botulinum C 2 Toxin and Clostridium petfringens Iota Toxin

Bacterial Toxins

Klaus Aktories Copyright 0 2002 WILEY-VCH Verlag GmbH & Co. KGaA

Clostridium difficile Toxins M. THELESTAM, I. FLORIN and E. CHAVES-OLARTE

12.1 Introduction Although Clostridium difficile had originally been described in 1935 as Bacillus difficilis (Hall and O’Toole, 1935), its role in human disease was appreciated only in the late 1970es, when a previously undescribed cytotoxin was detected in fecal samples from patients with antibiotic-associated pseudomembranous colitis (Larson et al., 1977). Antiserum against Clostridium sordellii neutralized the cytotoxic activity (Rifkin et al., 1977), but the species producing the toxin turned out to be C. difficile. The disease was elicited by giving animals filtered supernatants from cultures of this bacterium, indicating that toxin(s) could cause the symptoms in the absence of bacteria (Bartlett et al., 1977).A few years later the purification of this cytotoxin resulted in the detection of an additional toxin (Banno et al., 1981; Sullivan et al, 1982).This “new“ toxin, toxin A (ToxA) eluted from anion exchangers at a lower salt concentration than toxin B (ToxB),which was the highly potent cytotoxin originally detected in fecal samples. ToxA is also cytotoxic, although it is about 1000-fold less potent than ToxB. The characteristic morphological effect elicited in cells by both toxins has been termed “actinomorphic” (Chang et al., 1978) (sections 12.3.1 and 12.6).ToxA was shown also to be a potent enterotoxin, while ToxB was without enterotoxic activity in animal models. When given intragastrically ToxA causes hemorrhagic fluid secretion, severe inflammation and death, while ToxB does not cause any of these symptoms (Lyerly et al., 1985).Thus ToxA came to be considered the major virulence factor of C. difficile. However, both toxins have equal lethal potencies when injected parenterally (Arnon et al., 1984; Lyerly et al., 1985); about 50ng of either toxin injected intraperitoneally is sufficient to kill a 25 g mouse (Lyerlyet al., 1986). Furthermore, in the presence of sublethal amounts of ToxA, also ToxB is lethal when given by the intragastric route (Lyerly et al., 1985). Indeed both toxins seem to play a role in the disease, according to vaccination studies with toxoids (Libby et al., 1982).This is supported by a recent study on human colonic tissue (see section 12.5.1). It took 10 years from the separation of the two toxins in 1980 to their cloning and sequencing in 1990 (section 12.2),and another 5 years to

discovery of C. difficile toxins

K. Aktories (Ed.), Bacterial Toxins. 0Chapman & Hall, Weinheim, 1997. ISBN 3-8261-0080-8

the clarification in 1995 of the molecular mode of enzymatic action, which turned out to be the same for both toxins (section 12.4). This chapter will deal mainly with the molecular characteristics of these toxins, their cellular actions, and enzymatic activity, which should lay the foundation for understanding the molecular pathogenesis of the disease. Several recent reviews concerning effects of the C. difficile toxins in vivo are available (Bartlett, 1990; Bongaerts and Lyerly, 1994; Lyerly et al., 1988; Lyerly and Wilkins, 1995).

12.2 Molecular Characteristics of the Toxins

molecular weight of toxins

Purification of ToxA to homogeneity was achieved soon after the separation of the toxins (Banno et al., 1984),but with ToxB the purification problems were solved only several years later (Meador and Tweten, 1988). In fact, a variety of molecular sizes were reported for both toxins, which appeared to be large aggregates in their native form, based on their banding patterns on non-denaturing gels. O n SDSPAGE they banded at about 250-300 kDa, often with several bands of lower molecular weight, which were interpreted as subunits or breakdown products of the toxins (Kamiya et a/., 1989; Lyerly et al., 1986).This point was clarified only when both toxins had been cloned and sequenced (Barroso et al., 1990; Dove et al., 1990; Eichel-Streiber et al., 1990; Eichel-Streiber et al., 1992; Sauerborn and Eichel-Streiber, 1990).The molecules turned out to be single polypeptides of 308 kDa for ToxA and 269 kDa for ToxB, thus being the largest protein toxins known so far. The molecular characteristics of the C. difficile toxins have recently been reviewed in detail (Eichel-Streiber, 1993).What follows below is a brief summary of the extensive information now available. Neither toxin has any typical signal sequence. The two toxins have an overall homology of 6 3 % at the amino acid level. They contain four conserved cysteines, as well as a conserved putative nucleotidebinding site between two of the conserved cysteines in the N-terminal part. Moreover, a hydrophobic region near the center of each toxin is conserved. The C-terminal thirds of both toxins contain contiguous repeating units or CROPS (combined repetitive oligopeptides) which are partially homologous with each other, and also with the Cterminal carbohydrate-binding repeats of streptococcal glucosyltransferases (Eichel-Streiberand Sauerborn, 1990). Based on these findings a structural and functional domain structure was postulated for both toxins (schematic model depicted in Fig. 1 ) . The C-terminal repeat region is responsible for receptorbinding in the case of ToxA. This region of ToxB is supposed to have the same function, although a recent study showed that deletion mutants of ToxB, lacking the entire repeat structure, were only slightly less cytotoxic as long as the fourth conserved cysteine was retained (Barroso et al., 1994).The repeating units of ToxA were reported to be nontoxic (Price et al., 1987).For both toxins the N-terminal part is believed

M. THELESTAM. I. FLORIN and E. CHAVES-OLARTE

Fig. 1. Schematic model depicting the domain structure of the Clostridiurn difficile toxins. See text for further discussion

to contain the enzymatic activity. The central hydrophobic domain may be functional in translocating the molecules across membranes in order to reach the cytosol. Indeed, deletion of this region and the third cysteine from ToxB lowered the cytotoxic activity by 4 orders of magnitude (Barroso eta/., 1994). Polyclonal antibodies raised against the two toxins do not crossreact with the heterologous toxin (Libby and Wilkins, 1982), and the epitope domain structures of the two toxins differ from each other despite the high sequence homology. In ToxA the C-terminal is immunodominant, particularly due to two highly immunogenic sites ( h e y and Wilkins, 1992). When these are blocked with monoclonal antibody, the binding of polyclonal antibody to native ToxA is inhibited about 75 % (Lyerly and Wilkins, 1995). In contrast, epitopes on ToxB are located along the entire molecule (Sauerborn et a/., 1994). The two toxin genes probably represent gene repeats that have evolved different C-terminal nucleotide sequences encoding different receptor-binding portions.

12.3 Cellular Effects of the Toxins For detailed information concerning early studies of toxin-induced cellular effects we refer to previous reviews (Fiorentini and Thelestam, 1992; Thelestam and Florin, 1984; Thelestam and Gross, 1990). Here we will summarize only the general features of the intoxication process.

Clostridium difficile Toxins

12.3.1 Actin Cytoskeleton is the Toxin Target It was early recognized that the C. difficile toxins caused a cytoplasmic retraction leaving long projections on a rounded cell body (Chang et al., 1978). This characteristic so called "actinomorphic" cytopathogenic effect (CPE),seen in the early stages of cellular intoxication, resembled the CPE caused by cytochalasins, which were known to disrupt the actin cytoskeleton (ACTSK).We showed that the C. difficile cytotoxin disrupts the ACTSK of human fibroblasts as visualized by immunofluorescence with anti-actin antibodies (Thelestam and Bronneghrd, 1980).This was later confirmed with other cells by transmission electron microscopy (Wedel et al , 1983) as well as another immunofluorescence study, in which the other major cytoskeleta1 structures were found to be intact, while the actin filaments were disrupted (Ottlinger and Lin, 1988).This was the first bacterial protein toxin recognized to act on the ACTSK.

12.3.2 The Receptor for ToxA Contains Galactose The trisaccharide Gala1 -3Ga1@1-4GlcNAc, occurring on rabbit red blood cells and hamster brush border membranes, was identified as a receptor for ToxA (Krivan et al., 1986), but this structure cannot be the relevant ToxA-receptor in the human intestine, as the Galatransferase activity required for its formation is absent in humans. However, human tissues express the Galfil-4GlcNAc structure, for instance on the X, Y, and I blood group antigens, which also bind ToxA (Tucker and Wilkins, 1991). Recently ToxA was shown to bind with similar affinity to GalNAc@1-3GalP1-4GlcNAc, which is also present in human tissues (Karlsson, 1995). In conclusion, the disaccharide Gal@l-4GlcNAc appears to be the minimum structure required for binding of ToxA. No receptor structure for ToxB has yet been identified.

12.3.3 The Toxins Act lntracellularly after Endocytosis An effect on the ACTSK might be exerted either by surface-bound toxin via transmembrane signal transduction, or after internalization of toxin into the cytosol. The latter alternative turned out to be correct, as endocytosis was shown to be required for development of the CPE by both ToxA (Henriques et al., 1987) and ToxB (Florin and Thelestam, 1983).This conclusion was based on the observations that cells pretreated with compounds that raise the pH of endosomes and lysosomes were protected against the CPE, and that a mutant cell line with defective endosomal acidification was more resistant to the toxins than the wild type. Ultrastructural data supported the idea that ToxA enters cells by endocytosis via coated pits (Kushnaryov and Sedmak, 1989). In addition, intracellular processing of the toxins appears to be M. THELESTAM, I. FLORIN and E. CHAVES-OLARTE

required in post-endosomal compartments (Florin and Thelestam, 1986; Henriques et a/., 1987). Dithiothreitol-treatment of ToxB mimics the processing step, converting the large native aggregate to an active ToxB molecule banding at approximately 250kDa on nondenaturing gels (Shoshan et a/., 1993b). The nature of the putative post-endosomal enzymatic processing has not been clarified. Both toxins can also exert their cellular effects after microinjection into cells (Muller et a/., 1992), although much higher amounts of toxin are needed for intoxication by this route. Thus the required processing conditions also seem to exist in the cytosolic compartment, but the natural route via the endosomal vesicle system apparently offers far more efficient processing of the toxins. A recent report suggested that some kind of transmembrane signaling event, activating a calcium influx, occurs rapidly after ToxB contact with cells and is required for the disruption of actin filaments (Gilbert et a/., 1995). However, a calcium influx is required for endocytosis of ToxB (Caspar et a/., 1987))which may explain this observation. In conclusion, cellular internalization of the toxins is required for intoxication.This is consistent with the molecular mode of intracellular cytotoxic action of both toxins described below.

12.4 Molecular Mode of Action of the Toxins 12.4.1 C. difficile Toxins Modify Rho Proteins In the past decade, toxins produced by various species of Clostridium were reported to disrupt the ACTSK by ADP-ribosylation of either actin, as does the C2-toxin from C. botulinum (Aktories et a/., 1986; Reuner et a/., 1987), or the small GTPase Rho, as does exoenzyme C3 from the same bacterium (Aktories et al., 1987; Chardin et al., 1989). However, neither of the C. difficile toxins was found to have any ADPribosyltransferase activity (Florin and Thelestam, 1991; Just et al., 1994b; Popoff et a/., 1988). In 1992 proteins of the Rho subfamily of Ras-related GTPases were shown to control the integrity of focal contacts and stress fibers, as well as the process of membrane ruffling in Swiss 3T3 fibroblasts (Ridley and Hall, 1992; Ridley et a/., 1992). With this background a possible role for Rho proteins as targets for the ACTSK-disrupting C. difficile toxins became conceivable. Indeed, both toxins were shown to modify Rho in intact cells, in such a way that subsequent ADPribosylation by exoenzyme C3 was prevented (Just et al., 1994a,b). Rho proteins in cell lysates were also modified by both toxins in vitro, but no modification was detectable in an entirely cell-free buffer system with recombinant Rho. Further experiments made clear that the modification was strictly dependent on an unknown heat-stable cytosolic factor(s) of molecular mass between 500 and 3000 Da. The authors speculated that this factor might be covalently coupled to Rho Clostridium difficile Toxins

Fig. 2. Toxins A and B from Clostridium difficile are glucosyltransferases. They transfer one glucose-moiety from the co-substrate UDP-Glc to small G-proteins belonging to the Rho-subfamily of Ras-related GTPases

somewhere near Asn-41, which is the site of C3-mediated ADPribosylation, and is located in the putative effector region of this GTPase.

12.4.2 C. difficile Toxins are Glucosyltransferases The molecular mass of ToxB-modified recombinant Rho was subsequently shown by mass spectrometry to be 162 Da higher than unmodified Rho. Since this difference corresponds to the molecular size of hexoses, but the size of the required cytosolic factor was >500 Da, a number of sugar nucleotides were tested for cofactor activity. It turned out that UDP-glucose (UDP-Glc)was the essential cytosolic cosubstrate, while other UDP- or GDP-hexoses did not promote toxininduced Rho-modification. Both the C. difficile toxins were then shown to transfer in vitro one glucose moiety from UDP-Glc to recombinant Rho, Rac and Cdc42 (see Fig. 2), i.e., to each of the three subtypes of small G-proteins within the Rho family (Just et a/., 1995b,c). Other members of the Ras superfamily, such as Ras, Rub and Arf, were not glucosylated. Thr-37 in RhoA was shown to be the unique amino acid target for the glucosylation. Monoglucosylation of Rho inactivated M. THELESTAM, I. FLORIN and E. CHAVES-OLARTE

the molecule, which upon microinjection into cells behaved as a dominant negative Rho species, causing disassembly of actin stress fibers and the actinomorphic cell rounding characteristic of the C. difficile toxins. In contrast a [T37A] mutant Rho was inactive but not dominant negative in similar microinjection experiments (Self et a/., 1993), suggesting that the glucosylation confers this functional change. A speculative explanation is that glucosylated Rho may bind tightly to an effector or guanine nucleotide exchange factor, thereby sequestering the interacting protein (Aktories and Just, 1995).Whether Rac and Cdc42 are also attacked in vivo in cells remains to be seen; the morphological effects induced by microinjection of glucosylated Rac or Cdc42 have not yet been described. The interesting finding that the CPE caused by the C. difficile toxins is due to glucosylation of Rho proteins has been recently confirmed in our laboratory using a different approach. We used a C. difficile toxinresistant mutant cell line, which had been isolated after chemical mutagenesis and selection with ToxB (Florin, 1991), and showed that its toxin-resistance is due to a UDP-Glc deficiency. This protects the mutant cell against other glucosyltransferase toxins as well (see section 12.6). After microinjection of UDP-Glc into toxin-treated mutant cells they became sensitised to toxin B (Fig. 3), as well as the related lethal toxin (LT) from C. sordellii (Chaves-Olarte et a/., 1996). In conclusion, an exciting novel cytotoxic mechanism of action has been described for the C. difficile toxins. The possibilities of understanding the pathogenesis of C. difficile disease should increase, and the toxins should be useful tools in basic cell biology (section 12.7).

12.5 Biological Consequences of Toxin Action on Cells 12.5.1 In Cultured Cells Toxin-mediated glucosylation of Rho explains the cytoskeletal disruption seen in cells treated with either of the C. difficile toxins. Somewhat differing effects of both toxins have been reported on the cytoskeletal organization in Hep-2 cells (Fiorentini et a/., 1989). Cytoskeletal changes and a surface blebbing induced by ToxB were described in a variety of cell types, as determined by immunochemistry and electron microscopy (Malorni et a/., 1990). ToxA has been reported to cause a nuclear polarization (Fiorentini et a/., 1990), a multinucleation in the human leukemic T cell line Jurkat (Fiorentini et a/., 1992), as well as a surface blebbing with apoptosis-like cell death independent of calcium in rat intestinal cells (Fiorentini etal., 1993).All these effects are likely consequences of a disruption of the actin cytoskeleton. The subsequent cell rounding and inhibition of cell proliferation are logical general consequences of an irreversible progressive loss of functional Rho. Indeed a transient overexpression of Rho pro-

cytoskeletal disruption

Clostridium difficile Toxins

effects on tight junctions

teins can confer some resistance to the effects of both ToxA and B (Giry et al., 1995; Just et al., 1994a). Hyperphosphorylation of certain cellular proteins, as observed in ToxB-treated astrocytes (CiesielskiTreska et a/., 1991) and McCoy cells (Schue et al., 1994), might also occur after inactivation of Rho proteins and cytoskeletal disruption, although the signaling processes are not known. Of special interest with respect to the roles of the C. difficile toxins in disease are reports of effects on cultured intestinal cells. The T-84 cell line derived from human colon carcinoma, and cultured in vitro as a polarized epithelium, forms the tight junctions essential for epithelial barrier function. Both ToxA and B caused the disintegration of these tight junctions, measured as a loss of barrier function correlated with disruption of actin filaments (Hecht et al., 1988, 1992). This event occurred even at low toxin concentrations which did not cause rounding of the cells. Indeed, recent work with C. botulinum exoenzyme C3 demonstrated that Rho regulates the tight junctions and perijunctional actin organization in T-84 cells (Nusrat et al., 1995).This is consistent with the notion that the C. difficile toxins may disrupt epithelial barrier function due to glucosylation, and thereby inactivation, of Rho. The functions of the three subtypes of Rho proteins may vary in cells from different tissues, as well as in normal versus tumor cells (Coso et al., 1995; Nishiyama et al., 1994; Olson et al., 1995).If we assume that the C. difficile toxins attack Rac and Cdc42 as well as Rho in vivo, it is not surprising that the long-term consequences of toxin treatment differ between target cell types, as exemplified by the diversity of secondary effects described for both toxins. Thus the inhibition of macromolecular synthesis caused by ToxB in normal human fibroblasts (Florin and Thelestam, 1981) is expected, because cell rounding and the concomitant loss of cell surface fibronectin (Ahlgren et al., 1983) in anchorage-dependent cell lines will inhibit DNA-synthesis (Ben-Ze’ev et al., 1980). By contrast, ToxB did not affect DNA synthesis in transformed B cell lines, whereas it inhibited cytokinesis which depends directly on a functioning actin cytoskeleton (Shoshan et al., 1990). Finally, both toxins were reported to affect human monocytes causing cytokine release (Daubener et al, 1988; Flegel et al., 1991; Siffert et al., 1993), and ToxA was found to stimulate intracellular calcium release and a chemotactic response in human granulocytes (Pothoulakis et al., 1988).Whether these effects are due to glucosylation of Rho proteins or depend on some other separate effects of the toxins remains to be seen.

12.5.2 In Animal Models The in vivo effects of the C. difficile toxins on various targets in animal models have been detailed in a large number of reports (Castagliu010 et al., 1994; Gilbert et al., 1989a, b, Kelly et al., 1994; Kurose et al., 1994; Libby et al., 1982; Lima et al., 1988, 1989; Lyerly et al., 1985; Moore et al., 1990; Pothoulakis et al., 1994; Triadafilopoulos et al., 1989; and see also reviews cited in section 12.1). In particular the in M. THELESTAM, I. FLORIN and E. CHAVES-OLARTE

vivo effects of ToxA have been studied, since ToxB-induced intestinal effects in animals are negligible. Nevertheless, there is no simple model for the events that really take place in the animal intestine after ToxA-treatment, and there is even less understanding of the true C. difficile pathogenesis in huma ns. The most straightforward explanation for the intestinal symptoms elicited by ToxA would be that, after receptor-binding and endocytosis, the toxin glucosylates Rho proteins, leading to a loss of barrier function due to loss of integrity of the tight junctions. Later, a more general cytoskeletal breakdown would lead to disruption of microvilli and retraction of the cells from each other, causing the necrosis of villus cells actually observed after treatment with ToxA. The hemorrhagic fluid secretion and inflammation could be events secondary to the initial destruction of tight junctions and enterocytes, but would aggravate the mucosal necrosis. This simple cell model based on the enzymatic action of the toxins would also predict ToxB to be more potent than ToxA in the intestine, contrary to all previous data from animal studies (mostly on the small intestine). Indeed, in a recent investigation making use of mucosal sheets isolated from the human colon, ToxB was found to be approximately 10 times more potent than ToxA in causing epithelial cell necrosis, as well as a decreased barrier function (Riegler etal., 1995). Interestingly, there was an immediate onset of electrophysiological changes after luminal toxin exposure, suggesting direct damage to human colonic epithelium. These effects of the C. difficile toxins on the real target in human disease thus reflect more closely the different cytotoxic potencies of the two toxins. Another hypothetical model (Castagliuolo et al., 1994) suggests that ToxA, after receptor-binding on the intestinal mucosa, elicits a rapid transmembrane signal causing the release of a mediator(s) (of unknown nature) on the basolateral side of the epithelium. The mediator somehow activates neurons that trigger fluid secretion, as well as immune cells that initiate the strong inflammatory reaction. The two models d o not necessarily contradict each other, and a combination of them can be conceived. Due to its C-terminal ligand domain, ToxA might have a high transepithelial signaling capacity, with consequences according to the neuronal model, but it should also act according to the first model, by exerting its glucosyltransferase activity once it became internalized in enterocytes. ToxB has a shorter ligand domain, which in the tertiary structure of the toxin may not be exposed on the surface to the same extent as the ligand domain on ToxA. Thus ToxB might lack the capacity to induce the signaling events, explaining its lack of effect in animal intestines. However, its potent cytotoxic activity might be highly relevant in the human colon (Riegler et al., 1995),the site of C. difficile infection in man.

models of toxin action

Clostridiurn difficile Toxins

12.6 Relationship of the C. difficile Toxins to Other large Clostridial Cytotoxins A variant of ToxB produced by a serogroup F-strain of C. difficile (ToxBF) has recently been cloned and sequenced (Eichel-Streiber et al., 1995). Compared with classical ToxB, the variant toxin has 148 altered amino acid residues in the N-terminal third of the molecule, whereas the C-terminal repeat domain is highly conserved. There is a clear relationship between ToxA and B, and the C. sordellii hemorrhagic (HT) and lethal toxins (LT) respectively in their immunological (Martinez and Wilkins, 1992) and pathophysiological properties (Bette et al., 1991). The recently established sequence of LT (Green et al., 1995) confirms a close relationship with ToxB at the molecular genetic level. A sixth member of this family of large clostridial cytotoxins is the alpha-toxin from C. novyi, which appears to be immunologically unrelated to toxins A and B, although it shares 48 % sequence homology with these toxins (Hofmann et al., 1995). With regard to cellular morphologic effects, the C. novyi alphatoxin seems most closely related to toxins A and B (Bette et al., 1989), causing the characteristic actinomorphic response, i. e., cell retraction leaving long processes, as exemplified in Fig. 3. In contrast, the cytoskeletal effects of the C. sordellii toxins lead to cell rounding with formation of filopodia but no actinomorphic response (Giry et al.,

Fig. 3. Actinomorphic effect induced by Clostridium difficile ToxB in UDP-Glcdeficient mutant cells after microinjection of UDP-Glc. Chinese hamster lung mutant cells (Florin, 1991) were treated with ToxB (12ng/ml) for 1 h, after which some cells were microinjected with UDP-Glc (100mM). The microinjected cells developed the characteristic actinomorphic effect (Chang et al., 1978) within 30 min, whereas noninjected cells (arrows) were unaffected (Chaves-Olarte et al., 1996) M. THELESTAM, I. FLORIN and E. CHAVES-OLARTE

1995). Interestingly, the variant ToxBF resembles LT rather than ToxB with respect to phenotypic alterations induced in cells (Eichel-Streiber et a/., 1995).This suggests that the N-terminal amino acid alterations have somehow changed the activity of this toxin, and supports the assignment of the catalytic activity of all these toxins to the N-terminal domain. Taking into account the close structural and functional relationships between the large clostridial cytotoxins identified to date, one might expect them to have similar enzymatic activites. The finding that our UDP-Glc-deficientmutant cell is resistant to LT, HT and ToxBF (ChavesOlarte et al., 1996 and unpublished), in addition to ToxA and B (Florin, 1991), suggested that all these toxins may be glucosyltransferases. Indeed, the LT, HT and ToxBF were found to glucosylate small GTPases in cell lysates (Chaves-Olarte et a/., 1996; and Christoph von Eichel-Streiber, personal communication) but Rho is not one of the targets. The targets for LT were recently reported to be Ras, Rap and Rac (Popoff et a/., 1996). In contrast, the mutant cell is sensitive to the C. novyi alpha-toxin, which is consistent with the observation that this toxin does not depend on UDP-Glc, but instead uses UDP-GlcNAc as its co-substrate (Ingo Just, personal communication). As yet, no other bacterial toxin is known to act by glycosylation. Probably many other toxic proteins will turn out to have this activity, and it will be exciting to find out whether such toxins could also glycosylate cellular proteins other than the small GTPases targeted by the cytoskeleton-disruptingglycosyltransferase toxins known to date.

12.7 Conclusions and Perspectives for the Future During the past decade an increasing incidence of C. difficile disease has been evident worldwide. This bacterium is now recognized as a maior nosocomial pathogen in industrialized parts of the world (Lyerly and Wilkins, 1995).The pseudomembranous colitis caused by C. difficile was early recognized as a toxin disease, i.e., all symptoms can be evoked by the toxins. Understanding the disease thus requires an understanding of the toxins. Our knowledge of the molecular toxicology of C. difficile has been considerably improved as the enzymatic mode of action of the toxins have been identified. This knowledge also has the potential to help in answering a whole array of questions related to the functions of small G-proteins in cells. However, a large number of obvious questions remain both concerning the toxins per se, and their actions on single cells and in the intestine. One intriguing point is the significant differences in cytotoxic and enterotoxic potencies between toxins A and B. What determines these differences? Could the difference in cytotoxic potency simply reflect a difference in the affinity of the toxins for their cellular substrates? Preliminary observations in our laboratory suggest that ToxA, in glucosylation assays in vitro, using either cell lysates or recombinant Rho, is

comparison of Tox A and Tox B

Clostridiurndifficile Toxins

much less potent than ToxB with regard to glucosylation potency (Chaves Olarte et al., unpublished). However, the toxins obviously also have different cell surface receptors, and this could be part of the explanation. Despite the high homology at the primary sequence level, the native 3D-structures certainly differ. This is evident from the different epitope patterns, and also from the finding that ToxA is highly resistant to trypsin whereas ToxB is inactivated by this enzyme (Lyerly et al., 1989).The uneven epitope distribution on ToxA may imply that the native toxin is more or less covered by its C-terminal repeat structure, possibly by being dimerized in a manner that exposes predominantly the repeat structure. In contrast, ToxB is likely to have a 3Dconfiguration that also exposes some N-terminal parts of the molecule, as its epitopes appear to be distributed over the entire primary sequence of the molecule. Establishment of the 3D-structures for both toxins will be helpful in solving the receptor problem, and will answer several other questions as well. In the cellular intoxication steps occurring after receptor binding, both toxins require a low pH compartment and some kind of processing to be optimally active in the cytosol. The tertiary structures are likely to be unfolded at low pH, possibly exposing the common hydrophobic domain, which might promote toxin translocation across the endosomal membrane and delivery of (at least) the catalytically active part of the molecules to the cytosol. Nothing is known about how the toxins are processed in cells, or concerning the mode of toxin translocation across membranes. The 3D-structures created after these events most probably differ from the native ones. They should allow mapping of the amino acids forming the catalytic site, including the region(s) required for binding to the target GTPases as well as for binding the co-substrate UDP-Glc. Preparing fragments of the toxins for microinjection into cells will help to solve these latter questions, and also indicate whether the lower glucosylation potency of ToxA is due to a real difference in enzymatic potency or to a partial masking of the catalytic site by the C-terminal repeat structure. Another question to be explored is whether the C-terminal repeat regions alone, given extracellularly, could evoke some of the biological effects that have been described. For instance, an 873 amino acid repeat from ToxA can apparently elicit a rapid increase in cytosolic calcium in Chinese hamster ovary cells overexpressing sucrase isomaltase, a glycoprotein suggested as an intestinal receptor for ToxA (Thomas LaMont, personal communication). Would this same part of the ToxA molecule elicit fluid secretion in the intestine, explaining the postulated signaling capacity of ToxA? Does the ToxB repeat in isolation have any biological activity? Furthermore, high amounts of ToxB consistently cause a rapid "lytic" effect on cells, which has been correlated with a phospholipase A2 activation (Shoshan et al., 1993a). Since this phenomenon also occurred in the toxin-resistant UDP-Glc-deficient mutant cell, it is unrelated to the cytoskeletal effect mediated by Rho-glucosylation. How is the phospholipase A2 activation generated and does it have any implications for disease induced by C. difficile? Some of the answers to these questions may help to M. THELESTAM, I. FLORIN and E. CHAVES-OLARTE

clarify the true events in intestinal pathogenesis, and perhaps also give a clue to the intriguing phenomenon that neonates are resistant to C. difficile toxins in the intestine. Equally intriguing is the question of why and how animals die upon parenteral injection of low amounts of the toxins (Arnon et a/., 1984; Lyerly et al., 1986), and particularly the fact that the differences in enterotoxic and cytotoxic potencies are apparently not reflected in the lethal activities of the toxins. Defined fragments of the toxins will also be useful for clarifying this aspect of their biological actions. Obviously the C. difficile toxins will become extremely useful tools for further studies of how the Rho subfamily proteins control the ACTSK, as well as aspects of cell proliferation in different types of cells (Olson et al., 1995).These toxins will be easier to use for manipulation of small GTPases than the exoenzyme C3 from C. botulinurn, because they are internalized into cells and are more potent. Their drawback compared with C3 is that they attack more than one target protein. Preparation of mutant toxins, fragments of toxins or hybrid toxins which discriminate between various GTPase targets might solve this problem and allow sophisticated studies of the cellular cross-talk between small GTPases (see further discussion in Chapters 10 and 15). The use of the C. difficile toxins as tools should obviously not be restricted to basic cell biological applications. The possibility of using the toxins, or fragments of them, for vaccination purposes should be investigated. Moreover, it is tempting to suggest that potent cytotoxins like these might be useful in targeted tumor therapy. The recent study of Riegler and coworkers (1995) indicated that 3 nM ToxA had no effect on human colonic mucosal sheets, at least during a 5 h exposure. On the other hand as little as 0.01 -0.001 nM ToxA was shown to kill a variety of human colonic and pancreatic tumor cell lines, while 12-500 times more toxin was needed to kill cell lines derived from normal tissues (Kushnaryovet al., 1992). Is there a possibility of using ToxA locally as an antitumor agent directed against colon cancer?

applications of C. difficile toxins

Acknowledgements We are grateful to Drs Ove Lundgren, Christoph von Eichel-Streiber, Patrice Boquet, and Alberto Alape Giron for stimulating discussions and helpful suggestions. Studies from the authors' laboratory have been sponsored by the Swedish Medical Research Council (16X-05969)and Magnus Bergvalls Stiftelse.

References Ahlgren T, Florin I, Jarstrand C, et al. (1983): Loss of surface fibronectin from human lung fibroblasts exposed to cytotoxin from Clostridium difficile. In Infect. Immun. 39: 1470- 1472. Aktories K, Barman M, Ohishi I, et al. (1986): Botulinum C2 toxin ADP-ribosylates actin. In Nature (London) 322: 390-392. Aktories K, Weller U, Chatwal GS (1987):Clostridium botulinum type C produces a novel ADP-ribosyltransferase distinct from C2 toxin. In FEBS Lett. 212: 109- 113. Clostridiurn difficile Toxins

Aktories K, Just I (1995):Monoglucosylation of low-molecular-mass GTP-binding Rho proteins by clostridial cytotoxins. In Trends Cell Biol. 5: 441 -443. Arnon SS, Mills DC, Day PA, et al. (1984): Rapid death of infant rhesus monkeys injected with Clostridiurn difficile toxins A and B: Physiologic and pathologic basis. In J. Pediatr: 104: 34-40. BannoY, KobayashiT, Watanabe K, etal. (1981):Two toxins (D-1 and D-2) of Clostridiurn difficile causing antibiotic-associated colitis: purification and some characterization. In Biochern. Int. 2: 629-635. Banno V, KobayashiT, Kono H, et al. (1984):Biochemical characterization and biologic actions of two toxins (D-1 and D-2) from Clostridiurn difficile. In Rev. Infect. Dis. 6: Sll-S20. Barroso LA, Wang SZ, Phelps CJ, et al. (1990):Nucleotide sequence of the Clostridiurn difficile toxin B gene. In Nucl. Acids Res. 18: 4004. Barroso LA, Moncrief JS, Lyerly DM, et al. (1994):Mutagenesis of the Clostridiurn difficile toxin B gene and effect on cytotoxic activity. In Microb. Pathogen. 16: 297-303. Bartlett JG, Onderdonk AB, Cisneros RL, et al. (1977):Clindamycin-associatedcolitis due to a toxin-producing species of Clostridium in hamsters. In J. Inf. Dis. 136: 701 -705. Bartlett JG (1990):Clostridiurn difficile: Clinical considerations. In Rev. Inf. Dis. 12: S243-S251. Ben-Ze’ev A, Farmer SR, Penman S (1980): Protein synthesis requires cell-surface contact while nuclear events respond to cell shape in anchorage-dependent fibroblasts. In Cell, 21 : 365-372. Bette P, Frevert J, Mauler F, et al. (1989):Pharmacological and biochemical, studies of cytotoxicity of Clostridiurn novyi type A alpha-toxin. In Infect. Imrnun. 57: 2507-2513. Bette P, Oksche A, Mauler F, et al. (1991):A comparative biochemical, pharmacological and immunological study of Clostridiurn novyi alpha-toxin, C.difficile toxin B and C.sordellii lethal toxin. In Toxicon, 29: 877-887. Bongaerts GPA, Lyerly D M (1994):Mini-review Role of toxins A and B in the pathogenesis of Clostridiurn difficile disease. In Microb. Pathogen. 17: 1 - 12. Caspar M, Florin I, Thelestam M (1987):Calcium and calmodulin in cellular intoxication with Clostridiurn difficile toxin B. In J. Cell. Physiol. 132: 168-172. Castagliuolo I, LaMont JT, Letourneau R, etal. (1994):Neuronal involvement in the intestinal effects of Clostridiurn difficile toxin A and Vibrio cholerae enterotoxin in rat ileum. In Gastroenterol. 107: 657-665. Chang T-W, Bartlett JG, Gorbach SL, et al. (1978):Clindamycin-inducedenterocolitis in hamsters as a model of pseudomembranous colitis in patients. In Infect. Irnrnun. 20: 526-529. Chardin P, Boquet P, Madaule P, et al. (1989):The mammalian G-protein rhoC is ADP-ribosylated by Clostridiurn botulinurn exoenzyme C3 and affects microfilaments in Vero cells. In EM60 J. 8: 1087- 1092. Chaves Olarte E, Florin I, Boquet P, et al. (1996): UDP-glucose deficiency in a mutant cell line protects against glucosyltransferasetoxins from Clostridiurn difficile and Clostridium sordellii 271 : 6925-6932. Ciesielski-Treska J, Ulrich G, Baldacini 0, et al. (1991):Phosphorylation of cellular proteins in response to treatment with Clostridium difficile toxin B and Clostridiurn sordellii toxin L. In Eur. J. Cell 6i0l. 56: 68-78. Coso OA, Chiariello M, Yu J-C, et al. (1995):The small GTP-binding proteins Racl and Cdc42 regulate the activity of the JNK/SAPK signaling pathway. In Cell, 81: 1137- 1146. Dove CH, Wang SZ, Price SB, et al. (1990):Molecular characterization of the Clostridiurn difficile toxin A gene. In Infect. Irnrnun. 58: 480-488. Daubener W, Leiser E, von Eichel-Streiber Cv, et al. (1988): Clostridiurn difficile toxins A and B inhibit human immune response in vitro. In Infect. Imrnun. 56: 1107- 1112. Eichel-Streiber Cv, Sauerborn M (1990): Clostridiurn difficile toxin A carries a Cterminal repetitive structure homologous to the carbohydrate binding region of streptococcal glycosyltransferases. In Gene, 96: 107- 113. M. THELESTAM, I. FLORIN and E. CHAVES-OLARTE

Eichel-Streiber Cv, Laufenberg-Feldmann R, Sartingen S, et al. ( 1990): Cloning of Clostridium difficile toxin B gene and demonstration of high N-terminal homology between toxin A and B. In Med. Microbiol. Immunol. 179: 271 -279. Eichel-StreiberCv, Laufenberg-Feldmann R, Sartingen S, etal. (1992):Comparative sequence analysis of the Clostridium difficile toxins A and B. In M o l Gen Genet, 233: 260-268. Eichel-Streiber Cv (1993): Molecular biology of the Clostridium difficile toxins. In Genetics and Molecular Biology of Anaerobic Bacteria (Sebald M, ed) pp 264-289, New York: Springer Verlag. Eichel-Streiber Cv, Meyer zu Heringdorf D, Habermann E, et a/. (1995): Closing in on the toxic domain through analysis of a variant Clostridium difficile cytotoxin B. In Molec. Microbiol. 17: 313-321. Fiorentini C, Arancia G, Paradisi S, etal. (1989): Effects of Clostridium difficile toxins A and B on cytoskeleton organization in Hep-2 cells: A comparative morphological study. In Joxicon, 27: 1209-1218. Fiorentini C, Malorni W, Paradisi S, et al. (1990): Interaction of Clostridium difficile toxin A with cultured cells: cytoskeletal changes and nuclear polarization. In Infect. Immun. 58: 2329-2336. Fiorentini C, Thelestam M (1991):Review article Clostridium difficile toxin A and its effects on cells. In Toxicon, 29: 543-567. Fiorentini C, Chow SC, Mastrantonio P, et a/. (1992): Clostridium difficile toxin A induces multinucleation in the human leukemic T cell line JURKAT. In Eur. J. Cell Biol. 57: 292-297. Fiorentini C, Donelli G, Nicotera P, et a/. (1993): Clostridium difficile toxin A elicits Ca2+ -independent cytotoxic effects in cultured normal rat intestinal crypt cells. In Infect. Immun. 61 : 3988-3993. Flegel WA, Muller F, Daubener W, etal. (1991):Cytokine response by human monocytes to Clostridium difficile toxin A and toxin B. In Infect. Immun. 59: 3659-3666. Florin I, Thelestam M (1981): Intoxication of cultured human lung fibroblasts with Clostridium difficile toxin. In Infect. Immun. 33: 67-74. Florin I, Thelestam M ( 1983): Internalization of Clostridium difficile cytotoxin into cultured human lung fibroblasts. In Biochim. Biophys. Acta, 763: 383-392. Florin I, Thelestam M ( 1986): Lysosomal involvement in cellular intoxication with Clostridium difficile toxin B. In Microb. Pathogen. 1 : 373-385. Florin I (1991): Isolation of a fibroblast mutant resistant to Clostridium difficile toxins A and B. In Microb. Pathogen. 11: 337-346. Florin I, Thelestam M (1991): ADP-ribosylation in Clostridium difficile toxin-treated cells is not related to cytopathogenicity of toxin B. In Biochim. Biophys. Acta, 1091: 51 -54. h e y SM, Wilkins TD (1992): Localization of two epitopes recognized by monoclonal antibody PCG-4 on Clostridium difficile toxin A. In Infect. Immun. 60: 2488-2492. Gilbert RJ, Triadafilopoulos G, Pothoulakis C, et a/. (1989a): Effect of purified Clostridium difficile toxins on intestinal smooth muscle. I. Toxin A. In Am. J. Physiol. 256: G759-G766. Gilbert RJ, Pothoulakis C, LaMont JT (198913): Effect of purified Clostridium difficile toxins on intestinal smooth muscle. II. Toxin B. In Am. J. Physiol. 256: G767-G772. Gilbert RJ, Pothoulakis C, LaMont JT, et al. (1995): Clostridium difficile toxin B activates calcium influx required for actin disassembly during cytotoxicity. In Am. J. Physiol. 268: G487-G495. Giry M, Popoff MR, Eichel-Streiber Cv, et al. (1995):Transient expression of RhoA, -B, and -C GTPases in HeLa cells potentiates resistance to Clostridium difficile toxins A and B but not to Clostridium sordellii lethal toxin. In Infect Immun. 63: 4063-4071. Green GA, Schue V, Monteil H (1995): Cloning and characterization of the cytotoxin L-encoding gene of Clostridium sordellii: homology with Clostridium difficile cytotoxin B. In Gene, 161: 57-61. Hall JC, O’Toole E (1935): Intestinal flora in new-born infants with a description of a new pathogenic anaerobe Bacillus difficilis. In Am J. Dis. Child. 49: 390-402. Clostridium difficile Toxins

Hecht G, Pothoulakis C, LaMont JT, et a/. (1988): Clostridium difficile toxin A perturbs cytoskeletal structure and tight iuncion permeability of cultured human intestinal epithelial monolayers. In J. Clin. Invest. 82: 1516- 1524. Hecht G, Koutsouris A, Pothoulakis C, et al. (1992):Clostridium difficile toxin B disrupts the barrier function of T84 monolayers. In Gastroenterology, 102: 416-423. Henriques B, Florin I, Thelestam M (1987):Cellular internalizationof Clostridium difficile toxin A. In Microb. Pathogen. 2: 455-463. Hofmann F, Herrmann A, Habermann E, et a/. (1995):Sequencing and analysis of the gene encoding the alpha-toxin of Clostridium novyi proves its homology to toxins A and B of Clostridium difficile. In Mol Gen Genet, 247: 670-679. Just I, Fritz G, Aktories K, etal. (1994~):Clostridium difficile toxin B acts on the GTPbinding protein Rho. In J. Biol. Chem. 269: 10706-10712. Just I, Richter H-P, Prepens U, et a/. (1994b):Probing the action of Clostridium difficile toxin B in Xenopus laevis oocytes. In J. Cell Sci. 107: 1653-1659. The law molecular mass GTPJust I, Selzer J, Eichel-Streiber Cv, et a/. (1995~): binding protein Rho is affected by toxin A from Clostridium difficile. In J. Clin. Invest. 95: 1026-1031. Just I, Selzer J, Wilm M, etal. (199513):Glucosylation of Rho proteins by Clostridium difficile toxin B. In Nature, 375: 500-503. Just I, Wilm M, Selzer J, et a/. (1995~): The enterotoxin from Clostridium difficile (ToxA) monoglucosylates the Rho proteins. In J. Biol. Chem. 270: 13932-13936. Kamiya S, Reed PJ, Borriello SP (1989):Purification and characterisation of Clostridium difficile toxin A by bovine thyroglobulin affinity chromatography and dissociation in denaturing conditions with or without reduction. In J. Med. Microbiol.

30: 69-77. Karlsson K-A (1995):Microbial recognition of target-cell glycoconjugates. In Curr. Opin. Struct. Biol. 5:622-635. Kelly CP, Becker S, Linevsky JK, et a/. (1994):Neutrophil recruitment in Clostridium difficile toxin A enteritis in the rabbit. In J. Clin. Invest. 93: 1257-1265. Krivan HC, Clark GF, Smith DF, etal. (1986):Cell surface binding site for Clostridium difficile enterotoxin: Evidence for a glycoconjugate containing the sequence Galalphal-3Galbeta1-4GlcNAc. In Infect. Immun. 53: 573-581. Kurose I, Pothoulakis C, LaMont JT, et a/. (1994): Clostridium difficile toxin A-induced microvascular dysfunction. Role of histamine, In J. Clin. Invest. 94: 1919- 1926. Kushnaryov VM, Sedmak JJ (1989):Effect of Clostridium difficile enterotoxin A on ultrastructure of Chinese hamster ovary cells. In Infect. Immun. 57: 3914-3921. Kushnaryov VM, Redlich PN, Sedmak JJ, et a/. (1992):Cytotoxicity of Clostridium difficile toxin A for human colonic and pancreatic carcinoma cell lines. In Cancer Res. 52: 5096-5099. Larson HE, Parry JV, Price AB, et a/. (1977): Undescribed toxin in pseudomembranous colitis. In Brit. Med. J. 1: 1246- 1248. Libby JM, Wilkins TD (1982): Production of antitoxins to two toxins of Clostridium difficile and immunological comparison of the toxins by cross-neutralization studies. In Infect. Immun. 35: 374-376. Libby JM, Jortner BS, Wilkins TD (1982):Effects of the two toxins of Clostridium difficile in antibiotic-associated cecitis in hamsters. In Infect. Immun. 36: 822-829. Lima AA, Lyerly DM, Wilkins TD, et a/. (1988):Effects of Clostridium difficile toxins A and B in rabbit small and large intestine in vivo and on cultured cells in vitro. In Infect. Immun. 56:582-588. Lima AA, lnnes DJ, Chadee K, etal. (1989):Clostridium difficile toxin A. Interaction with mucus and early sequential histopathologic effects in rabbit small intestine. In Lab. Invest. 61 : 419-425. Lyerly DM, Saum KE, MacDonald DK, et a/. (1985): Effects of Clostridium difficile toxins given intragastrically to animals. In Infect. Immun. 47: 349-352. Lyerly DM, Roberts MD, Phelps CJ, et al. (1986):Purification and properties of toxins A and 6 of Clostridium difficile. In FEMS Microbiol. Lett. 33: 31 -35. Lyerly DM, Krivan HC, Wilkins TD (1988):Clostridium difficile: its disease and toxins. In Clin. Microbiol. Rev. 1: 1-18. Lyerly DM, Carrig PE, Wilkins TD (1989):Susceptibility of Clostridium difficile toxins A and B to trypsin and chymotrypsin. In Microb. Ecol. Health & Dis. 2: 219-221. M. THELESTAM. I. FLORIN and E. CHAVES-OLARTE

Lyerly DM, Wilkins TD (1995):Clostridium difficile. In Infections of the gastrointestinal tract (Blaser MJ, Smith PD, Ravdin JI, et a/. eds) pp 867-891, New York: Raven Press, Ltd Malorni W, Fiorentini C, Paradisi S, et al. (1990):Surface blebbing and cytoskeletal changes induced in vitro by toxin B from Clostridium difficile: An immunochemical and ultrastructural study. In Exp. Molec. Pathol. 52: 340-356. Martinez RD, Wilkins TD (1992):Comparison of Clostridium sordellii toxins HT and LT with toxins A and B of C.difficile. In J. Med. Microbiol. 36: 30-36. Meador Ill J, Tweten RK (1988): Purification and characterization of toxin B from Clostridium difficile. In Infect. Immun. 56: 1708- 1714. Moore R, Pothoulakis C, LaMont JT, et a/. (1990): C.difficile toxin A increases intestinal permeability and induces CI- secretion. In Am. J. Physiol. 259: G165G172. Muller H, Eichel-Streiber Cv, Habermann E (1992):Morphological changes of cultured endothelial cells after microinjection of toxins that act on the cytoskeleton. In Infect. Immun. 60: 3007-3010. Nishiyama T, Sasaki T, Takaishi K, et a/. (1994):rac p21 is involved in insulin-induced membrane ruffling and rho p21 is involved in hepatocyte growth factor- and 120-tetradecanoylphorbol-13-acetate (TPA)-induced membrane ruffling in KB cells. In Mol. Cell. Biol. 14: 2447-2456. Nusrat A, Giry M, Turner JR, etal. (1995):Rho protein regulates tight junctions and perijunctional actin organization in polarized epithelia. In Proc. Natl. Acad. Sci. 92: 10629-10633. Olson MF, Ashworth A, Hall A (1995):An essential role for Rho, Rac, and Cdc42 GTPases in cell cycle progression through G1. In Science, 269: 1270-1272. Ottlinger ME, Lin S (1988): Clostridium difficile toxin B induces reorganization of actin, vinculin, and talin in cultured cells. In Exptl Cell Res. 174: 215-229. Popoff MR, Rubin EJ, Gill DM, et a/. (1988):Actin-specific ADP-ribosyl-transferase produced by a Clostridium difficile strain. In Infect. Immun. 56, 2299-2306. Popoff MR, Chaves-Olarte E, Lemichez E., et a/. (1996): Ras, Rap, and Rac small GTP-binding proteins are targets for Clostridium sordellii lethal toxin glucosylation. In J. Biol. Chem. 271, 10217-10224. Pothoulakis C, Sullivan R, Melnick DA, et a/. (1988): Clostridium difficile toxin A stimulates intracellular calcium release and chemotactic response in human granulocytes. In J. Clin. Invest. 81 : 1741- 1745. Pothoulakis C, Castagliuolo I, LaMont JT, et a/. (1994):CP-96,345, a substance P antagonist, inhibits rat intestinal responses to Clostridium difficile toxin A but not cholera toxin. In Proc. Natl. Acad. Sci. 91 : 947-951. Price SB, Phelps CJ, Wilkins TD, et al. (1987): Cloning of the carbohydrate-binding portion of the toxin A gene of Clostridium difficile. In Curr. Microbiol. 16: 55-60. Reuner KH, Presek P, Boschek CB, etal. (1987):Botulinum C2 toxin ADP-ribosylates actin and disorganizes the microfilament network in intact cells. In Eur. J. Cell Biol. 43: 134-140. Ridley AJ, Hall A (1992):The small GTP-binding protein rho regulates the assembly of focal adhesion and actin stress fibers in response to growth factors. In Cell, 70: 389-399. Ridley AJ, Paterson HF, Johnston CL, etal. (1992):The small GTP-binding protein rac regulates growth factor-induced membrane ruffling. In Cell, 70: 401 -410. Riegler M, Sedivy R, Pothoulakis C, et al. (1995):Clostridium difficile toxin B is more potent than toxin A in damaging human colonic epithelium in vitro. In J. Clin. Invest. 95: 2004-2011. Rifkin GD, Fekety FR, Silva J, etal. (1977):Antibiotic-induced colitis implication of a toxin neutralised by Clostridium sordellii antitoxin. In Lancet, ii: 1103-1106. Sauerborn M, Eichel-Streiber Cv (1990):Nucleotide sequence of Clostridium difficile toxin A. In Nucl. Acids Res. 18: 1629-1630. Sauerborn M, Hegenbarth S, Laufenberg-Feldmann R, et a/. (1994):Monoclonal antibodies discriminating between Clostridium difficile toxins A and B. In Bacterial protein toxins (Freer J, Aitken R, Alouf JE, et al. eds) pp 510-511, Stuttgart Jena New York: Gustav Fischer Verlag.

Clostridiurn difficile Toxins

Schue V, Green GA, Girardot R, et a/. (1994):Hyperphosphorylation of calnexin, a chaperone protein, induced by Clostridium difficile cytotoxin. In Biochem. Biophys. Res. Commun. 203: 22-28. Self AJ, Paterson HF, Hall A (1993):Different structural organization of Ras and Rho effector domains. In Oncogene, 8: 655-661. Shoshan MC, Aman P, Skog S, et a/. (1990):Microfilament-disrupting Clostridium difficile toxin B causes multinucleation of transformed cells but does not block capping of membrane lg. In Eur. J. Cell Biol. 53: 357-363. Activation of cellular phospholipase Shoshan MC, Florin I, Thelestam M (1993~): A2 by Clostridium difficile toxin B. In J. Cell. Biochem. 52: 116-124. Shoshan MC, Bergman T, Thelestam M, et a/. (1993b): Dithiothreitol generates an activated 250,000 mol.wt form of Clostridium difficile toxin B. In Toxicon, 31: 845-852. Siffert J-C, Baldacini 0, Kuhry J-G, et a/. (1993):Effects of Clostridium difficile toxin B on human monocytes and macrophages: possible relationship with cytoskeleta1 rearrangement. In Infect. Immun. 61 : 1082-1090. Sullivan NM, Pellet S, Wilkins TD (1982):Purification and characterization of toxins A and B of Clostridium difficile. In Infect. Immun. 35: 1032-1040. Thelestam M, Bronneg6rd M (1980): Interaction of cytopathogenic toxin from Clostridium difficile with cells in tissue culture. In Scand. J. Infect. Dis. Suppl. 22: 16-29. Thelestam M, Florin I ( 1984):Cytopathogenic action of Clostridium difficile toxins. In J. Toxicol. Toxin Rev. 3:139-180. Thelestam M, Gross R (1990):Toxins acting on the cytoskeleton. In Handbook of toxinology (Shier WT, Mebs D, eds) pp 423-492, New York and Basel: Marcel Dekker, Inc. Triadafilopoulos G, Pothoulakis C, Weiss R, et a/. (1989): Comparative study of Clostridium difficile toxin A and cholera toxin in rabbit ileum. In Gastroenterol.97: 1186-1192. Tucker KD, Wilkins TD (1991):Toxin A of Clostridium difficile binds to the human carbohydrate antigens I, X, and Y. In Infect. Immun. 59: 73-78. Wedel N, Toselli P, Pothoulakis C, et a/. (1983):Ultrastructural effects of Clostridium difficile toxin B on smooth muscle cells and fibroblasts. In Exptl Cell Res. 148: 413 -422.

M. THELESTAM, I, FLORIN and E. CHAVES-OLARTE

Bacterial Toxins

Klaus Aktories Copyright 0 2002 WILEY-VCH Verlag GmbH & Co. KGaA

CHAPTER 13

Clostridium difficile Toxin B as a Probe for Rho GTPases I. JUST, J. SELZER, F. HOFMANN, and K. AKTORIES

13.1 Int roduction Clostridium difficile toxin B exhibits cytotoxic activity which leads to preferential destruction of the microfilament system of cell monolayers (Bongaerts and Lyerly, 1994; Wolfhagen et al., 1994).These effects are due to the mono-glucosyltransferase activity of toxin B which modifies the low molecular mass GTPases of the Rho subfamily (Just et a/., 1995a; Aktories and Just, 1995). ToxinB uses selectively the cosubstrate UDP-glucoseto transfer the glucose moiety to the Rho, Rac and Cdc42 proteins. The acceptor amino acid of glucose (Thr-37 in Rho and Thr-35 in Rac / Cdc42) is located in the effector domain of the Rho GTPases, and glucosylation renders the GTPases inactive (Just et a/., 1995a).The Rho subfamily GTPases are involved in the regulation of the submembranous and cytoplasmatic actin cytoskeleton (Hall, 1994). Whereas Rho controls the formation of focal adhesions and stress fibers (Ridley and Hall, 1992), Rac participates in membrane ruffling (Ridley et a/., 1992) and Cdc42 in formation of filopodia (Nobes and Hall, 1995; Kozma et a/., 1995). Recently, the Rho subfamily GTPases have been identified as being involved in the activation of transcription factors via the Ras-regulated pathway (Minden et a/., 1995) and in a Ras independent signal cascade (Hill et a/., 1995; Coso et al., 1995; Olson et a/., 1995). Clostridium difficile toxin B (cytotoxin) is coexpressed with toxin A (enterotoxin). Both toxins are single-chained exotoxins which have molecular weights of 270 and 308 kDa, respectively (Barroso et a/., 1990; Dove et a/., 1990; Sauerborn and Eichel-Streiber, 1990). Both toxins show 45% identity (63% homology) at the amino acid level (Eichel-Streiber et a/., 1992; Eichel-Streiber, 1993), and they share common structural features: The C-terminal part contains repetitive peptide structures, which are most likely involved in cell receptor binding, followed by a small hydrophobic region in the center of the toxin molecule which putatively participates in the transport of the toxins into the cytoplasm of the target cell (Eichel-Streiber and Sauerborn, 1990; Hofmann et al., 1995; Dove et al., 1990).The N-terminal part is probably responsible for the biological activity of the cytotoxins (Eichel-Streiber, 1993; Barroso et al., 1994). Both toxins are mono-

mode of action of toxin B

structure of toxins A and B

K. Aktories (Ed.),Bacterial Toxins. @ Chapman & Hall, Weinheim, 1997. ISBN 3-8261-0080-8

glucosyltransferases which share the identical target proteins, the Rho GTPases (Just etal., 1995a; Just etal., 199513; Aktories and Just, 1995).

13.2 Purification of Toxin A and Toxin B Purification of Clostridium difficile toxins A and B is performed using a modified procedure according to Eichel-Streiber et al., 1987.

1. A dialysis bag containing 900 ml of 0.9 % NaCl in a total volume of 4 I brain heart infusion (BHI, Difco) is inoculated with 100 ml of an overnight BHI culture of C. difficile strain VPI 10463. 2. After incubation under microaerophilic conditions at 37°C for 72 h the culture is centrifuged for 20 min at 8000g in a Sorvall GSA rotor to obtain the culture supernatant.

3. Ammonium sulfate is slowly added to the culture supernatant to reach a final concentration of 70 %. 4. After stirring for 3 h at 4"C, the suspension is centrifuged at 8000 g for 30 min at 4°C. 5. The precipitate is dissolved in 15 mI of 50 mM Tris-HCI (pH Z5) followed by overnight dialysis against 2 x 1 I of 50 mM Tris-HCI at 4°C. 6. The dialyzed sample is cleared by centrifugation (8000g for 30min at 4°C) and is then directly applied onto a MonoQ column (HR 10/10; Pharmacia, Freiburg, Germany), previously equilibrated with 50 mM Tris-HCI (pH 7.5).

7. The bound proteins are eluted by using a linear gradient from 50 mM to 700 mM NaCI. A typical chromatogram is shown in Fig 1.

8. The fractions (1.5 ml each) are analyzed by 7 % SDS-PAGE followed by Coomassie blue staining to test the purity of the toxins.

assaying cytotoxic activity

The cytotoxic activity of the purified toxins is tested by applying toxin dilutions ranging from to to Chinese hamster ovary (CHO) cells cultured in 96 well plates, and monitoring the morphological changes of the cells. The morphological changes are characterized by rounding of the cells accompanied by formation of arborescent extensions. These features occur within 3 h at concentrations of approximately 1 ng/ml for toxin B and 1 pg/ml for toxin A. Storage and stability: Toxin B is dissolved in 550mM NaCl plus 50 mM Tris-HCI (pH 7.5), and toxin A in 250 mM NaCl plus 50 mM TrisHCI (pH 7.5). After addition of glycerol (20%, v/v) the toxins are

storage

I. JUST eta/.

stored at -80°C. Repeated thawing and freezing should be avoided because of increased denaturation / inactivation of the toxins. At 4°C the toxins are stable for 2-3 weeks.

ToxA

/TOXB

. l

0

'

l

20

'

l

40

'

t

'

60

l

80

'

l

'

100

l

'

120

l

'

1

140

ml Fig. 1. Purification of Clostridium difficile toxin A and toxin B by FPLC chromatography. 20ml of the resuspended and dialyzed ammonium sulfate precipitate of the culture supernatant was loaded onto a MonoQ HR 10/10 column (Pharmacia, Freiburg, Germany). Elution was performed with a linear NaCl gradient (0.05 to l.0M) at a flow rate of 1 ml/min. Extinction was measured at 280nm in arbitrary units with an amplification factor of 0.5. The toxins eluted at either 25 % buffer B (1 M NaCl / 0.05 M Tris-HCI; pH Z5) for toxin A (ToxA) or at 60 % buffer B for toxin B (ToxB)

13.3 Glucosyltransferase Activity of Toxin B Both toxin A and toxin B catalyze 0-glucosylation of the Rho subfamily proteins at a threonine residue. UDP-glucose serves as cosubstrate, and is selectively used by the .toxins, whereas other nucleotide sugars are not cosubstrates. Toxin-catalyzed monoglucosylation is almost complete in the GDP-bound form. Bound GTP causes a decrease in incorporation of glucose, and the bound nonhydrolyzable analogue GTP[S] completely blocks glucosylation. Besides transferase activity, both toxins exhibit, in the absence of the protein substrates, UDP-glucose glycohydrolase activity that cleaves Clostridiurn difficile Toxin B 0 s o Probe for Rho GTPoses

the cosubstrate into UDP and glucose. Glycohydrolase activity is much lower than transferase activity. In cell-free systems (recombinant GTPases or cell lysates), both toxins exhibit their enzymic effects only at a concentration 10-100 times higher than that applied to intact cells. This difference in concentration may be due to failure in toxin activation, which most likely occurs when the toxins enter the cell by receptor-mediated endocytosis. The same is true when the toxins are microiniected, thereby bypassing the entry (and activation) mechanism.

13.3.1 Glucosylation in Cell Lysates cell lysis

Before cell lysis, the cell monolayer is rinsed with ice cold phosphatebuffered saline (PBS) and the cells are then mechanically removed in the presence of lysis buffer (2 m M MgCI2, 0.1 m M phenylmethylsulfonyl fluoride, 20 pg/ml leupeptin, 80 pg/ml benzamidine in 50 mM HEPES, pH 7.4) followed by sonication five times on ice. After centrifugation for 10 min at 2000 g to remove the nuclei and cell debris, the supernatant is used for the glucosylation reaction.

1. 40pl of cell lysate plus 10pM of UDP-['4C]glucose (300mCi/ mmol) and 20 y M unlabeled UDP-glucose, is incubated with 1 pg/ml of toxin B (total volume 50 pl) for 45 min at 37°C. If UDPglucosylation reaction

['4C]glucose is purchased in ethanolic solution, a sample is freeze dried and dissolved in 50 m M HEPES, pH 7.4 to obtain a concentration of 100 pM.

2. The glucosylation reaction is terminated by addition of 1OpI Laemmli sample buffer followed by incubation for 5 min at 95°C. Alternatively, the reaction is stopped by addition of 1 ml of trichloroacetic acid (20 %, w/v). 3. The proteins are separated by 12.5 % SDS-gel electrophoresis and the ['4C]labeled proteins are analyzed using a phosphorimager system (Molecular Dynamics) (Fig.2). The exposure time for the gels varies from 6 to 24 h, depending on the concentration of the Rho subfamily proteins and the concentration of unlabeled UDP-glucose. For autoradiography, the exposure time is 5 to 14 days.

glucosylation with toxin A

I. JUSTetal.

Toxin A glucosylates the identical substrate proteins (Rho, Rac, Cdc42) that are glucosylated by toxin B. Thus, the glucosylation reaction can also be performed with toxin A. However, the toxin A concentration has to be 10 pg/ml (instead of 1 yglml for toxin B).

Fig. 2. Glucosylation of cell lysates with Clostridium difficile toxin B. For [32P]ADPribosylation, lysates from NIH3T3 cells (1.5 mg/ml of protein) were incubated in the presence of 10pM [32P]NADplus 10mM thymidine with 1 pg/ml C3 for 30min at 37°C as described in section 13.4.2.2. For [ I C]glucosylation, lysates from NIH3T3 cells (1.5mg/ml of protein) were incubated in the presence of 30pM UDP['4C]glucosewith 1 pg/ml toxin B for 45 min at 37°C as described in Section 13.3.1. The proteins were separated on 12.5 % SDS-PAGE and the labeled proteins were analyzed using a phosphorimager system (shown).ADP-ribosylation, but not glucosylation, alters the migration behaviour of Rho

13.3.2 Glucosylation of Recombinant Rho Proteins or Membranous Fractions Recombinant GTPases (50 yg/ml) or membranous fractions (1 mg/ ml) are glucosylated in a buffer containing 2 m M MgCI2, lOOmM KCI, 0.1 m M phenylmethylsulfonyl fluoride, 0.1 m M GDP, 5 0 m M HEPES (pH Z4),30 y M UDP-['4C]glucoseand 1 yg/ml toxin B (total volume 50 yl) for 45 min at 37°C. The concentration of UDP-glucose should exceed the concentration of the GTPases at least five-fold. The glucosylation reaction is terminated by addition of 10 yl Laemmli sample buffer, incubated for 5min at 95"C, followed by separation of the proteins by electrophoresis on a 12.5 % SDS-gel. Labeled proteins are analyzed using a phosphorimager system.

13.3.3 Glucosylation of Recombinant GTPases for Microinjection Recombinant Rho proteins are glucosylated in the presence of unlabeled UDP-glucose. The reaction is terminated by passing the reaction mixture through a membrane with an exclusion molecular weight of 100kDa to remove toxin B (270kDa). As a control, the GTPase is incubated with toxin B but in absence of the cosubstrate UDP-glucose.

Clostridium difficile Toxin B as a Probe for Rho GTPases

@

Recombinant Rho proteins (400 pg/ml) are glucosylated in a buffer containing 2 m M MgCI2, 0.1 mM GDP, 50mM HEPES ( ~ H 7 . 4 ) ~ 0.1 mM UDP-glucose and 5 yglml toxin B (total volume 100 pl) for 45min at 37°C. Thereafter, the reaction mixture is centrifuged at 5000g in a Microcon 100 (Amicon). The filtrate (<100 kDa) contains the GTPase but not toxin B. The sample is now ready for microinjection or can be concentrated further prior to microinjection using Microcon 10 (Amicon). The effects of microinjected glucosylated Rho GTPases are monitored by detection of morphological changes (see section 13.4) or by visualization of the microfilament system with rhodamine- or FITCphalloidin (for staining see Chapter 10).

13.4 Toxin B Action on Intact Cells Toxin B is an intracellularly acting cytotoxin and enters the cell via a receptor-mediated endocytosis pathway to reach the endosomes, from which the toxin is translocated to the cytoplasm (Florin and Thelestam, 1986; Henriques et al., 1987).Because of this specific mode of entry, the toxin concentration needed for intoxication of cells is low (100 ng/ml for about 4 h). In contrast, Clostridium botulinum exoenzyme C3 (23.5 kDa), which ADP-ribosylates the Rho subtype proteins RhoA, B and C only, enters the cells by a non-specific uptake process, possibly by pinocytosis. Therefore, C3 has to be applied in high concentrations (about 30 pg/ml) for 24 h or longer.

13.4.1 Treatment of Intact Cells with Toxin B For intoxication of cell monolayers, toxin B stock solution (200-500pg/ml) is diluted 1 : 100 with 0.1 mg/ml bovine serum albumin in 50 mM HEPES, pH 7.5. This working solution is used for intoxication of cells. Final concentration of toxin B in the medium is 10- 100 ng/ ml, which induces morphological changes during a period between 2 and 10 h. By phase-contrast microscopy, the cytotoxic features are characterized by cell shrinkage and formation of neurite-like extensions. Eventually, the cells become round and detached. Staining of the fixed cells with rhodamine- or FITC-phalloidin shows disintegration of the stress fibers. During the process of intoxication, condensed plaques of actin localized in the perinuclear space are the predominant stainable form of actin (for actin staining see Chapter 10).

13.4.2 Assessment of the Extent of Glucosylation The extent of glucosylation of the Rho GTPases can be assessed by using either of two approaches: I. JUST et al.

73.4.2.7 Differential Glucosylation by Toxin 6 If toxin B has catalyzed glucosylation of the Rho GTPases in intact cells, subsequent ['4C]glucosylation of the lysates should result in a decreased incorporation of ['4C]glucose into the GTPases. For differential glucosylation, lysates from toxin B-treated cells and nontreated cells are subjected to toxin B-catalyzed ['4C]glucosylationas described in Section 13.31. Incorporation of ['4C]glucose into the samples is measured and compared.

73.4.2.2 ADP-ribosylation of Rho by Clostridium botulinum Exoenzyme C3 Glucosylation of Rho at Thr-37 prevents subsequent C3-catalyzed ADP-ribosylation of Rho at Asn-41 (Just et al., 1994; Just et al., 1995a). Therefore, C3-catalyzed ADP-ribosylation can be used to quantify the amount of glucosylation of Rho by comparing ADP-ribosylation in lysates from control cells and toxin B-treated cells (Fig.2). However, only Rho but not Rac and Cdc42 are ADP-ribosylated.

1. 40 pI of cell lysate or membranous fraction (for preparation see Section 15.3.1) plus 10 pM of [32P]NAD+(0.3 pCi) plus 10 mM thymidine is incubated with 1 pg/ml of C3 (total volume 50p.1) for 30 min at 37°C. Thymidine blocks the poly(ADP-ribose)-polymerase (approx. 120 kDa), thereby preventing consumption of NAD' which is essential for quantitative ADP-ribosylation of Rho. 2. The ADP-ribosylation reaction is terminated by addition of 10 pI Laemmli sample buffer or of 1 ml of trichlororacetic acid (20 %, WIV)

.

3. The proteins are separated by 12.5 % SDS-gel electrophoresis, and the labeled proteins are analyzed using a phosphorimager system. The exposure time of the gels varies from 1 h up to 4 h. For autoradiography, the exposure time is 12 to 24 h.

A small proportion of the Rho subtype proteins is located at the membranes whereas most (about 90 %) is in the cytosol (Adamson et al., 1992). Functionally active Rho, however, appears to be bound to the membranous fraction. In toxin-treated cells, ADP-ribosylation of membranous Rho and glucosylation of membranous Rho / Rac / Cdc42 decrease prior to the appearance of morphological effects, indicating that inactivation of less than 10 % of cellular Rho is sufficient to cause maximal morphological effects. ADP-ribosylation and glucosylation of total cellular Rho (cell lysates) is delayed compared with the appearance of morphological effects (Fig.3). Depending on the issue addressed, the data of ADP-ribosylation / glucosylation of cellular subfractions may be more significant. Clostridiurndifficile Toxin B as a Probe for Rho GTPases

- 100

100

-- 80 -- 60 -- 40 -- 20

A cell lysate membrane fraction I

- 0 I

0

I

I

I

I

I

I

I

I

I

15 30 45 60 75 90 105120 time [min]

Fig. 3. Correlation between cell rounding and toxin B-catalyzed glucosylation. NIH3T3 cells were treated with toxin B (100 ng/ml) for the times indicated. The number of cells completely rounded was determined from photographs taken at the times indicated ( 0 ) .The cells were lysed and the lysates (A) and the membranous fractions (m) (prepared by ultracentrifugation), were ['4C]glucosylatedwith toxin B as described in Section 13.3.1

13.5 Toxin B a Tool in Cell Biology

comparison with c2 toxin I. JUST etal.

Use of the ADP-ribosyltransferase C3 from Clostridium botulinum resulted in the identification of the involvement of the Rho proteins in the regulation of the microfilament system. The advantage of C3 (selective modification of RhoA, B and C) is offset by the disadvantage of poor cell accessibility. In contrast, toxin B can enter the cells by a specific mechanism. Therefore, the concentration needed is quite low and the incubation times are in a moderate range (2-6 h). However, toxin B glucosylates not only Rho subtype proteins, but also all members of the Rho subfamily (Rho, Rac and Cdc42). Although these GTPases are involved in the control of the actin cytoskeleton, each of them exhibits a specialized function in the regulation of the complex microfilament system. Furthermore, these Rho GTPases exhibit functions which are apparently not related to the cytoskeleton; for example, they participate in the regulation of transcription factors (Minden etal., 1995; Hill et a/., 1995; Coso etal., 1995; Olson et a/., 1995),and control exocytosis of transmitters in rat basophilic leukemia cells (Prepens et a/., 1996). It is therefore necessary to distinguish between cellular effects caused merely by depolymerization of the actin filaments and effects caused by inactivation of the Rho GTPases which are independent of the actin system. TO this end, it is very helpful to compare effects of toxin B with those of Clostridium botulinum C2 toxin. C2 toxin directly acts

on the cellular G-actin by ADP-ribosylating actin at Arg-177. This modification blocks the ability of actin to polymerize, and makes it a capping protein. Both mechanisms eventually result in almost complete depolymerization of the cellular actin cytoskeleton (for review see references Aktories and Just, 1990; Aktories et al., 1992; Aktories and Wegner, 1992; and see Chapters 8 and 11). By using C2 toxin, the direct effects of the actin cytoskeleton on the functions studied can be monitored.

13.6 Reagents and Chemicals Materials

Supplier

Cat-No.

U DP-[''C]glucose

NEC-403 DuPont DuPont Str.1, D-61343 Bad Homburg, Germany ARC-154 Bio Trend Eupener Str. 159, D-50876 Koln, Germany GDP Boehringer Mannheim GmbH 106208 Sandhofer Str. 116, D-68298 Mannheim, Germany 106372 GTP Boehringer 220647 GTP[S] Boehringer H-3375 HEPES Sigma-Aldrich Chemie GmbH Postfach, D-82039 Deisenhofen, Germany 8-6506 benzamidine Sigma L-2884 Sigma leupetin P-7626 phenylmethylsulfonyl fluoride Sigma R415 rhodamine-phalloidine Molecular Probes Inc. 4849 Pitchford Av., Eugene, OR, USA 0037-07-0 Difco Laboratories brain heart infusiuon (BHI) PO. Box 331058, Detroit, MI, 48232-7058 USA 17-0556-01 Mono Q column (HR 10/10) Pharmacia Biotech AB S-75182 Uppsala, Sweden

References Adamson P, Paterson HF, Hall A (1992): lntracellular localization of the P21'h0 proteins. In J. Cell Biol. 119: 617-27 Aktories K, Wille M, Just I (1992): Clostridial actin-ADP-ribosylating toxins. In Curr. Top. Microbiol. Immunol. 175: 97- 113 Aktories K, Just I (1990): Botulinum C2 Toxin. In ADP-ribosylating toxins and Gproteins, (Moss J, Vaughan M. 79-95 Washington, D.C. American Society for Microbiology. Aktories K, Just I (1995): Monoglucosylation of low-molecular-mass GTP-binding Rho proteins by clostridial cytotoxins. In Trends in Cell Biology 5: 441 -3 Aktories K, Wegner A (1992): Mechanisms of the cytopathic action of actin-ADPribosylating toxins. In Mol. Microbiol. 6: 2905-8 Barroso LA, Wang S-Z, Phelps CJ, et al. (1990): Nucleotide sequence of Clostridium difficile toxin B gene. In Nucl. Acids Res. 18: 4004 Barroso LA, Moncrief JS, Lyerly DM, et al. (1994): Mutagenesis of the Clostridium difficile toxin B gene and effect on cytotoxic activity. In Microb. Pathog. 16: 297-303 Clostridiurn difficile Toxin B as o Probe for Rho GTPases

Bongaerts GPA, Lyerly DM (1994): Role of toxins A and B in the pathogenesis of Clostridium difficile disease. In Microb. Pathog. 17: 1-12 Coso OA, Chiariello M, Yu J-C et a/. (1995):The small GTP-binding proteins Racl and Cdc42 regulate the activity of the JNK/SAPK signaling pathway. In Cell 81: 1137-46 Dove CH, Wang SZ, Price SB etal. (1990):Molecular characterization of the Clostridium difficile toxin A gene. In Infect. Immun. 58: 480-8 Eichel-Streiber C, Harperath U, Bosse D et a/. (1987):Purification of two high molecular weight toxins of Clostridium difficile which are antigenetically related. In Microb. Pathogen. 2: 307- 18 Eichel-Streiber C, Laufenberg-FeldmannR, Sartingen S et al. (1992):Comparative sequence analysis of the Clostridium difficile toxins A and B. In Mol. Gen. Genet. 233: 260-8 Eichel-Streiber C (1993): Molecular Biology of the Clostridium difficile Toxins. In Genetics and Molecular Biology of Anaerobic Bacteria, (Sebald M. 264-89 New York: Springer-Verlag. Eichel-Streiber C, Sauerborn M (1990): Clostridium difficile toxin A carries a Cterminal structure homologous to the carbohydrate binding region of streptococcal glycosyltransferase. In Gene 96: 107- 13 Florin I, Thelestam M (1986): Lysosomal involvement in cellular intoxication with Clostridium difficile toxin B. In Microb. Pathogen. 1: 373-85 Hall A (1994):Small GTP-binding proteins and the regulation of the actin cytoskeleton. In Annu. Rev. Cell 6iol. 10: 31 -54 Henriques B, Florin I, Thelestam M (1987):Cellular internalisationof Clostridium difficile toxin A. In Microb. Pathogen. 2: 455-63 Hill CS, Wynne J, Treisman R (1995):The Rho family GTPases RhoA, Racl, and CDC42Hs regulate transcriptional activation by SRF. In Cell 81: 1159-70 Hofmann F, Herrmann A, Habermann E et a/. (1995): Sequencing and analysis of the gene encoding the a-toxin of Clostridium novyi proves its homology to toxins A and B of Clostridium difficile. In Mol. Gen. Genet. 247: 670-9 Just I, Fritz G, Aktories K et al. (1994):Clostridium difficile toxin B acts on the GTPbinding protein Rho. In J. 6iol. Chem. 269: 10706-12 Just I, Selzer J, Wilm M etal. (1995~): Glucosylation of Rho proteins by Clostridium difficile toxin B. In Nature 375: 500-3 Just I, Wilm M, Selzer J et a/. (l995b): The enterotoxin from Clostridium difficile (ToxA) monoglucosylates the Rho proteins. In J. 6iol. Chem. 270: 13932-6 Kozma R, Ahrned S, Best A et al. (1995):The Ras-relatedprotein Cdc42Hs and bradykinin promote formation of peripheral actin microspikes and filopodia in Swiss 3T3 fibroblasts. In Mol. Cell. 6iol. 15: 1942-52 Minden A, Lin A, Claret F-X et a/. (1995):Selective activation of the JNK signaling cascade and c-Jun transcriptional activity by the small GTPases Rac and Cdc42Hs. In Cell 81 : 1147-57 Nobes CD, Hall A (1995):Rho, Rac, and Cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. In Cell 81 : 53-62 Olson MF, Ashworth A, Hall A (1995):An essential role for Rho, Rac, and Cdc42 GTPases in cell cycle progression through G,. In Science 269: 1270-2 Prepens U, Just I, Von Eichel-Streiber C et a/. (1996): Inhibition of FcIRI-mediated activation of rat basophilic leukemia cells by Clostridium difficile toxin B (monoglucosyltransferase). In J. 6i0l. Chem. 271 : 7324-9 Ridley AJ, Paterson HF, Johnston CL et al. (1992):The small GTP-binding protein rac regulates growth factor-induced membrane ruffling. In Cell 70: 401 -10 Ridley AJ, Hall A (1992):The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. In Cell 70: 389-99 Sauerborn M, Eichel-Streiber C (1990):Nucleotide sequence of Clostridium difficile toxin A. In NucleicAcids Res. 18: 1629-30 Wolfhagen MJHM, Torensen R, Fluit AC et al. (1994):Toxins A and B of Clostridium difficile. In FEMS Microbiol. Reviews 13: 59-64

I. JUST et a/.

Bacterial Toxins

Klaus Aktories Copyright 0 2002 WILEY-VCH Verlag GmbH & Co. KGaA

CHAPTER 14

CIost ridia I Neurotoxins G. SCHIAVO and C. MONTECUCCO

14.1 The Origin of Clostridial Neurotoxins Tetanus is a syndrome that is often lethal, characterized by a spastic paralysis. Death follows bodily exhaustion and occurs by respiratory failure or circulatory collapse. For twenty four centuries tetanus had been considered a neurologic disease until the identification of Clostridium tetani, the bacterium that causes tetanus by the release of a protein toxin, named tetanus neurotoxin (TeTx)(Faber, 1890; Kitasato, 1891; Tizzoni and Cattani, 1890a,b). Botulism, on the other hand, is characterized by a generalized muscular weakness. In its severe form, a generalized flaccid paralysis becomes evident in the victim and death results from respiratory failure (Hatheway, 1995).The causative agents of botulism are the neurotoxigenic strains of Clostridium botulinum (van Ermengem, 1897), C. barati and C. butyricum (Hall et al., 1985; Aureli et al., 1986). So far, seven different serotypes of botulinum neurotoxin (BoNT), called A to G, have been identified. TeTx and BoNTs are the most potent toxins known. In fact, the 50 % lethal dose (LDS0)in mice, human and horses varies between 0.1 ng and 1 ng of toxin per kg of body weight. lnterestingly, different animal species show a great range of sensitivity to TeTx and to BoNTs. While mice are exquisitely sensitive to TeTx, rats and birds are quite resistant, and turtles are completely insensitive to TeTx effects (Payling-Wright, 1955). The extreme toxicity of clostridial neurotoxins (CNTs) derives from their absolute neurospecificity as well as from catalytic activity. TeTx and BoNTs bind specifically to the neuromuscular junction (NMJ)of motor neurons. The identity of the receptor(s) on the presynaptic membrane is unknown, but their extreme toxicity suggests that the binding affinity to the cognate receptor must be very high. The receptor-bound toxin is internalized at the presynaptic membrane of the NMJ and gains access to the neuronal cytosol. Here it blocks the release of acetylcholine (ACh), causing a flaccid paralysis (Simpson, 1989).TeTx also binds to the presynaptic membrane of the motor neuron, but its action is limited to the level of the central nervous system. TeTx undergoes retrograde transport inside the motor neuron to the spinal cord (Bruschettini, 1892; Vallee and Bloom, 1991), where it migrates trans-synaptically into inhibitory interneurons (Simpson, 1989).

diseases caused by clostridial neurotoxins

K. Aktories (Ed.),Bacterial Toxins. 0 Chapman & Hall, Weinheim, 1997. ISBN 3-8261-0080-8

The blockade of inhibitory synapses at the spinal cord impairs the neuronal circuitry that controls voluntary muscle contraction, thereby causing the spastic paralysis characteristic of tetanus (Simpson,

1989). The opposite clinical symptoms of tetanus and botulism result from the sites of action of TeTx and BoNTs within the nervous system, rather than from a different pathophysiological mechanism. TeTx acts on the central nervous system, while BoNTs exert their action on the peripheral nervous system. The only known activity of TeTx and BoNTs is a persistent inhibition of neurotransmitter release, including glycine, GABA, ACh, glutamate and norepinephrine. The release of opioids, oxytocin and vasopressin is also inhibited by CNTs (Wellhoner, 1992). The action of clostridial neurotoxins can be extended to different exocytotic events in a wide range of non-neuronal cells if the neurospecific binding and uptake processes are circumvented (Penner et al., 1986; Bittner and Holz, 1988; Ahnert-Hilger et al., 1989 a,b; Bittner et al., 1989 a,b; Stecher et al., 1989; Mclnnes and Dolly, 1990; Dayanithi et al., 1992, 1994; lkonen et al., 1995; Regazzi et al., 1995; Sadoul et al., 1995).

14.2 Structure of Clostridial Neurotoxins

mechanism of action of neurotoxins

structure of clostridial neurotoxins

Clostridial neurotoxins share a common structural organization. They are produced as inactive polypeptide chains of 150 kDa. Upon bacterial lysis, the CNTs are released and cleaved by endogenous or exogenous proteases at an exposed loop. An active di-chain neurotoxin is thus generated (Fig. 1) (DasGupta, 1994). The heavy chain (H, 100 kDa) and the light chain (L, 50 kDa) are bridged by a single interchain disulfide bridge. This interchain bond is essential for neurotoxicity of CNTs when the toxins are applied to the extracellular space (Schiavo et al., 1990; De Paiva et al., 1993). Biochemical studies, as well as low resolution electron microscopic analysis, led to the hypothesis that TeTx and BoNTs are folded into three distinct 5OkDa domains, each with a different set of physiological functions (Fig. 1). The mechanism of neurotoxin cell intoxication consists of four distinct steps: (a) binding to the neuronal membrane, (b) internalization into an acidic compartment, (c) membrane translocation and (d) enzymatic target modification (Montecucco etal., 1994).The L chain is responsible for the intracellular catalytic activity of CNTs. The aminoterminal 50 kDa domain of the H chain (HN)is implicated in membrane translocation, while the carboxy-terminal part ( Hc) is mainly responsible for the neurospecific binding. The amino acid sequence of all eight CNTs has been derived from their corresponding genes (Minton, 1995).The L chains and H chains are composed on average of 439 and 843 residues, respectively. Both chains contain homologous domains separated by regions of very little similarity (Fig. 2). The most conserved portions of the L chains are the amino-terminal and central regions (residues216-244,

G. SCHIAVO and C. MONTECUCCO

Fig. 1. The mechanism of activation of tetanus and botulinum neurotoxins. The toxins are produced as an inactive single polypeptide chain of 150 kDa, composed of three 50 kDa domains, connected by protease-sensitive loops. The toxins are activated by selective proteolytic cleavage which generates two disulfide-linked chains: L (50 kDa) and H (100kDa). The three domains play different functional roles in cell penetration: Hc is responsible for cell binding and HNfor cell penetration. Reduction takes place inside the nerve cells and liberates the metalloprotease activity of the L chain in the cytosol

Fig. 2. Structure and active site of clostridial neurotoxins. The upper panel shows the structure of CNTs, and the segments that show significant homology between the different serotypes are in black (Minton, 1995).The highest homology is shown by a short segment corresponding to the amino acid residues 216-244 in TeTx. This segment contains the zinc-binding motif of metallo-proteinases (zincins) and it is dissimilar to the consensus sequence of the metzincin metallo-proteinase family (Jiang and Bond, 1992)

Clostridial Neurotoxins

numbering of TeTx). The latter region contains the His-Glu-Xaa-XaaHis binding motif of zinc-endopeptidases (Fig. 2 ) (Jongeneel et a/., 1989; Jiang and Bond, 1992).This observation led to the demonstration that CNTs are zinc-containing proteins (Schiavo et al., 1992 b, c, 1993 c, 1994, 1995 a; Wright et al., 1992; Yamasaki et al., 1994 b).One atom of zinc is bound to the L chain of TeTx, BoNT/A, B, and F. These neurotoxins show a single zinc binding site with a dissociation constant (Kd) of 50-100 nM. In addition, multiple divalent cation binding sites with a lower affinity are also present (Schiavo et al., 1992 c; Wright et al., 1992). BoNT/C binds two atoms of zinc (Schiavo et al., 1995 a ) , similar to the neutrophil collagenase, whose threedimensional structure has been recently determined (Lovejoy et a/., 1994). One atom of zinc is present at the active site of this metalloproteinase and is exchangeable, while the second one is bound very strongly and is thought to play a structural role. Zinc can be removed from the CNTs with heavy metal chelators, thus generating an inactive apo-neurotoxin. The active holo-toxin can be reformed upon incubation of the apo-neurotoxin in zinc-containing buffers (Schiavo et a/., 1992 b, c, 1993 c, 1994, 1995 a; Simpson et al., 1993; Hohne-Zell et al.,

1994). The zinc atom of zinc-endopeptidases is coordinated by either two or three histidine residues (Jiang and Bond, 1992). In thermolysin-like enzymes, the zinc atom is coordinated by two histidines within the consensus sequence of zinc-endopeptidases, by a water molecule bound to the glutamic acid of the same motif and by another (distal) glutamate residue. Astacin, a crayfish metallo-protease, pentacoordinates zinc via three histidine residues, one tyrosine residue and one water molecule. Adamalysin, the alkaline protease of Pseudomonas aeruginosa, and collagenase adopt a tetrahedral zinc coordination via three histidines and a glutamate-bound water molecule. The active site of zinc-endopeptidases resides in a cleft with the zinc atom in its center and the residues of the zinc-binding motif forming an ahelix. The orientation and volume of the amino-acid side-chains at the active site determine the peptide bond specificity of the toxin cleavage. The water molecule bound to the glutamate residue of the motif is involved in peptide bond hydrolysis by a mechanism that has been studied in detail only for thermolysin (Matthews, 1988). In order to determine the number of histidine residues involved in zinc coordination, the L chains of TeTx and BoNT/A, B and E were modified with diethyl pyrocarbonate (DEPC), a reagent that specifically modifies histidine residues. In each case, two additional histidines were modified in the apo-toxin that were not affected in the holo-neurotoxin (Schiavo et al., 1992 b, c). These results indicate that the zinc atom of CNTs is coordinated via two histidines and a Glubound water molecule, as in thermolysin. Mutations at the two histidines of the motif inactivate TeTx and suppress its ability to bind radiolabeled 65Zn2+(Yamasaki et a/., 1994 b). In addition, mutations of the conserved Glu-271 and Glu-272 of TeTx, predicted to be in an ahelical segment (Lebeda and Olson, 1994), result in decreased zinc binding and loss of activity. Based on these experimental results, it has

G.SCHIAVO and C.MONTECUCCO

been suggested that CNTs are thermolysin-like proteases and that one of these two Glu residues represents the fourth zinc ligand (Yamasakietal., 1994 b). In contrast, a comparison of the extended Xray absorption fine structure (EXAFS) spectra of TeTx, astacin, alkaline protease and thermolysin shows a close similarity of TeTx with astacin and alkaline protease (Morante et al., 1996).This result indicates that at least one additional aromatic residue is present around the zinc atom of TeTx, in addition to the two histidines required for cation coordination. Sequence comparison indicates that a Tyr residue (Tyr-243in TeTx), conserved among all CNTs, is located in the same position as the third histidine in astacin and astacin-like proteinases. Moreover Tyr-243 replacement in TeTx leads to a great loss of toxicity (Yamasaki etal., 1994 b ) .Taken together, these results suggest a novel manner of zinc coordination among metallo-proteinases. In addition, studies on the denaturant sensitivity of the holo- and apo-L chain indicate that the zinc atom does not contribute significantly to the structural stability of the CNTs (De Filippis et al., 1995). The H chains are less conserved than the L chains and the carboxyl-terminal part of the H chain (Hc)is the most variable region of the toxin (Fig. 2) (Minton, 1995). This is consistent with the notions that the Hc domain is involved in binding to the nerve terminals and that different neurotoxins bind to different cognate receptors. O n this basis it may be suggested that the receptor binding regions of TeTx and BoNTs are mainly located within the 180 carboxy-terminal residues of the H chain. Nucleotide and amino acid sequence comparisons of the CNTs clearly indicate that they derive from a common ancestral gene. In this respect, it is significant that the CNT genes are located on mobile genetic elements (Minton, 1995).Bacteriophages, plasmids, and conjugation transposons may have spread these genes among bacteria of the Clostridiurn genus. Mutations in CNT genes are apparent from the discovery of variants of the seven BoNTs among the same serotypes, which have being detected with the methods of modern molecular genetics (Minton, 1995). Moreover, strains that harbour more that one BoNT gene have been identified (Hatheway, 1995). The scant knowledge about the ecology of toxigenic strains of Clostridia allows us only to speculate on the role of CNTs in Clostridia life cycles. A successful bacterium is capable of multiplying and spreading rapidly. In general, well-established infectious agents cause the smallest alteration in host physiology compatible with their need to enter and multiply in the host and spread to other individuals. This state is defined as “balanced pathogenicity” and is of paramount importance for the ecology of the infectious agent (Mims, 1987). Living vertebrates offer only very small anaerobic habitats within their bodies where Clostridia can survive. The release of a neurotoxin that kills the animal host converts it into an anaerobic fermentor able to support the massive growth of Clostridia of endogenous as well as exogenous origin. In this simplified view, the production of neurotoxins is crucial to create a new habitat. Since the cadaver cannot support bacterial spread to other hosts, Clostridia sporulate and

natural history of clostridia

Clostridial Neurotoxins

the spores are dispersed. O n this basis toxigenic Clostridia do not appear to be “balanced pathogens”. However, it should be considered that killing the host is necessary to the life cycle of a strictly anaerobic organism in an oxygen-rich habitat, and that the production of spores is essential to their survival and spreading in the environment. The finding that CNTs are zinc-endopeptidases specific for different proteins of the neuroexocytotic apparatus suggests a possible evolutionary origin of these neurotoxins. Clostridia produce a variety of proteinases that act outside cells. At a certain stage of evolution a metallo-proteinase gene fused with another gene, giving rise to a protein able to act specifically at the level of the nervous system. Further genetic rearrangements may have led to a molecule able to cleave selected proteins of the exocytotic apparatus. Different sites of attack on the same supramolecular structure ensures that an animal species cannot become resistant to all CNTs by single point mutations of the target.

14.3 Mechanism of Action of Clostridial Neurotoxins 14.3.1 Cell Binding After diffusion into the body fluids from the site of production or adsorption, BoNTs and TeTx bind to the presynaptic membrane at the NMJ of a-motor neurons (Black and Dolly, 1986 a; Halpern and Neale, 1995).Despite many efforts, the chemical nature of the molecule(s) responsible for the high affinity neurospecific binding of the CNTs to the presynaptic terminal has not been identified. A large number of studies have been devoted to assessing the role of polysialogangliosides in neurotoxin binding (Montecucco, 1986; Wellhoner, 1992; Halpern and Neale, 1995).The outer leaflet of the presynaptic membranes contains a large proportion of polysialogangliosides, providing a large number of acceptor sites for clostridial neurotoxins that are known to adsorb on negatively charged membrane surfaces (Montecucco, 1986; Wellhoner, 1992; Halpern and Neale, 1995). Incubation of cultured chromaffin cells with polysialogangliosides increases their sensitivity to TeTx and BoNT/A. Preincubation of these neurotoxins with ganglioside mixtures prior to neuronal exposure reduces TeTx toxicity and intra-axonal toxin transport in neurons (Wellhoner, 1992).In addition, treatment of membranes with neuraminidase, which removes the negatively charged sialic acid residues, decreases toxin binding (Montecucco, 1986; Wellhoner, 1992; Halpern and Neale, 1995). Although these studies have clearly established that polysialogangliosides interact with the various CNTs, no clear demonstration of their direct involvement in the neurospecific binding of these toxins in vivo has been provided. As discussed in detail elsewhere (Montecucco, 1986; Wellhoner, 1992; Halpern and G. SCHIAVO and C. MONTECUCCO

Neale, 1995), it is very unlikely that polysialogangliosides are the sole receptors of the CNTs at synaptic terminals. Parallel experiments indicate that cell surface proteins may be involved in toxin binding (Yavin and Nathan, 1986; Pierce et al., 1986; Parton et al., 1988; Schiavo et al., 1991). A “double receptor” model for clostridial neurotoxin binding to neuronal cells has emerged to account for the action of polysialogangliosides and the putative receptor protein (Montecucco, 1986).In this model, the neurotoxin first comes into contact with the negatively charged lipids of the presynaptic membrane via a high capacity interaction(s).This mode of binding would constitute a large trapping device for the minute amounts of neurotoxin known to cause disease. The toxin bound to the presynaptic membrane surface could then interact with a protein receptor that is responsible for neurospecificity and uptake at the NMJ. Specific interactions of each neurotoxin with its protein receptor is greatly favored by the bi-dimensionality of the cell surface membrane, which greatly reduces the reaction volume and increases the rate of neurotoxin binding. Nishiki and colleagues have recently identified a protein receptor of BoNT/B as synaptotagmin, a transmembrane protein of small synaptic vesicles (Nishiki et al., 1994).The protein receptor of the other clostridial neurotoxins is not yet known.

receptors for clostridial neurotoxins

14.3.2 Internalization and Membrane Translocation Since the L chain of TeTx and BoNTs is responsible for the cytosolic activity of the CNTs, at least this domain of the toxin molecule must reach the cytosol. Pharmacological and morphologic evidence indicates that the CNTs enter the cell by endocytosis (Black and Dolly, 1986 b) and that TeTx and BoNTs have to pass through a low pH step for neuron intoxication to occur (Williamson and Neale, 1992; Simpson et a/., 1994). Acidic pH does not activate the toxin directly via a structural change, since the direct introduction of the L chain in the neutral pH environment of the cytosol is sufficient to block exocytosis (Penner et al., 1986; Anhert-Hilger et al., 1989 b; Bittner et al., 1989 a, b; Mochida et al., 1989; Weller et al., 1991). Hence, low pH is necessary for the process of L chain membrane translocation from the vesicle lumen into the cytosol, by analogy with the other bacterial protein toxins with a three-domain structure (Montecucco et al., 1994). The interaction of CNTs with membrane bilayers has been studied mainly with model membrane systems, and limited data have been obtained in vivo. Available evidence indicates that at low pH TeTx and BoNTs undergo a conformational change from a water soluble “neutral” form to an “acidic” form, the latter characterized by the exposure of hydrophobic segments. This increase in hydrophobicity allows penetration of both the H and L chains into the hydrocarbon core of the lipid bilayer (Montecucco et al., 1994).Following this low pH-induced membrane insertion, TeTx and BoNTs form ion channels in planar lipid bilayers (Beise et al., 1994; Montecucco et al., 1994).These channels are cation-selective, have few tens of pS conductance and are per-

interaction of clostridial neurotoxins with membranes

Clostridial Neurotoxins

meable to molecules smaller than 700 daltons. The HN domain of CNTs includes several segments which may form amphipathic ahelices and thus may be candidate regions for channel formation after toxin oligomerization (Lebeda and Olson, 1994; Beise et al., 1994).Three-dimensional image reconstruction of the channel formed by BoNT/B in phospholipid bilayers (Schmid et al., 1993) is consistent with this model. A general consensus exists that these toxin channels are related to the translocation process of the enzymatic domain across the vesicle membrane into the neuronal cytosol. The most likely possibility is that the L chain translocates into the cytosol at the lipidprotein boundary, rather than inside a proteinaceous pore (Montecucco et al., 1994).This cleft model proposes that the two toxin subunits change conformation at low p H so that both expose hydrophobic surfaces and both contact the hydrophobic core of the lipid bilayer. In its "acidic" conformation, it is suggested that the H chain forms a transmembrane hydrophilic cleft that allows the passage of the partially unfolded acidic form of the L chain through the membrane. The neutral pH environment found in the cytosol promotes the refolding of the L chain to its water-soluble *neutralN conformation. While the L chain is leaving the vesicle membrane, the transmembrane hydrophilic cleft of the H chain tightens up to reduce the amount of hydrophilic protein surface exposed to the hydrophobic core of the lipid bilayer. However, this process leaves a channel in the membrane with two rigid protein walls and a small mobile lipid seal on one side. This is proposed to be the structure responsible for the ion-conducting properties of TeTx and BoNTs. In the cleft model, the ion channel is a consequence of membrane translocation, rather than a prerequisite for this process.

14.3.3 lntracellular Activity

proteolytic activity of clostridial neurotoxins

The most conserved segment of the L chain of CNTs is a central region that contains a His-Glu-Xaa-Xaa-His zinc-binding motif characteristic of zinc-endopeptidases, thus suggesting that TeTx and the BoNTs may inhibit neuroexocytosis through a zinc-endopeptidase activity. This hypothesis was confirmed with two experimental approaches in Aplysia neurons. First, the lack of toxicity of the apo-TeTx L chain demonstrated the essential role of the metal atom in toxin activity (Schiavo et al., 1992 a, b ) . Second, phosphoramidon, a very specific inhibitor of zinc-endopeptidases, was shown to inhibit TeTx-induced blockade of ACh release (Schiavo et al., 1992 b).These results were the first clear evidence that the L chain of TeTx was acting via a metallo-protease activity. Protease-free preparations of TeTx and BoNT/B, D, F and G cleave a membrane protein of small synaptic vesicles (SSV) called VAMP or synaptobrevin (Schiavo et al., 1992 a, 1993 a,c, Yamasaki et al., 1994 a, b). Conversely, BoNT/A, C and E act o n proteins associated with the presynaptic membrane: BoNT/A and E cut SNAP-25, while serotype C cleaves syntaxin, in addition to SNAP-25 (Blasi et al., 1993 a,b;

G. SCHIAVO and C.MONTECUCCO

BoNTD

BoNTlG

VAMP 11.8 kDa E R D Q a KLSE ratVAMP2

GASQ76 FETS rat VAMP 2

BoNTF

TeNT BoNTB

BONTIA

EANQ187RATK rat SNAP-25

4

SNAP-2 5 BoNTIC

25 kDa

SyntaxWHPC- 1 34 kDa BoNT1c

DTKK '@AVKY

rat syntamn IA

DTKPAVKY

rat syntaxin 18

Fig. 3. Specificity and sites of cleavage of the clostridial neurotoxins. VAMP is bound to the SSV membrane through a single transmembrane domain (black box), with the majority of the protein exposed to the cytoplasm. In addition, VAMP contains an amino-terminal domain rich in proline (hatched box). SNAP-25 and syntaxin are bound to the target membrane via palmitoylation (SNAP-25) or via a single transmembrane domain (syntaxin).TeTx and BoNT/B, D, F or G act on the conserved central portion of VAMP and release its amino-terminal part into the cytosol. The sequences indicate the peptide bonds cleaved by CNTs on rat VAMP-1 and VAMP-2, BoNT/A and E cleave SNAP-25 at the carboxyl terminus, with the release of nine and twenty-six residues peptides respectively. BoNT/C also cleaves SNAP-25 at the carboxy-terminus, and cleaves syntaxin at a single site near the cytosolic membrane surface. The action of TeTx and BoNT/B, C, D, F and G causes the release of a large portion of the cytosolic domain of VAMP and syntaxin. Conversely, only a small segment of SNAP-25 is released by the selective proteolysis of BoNT/A, C and E

Schiavo et al., 1993 a, b, 1995 b; Foran et al., 1996; Osen-Sand et al., 1996;Williamson etal., 1996).Fig. 3 shows schematically the cleavage sites of the various CNTs on VAMP, SNAP-25 and syntaxin. Recombinant VAMP, SNAP-25 and syntaxin are cleaved at the same peptide bonds as the corresponding cellular proteins. This indicates that no Clostridial Neurotoxins

Table 1. Tetanus and Botulinum Neurotoxins Target and Peptide Bond Specificities Toxin type

Target

peptide bond cleaved P3-P2-P, P,'-Pz'-P3'

TeTx BoNT/A BoNT/B BoNTIC BoNTIC BoNTID BoNT/E BoNTIF BoNTIG

VAMP

A-S-Q-F-E-T A- N-Q-R-A-T A- S- Q-F- E-T T- K- K-A-V- K N-Q-R-A-T- K~ D-Q-K-L-S-E I- D- R-I- M - E R- D-Q-K- L-S T-S-A-A-K-L

SNAP-25

VAMP syntaxin SNAP-25"

VAMP S NAP-25 VAMP VAMP

-

SNAP-25 is also cleaved in neurons exposed to BoNTIC (Foran et al., 1996; Osen-Sand et al., 1996; Williamson eta/., 1996). Niemann et al., personal communication.

a

additional endogenous factors are involved in neurotoxin proteolytic activity. The peptide bonds hydrolyzed by each neurotoxin have been identified (Table 1). With the exception of TeTx and BoNT/B, every other CNT catalyzes the hydrolysis of a different peptide bond. The amino acid residues flanking the cleavage sites differ in terms of charge, polarity and size. Hence, the active sites of these metalloproteinases must all differ in their spatial organization in order to accommodate and cleave different peptide bonds with a variety of disparate flanking sequences. The finding that VAMP, SNAP-25 and syntaxin are the only known substrates of these neurotoxins clearly demonstrates that these three proteins play a central role in neuroexocytosis.

74.3.3.7 VAMP/Synaptobrevin VAMP (vesicle-associated membrane protein) or synaptobrevin (Bennett and Scheller, 1994; Sudhof, 1995) is about 120 residues long and the exact number of residues depends on its source and isotype. As shown in Fig. 3, VAMP contains a short carboxyl-terminal segment located inside the vesicle lumen, while most of the molecule is exposed to the cytosol. The thirty residue long amino-terminus diverges considerably between species and isoforms, and has a high proline content. The highly conserved central part of VAMP (residues 30-96), rich in charged and hydrophilic residues, includes the known cleavage sites of the neurotoxins. O n the synaptic vesicle membrane, VAMP is associated with synaptophysin (Calakos and Scheller, 1994; Edelmann et al., 1995; Washbourne et a/., 1995), a maior membrane component of SSV. Synaptophysin is capable of forming oligomers endowed with channel properties, that may be directly involved in neurotransmitter release (Bennett and Scheller, 1994; Sudhof, 1995). Additionally, VAP-33, another integral membrane protein of small synaptic vesicles, interacts functionally with VAMP in Aplysia neurons (Skehel et al., 1995).Three isoforms of VAMP have so far been identified: VAMP-1, VAMP-2 and cellubrevin (Bennett and Scheller, 1994; G. SCHIAVO and C. MONTECUCCO

Sudhof, 1995).VAMP appears to be present in all vertebrate tissues, but the distribution of VAMP-1 and -2 varies among different cell types (Rossetto et al., 1996).The reason for this differential distribution of VAMP isotypes is unknown, but suggests that VAMP plays a role in distinct events of vesicle fusion and not only in neuroexocytosis. Chicken and rat VAMP-1 is not cleaved by CNTs (Schiavo et al., 1992 a, 1994; Patarnello et al., 1993).These VAMP isotypes contain a Val residue in place of the Gln residue at the cleavage site of TeTx and BoNT/B in human and mouse VAMP-1. It has been proposed that this single amino acid replacement at the cleavage site is associated with the resistance of rats and chickens to tetanus and type B botulism (Patarnello et al., 1993). The differential susceptibility to TeTx and BoNT/B proteolysis of rat VAMP-1 and -2 has been used to identify the VAMP isotypes (Braun et al., 1994; Steinhardt et al., 1994). O n the other hand, BoNT/D, F and G cleave both VAMP-1 and -2 at similar rates.

74.3.3.2 SNAP-25 SNAP-25 (synaptosomal-associated protein of 25 kDa) is a 206 residue protein that lacks a classical transmembrane segment, but is bound to the cytosolic surface of the neuronal plasmalemma via palmitoylation of four cysteine residues located at the center of the molecule (Fig. 3) (Bennett and Scheller, 1994; Sudhof, 1995). SNAP-25 is required for axonal growth during development, and for nerve terminal plasticity in the mature nervous system (Osen-Sand et a/., 1993). The tissue distribution of SNAP-25 is less well characterized than that of VAMP; however, its presence in pancreatic cells (Jacobson et al., 1994; Sadoul eta/., 1995) may indicate that it is also expressed outside the nervous system. A SNAP-25 related protein required for postGolgi transport has been cloned from yeast (Brenwald et al., 1994).

74.3.3.3 Syntaxin Syntaxin is located on the cytosolic surface of the neuronal plasmalemma (Bennett and Scheller, 1994; Sudhof, 1995).It is a typical type II membrane protein with the greater part of its molecule exposed to the cytosol (Fig. 3). Syntaxin is associated with calcium channels in the active zones of the presynaptic membrane, where neurotransmitter release takes place. Syntaxin also interacts with synaptotagmin, the putative high affinity calcium sensor on SSV (Sudhof, 1995). In addition, syntaxin was recently localized in chromaffin granules (Tagaya et al., 1995). Syntaxin undergoes, together with SNAP-25, a complex recycling process in organelles indistinguishable from synaptic vesicles (Walch-Solimena et al., 1995). Two isoforms of slightly different length have been identified in neurons (isotypes 1A and 1 B) and a vast polymorphism is present in other tissues (Bennett et al., 1993). In addition, syntaxin-related proteins have been identified in yeast and Clostridial Neurotoxins

in plants (Bassham et al., 1995).The various syntaxin isoforms show different sensitivity to BoNT/C: isoforms 1A, 1 B, 2, 3 are sensitive, while isoforms 4 and 5 are resistant to toxin cleavage (Blasiet al., 1993 b; Schiavo et a/., 1995 b ) .

74.3.3.4 Target Recognition

recognition motifs in target proteins

The specificity of the various CNTs was tested by using a vast selection of synthetic peptides as possible proteolytic substrates. Some of them were designed on the basis of the known protein sequence flanking the cleavage sites of the various toxins. Invariably, none of these peptides were cleaved by CNTs (Schiavo et al., 1992 b; Yamasaki et al., 1994 a;Shone and Roberts, 1994; Shone et al., 1993; Cornille et al., 1994; Foran et al., 1994). In contrast, peptides encompassing the cleavage sites do bind to the respective toxins and inhibit their proteolytic activity against endogenous substrates (Schiavo et al., 1992 a, 1994).BoNT/B proteolysis of VAMP peptides requires a fortyresidue-long peptide, and maximal cleavage rates were observed with a VAMP segment spanning residues 33 to 96 of the native protein (Shone et al., 1993). BoNT/A, D, E and F also required long peptides for cleavage (Binz eta/., 1994; Yamasaki et al., 1994 a).Another peculiarity of the CNTs is that they hydrolyze only one out of several identical peptide bonds present in the target protein sequence. These features suggest that TeTx and BoNTs recognize the tertiary structure of their targets and that the cleaved peptide bond and flanking residues are not the sole determinant for target recognition and cleavage by CNT (Rossetto et al., 1994). Close inspection of the sequences of VAMP, SNAP-25 and syntaxin, reveals that all three proteins contain a short motif predicted to adopt an a-helical conformation (Rossetto et a/., 1994). Helical wheel projections of this motif show a face with three negative charges next to a hydrophobic region. This motif is present in all VAMP, SNAP-25 and syntaxin isoforms sensitive to the neurotoxins.Variations are present in VAMP and syntaxin of Drosophila and yeast, species which are resistant to these neurotoxins, and in syntaxin isoforms known not to be involved in exocytosis. There are two copies of this motif in VAMP, four copies in SNAP-25 and two copies in syntaxin (Rossetto et al., 1994; Pellizzari et al., 1996). Peptides corresponding to the specific sequences of this motif from VAMP, SNAP-25 and syntaxin inhibit neurotoxin activity in vitro and in vivo, irrespective of their origin and toxin isoform. In VAMP, this toxin-binding region is essential for the correct targeting of VAMP to small synaptic vesicles (Grote et al., 1995). In summary, CNTs recognize their protein substrates via two distinct regions: (a) a segment that includes the peptide bond to be cleaved, and (b) another region conserved in VAMP, SNAP-25 and syntaxin, which accounts for antibody cross-reactivity and cross-inhibition of the different neurotoxin types.

G. SCHIAVO and C. MONTECUCCO

14.4 Clostridial Neurotoxins and the Blockade of Neurotransmitter Release Recently, Sollner and colleagues (Sollner et al., 1993 a, b) have shown that VAMP, SNAP-25 and syntaxin, together with a group of cytosolic proteins (NSF,a- and y-SNAP), form a 20s protein complex involved in the docking and fusion of SSV with the presynaptic membrane (Rothman, 1994; Sollner, 1995). The specific targeting of a vesicle is ensured by the recognition between a vesicle receptor protein (termed V-SNARE)and a complementary receptor protein located on the target membrane (t-SNARE). In neurons, VAMP and synaptotagmin appear to be V-SNARES,and syntaxin and SNAP-25 are the t-SNARES (Sollner et al., 1993 a; Schiavo et al., 1995 a). Specific SNARE isoforms couple and control each vesicle docking event within the cell (Rothman, 1994; Sollner, 1995). Experimental evidence supporting the SNARE hypothesis is rapidly accumulating and suggests that the SNAREs functionally define the borders of intracellular transport units (Sollner, 1995). SNAP-25 and syntaxin form a stoichiometric complex. This complex can bind one molecule of VAMP with high affinity. This trimeric SNARE complex is stable in sodium dodecylsulfate (Chapman et al., 1994; Hayashi et al., 1994, 1995).In the process of neuroexocytosis, SNARE complex formation precedes the recruitment of cytosolic and membrane protein components required for fusion of the lipid bilayers (Rothman, 1994; Sollner, 1995).It is likely that NSF-mediated hydrolysis of ATP provides energy for priming the neuroexocytotic apparatus. The primed system is now ready to trigger exocytosis upon calcium influx into the synapse. It is not yet established if the last step of neurotransmitter release takes place via a fusion pore or through a complete membrane fusion with lipid intermixing (Monk and Fernandez , 1994; Bruns and Jahn, 1996). BoNT/A removes only nine residue from the SNAP-25 carboxyterminus and yet this cleavage is sufficient to impair neuroexocytosis. This indicates that the extreme carboxy-terminal portion of the molecule is essential for SNAP-25 folding or for correct interaction with other SNARES. The functional intoxication with BoNT/A can be reversed by a-latratoxin, or by increasing the intracellular calcium concentration. This rescue process is not possible when SNAP-25 is cleaved with BoNT/E, a process that removes another eighteen amino acid residues from its carboxy-terminus, or by intoxication of the synapse with other CNTs. Taken together, these results suggest that SNAP-25 may have an additional modulatory role, strictly related to the calcium-sensitive step. The association of SNAP-25 with the vesicle membrane protein and the likely calcium sensor synaptotagmin (Schiavo et al., 1997) may represent the molecular basis of these findings. Cleavage of VAMP and syntaxin by TeTx, BoNT/B, D, F, G and C leads to the release of a large part of the target molecule into the cytosol. These fragments, as well the fragments of SNAP-25 gener-

vesicle docking and fusion

Clostridial Neurotoxins

ated by BoNT/A and E, can still form a SNARE complex capable of recruiting SNAPS and NSF. In the presence of ATP, the 20s particle is able to dissociate with a kinetic rate similar to that of the native complex (Hayashi et a/., 1995; Pellegrini et al., 1995).Ultrastructural analysis of electrically silent synapses poisoned by CNTs show a similar number of docked vesicles to control synapses (Hunt et al., 1994; Broadie et a/., 1995).This suggests that the docking step is a multiprotein mediated process involving additional components. However, these docked vesicles are unable to fuse with the plasma membrane, even after prolonged stimulation. Taken together, these results indicate that VAMP, SNAP-25 and syntaxin may also be involved in the fusion process, in addition to their proposed role in docking.

14.5 Biomedical Applications of Clostridial Neurotoxins 14.5.1 Safety Warnings

A

vaccination advisable

A

work in contained space, and decontaminate afterwards

Clostridial neurotoxins are very toxic. However they are ineffective in individuals immunized with the corresponding toxoids. In most countries children are vaccinated with tetanus toxoid and this is sufficient to provide full protection against tetanus for decades. A booster injection of tetanus toxoid (available from health authorities) before starting research with TeTx is advisable. O n the other hand, the vaccine for BoNT/A, B, C, D and E is not commercially available, but can be obtained from the Center for Disease Control (CDC, Atlanta, G A ) . Due to the rather low efficacy of the BoNTs vaccine, a protective serum anti-BoNT titre is generally, but not always, achieved. Human anti-TeTx antibodies and horse anti-BoNT antibodies are also available from health authorities, and their injection immediately after accidental penetration of the toxin into the circulatory system is sufficient to prevent the disease. Work with the toxin should be performed in a contained space and all the materials contaminated with CNTs should be autoclaved or washed at the end of the experiment with dilute sodium hypochloride, due to the extreme sensitivity to oxidants of CNTs (see Chapter 20).

14.5.2 Purification of Clostridial Neurotoxins TeTx is isolated from culture supernatants of Clostridium tetani by ammonium sulfate precipitation followed by chromatography (Matsuda and Yoneda, 1975; Schiavo and Montecucco, 1995).Single chain TeTx is released by bacterial lysis and is rapidly converted by bacterial proteases into the di-chain TeTx form (Krieglstein et al., 1991; DasGupta, 1994). Different growth conditions and extraction procedures G. SCHIAVO and C. MONTECUCCO

are followed for the preparation of the single chain TeTx and di-chain TeTx (Matsuda and Yoneda, 1975; Schiavo and Montecucco, 1995). With this procedure it is also possible to obtain some free L chain, which can be purified to homogeneity via isoelectrofocusing or HPLC (Weller etal., 1989; De Filippis etal., 1995).The L chain of TeTx has also been produced by expression in E. coli (66). Hc is obtained by selective proteolysis of TeTx with papain followed by chromatography (Helting and Zwisler, 1977; Matsuda et al., 1989). BoNTs can be isolated from bacterial culture by acid precipitation and citrate extraction of the pellet, followed by ammonium sulfate fractionation and chromatography on SP-Sephadex (Shone and Tranter, 1995).Alternatively, CNTs can also be obtained from different commercial sources (see 14.8). The percentage of single chain and di-chain CNTs in the final toxin preparation may vary depending on the growth conditions, the age of the bacterial culture and the bacterial strain. In order to obtain the dichain toxin, single chain TeTx and BoNTs are treated with protease(s). It is advisable to perform first an analytical test to optimise nicking conditions. s-TeTx is cleaved with TPCK-treated trypsin at 25°C for 60 min with a toxin/protease ratio of 1OOO:l (w/w) (Schiavoand Montecucco, 1995).Proteolysis is terminated by addition of soybean trypsin inhibitor at a protease-to-inhibitor ratio of 1:4 (w/w). This procedure is applicable with minor modifications to the nicking of single chain BoNTs (DasGupta, 1994). Protease-free preparation of CNTs can be obtained using an immobilized-metal-ion affinity chromatography (IMAC) step (Rossetto et al., 1992).This procedure is also useful for the purification of Hc, the 50 kDa carboxy-terminal part of the heavy chain of TeTx, which shows an identical retention time. However, IMAC-chromatography cannot be used for the purification of BoNT/D because this serotype is not retained. To obtain a protease-free BoNT/D preparation, an ion-exchange chromatography procedure was used (Schiavo and Montecucco, 1995).After freezing in liquid nitrogen, purified CNTs are stored at -80°C. Toxicity is routinely tested by intraperitoneal injection of different amounts of toxin. Mouse LD5,, is 0.1-1 ng/kg (Payling-Wright, 1955; Gill, 1982). Toxicity of CNTs varies in different animal species (Gill, 1982),and this is due to different binding at the neuromuscular junction and/or to mutations at the site of action inside cells.

e\ toxicity testing

14.5.2 Functional Assay of Clostridial Neurotoxins TeTx and BoNTs are zinc-endopeptidases highly specific for their substrates and do not cleave short peptides spanning the respective cleavage sites. As a consequence, a simple spectrophotometric or fluorometric assay of their activity is not yet available. Their zincdependent activity must be assayed with the target protein or a substantial portion of it. The target proteins can be obtained by subClostridial Neurotoxins

cellular fractionation of the nervous tissue or alternatively, by expression as recombinant protein in bacterial or insect cells.

74.5.2.7 In vitro Assay of Clostridial Neurotoxins The procedure for the preparation of synaptosomes and small synaptic vesicles (SSV) from rat brain cortex follows established methods with minor modifications (Schiavo and Montecucco, 1995).

@

in vitro assay

1. Di-chain CNTs are reduced for 30 min at 37°C in the presence of 10 mM dithiothreitol.

2. SSV (30 pg) are incubated with 5-20 n M of the reduced TeTx or BoNT/B, D, F or G for 30- 120 min at 37°C. Alternatively, 50 pg of synaptic membrane fraction (Schiavo and Montecucco, 1995) made 0.4 Yo in octylglucoside is incubated with 5-20 n M BoNT/ A, C or E, reduced as above, for 30-120 min at 37°C. 3. Reactions are stopped by addition of 2 % in SDS, 5 % 2mercaptoethanol, 5 mM EDTA, 5 % glycerol and boiling. 4. Proteins are analyzed in SDS-PAGE and stained with Coomassie Blue or, after transfer onto nitrocellulose membrane, by Western Blotting with antibodies against VAMP/synaptobrevin (for TeTx, BoNT/B, D, F and G) or SNAP-25 (for BoNT/A, C and E ) or syntaxin (for BoNT/C) (see Table 2).

5. Quantitative analysis of the proteolytic cleavage is performed by densitometric analysis.

74.5.2.2 Use in Cell Biology Neuronal cells in culture, including synaptosomes, internalize CNTs when present in the extracellular at concentrations of about 1-100 n M (Sanchez-Prietoet a/., 1987; Blasi et a/., 1993 a, b; Schiavo et a/., 1993 a; Osen-Sand et a/.,1996; Williamson et a/., 1996). Nonneuronal cells have to be permeabilized or injected to allow access of the L chain to the cytosol (Penneret a/., 1986; Ahnert-Hilger et a/., 1989 a; Boyd et a/., 1995; Sadoul et a/., 1995; Foran et a/., 1996).An alternative is to express a gene encoding the L chain in a cell or in a transgenic animal (Eisel et a/., 1993; Sweeney et a/., 1995). In vivo, activity of CNTs is assayed by immunoblotting or immunofluorescence, and following the disappearance of the target staining (Galli et a/., 1994; Sadoul et a/., 1995; Sweeney et a/., 1995; Osen-Sand et a/., 1996; Williamson et a/., 1996). Because of its ability to perform a trans-synaptic migration, H, is frequently used as a marker of retroaxonal transport to map neuronal routes from the peripheral nervous system to the CNS by coupling it to horseradish peroxidase or gold particles (Cabot et a/., 1991). G. SCHIAVO and C. MONTECUCCO

14.6 Therapeutic Uses of Botulinum Neurotoxins The demonstration that BoNTs block acetylcholine release at the neuromuscular junction (Burgen et al., 1949), and subsequent studies on the histologic effects caused by BoNT in animal muscles (Jankovic and Hallett, 1994), provided the basis for the use of these toxins as therapeutic agents. Nowadays, BoNT is used in the therapeutic management of several focal or segmental dystonias, of strabismus, and also in a growing number of situations where a functional and reversible depression of a particular neuromuscular function is desired (Montecucco et al., 1996). Available therapeutic protocols were developed largely on the basis of clinical experience, and may vary considerably. The toxin that is currently used is BoNT/A in association with its agglutinins. This complex is more stable than the toxin alone and resists lyophilization. However, a large variation of potency between different lots is experienced. Moreover, most of the protein mass present in the preparation is accounted for by BoNT/A accessory proteins. Since the maior negative consequence that may arise from repeated treatments is the formation of antitoxin complex antibodies, the elimination of the agglutinins should reduce the chance of stimulating the immune system. Another line of improvement concerns the L chain of BoNTs. Since SNAP-25, VAMP and syntaxin are essential for neuroexocytosis, one may assume that the re-establishment of a functional synapse follows the inactivation of the L chain by the cell and the reformation of their physiological synaptic pool. Clearly, any modification of the L chain, by genetic engineering or chemical methods, that would prolong its life within the cell will extend the time period for the beneficial effects of this therapy. So far, BoNT/A has almost invariably been the toxin used (Jankovic and Hallett, 1994). In some cases, BoNT/A is not effective at the first attempt, but the reason of these failures have not been investigated. Since the symptoms of botulism are not evident and the disease may go unnoticed, there is the possibility that some dystonia patients may have been immunized against BoNT/A earlier in their life. The availability of other BoNTserotypes overcomes this problem. In addition to BoNT/B and F, already used experimentally in humans, BoNT/C could represents a valid alternative to BoNT/A. In fact, BoNT/C causes a paralysis that lasts as long as that caused by BoNT/A (Eleopra et a/., submitted) and hence represents a new potent biomedical tool whose potential has yet to be fully explored.

14.7 Concluding Remarks The clostridial neurotoxins responsible for tetanus and botulism form a new group of zinc-endopeptidases endowed with peculiar properties. They are produced as inactive precursors which are activated by specific proteolysis, followed by intracellular reduction of a single diClostridial Neurotoxins

sulfide bond. The amino acid sequence around the zinc binding motif is different from that of any other group of zinc-endopeptidases. They act in the neuronal cytosol and are very specific in terms of their protein target and of the peptide bond hydrolyzed. Their exquisite specificity for proteins of the neuroexocytotic apparatus makes them valuable tools in modern neuroscience research and in human therapy. Future important discoveries will be the identification of the neuronspecific receptors of CNTs and of the mode of internalization and membrane translocation of the neurotoxins. Another important line of research is aimed at finding specific inhibitors of these metalloproteinases. Inhibitors which can cross the neuronal plasmalemma into the cytosol would be potential therapeutic agents in the treatment of tetanus and botulism. The modification of BoNTs to prolong their life time inside the NMJs would be an important research goal to improve the treatment of dystonias. The determination of the threedimensional structure of these neurotoxins will greatly accelerate the research on these fronts.

14.8 Chemicals Tetanus and Botulinum Neurotoxins Commercial Sources Toxin type TeTx BoNTIA BoNT/B BoNTIC BoNT/D BoNTIE BoNT/F BoNT/G a

Supplier

Catalog number

Alomone Labs

T-150

Calbiochem Boehringer Mannheim Calbiochem Sigma Wa ko Calbiochem Sigma Wa ko Calbiochem Sigma Wa ko Calbiochem Sigma Wa ko Calbiochem Sigma Wa ko Calbiochem Sigma Wa ko not available

582235 (fragment C) 1 348 655 (fragment C) 203652" B8776 980- 10141 203672" 86403 988- 10142 203676" B 1036 986- 10143 203677 B 1397 984- 10144 203673 86528 982- 10145 203679 89152 980- 10146

purified L and H chains also available

Acknowledgments Work in the author laboratory (C.M.)is supported by CNR-Biotecnologie,Telethonltalia grant 473 and MURST. G. SCHIAVO and C. MONTECUCCO

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Schiavo G, Benfenati F, Poulain B, Rossetto 0, Polverino de Laureto P, DasGupta BR, Montecucco C 11992 al: Tetanus and botulinum-B neurotoxins block neurotransmitter release by a proteolytic cleavage of synaptobrevin. In Nature 359: 832-5 Schiavo G, Poulain B, Rossetto 0, Benfenati F, Tauc L, Montecucco C (1992 b):Tetanus toxin is a zinc protein and its inhibition of neurotrasmitter release and protease activity depend on zinc. In EM60 J. 11: 3577-83 Schiavo G, Rossetto 0, Santucci A, DasGupta BR, Montecucco C (1992 c): Botulinum neurotoxins are zinc proteins. In J. Biol. Chern. 267: 23479-83 Schiavo G, Rossetto 0, Catsicas S, Polverino de Laureto P, DasGupta BR, Benfenati F, Montecucco C (1993 a): Identification of the nerve-terminal targets of botulinum neurotoxins serotypes A, D and E. In J. Biol. Chern. 268: 23784-7 Schiavo G, Santucci A, DasGupta BR, Metha PP, Jontes J, Benfenati F, Wilson MC, Montecucco C (1993 b): Botulinum neurotoxins serotypes A and E cleave SNAP25 at distinct COOH-terminal peptide bonds. In FEBS Lett. 335: 99-103 Schiavo G, Shone CC, Rossetto 0, Alexandre FCG, Montecucco C (1993 c): Botulinum neurotoxin serotype F is a zinc endopeptidase specific for VAMP/synaptobrevin. In J. Biol. Chern. 268: 11516-9 Schiavo G, Malizio C, Trimble WS, Polverino de Laureto P, Milan G, Sugiyama H, Johnson EA, Montecucco C (1994): Botulinum G neurotoxin cleaves VAMP/synaptobrevin at a single Ala/Ala peptide bond. In J. Biol. Chem. 269:20213-6 Schiavo G, Gmachl MJ, Stenbeck G, SollnerTH, Rothman JE (1995 a): A possible docking and fusion particle for synaptic transmission. In Nature 378: 733-6 Schiavo G, Shone CC, Bennett MK, Scheller RH, Montecucco C (1995 b) Botulinum neurotoxin type C cleaves a single Lys-Ala bond within the carboxyl-terminal region of syntaxins. In J. Biol. Chern. 270: 10566-70 Schiavo G, Stenbeck G, Rothman J.E., Sollner T.H. (1997) Binding of the synaptic vesicle SNARE, synaptotagmin, to the plasma membrane, target-localized SNARE, SNAP-25, can explain docked vesicles at neurotoxin-treatedsynapses. froc. Natl. Acad. Sci. USA, in press Schmid MF, Robinson JP, DasGupta BR (1993): Direct visualization of botulinum neurotoxin-induced channels in phospholipid vesicles. In Nature 364: 827-30 Shone CC, Roberts AK (1994):Peptide substrate specificity and properties of the zinc-endopeptidase activity of botulinum type B neurotoxin. In Eur. J. Biochern. 225: 263-70 Shone CC, Tranter HS (1995): Growth of Clostridia and preparation of their neurotoxins. In Curr. Top. Microbiol. Irnrnunol. 19: 143-60 Shone CC, Quinn CP, Wait R, Hallis B, Fooks SG, Hamblen P (1993): Proteolytic cleavage of synthetic fragments of vesicle-associated membrane protein, isoform-2 by botulinum type B neurotoxin. In Eur. J. Biochern. 217: 965-71 Simpson LL (ed)(1989):Botulinurn neurotoxin and tetanus toxin. Sun Diego: Academic Press Simpson LL, Coffield JA, Bakry N (1993):Chelation of zinc antagonizes the neuromuscular blocking properties of the seven serotypes of botulinum neurotoxin as well as tetanus toxin. In J. Pharrnacol. Exp. Ther. 267: 720-7 Simpson LL, Coffield JA, Bakry N (1994):lnhibiton of vacuolar adenosine triphosphatase antagonizes the effects of clostridial neurotoxins but not phospholipase A2 neurotoxins. J. fharrnacol. Exp. Ther: 269: 256-62 Skehel PA, Martin KC, Kandel ER, Bartsch D (1995) A VAMP-binding protein froim Aplysia required for neurotransmitter release. In Science 269: 1580-3 Sollner TH (1995):SNARESand targeted membrane fusion. In FEBS Lett. 369: 80-3 Sollner TH, Whiteheart SW, Brunner M, Erdjument-Bromage H, Geromanos S, Tempst P, Rothman JE (1993 a): SNAP receptors implicated in vesicle targeting and fusion. In Nature 362: 318-24 Sollner TH, Bennett MK, Whiteheart SW, Scheller RH, Rothman JE (1993 b): A protein assembly-disassembly pathway in vitro that may correspond to sequential steps of synaptic vesicle docking, activation, and fusion. In Cell 75: 409-18 Stecher B, Weller U, Habermann E, Gratzl M, Anhert-Hilger G (1989):The light chain but not the heavy chain of botulinum A toxin inhibits exocytosis from permeabilized adrenal chromaffin cells. In FEBS Lett. 255: 391 -4

Clostridial Neurotoxins

Steinhardt RA, Bi G, Alderton JM (1994): Cell membrane resealing by a vesicular mechanism similar to neurotransmitter release. In Science 263: 390-3 Sudhof TC (1995):The synaptic vesicle cycle: a cascade of protein-protein interactions. Nature 375: 645-53 Sweeney ST, Broadie K, Keane J, Niemann H, O’Kane J (1995):Targeted expression of tetanus toxin light chain in Drosophila specifically eliminates synaptic transmission and causes behavioral defects. In Neuron 14: 341 -51 Tagaya M, Toyonaga S, Takahashi M, Yamamoto A, Fuiiwara T, Akagawa K, Moriyama Y, Mizushima S (1995): Syntaxin 1 (HPC-1) is associated with chromaffin granules. In J. Biol. Chem. 270: 15930-3 Tizzoni G, Cattani G (1890 a): Uber das Tetanusgift. In Zentralbl. Bakt. 8: 69-73 Tizzoni G, Cattani G. (1890 b): Untersuchungen uber das Tetanusgift. In Arch. exp. fathol. fharrnakol. 27: 432-50 Vallee RB, Bloom GS (1991):Mechanisms of fast and slow axonal transport. In Annu. Rev. Neurosci. 14;.59-92 van Ermengem E (1897): Uber ein neuen anaeroben Bacillus und seine Beziehungen zum Botulismus. In Ztsch. Hyg. Infektkrh. 26: 1-56 Walch-Solimena C, Blasi J, Edelmann L, Chapman ER, von Mollard GF, Jahn R (1995): The t-SNARES syntaxin 1 and SNAP-25 are present on organelles that participate in synaptic vesicle recycling. In J. Cell Biol. 128: 637-45 Washbourne P, Schiavo G, Montecucco C (1995): VAMP-2 forms a complex with synaptophysin. In Biochem. J. 305: 721 -4 Weller U, Dauzenroth M-E, Meyer Heringdorf D, Habermann E (1989):Chains and fragments of tetanus toxin. In Eur. J. Biochern. 182: 649-56 Weller U, Dauzenroth M-E, Gansel M, Dreyer F (1991): Cooperative action of the light chain of tetanus toxin and the heavy chain of botulinum toxin type A on the transmitter release of mammalian motor endplates. In Neurosci. Lett. 122: 132-4 Wellhoner HH (1992):Tetanus and botulinum neurotoxins. In Handbook of Experimental Pharmacology, vol 102 (Herken H, Hucho F, eds), pp 357-417, Ber1in:Springer-Verlag Williamson LC, Neale EA (1992): Bafilomicin A1 inhibits the action of tetanus toxin in spinal cord neurons in cell culture. InJ. Neurochern. 63: 2342-45 Williamson LC, Halpern JL, Montecucco C, Brown JE, Neale EA (1996):Clostridial neurotoxins and substrate proteolysis in intact neurons: botulinum neurotoxin C acts on synaptosomal-associated protein of 25 kDa. In J. Biol. Chem. 271: 7694-9 Wright JF, Pernollet M, Reboul A, Aude C, Colomb M (1992): Identification and partial characterization of a low affinity metal-binding site in the light chain of tetanus toxin. In J. Biol. Chem. 267: 9053-8 Yamasaki S, Baumeister A, Binz T, Blasi J, Link E, Cornille F, Roques B, Fykse EM, Sudhof TC, Jahn R, Niemann H (1994 a): Cleavage of members of the synaptobrevin/VAMP family by types D and F botulinal neurotoxins and tetanus toxin. In J. Biol. Chem. 269: 12764-72 Yamasaki S, HuY, BinzT, Kalkuhl A, Kurazono,TamuraT, Jahn R, Kandel E, Niemann H (1994 b): Synaptobrevin/VAMP of Aplysia californica: structure and proteolysis by tetanus and botulinal neurotoxins type D and F. In Proc. Natl. Acad. Sci. USA 91 : 4688-92 Yavin E, Nathan A (1986):Tetanus toxin receptors on nerve cells contain a trypsin sensitive component. In Eur. J. Biochem. 154: 403-7

G . SCHIAVO and C.MONTECUCCO

Bacterial Toxins

Klaus Aktories Copyright 0 2002 WILEY-VCH Verlag GmbH & Co. KGaA

CHAPTER 15

Isolated Nerve Terminals as a Model System for the Study of Botulinum and Tetanus Toxins J. BLASI, E. LINK and R. JAHN

15.1 Introduction For many years, clostridial neurotoxins, including tetanus toxin and botulinum neurotoxins, have been applied as tools in a variety of areas of neurobiology and neurology. All of these toxins ultimately block neurotransmitter release from presynaptic nerve endings. Thus, depending on the type of neurotoxin, they cause functional denervation at the neuromuscular junction (Frangez et al., 1994) which leads to sprouting of new axon collaterals (Angaut-Petit et al., 1990). The toxins have also been used as therapeutic agents in muscle contraction disorders (Davis, 1993; Jankovic, 1994), and their fragments as molecular tracers for retrograde axonal transport (Manning et al., 1990, Cabot et al., 1991). Recently, the molecular mechanisms of action of these toxins have been elucidated. Each of these toxins functions as a metalloendoprotease that selectively cleaves a set of conserved proteins involved in exocytosis of synaptic vesicles (Montecucco and Schiavo, 1993; Niemann et al., 1994).Therefore, they provide the researcher with a unique set of tools of exquisite specificity in the study of neurotransmitter release. In the following paragraphs, we will first briefly review the mechanism of action of these toxins (for a more comprehensive discussion see this volume, Chapter 14; Montecucco and Schiavo, 1994, Niemann et al., 1994). We will then give a short overview over the preparations that have been used in the study of toxin action. Finally, we will describe in detail the procedures required for the study of these toxins using isolated nerve terminals (synaptosomes) as an experimental model system.

15.2 Clostridial Neurotoxins: How Do They Work? Clostridial neurotoxins are proteins that are produced by the anaerobic bacteria Clostridiurn tetani (tetanus toxin) and Clostridiurn botulinurn (botulinum neurotoxins). Whereas tetanus toxin (TeTx) comprises a single molecular species, different strains of Clostridiurn botulinurn produce seven different types of botulinum neurotoxin (desigK. Aktories (Ed.), Bacterial Toxins. 0 Chapman & Hall, Weinheim, 1997. ISBN 3-8261-0080-8

clinical symptoms

structure

poisoning pathway

nated BoNTIA , B, C1, D, E, F, and G) that were originally differentiated using immunological methods, and that represent different proteins. TeTx and BoNTs differ in their clinical manifestations. Tetanus toxin poisoning produces tetanus, i. e. muscle contractions resulting in spastic paralysis. In contrast, botulinum neurotoxins cause botulism, which is characterized by flaccid paralysis. This difference reflects differences in the anatomical level of action of these toxins. TeTx acts primarily on the CNS where it blocks exocytosis from inhibitory glycinergic synapses in the spinal cord. Loss of inhibitory control results in motoneuron firing. BoNTs act primarily in the periphery where they inhibit acetylcholine release at the neuromuscular junctions. Despite the differences in their clinical manifestations, the clostridial neurotoxins comprise a group of homologous proteins with similar mechanisms of action. They are synthesized as single-chain precursors of 150 kDa which are biologically inactive. Active toxin is generated by proteolytic cleavage at a position approximately one third downstream from the N-terminus. A di-chain molecule is generated that consists of the smaller N-terminal portion (approx. 50 kDa, designated light, or L chain) and the larger C-terminal fragment (approx. 100 kDa, designated heavy, or H chain). The chains are held together by a single disulfide bond and by non-covalent forces. During poisoning of neurons, each of the two chains has a different function. The H chain is responsible for recognition and uptake of the toxin by the peripheral nerve terminal, whereas the L chain is responsible for the final toxic effect (Mochida et al., 1989; Ahnert-Hilger et al., 1990; Poulain et al., 1990; Niemann, 1991). Numerous studies using a variety of experimental model systems have contributed to the elucidation of the poisoning pathway of the toxins (see Niemann et al., 1994; Ahnert-Hilger and Bigalke, 1995; and this volume, Chapter 14, for more details). Briefly, the following steps can be summarized:

1. Recognition and binding of the toxin to the plasma membrane of the nerve terminal: Binding occurs spontaneously and with high affinity to specific receptors which include both ganglioside and protein components (Montecucco, 1986; Middlebrook, 1989; Niemannn, 1991; Nishiki et al., 1994).

2. Entry of the toxin into the nerve terminal: Both TeTx and BoNTs enter the nervous system preferentially at the neuromuscular junction. Bound toxins are internalized by the nerve terminal, by means of endocytosis. This process requires energy and probably delivers the toxin to an endosomal compartment within the terminal. H chains mediate specific binding, internalization and intra-neuronal sorting (Niemann, 1991).

3. Sorting to the final destination: TeTx is translocated predominantly by retrograde axonal transport to the axodendritic area of the motoneurons in the spinal cord. Here, the toxin is released, probably by a transcytotic mechanism, crosses the synaptic cleft, J. BLASI, E. LINK and R. JAHN

and poisons the nerve endings of inhibitory interneurons. BoNTs mostly remain in the peripheral nerve terminals.

4. Reduction of the toxin and translocation of the 1 chain into the cytoplasm: To gain access to the cytoplasm, the L chain needs to cross the membrane of the endocytic compartment. For this translocation, the H chain is required, probably by forming a proteinaceous translocation complex in the membrane that exposes (and possibly releases) the L chain to the cytoplasm. In the reductive intracellular environment, the disulfide bond linking the H and L chains is reduced (Kistner and Habermann, 1992).

5. Inhibition of exocytosis by cleaving essential proteins of the exocytotic fusion complex: The reduced L chains are Zn2+-dependent metalloendoproteases with exquisite substrate specificities.

The molecular targets for these toxins are synaptobrevin (TeTx, BoNT/B, D, F and G; Schiavo et al., 1992, Link et al., 1992; Schiavo et al., 1993c; Yamasaki et al., 1994a, b), SNAP-25 (BoNT/A, E, and C1; Blasi et al., 1993a; Binz et al., 1994, Schiavo et al., 1993a, b; Foran et al., 1996; Williamson et al., 1996), and syntaxin (BoNT/Cl; Blasi et al., 199313; Schiavo et al., 1995). Cleavage results in an essentially irreversible block of membrane fusion, whereas vesicle docking and ion currents remain unaffected (for a comprehensive overview see Simpson (1989), DasGupta (1993)). The poisoning sequence outlined above defines the conditions that are required for using the toxins as tools to block exocytosis. Accordingly, the following points should be taken into account:

molecular targets for toxins

Cell entry: Toxins enter into the cell that contains the respective membrane receptor. The presence of the receptor (and not the mechanism of action) is responsible for the high selectivity with which peripheral motoneurons are targeted. As discussed below in more detail, CNS synaptosomes also contain toxin receptors.

1 chain or Di-chain-toxin: If the biological poisoning pathway is being used, the toxins must be in their di-chain form. Isolated L chains cannot enter the cell and are thus ineffective when added to intact cells or isolated nerve terminals. However, isolated L chains block exocytosis if they are introduced directly into the cytoplasm, e.g., by microinjection (Penner et al., 1986; Mochida et al., 1989; Hunt et al., 1994))by permeabilization of the cells prior to toxin exposure (Bittner and Holz, 1988; Ahnert-Hilger et al., 1989a,b, and this volume, Chapter 18; Dayanithi et al., 1992), or by expression of recombinant L chains (Mochida et al., 1990, Sweeney et al., 1995).These techniques allow the inhibition of exocytosis in neurosecretory cells that are toxinresistant due to a lack of receptors. Likewise, only the free L chains (reduced di-chain toxin or isolated L chain) are active towards the respective substrates in vitro (Schiavo et al., 1992; Link et al., 1992; Schiavo et al., 1993c; Link et al., 1994).

Isolated Nerve Terminals as a Model System for the Study of Botulinum and Tetanus Toxins

Substrate specificity: When an L chain has reached the cytoplasm its potency is determined by the availability and functional relevance of cleavable substrates. As discussed above, the neuronal substrates are the synaptic vesicle protein synaptobrevin (also referred to as VAMP) and the synaptic membrane proteins syntaxin and SNAP-25. Each of these proteins is a member of a protein family with ubiquitously expressed members that function in many intracellular membrane fusion events (reviewed by Ferro-Novick and Jahn, 1994).Furthermore, the cleavage sites of all toxins are known (they are all different, with the exception of BoNT/B and TeTx), and major progress has been made recently concerning the structural and sequence requirements for toxin-mediated cleavage of the substrate proteins (Niemann et al., 1994; Yamasaki et al., 1994; Hayashi et al., 1994, 1995; Blasi et al., 199313; Schiavo et al., 1995; Otto et al., 1995; Pellegrini et al., 1995). Although a full discussion of this topic is beyond the scope of this chapter, several points of importance have begun to emerge. First, synaptobrevin, SNAP-25 and syntaxin are highly conserved during evolution, with up to 80 % sequence identity between vertebrates and invertebrates. Thus, most of the toxins are active in all species tested. However, even minor sequence alterations at or near the cleavage sites are not well tolerated. For instance, synaptobrevin 1 is far less susceptible to these toxins than its isoform synaptobrevin 2, due to a single amino acid alteration at the cleavage site. In contrast, BoNT/D (which cleaves at an adjacent site that is identical in the isoforms) is equally potent. Another example is the resistance of SNAP-25 in some invertebrates towards BoNT/A and E, which is also due to substitutions around the respective cleavage sites. Second, some of the nonneuronal isoforms of the substrate proteins are susceptible to toxin cleavage including, cellubrevin (McMahon et al., 1993) and syntaxins 2 and 3 (Schiavo et al., 1995).This offers the exciting possibility of using toxin L chains in a wide variety of non-neuronal cells (Link et al., 1993; Galli et al., 1994; Gaisano et al., 1994 ; Steinhardt et al., 1994, lkonen et al., 1995 ; Sadoul et al., 1995).Third, it was recently discovered that synaptobrevin 2 is expressed in all cells, where it is apparently specific for specialized pathways whose functions are not yet fully understood (Ralston et a/., 1994). Although it cannot be predicted which of the members of these growing protein families will be susceptible to toxin cleavage (e.g., none of the yeast proteins can be cleaved), one can safely predict that the toxins will become established tools for the highly selective dissection of exocytotic pathways in non-neuronal cells (Volchuk et al., 1994; Galli et al., 1994; lkonen et al., 1995).

15.3 Model Systems for the Study of Toxin Action

sensitivity to neurotoxins J. BLASI, E. LINK and R. JAHN

In general, sensitivity of a given preparation to clostridial neurotoxins depends on the type of neurotoxin. For instance, BoNT/A is particularly potent in poisoning peripheral cholinergic nerve terminals (i.e. neuromuscular junctions), whereas TeTx is most potent for CNS preparations. However, all eight neurotoxins are able to inhibit exocy-

tosis in virtually every neuron. So far, each toxin also appears to block secretion in neuroendocrine and endocrine cells, provided that the L chain can gain access to the cell (e.g., by cell permeabilization or microinjection). Before discussing synaptosomes in greater detail, we will briefly review experimental approaches that have contributed to the elucidation of toxin action.

15.3.1 Bioassay This assay is based on injection of toxin into live mammals (usually mice) and is used to check the potency (lethality) of a given toxin preparation. It has also been applied to the study of substances that antagonize the effect of the toxin by interfering with the initial steps of poisoning (Bakry et a/., 1991; Simpson etal., 1990).Normally, the LD50 or the "time to death" is measured (Boroff and Fleck, 1966) and compared with non-treated control animals. It is also possible to inject small amounts of toxin into mammals (mainly BoNT/A) to produce local paralysis which, may result in long term morphological and functional changes (Angaut-Petit et a/., 1990). The highly localized and long-lasting action of the botulinum neurotoxins led to the widespread use of BoNT/A in the symptomatic treatment of local muscle spasms (Hughes, 1994).

15.3.2 Neuromuscular Junction Preparations Most of the general aspects of the action of clostridial neurotoxin have been established using neuromuscular junction preparations as experimental model systems (Habermann and Dreyer, 1986). The neuromuscular endplate is the principal target in the "natural" poisoning process of botulinum neurotoxins. As little as 0.1 n M of BoNT/A is sufficient to completely block acetylcholine release within 90 min (Dolly et a/., 1984; Gansel et a/., 1987). With TeTx, higher concentrations are required to achieve the same result (Dreyer, 1989). Several different neuromuscular junction preparations have been used, such as mouse hemidiaphragm, the levator auris longus muscle, the triangularis sterni nerve-muscle of mice, or the extensor digitorurn longus muscle from rat. The mouse hemidiaphragm preparation is one of the most widely used systems. Inhibition of transmitter release can be conveniently monitored by recording from the muscle fiber or by measuring the strength of contraction. Furthermore, studies were also performed on frog neuromuscular junctions (Molgo and Thesleff, 1984) since they are easily accessible and can be used for long term experiments (Molgo et a/., 1987).

Isolated Nerve Terminals as a Model System for the Study of Botulinum and Tetanus Toxins

15.3.3 Synaptic Preparations Derived from the Autonomic Nervous System Synapses of the autonomic nervous system are vulnerable to poisoning by botulinum neurotoxins (Dickson and Shevky, 1923).One of the most frequently used preparation is the superior cervical ganglion (Eccles and Libet, 1961; Mochida et al., 1995).

15.3.4 Invertebrate Neurons large neurons

A major advantage of some invertebrate model synapses is the large size of the neurons, allowing easy access with microelectrodes to preand postsynaptic cells. For instance, in the buccal ganglion of Aplysia californica two large cholinergic neurons contact the same postsynaptic neuron and form a chloride-dependent inhibitory synapse. With this preparation it is possible to microinject toxin into one neuron while the second neuron serves as a control for the excitability of the postsynaptic neuron (Tauc et al., 1974; Poulain et a/., 1986).This preparation is almost as sensitive to botulinum toxins as the vertebrate neuromuscular junction, and the mechanism of poisoning has been extensively investigated (Poulain et al., 1989). Recently, toxins have also been shown to be active when microinjected into presynaptic compartments of the leech Retzius cell and the squid giant synapse (Bruns and Jahn, 1995, Bruns and Jahn, in preparation; Hunt et al., 1994; Llinas eta/., 1994). Finally, in a recent elegant study, TeTx-L chain was expressed in Drosophila using a promoter that contained the yeast UASGAL regulatory-element. TeTx-L chain was thus dependent on the presence of the yeast GAL4 transcription factor. Toxin expression in embryonic neurons eliminated both synaptobrevin and the evoked neurotransmitter release at the neuromuscular junction (Sweeney et al., 1995). Axonal outgrowth and synapse formation were not affected, suggesting an exclusive role of synaptobrevin in synaptic transmission. This approach has the advantage that numerous GAL4 enhancer-trap lines with different GAL4 expression patterns are available, allowing for the directed expression of TeTx L chain in selected cell types.

15.3.5 Primary Cultures of CNS Neurons Neurons in primary cultures are extremely sensitive to poisoning by clostridial toxins (Habermann et al., 1988; Williamson et al., 1996). Moreover, primary neuronal cultures mimic many basic features of neural tissue in vivo, providing much easier access of toxins to neuronal membranes and synaptic structures. By applying clostridial toxins to primary cultured neurons it is also possible to study the possible role of the target proteins in axon elongation, synaptogenesis or neuronal survival. J. BLASI, E. LINK and R. JAHN

15.3.6 Endocrine Cells in Culture and Endocrine Cell Lines Chromaffin cells have been a useful and important model in the study of clostridial toxins. Chromaffin cells do not have toxin receptors on their surface, and, therefore, relatively high concentrations of toxins and long exposure times are needed to block secretion of catecholamines. However, susceptibility to poisoning can be greatly accelerated when the cells are preincubated with gangliosides, or in a low ionic strength buffer (Marxen and Bigalke, 1989), a procedure that may also be useful for non-neuronal cells. Permeabilization of endocrine cell lines, such as PC12 (Ahnert-Hilger et al., 1989a) and insulinoma cells (Sadoul et al., 1995), by the pore-forming toxin streptolysin 0 allows the direct inhibition of exocytosis by toxin L chains.

15.3.7 Synaptosomes from Mammalian Brain Synaptosomes are pinched off nerve terminals, which reseal immediately. They have a diameter of approx. 1 pm and contain all necessary components for the release of neurotransmitters and the recycling of synaptic vesicles (Whittaker et al., 1964). For several hours after isolation, they maintain a stable membrane potential and the capacity to release neurotransmitter upon stimulation. A thorough investigation of their bioenergetic parameters by Nicholls and co-workers has demonstrated that synaptosomes are, in fact, autonomous units that perform essentially all functions of nerve terminals in intact neurons (for a review, see Nicholls, 1989). Electron microscopy shows that they contain mitochondria and numerous synaptic vesicles (Fig. 1). Release of transmitter can be evoked in various ways. In many cases, they are depolarized by increasing the extracellular K+ concentration in Ca2+-containingbuffers. This treatment leads to the activation of voltage-gated Ca2+ channels in the synaptosomal plasma membrane. The resulting Ca2+influx triggers exocytosis of synaptic vesicles. Alternatively, intraterminal Ca2+concentration can be increased by calcium ionophores such as ionomycin or A 23187. Nicholls and coworkers have shown that a partial inhibition of synaptosomal K+ channels (by 4-aminopyridine or by dendrotoxin) leads to a sustained release that is sensitive to tetrodotoxin, indicating that these inhibitors cause repetitive firing in the isolated terminals (Tibbs et al., 1989). These compounds provide a gentler alternative to the grossly nonphysiological stimulation by K' depolarization or by ionophores. Finally, release can also be triggered by a-latrotoxin, the active ingredient of black widow spider venom, although caution is required due to its nonspecific side-effects (McMahon et a/., 1990; Stahl et al., 1994 and in press). Due to their availability and ease of use, synaptosomes have been widely used in the study of clostridial neurotoxins. These advantages clearly outweigh the disadvantages of the preparation (e.g., heteroIsolated Nerve Terminals as a Model System for the Study of Botulinum and Tetanus Toxins

Fig. 1. Electron micrograph of synaptosomes isolated from rat brain by differential and Ficoll-gradientcentrifugation (Courtesy of Christiane Walch-Solimena)

geneity with respect to neurotransmitter, low time-resolution of the release event). Synaptosomes have been instrumental in the elucidation of the molecular target of the toxin in our laboratories, since they are ideally suited for subsequent analysis by subcellular fractionation and by further biochemical and immunological tests.

J. BLASI, E. LINK and R. J A H N

15.4 Practical Considerations 15.4.1 Sources for Clostridial Neurotoxins Although the toxins are produced in large amounts by the relevant bacterial strains and are not difficult to purify, until recently most of them have been available only to a limited number of laboratories. The main reason for this is their extreme toxicity, particularly in the case of the botulinum neurotoxins. These toxins are, by several orders of magnitude, the most potent poisons known to man. Due to the recent increase in their clinical use (mostly BoNT/A) and the high demand for scientific research, several of the toxins are now commercially available through specialized companies, for instance Alomone Labs (Jerusalem, Israel), Calbiochem (San Diego, CA, USA), Sigma (St. Louis, MO, USA), and Wako (Osaka, Japan). Depending on the type and source of the toxin, it may be supplied as a complex of neurotoxin with non-neurotoxic proteins, or as a pure neurotoxin. If possible, purity should be checked by electrophoresis. Quantities provided are usually low, due to the potency of these toxins. Recently, recombinant L chains have been used in some laboratories although, due to the unusual codon usage (very AT-rich), the yields in E. coli are low.

15.4.2 Precautions When using these toxins, one should be aware that these are the most potent known poisons. Exposure is less a problem for TeTx since protection through immunization is easily available (most people in developed countries are immunized) and since the toxin is not taken up by the gastrointestinal tract. Care should be used in manipulating these toxins (particularly botulinum toxins, which can resist the acidic and protease-rich environment within the gastric iuice when complexed with the hemagglutinating and non-toxic proteins) and in handling the material that has been in contact with them. Always use gloves, protective goggles and appropriate clothes. After using toxins, wash every working surface with bleach, which is an efficient decontaminant. During the experiments, disposable materials should be used that can be eliminated as a biological hazard. As proteins, these toxins are heat labile and are completely inactivated by heating at 80°C for lOmin, i.e., conditions met by most standard decontamination treatments (heat autoclave) of medical infectious waste. Clostridia are spore forming bacteria. Their spores are more resistant and higher temperatures for a longer time must be used for inactivation (autoclave for 1 h at 130°C). Refer to an appropriate laboratory safety book, and see also Chapter 20.

A

extreme toxicity

A

most potent poisons known

A

protective clothing and decontamination

Isolated Nerve Terminals as a Model System for the Study of Botulinum and Tetanus Toxins

15.4.3 Preparation of Synaptosomes Synaptosomes were isolated for the first time from guinea pig forebrains by differential and sucrose density gradient centrifugation (Gray and Whittaker, 1962, Whittaker et al., 1964). Small changes have been introduced into the original protocol in order to increase purity and responsiveness of the synaptosomal fractions (Nicholls, 1978, 1989). For most purposes, synaptosomes can be simply prepared by differential centrifugation, but it should be kept in mind that this fraction (P2-fraction) is heavily contaminated with mitochondria and myelin. For these reasons, further purification by Ficoll gradient centrifugation is recommended. The protocol described below is based on the procedure of Nicholls and coworkers (see e.g., Nicholls, 1978; McMahon et al., 1992). Figure 2 summarizes the procedure to monitor the blockade of neurotransmitter release and the specific proteolytic activity of clostridial toxins on brain synaptosomes. Solutions Solution 1 : Sucrose, 320 mM. Solution 2: Sucrose, 320 mM; in HEPES/NaOH, 10 mM, p H 7.4. Solution3: 6%, 9 % and 12% Ficoll (Pharmacia), prepared in 320 mM sucrose ; HEPES 5 mM pH 7.4. Solution 4: Assay buffer: glucose, 10 mM; KCI, 5 mM; NaCI, 140 mM; NaHCOa 5mM; MgC12, 1 mM; Na2HP04, 1.2mM; HEPES, 20 mM, pH 74.

00 + +:':

00 rat brain synaptosomes

toxin

- toxin

\

Fl/

glutamate release

toxin

immunoblotting

4--

\

-

G

i - F

Fig. 2. Schematic drawing that summarizes the procedure for monitoring the blockade of neurotransmitterrelease from brain synaptosomes, followed by immunoblot analysis to detect specific proteolytic activity of clostridial neurotoxins on poisoned synaptosomes J. BLASI, E. LINK and R. JAHN

All these solutions can be stored frozen and should be put on ice before use. Procedure

1. Decapitate two rats, remove the cerebral cortices and place them in 30 ml of ice cold 320 m M sucrose (solution 1). Scraping off the white matter helps by reducing myelin, one of the main contaminants of synaptosomal preparations. All the following steps should be carried out at 4°C.

synaptosome preparation

2. Homogenize the tissue in a glass-Teflon homogenizer, with 10 strokes (up and down) at 800 r.p.m. Avoid formation of foam. 3. Centrifuge the homogenate for 2min at 3000 g (e.g., Sorvall SS34 rotor at 5000 r.p.m).

4. Decant the supernatant into a fresh tube and spin for 12 min at 14 000 g (e.g., SS34 rotor at 11 000 r.p.m). A pellet is generated in which three layers can be distinguished: a dark brown bottom part (mostly mitochondria), a lighter brown middle part (synaptosomes) and a whitish top layer(most1y myelin).

5. Resuspend the pellet in approximately 7-8 ml of solution 2. Try to avoid resuspending too many of the mitochondria. This fraction (P2) can be used for poisoning and release experiments if high purity of the synaptosomal fraction is not required. In this case the protein concentration should be determined. Then, the sample should be divided into aliquots containing the desired amount of protein (usually 1-3 mg), recentrifuged at 14 000 g for 12 min and stored as pellets on ice until use. Further purification by Ficoll gradient centrifugation

1. Prepare Ficoll step-gradients in two centrifuge tubes by adding sequentially e.g., 4ml of 12%, 1 ml of 9 % and 4ml of the 6 % Ficoll solutions in 0.32 M sucrose (solutions 3). Leave enough room in the tube (3-4ml) to load the resuspended synaptosoma1 pellet (P2). We use a Beckman SW41 rotor but other swing-out rotors with similar geometries are also acceptable.

further purification

2. Gently overlay the gradient with the resuspended synaptosomal pellet (P2) and centrifuge at 64 000 g,, (SW 41 rotor; 22 500 r.p.m) for 35 min at 4°C. 3. Synaptosomes are enriched in the 9 % Ficoll layer, or more specifically, at the interfaces between the 12% and 9%, and the 9 % and 6 % Ficoll layers. Myelin is enriched at the top of the gradient whereas most of the mitochondria are in the pellet. Collect the fraction enriched in synaptosomes and dilute to a final volume of 10 ml in assay buffer (solution 4). At this point, the protein concentration should be determined, e.g., by the method of Bradford (1976). Isolated Nerve Terminals as a Model System for the Study of Botulinum and Tetanus Toxins

4. Divide into aliquots (e.g., 1-3mg protein) and centrifuge (14 000 g; 12 min) to pellet the synaptosomes. Notes

1. All glass- and plasticware should be completely free of traces of detergent.

2. Ficoll dissolves slowly and the solutions should be prepared in advance. It is possible to freeze aliquots. 3. The assay buffer can be prepared without glucose and stored at 4"C, or the complete buffer (including glucose) can be stored frozen.

4. The above procedure is scaled for two rat brains. However, it can be scaled up (or down) according to your requirements. scale procedure to your needs

5. To obtain an active preparation it is essential to perform all steps without delay. Furthermore, avoid keeping synaptosomes resuspended for prolonged periods of time (e.g., use a fast dye-binding assay for protein determination that gives results in 10 min or less, such as that described by Bradford, 1976).The aliquoted synaptosoma1 pellets should not be resuspended until immediately before being used in the experiment.

15.4.4 Monitoring Release of Neurotransmitter A variety of methods are available to monitor release of transmitter. In many studies, synaptosomes are preloaded with radioactive transmitter or precursors, and the release is measured by scintillation counting of media aliquots. This method has the disadvantage that the synaptosomes must first be washed to remove excess label and then again be separated from the incubation medium in order to determine the amount of release. Numerous procedures for measuring release of radiolabel have been developed, some of which are fairly sophisticated (perfusion chamber systems, see e.g., Turner and Dunlap, 1995). If a fluorometer or dual-wavelength photometer suitable for enzyme kinetic measurements is available (thermoequilibrated chamber with magnetic stirrer and on-line recording with a chart recorder or computer), we recommend the enzymatic procedure developed by Nicholls and colleagues (Nicholls and Sihra, 1986, Nicholls etal., 1987; Sanchez-Prieto etal., 1987a).The method is based on the oxidative deamination of glutamate by glutamate dehydrogenase, which involves the reduction of NADP+ to NADPH. Generation of NADPH can be monitored either by absorbance (high sensitivity required, e.g., in dual wavelength mode: hobs360 nm, href 390 nm) or by fluorescence (hex340 nm, he, 460 nm). In addition to being able to monitor neurotransmitter release on-line, the method offers the advantage that the sample can be removed from the cuvette at the end of the experiment and subjected to further bioJ. BLASI,

E. LINK and R. JAHN

chemical analysis. Here we give a description of the basic procedure as we have used it in the study of clostridial neurotoxins. For more information on this assay the reader is referred to the thorough characterization by Nicholls and coworkers (Nicholls and Sihra, 1986; Nicholls et al., 1987; Sanchez-Prieto et al., 1987a; Nicholls, 1989). Material and solutions for glutamate release

Assay buffer (see solution 4 above)

KCI CaCI2 EGTA NADP GDH (Sigma G-2525) L- GIuta mate

2M 0.5 M 100mM p H 7 100mM pH 7

1 mM

1 mg protein) Fluorometer or dual-wavelength spectrophotometer with magnetic stirrer and thermostatically controlled chamber (37°C) Cuvettes Small stirring bars Water bath Magnetic stirrer

- Synaptosomes (stored as pellets on ice, e.g., in aliquots of 0

0 0 0 0

1. Resuspend 1 mg of synaptosomes with 1 ml of prewarmed assay buffer and stir the solution with a magnetic stirrer in a water bath at 37°C for 15 min. 2. Transfer synaptosomal suspension to a cuvette, place the cuvette in the thermoequilibrated chamber and stir.

monitoring release of neurotransmitter

3. Unless added previously, add Ca2+or EGTA to final concentrations of 1.3 m M or 0.5- 1 m M respectively. 4. Add NADP and glutamate dehydrogenase, switch on the recorder and wait for a flat baseline (it can take 1 to 5 min). 5. Trigger exocytosis by adding KCI to a final concentration of 30-50 mM. Record for 5-10 min.

6. Add glutamate as internal standard for calibration (10 nmols is adequate, see Sanchez-Prieto et al., 1987a, for discussion of linearity and evaluation). The table below shows the amounts and final concentrations of the additions listed above using sample volumes of 1 and 1.6 ml of synaptosomal suspension:

Isolated Nerve Terminals as a Model System for the Study of Botulinum and Tetanus Toxins

CaCI2 (0.05 M) EGTA (100 mM) GDH NADP (100 mM) KCI (2 M) 1-Glutamate (1 mM)

volume 1 ml

volume 1.6ml

final concentration

2.4 pI 10 pI

1.3m M 1 mM

50 U 10 pI

3.8 pI 16 pl 80 U 16 p1

25 p1

40 p1

50 m M

5-10 pI

5- 10 pI

1 mM

Notes

1. Higher amounts of synaptosome protein can be used if required

calibrate for changes in amount of protein

(e.g., for subsequent subfractionation) but maintaining the protein concentration (see table). When high amounts of synaptosomes are used, a glutamate calibration curve should be performed for a proper calculation of the amount of neurotransmitter released (Nicholls et al., 1987).

2. Make sure that the stirring bar does not interfere with the light path. Red stirring bars may be useful to reduce noise due to light scattering (Nicholls et a/., 1987).

3. As discussed by Nicholls eta/. (1987)depolarizing agents cause an

correct for non-exocytotic release

increase of glutamate release in the absence of Ca2+,which is due to non-exocytotic release from the cytoplasm. The reason for this is that depolarization changes the equilibrium of the glutamate transporter in the plasma membrane, resulting in net efflux instead of uptake (see Fig. 3). To determine the exocytotic portion of release, appropriate corrections are required. The net Ca2+dependent component of release is calculated as the difference between the Ca2+ -dependent and Ca2+-independent (EGTA) releases following depolarization (see Nicholls et a/., 1987 for details).

Troubleshooting Problems can occur at different levels. If no release can be measured, check first whether the assay mixture is active by adding glutamate standard (in this case, the reason may be an inactive batch of glutamate dehydrogenase). If the glutamate standard gives a signal, start a new sample and add a small aliquot of detergent (e.g., Triton X-100 at a final concentration of 0.1 %) after adding all other components and after having obtained a stable baseline. Detergent will release all internal glutamate (and should give a large signal). This ensures that your synaptosomes are capable of storing glutamate against a concentration gradient. If this test is positive, then it is likely that your batch of synaptosomes is inactive. When setting up the system, it is therefore advisable to practice by preparing P2 fractions from single animals before proceeding to the Ficoll gradient method.

J. BLASI, E. LINK and R. JAHN

15.4.5 Poisoning of Synaptosomes Although all toxins will block neurotransmitter release from synaptosomes, their potency varies depending on which toxin type is being used (McMahon et al., 1992, Blasi et al., 1993a, 1993b) and on the source and quality of the toxin. The differences between the individual toxins may be due to differences in receptor binding, uptake by the nerve terminal, and potency of the L chain towards its intraterminal target. When poisoning synaptosomes, a compromise between toxin concentration and incubation time must be made. Synaptosomes lose activity when kept at 37°C in a stirred cell but some time is required for the toxins to be endocytosed and to cleave their intracellular targets. We usually use preincubation times of 60 min, with control (non-poisoned) samples always run in parallel. Up to this time, the release activity of the control samples is more than 80 % of the samples that are measured immediately after resuspension. An example of glutamate recording is shown in Figure 3. In this particular case,

-

NT/C1 100 nM

E 0.021 c

-

0.00

0

rnin.

2

Fig. 3. Release of glutamate from rat brain synaptosomes with 50 mM KCI depolarization using the fluorometric assay described in the text. Ca2+-dependentglutamate release was inhibited by pre-incubating the synaptosomes with active BoNT/Cl (100 nM) for 90min at 37°C Isolated Nerve Terminals as a Model System for the Study of Botulinum and Tetanus Toxins

synaptosomes were incubated in the presence of 100 n M BoNT/Cl. Heat inactivated BoNT/Cl was used as control. The standard has been omitted in the Figure.

1. Resuspend synaptosomes in prewarmed (37°C) assay buffer (solution 4). Neurotoxins are added at this time. Ca2+or EGTA may be also included at this step or, alternatively, added just before measuring glutamate release. Our standard procedure consists of resuspending 1 mg of the synaptosomal pellet in 1 ml assay buffer, but you may decrease the initial volume down to 200 pl, to save the amount of toxin you have to add. In this case, add 800 pl of pre-warmed assay buffer before measuring glutamate release. Incubate at 37°C with constant vigorous stirring. Incubation may last from 15 to 90 min (normally 60 min). Prepare also a control sample without any toxins, to check the activity of the synaptosomes.

2. Follow the protocol above for monitoring neurotransmitter release, starting at step 2 (Section 15.4.4). Notes No reducing agent should be added, to avoid reduction of the neurotoxins (di-chain form required). The final toxin concentration varies depending on the type and source of neurotoxin. In our hands, 5 n M TeTx efficiently blocks glutamate release (Link et al., 1992) whereas 100-150 n M of BoNT/A is required for maximal inhibition (maximum only 70-80 % inhibition, in contrast to TeTx and BoNT/Cl, D, F, G) with BoNT/D being the most potent of the botulinum neurotoxins (Sanchez-Prieto et al., 1992, Blasi et al., 1993a, 1993b, Yamasaki et al. 1994a). Troubleshooting If you d o not detect any inhibition of the transmitter release from the poisoned synaptosomes check the activity of the neurotoxins using one of the following methods:

1. Bioassay. This assay is sensitive, direct and reliable but it requires the sacrifice of many laboratory mice. Various dilutions of the neurotoxin are injected intraperitoneally into mice. The amount of toxin that kills half a population of mice in four days is calculated (LD,,). Toxin can also be injected intravenously. The delay time between injection and death can then be used as a parameter to evaluate the potency of the toxin, allowing calculation of the LDS0(Boroff and Fleck, 1966).

2. In viiro Assay of 1 Chain Activity. Since the elucidation of the mocheck proteolytic activity

J. BLASI. E. LlNKand R. JAHN

lecular mechanisms of all clostridial neurotoxins, it has become convenient to test the proteolytic activity of the L chain in vitro. For this purpose, L chain or reduced di-chain toxin is incubated with substrate protein. The cleavage products are then separated by SDS-PAGE and analyzed by immunoblotting or autoradiography.

This procedure is the most direct and simple way to test toxin activity. It should be borne in mind, however, that it does not give any information about H chain-related activities (binding, internalization and sorting). Synaptobrevin, SNAP-25, and syntaxin are all highly abundant membrane proteins in the mammalian CNS. Thus, a sufficiently enriched membrane preparation can be obtained by crude subfractionation techniques. While this approach is convenient, particularly for laboratories with no expertise in molecular techniques, it has a number of shortcomings due to the inherent property of the proteins to form toxin-resistant complexes (Hayashi et.al., 1994). A procedure based on a crude tissue extract is given below. Better results can be achieved when using more highly purified subcellular fractions, e.g., synaptic vesicle fractions, for the assay of synaptobrevin-cleaving toxins.

use of crude tissue extract

Analysis of 1 Chain Activity Using Tissue Extracts as a Source of Substrate Protein

1. The simplest method is to use freshly made postnuclear supernatant from a brain homogenate (spin a crude brain homogenate for 10 min at 1000 g and take the supernatant). Determine protein concentration and prepare 15 to 20 pg of protein aliquots in small volumes (10-15 PI), using buffered saline for dilution

2. Add DTT (1 M stock solution) to a final concentration of 10 m M 3. Add di-chain toxin and incubate for 1 h at 37°C 4. Add SDS-sample buffer and boil the sample for 3-5 min.

5. Analyze samples by SDS-PAGE and immunoblotting. Since in crude extracts toxin-generated fragments are difficult to detect (exception: SNAP-25), activity is measured by the reduction in the amount of substrate protein. Notes

1. To control for nonspecific proteolysis, parallel incubations should

be performed in which toxin is omitted or heat-inactivated before addition.

2. Do not expect complete degradation. It has been shown that synaptobrevin, SNAP-25, and syntaxin spontaneously form complexes that are toxin-resistant (Hayashi et al., 1994; Pellegrini et al., 1994). Therefore use fresh material and avoid prolonged storage, particularly in detergents. Addition of small amounts of Triton X-100 (0.05-0.5 % final concentration) immediately before the assay, however, improves accessibility of the substrates. Exception: Syntaxin cleavage by BoNT/Cl. Here, detergents should be kept below 0.1 % final concentration or be avoided altogether (Blasi et al., 1993b).

controls

incomplete degradation

Isolated Nerve Terminals as a Model System for the Study of Botulinum and Tetanus Toxins

3. Mg-ATP can be used to disaggregate protein complexes (Otto et al., 1995; Hayashi et al., 1995) so as to obtain higher amounts of protein cleavage (Pellegrini et al., 1994).

4. DTT does not need to be added when the L chain of toxin is used instead of the di-chain form. However, toxin in its di-chain form should be reduced before the assay in order to liberate the L chain. This can be done by incubating the toxin for 30min at 37°C in a 10 m M DTT containing buffer.

5. If there is any problem with protein detection, enriched synaptic protein preparations (synaptosomes, presynaptic plasma membrane of synaptic vesicle preparations, depending of the toxin and the substrate) can be better starting material than brain homogenate to check the proteolytic activity of clostridial neurotoxins L chains.

recombinant proteins

Alternatively, recombinant proteins, expressed in vitro or in bacteria, can be used as substrates. This approach has many advantages since there is no interference with other proteins as in a crude extract, the assays are more sensitive and a variety of different techniques can be used to analyze the proteins after SDS-PAGE ( e.g., protein staining or autoradioraphy when labeled substrates are used). Note that it remains important to check for nonspecific proteolysis by appropriate control incubations. One of the fastest (but not necessarily cheapest) methods is to generate the substrate protein by in vitro translation in the presence of radioactive amino acids, incubate with toxin, and then analyze the products by SDS-PAGE and autoradiography (see e.g., Blasi et al., 1993a, 1993b, Binz et al., 1994; Yamasaki et al., 1994). With recombinant synaptobrevin and SNAP-25, quantitative cleavage can be achieved in solution, and the transmembrane domain of synaptobrevin is not required for toxin cleavage. However, syntaxin cleavage by BoNTK1 requires special conditions. Syntaxin must be anchored to the membrane via its transmembrane domain in order to be proteolyzed. This can be achieved by incorporating the protein post-translationally into liposomes or by adding microsomes (e.g., canine pancreatic microsomes) during in vitro translation (Blasi et al., 199313, Schiavo et al., 1995). Even under these conditions, cleavage is usually not quantitative. It is hoped that a more convenient assay for BoNTKl, based on recombinant syntaxin, will become available as soon as the cleavage conditions can be better defined.

15.4.6 Subfractionation of Synaptosomes Synaptosomes can be subfractionated into a heavy membrane fraction that contains plasma membranes, synaptic vesicle clusters and most of the contaminating membranes, a light membrane fraction that is enriched in synaptic vesicles and devoid of measurable contamination by mitochondria or neuronal plasma membranes, and a J. BLASI, E. LINK and R. JAHN

cytosolic fraction (Fischer von Mollard et al., 1991).Subfractionation is useful if different subcellular pools of the substrate proteins are to be analyzed (keep in mind, however, that the toxins may be active during fractionation, causing further breakdown of substrate proteins after osmotic lysis). The procedure is as follows:

1. At the end of the photometric recording, synaptosomes are chilled on ice and centrifuged at 4°C for 10 min at 12 000 g. The pellet is resuspended in 300 PI of ice-cold assay buffer.

2. To osmotically lyse the synaptosomes, 2.7 ml of ice-cold distilled water are added, followed by rapid homogenization in a glass teflon homogenizer (6 strokes at 2000 r.p.m).

3. The sample is centrifugued at 12 000 g for 10 min at 4°C to yield the heavy membrane fraction.

4. The remaining supernatant is centrifugued at 100 000 g for 1 h (shorter times may be used if small rotors, e.g., Beckman Tla 100.3, are used) yielding a pellet enriched in synaptic vesicles, and a cytosol fraction in the supernatant. Breakdown of the substrates can be checked by SDS-PAGE and immunoblotting. Monoclonal antibodies for syntaxin 1a and 1 b, as well as for SNAP-25 are available from commercial sources (Sigma, Sternberger Monoclonals). Antibodies for synaptobrevin have been raised by many laboratories and can usually be obtained from one of them.

15.5 Relevant Chemicals Material

Supplier

Cat-No.

Ficoll 400 L-Glutamic Dehydrogenase (GDH) (EC 1.4.1.3) L-Glutamic Acid NADP

Pharmacia Biotech Sigma

G 2626

Sigma Boehringer Mannheim

17-0400-01

G 1626 128040

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Bakry N, Kamata Y, Simpson LL (1991):Lectins from Triticum vulgaris and Limax flavus are universal antagonists of botulinum neurotoxin and tetanus toxin. J. Pharmacol. Exp. lher. 258: 830-6. Binz T, Blasi J, Yamasaki S et a/. (1994):Proteolysis of SNAP-25 by types E and A batulinal neurotoxins. J. Biol. Chem. 269: 1617-20. Bittner M A , Holz RW (1988):Effects of tetanus toxin on catecholamine release from intact and digitonin-permeabilized chromaffin cells. J. Neurochem. 51 : 451 -6. Blasi J, Chapman ER, Link E et a/. (1993~)Botulinum neurotoxin A selectively cleaves the synaptic protein SNAP-25. Nature 365: 160-3. Blasi J, Chapman ER, Yamasaki S etal. (1993):Botulinum neurotoxin C1 blocks neurotransmitterrelease by means of cleaving HPC-l/syntaxin. EMBOJ. 12: 4821 -8. Boroff DA, Fleck U (1966):Statistical analysis of a rapid ”in vivo” method for the titration of the toxin of Clostridium botulinum. J. Bacteriol. 92: 1580-1. Bradford M.M. (1976):A rapid and sensitive method for the quantification of micrograms quantities of protein utilizing the principe of protein-dye binding. Anal. Biochem. 72: 248-54. Cabot JB, Mennone A, Bogan N etal. (1991)Retrograde, trans-synaptic and transneuronal transport of fragment C of tetanus toxin by synpathetic preganglionic neurons. Neuroscience. 40: 805-23. Dayanithi G, Weller U, Ahnert-Hilger G etal. (1992):The light chain of tetanus toxin inhibits calcium-dependent vasopressin release from permeabilized nerve endings. Neuroscience 46: 489-93. DasGupta BR, editor (1993):Botulinum and tetanus neurotoxins. Plenum Press. New York. Davis LE ( 1993): Botulinum toxin. From poison to medicine. Western Journal of Medicine. 158: 25-29. Dickson EC, Shevky R (1923):Botulism, studies on the manner in which the toxin of Clostridium botulinum acts upon the body. I. The effect upon the autonomic nervous system. J. Exp. Med. 37: 711 -31 Dolly JO, Black J, Williams RS et a/. (1984):Acceptors for botulinum neurotoxin reside on motor nerve terminals and me4diate its internalization. Nature. 307: 457-60. Dreyer F. (1989): Peripheral action of tetanus toxin. In: Botulinum Neurotoxin and Tetanus Toxin, (Simpsan LL, ed) pp 179-202. Sun Diego: Academic Press. Eccles RM, Libet B (1961):Origin and blockade of the synaptic responses of curarized sympathetic ganglia. J. Physiol (Lond) 157: 484-503. Ferro-Novick S, Jahn R (1994): Vesicle fusion from yeast to man. Nature 370:

191- 193. Fischer von Mollard G, Sudhof TC , Jahn R (1991):A small GTP-binding protein dissociates from synaptic vesicles during exocytosis. Nature. 349: 79-81. Foran P, Lawrence GW, Shone CC et al. (1996):Botulinum neurotoxin C1 cleaves both syntaxin and SNAP-25 in intact and permeabilized chromaffin cells: correlation with its blockade of catecholamine release. Biochemistry 35: 2630-6. Frangez R, Dolinsek J, Demsar F etal. (1994):Chronic denervation caused by botulinum neurotoxin as a model of a neuromuscular disease.Annals of the NewYork Academy of Sciences. 710: 88-93. Gaisano HY, Sheu L, Foskett JK et a/. (1994):Tetanus toxin light chain cleaves a vesicle-associated membrane protein (VAMP) isiform 2 in rat pancreatic zymogen granules and inhibits enzyme secretion. J. Biol. Chem. 269: 17062-6. Galli T, Chilcote T, Mundigl 0 et a/. (1994):Tetanus toxin-mediated cleavage of cellubrevin impairs exocytosis of transferrin receptor-containing vesicles in CHO cells. J. Cell Biol. 125: 1015-24. Gansel M, Penner R, Dreyer F (1987):Distinct sites of action of clostridial neurotoxins revealed by double-poisoning of mouse motor nerve terminals. Pflugers Arch. Eur. J. Physiol. 409: 533-9. Gray EG, Whittaker VP (1962): The isolation of nerve endings from brain: an electron-microscopic study of cell fragments derived by homogeneization and centrifugation.J. Anat. Lond. 96: 79-88. Habermann E, Dreyer F (1986):Clostridial neurotoxins: handling and action at the cellular and molecular level. Current Topics Microbiol. Immunol. 129: 93- 179.

J. BLASI, E. LINK and R. J A H N

Habermann E, Muller H, Hudel M (1988): Tetanus toxin and botulinum A and C neurotoxins inhibit noradrenaline release from cultured mouse brain. J. Neurochem. 51 : 522-7. Hayashi T, McMahon H, Yamasaki S et a/. (1994): Synaptic vesicle membrane fusion complex: action of clostridial neurotoxins on assembly. EMBO J. 13: 5051-

61. HayashiT, Yamasaki S, Nauenburg S etal. (1995):Disassembly of the reconstituted synaptic vesicle membrane fusion complex in vitro. E M 6 0 J. 14: 2317-25. Hughes AJ (1994): Botulinum toxin in clinical practice. Drugs. 48: 888-93. Hunt JM, Bommert K, Charlton MP et al. (1994):A Post-docking role for synaptobrevin in synaptic vesicle fusion. Neuron. 12: 1269-79. lkonen E, Tagaya M, Ullrich 0 et a/. (1995): Different requirements for NSF, SNAP, and rub proteins in apical and basolateral transport in MDCK cells. Cell. 81: 571 -50. Jankovic J (1994):Botulinum toxin in movement disorders. Current Opinion in Neurology. 7: 358-66. Kistner A, Habermann E (1992):Reductive cleavage of tetanus toxin and botulinum neurotoxin A by the thioredoxin system from brain. Evidence for two redox isomers of tetanus toxin. Naunyn. Schmiedebergs Arch. fharmacol. 345: 227-34 Link E, Edelmann L, Chou et a/. (1992): Tetanus toxin action: inhibition of neurotransmitter release linked to synaptobrevin proteolysis. Biochem. Biophys. Res. Comm. 189: 1017-23. Link E, McMahon H, Fischer von Mollard G et a/. (1993): Cleavage of cellubrevin by tetanus toxin does not affect fusion of early endosomes. J. Biol. Chem. 268: 18423-6. Link E, Blasi J, Chapman ER etal. (1994):Tetanus and botulinal neurotoxins: tools to understand exocytosis in neurons. Advances in Second Messenger and fhosphoprotein Research 29: 47-58. Llinas R, Sugimori M, Chu D etal. (1994):Transmission at the squid giant synapse was blocked by tetanus toxin by affecting synaptobrevin, a vesicle-bound protein. J. fhysiol. (London) 477.1 : 129-33. Manning KA, Erichsen JT, Evinger C (1990): Retrograde transneuronal transport properties of fragment C of tetanus toxin. Neuroscience. 34: 251 -63 Marxen P, Bigalke H (1989):Tetanus toxin: inhibitory action in chromaffin cells is initiated by specified types of gangliosides and promoted in low ionic strength solution. Neurosci. Lett. 107: 261 -6. McMahon HT, Rosenthal L, Meldolesi J et al. (1990): a-Latrotoxin releases both vesicular and cytoplasmic glutamate from isolated nerve terminals. J. Neurochem. 55: 2039-47. McMahon HT, Foran P, Dolly JO et a/. (1992):Tetanus toxin and botulinum toxins type A and B inhibit gltamate, gamma-aminobutyric acid, aspartate, and metenkephalin release from synaptosomes. Clues to the focus of action. J. 6/01. Chem. 267: 21338-43. McMahon HT, Ushkaryov YA, Edelmann L et al. (1993): Cellubrevin is a ibiquitous tetanus-toxin substrate homologous to a putative synaptic vesicle fusion protein. Nature. 364: 346-9. Middlebrook JL. (1989): Cell surface receptors for protein toxins. In Botulinum neurotoxin and tetanus toxin ( Simpson LL, ed) Sun Diego: Academic Press. Mochida S, Poulain B, Weller U etal.. (1989): Light chain of tetanus toxin intracellularly inhibits acetylcholine release at neuro-neuronal synapses, and its internalization is mediated by heavy chain. FEBS Lett. 253: 47-51. Mochida S, Poulain B, Eisel U et a/. (1990):Exogenous mRNA encoding tetanus or botulinum neurotoxins expressed in Aplysia neurons. froc. Acad. Sci USA. 87: 7844-8) Mochida S, Saisu H, Kobayashi H etal. (1995):Impairment of synatxin by botulinum neurotoxin C, or antibodies inhibits acetylcholine release but not Ca2+channel activity. Neuroscience. 65: 905-15. Molgo J, Lemeignan M, Thesleff S (1987): Aminoglycosides and 3,4-diaminopyridine on neuromuscular block caused by botulinum type A toxin. Muscle Nerve. 10: 464-70. Isolated Nerve Terminals as a Model System for the Study of Botulinum and Tetanus Toxins

Molgo J, Thesleff S (1984):Studies on the mode of action of botulinum toxin type A at the frog neuromuscular junction. Brain Res. 297: 309-16. Montecucco C (1986): How do tetanus and botulinum toxins bind to neuronal membranes? Trends Biochem Sci. 11: 314-7. Montecucco C, Schiavo G (1993):Tetanus and botulism neurotoxins: a new group of zinc proteases. Trends Biochem. Sci. 18: 324-7. Montecucco C, Schiavo G. (1994):Mechanism of action of tetanus and botulinum neurotoxins. Mol Microbiol. 13: 1-8. Nicholls D.G. (1978): Calcium transport and proton electrochemical potential potential gradient in mitochondria from guinea-pig cerebral cortex and rat heart. Biochem. J. 170: 511 -22. Nicholls DG, Sihra TS (1986): Synaptosomes posses and exocytotic pool of glutamate. Nature 321 : 772-3. Nicholls DG, Sihra TS, Snachez-Prieto J (1987): Calcium-dependent and -independent release of glutamate from synaptosomes monitored by continuous fluorometry. J. Neurochem. 49: 50-7. Nicholls DG. (1989): Release of glutamate, aspartate, and y-aminobutyric acid from isolated nerve terminals. J. Neurochem. 52: 331-41. Niemann H (1991):Molecular biology of clostridial neurotoxins. In: Sourcebook of Bacterial Toxins (Alouf JE, Freer JH, ed) pp303-48, New York: Academic Press. Niemann H, Blasi J, Jahn R (1994):Clostridial neurotoxins: new tools for dissecting exocytosis. Trends Cell Biol. 4: 179-85. Nishiki T, Kamata Y, Nemoto Y et al. (1994): Identification of protein receptor for clostridium botulinum type B neurotoxin in rat brain synaptosomes.J. Biol. Chem. 269: 10498-503. Otto H, Hanson PI, Chapman ER et al. (1995):Poisoning by botulinum neurotoxinA does not inhibit formation or disassembly of the synaptosomal fusion complex. Biochem. Biophys. Res, Commun. 212: 945-52. Pellegrini LL, O'Connor V, Betz H. (1994): Fusion complex formation protects synaptobrevin against proteolysis by tetanus toxin light chain. FEBS Lett. 353: 319- 23. Pellegrini LL, Oconnor V, Lottspeich F et al. (1995):Clostridial neurotoxins compromise the stability of a low-energy SNARE complex mediating NSF activation of synaptic vesicle fusion EM60 J. 14: 4705-13. Penner R, Neher E, Dreyer F (1986): Intracellularly injected tetanus toxin inhibits exocytosis in bovine adrenal chromaffin cells. Nature 324: 76-8. Poulain B, Baux G, Tauc L (1986):Presynaptic transmitter content controls the number of quanta released at the neuro-neuronal cholinergic synapse froc. Natl. Acad. Sci. USA 83: 170-3. Poulain B, Mochida S, Wadsworth JD et al. (1990): Inhibition of neurotransmitter release by botulinum neurotoxins and tetanus toxin at Aplysia synapses: role of the constituent chains. J. fhysiol. (Paris) 84: 247-61. Poulain B, Wadsworth JDF, Maisey EA et al. (1989):Inhibition of transmitter release by botulinum neurotoxin A. Eur. J. Biochem. 185: 197-203. Ralston E, Beushausen S, Ploug T. (1994):Expression of the synaptic vesicle proteins VAMPs/synaptobrevins 1 and 2 in non-neural tissues. J. Biol. ,Chem. 269: 15403-6. Sadoul K, tang J, Montecucco C et al. (1995):SNAP-25 is expressed in isletd of Langerhans and is involved in insulin release J. Cell Biol. 128: 1019-28. Sanchez-Prieto J, Sihra TS, Nicholls DG. (1987~): Characterization of the exocytotic release of glutamate from guinea pig cerebral cortical synaptosomes. J. Neurochem. 49: 58-64. Sanchez-Prieto J, Sihra TS, Evans D et al. (198713):Botulinum toxin A blocks glutamate exocytosis from guinea-pig cerebral cortical synaptosomes. Eur. J. Biochem. 165: 675-81. Schiavo G, Benfenati F, Poulain B etal. (1992):Tetanus and botulinum-B neurotoxins block neurotransmitterrelease by proteolytic cleavage of synaptobrevin. Nature 359: 832-5. J. BLASI, E. LINK and R. JAHN

Schiavo G, Rossetto 0, Catsicas S et a/. (1993~): Identification of the nerve terminal targets of botulinum neurotoxin serotype-A, serotype-D, and serotype-E. J. Biol. Chem. 268: 23784-7. Schiavo G, Santucci A, DasGupta BR et a/. (199313): Botulinum neurotoxins serotypes A and E cleave SNAP-25 at distinct COOH-terminal peptide bonds. FEBS Lett 335: 99-103. Schiavo G, Shone CC, Rossetto 0 et a/. (1993~):Botulinum neurotoxin serotype F is a zinc endopeptidase specific for VAMP/synaptobrevin. J. Biol. Chem. 268: 11516-9. Schiavo G, Shone CC, Bennett MK et a/. (1995): Botulinum neurotoxin type C cleaves a single Lys-Ala bond within the carboxyl-terminal region of syntaxins. J. Biol. Chem. 270: 10566-70. Simpson LL (ed) (1989): Botulinum neurotoxin and tetanus toxin. Sun Diego: Academic Press. Simpson LL, Lake P, Kozaki S (1990): Isolation and characterization of a novel human monoclonal antibody that neutralizes tetanus toxin. J. Pharmacol. Exp. Jher. 254: 98-103. Stahl B, Fischer von Mollard G, Walch-Solimena C et a/. (1994):GTP-cleavage by the small GTP-binding protein Rab3A is associated with exocytosis of synaptic vesicles induced by a-latrotoxin. J. Biol. Chem. 269: 24770-6. Stahl B, Chou JH, Li C et a/. (1996): Rub3 reversibly recruits rabphilin to synaptic vesicles by a mechanism analogous to raf recruitment by ras. EM60 J. 15: 1799-809. Steinhardt RA, Bi G, Alderton JM (1994): Cell membrane resealing by a vesicular mechanism similar to neurotransmitter release. Science 263: 390-3. Sweeney ST, Broadie K, Keane J etal. (1995):Targeted expression of tetanus toxin light chain in Drosophila Specifically eliminates synaptic transmission and causes behavioral defects. Neuron. 14: 341 -51. Tauc L, Hoffmann A, Tsuiii S et a/. (1974): Transmission abolished on a cholinergic synapse after injection of AChE into the presynaptic neuron. Nature. 250: 496-8. Tibbs GR, Barrie AP, Van Mieghem FJE et a/. (1989): Repetitive action potentials in isolated nerve terminals in the presence of 4-aminopyridine: effecs on cytosolic free Ca2+ and glutamate release. J. Neurochem. 53: 1693-9. Turner TJ, Dunlap K (1995):Prolonged time course of glutamate release from nerve terminals: relationship between stimulus duration and the secretory event. J. Neurochem. 64: 2022-33 Volchuk A, Mitsumoto V, He L et a/. (1994): Expression of veicle-associated membrane protein 2 (VAMP-2)/synaptobrevin II and celubrevin in rat skeletal muscle and in a muscle cell line. Biochem. J. 304: 139-45. Whittaker VP, Michaelson IA, Kirklan RJA (1964):The separation of synaptic vesicles from nerve-endings particles ("synaptosomes"). Biochem J. 90: 293-303. Williamson LC, Halpern JL, Montecucco C etal. (1996):Clostridial neurotoxins and substrate proteolysis in intact neurons. Botulinum neurotoxin C acts on synaptosomal-associated protein of 25 kDa. J. Biol. Chem. 271 : 7694-9. Yamasaki S, Baumeister A et a/. (1994~): Cleavage of members of the synaptobrevin/VAMP family by types D and F botulinal neurotoxins and tetanus toxin. J. &o/. Chem. 269: 12764-72. Yamasaki S, Binz T, Hayashi T et a/. (199413): Botulinum neurotoxin type G proteolyses the AIa8'-AlaE2bond of rat synaptobrevin 2. Biochem. Biophys. Res. Comm. 200: 829-5.

Isolated Nerve Terminals as a Model System for the Study of Botulinum and Tetanus Toxins

Bacterial Toxins

Klaus Aktories Copyright 0 2002 WILEY-VCH Verlag GmbH & Co. KGaA

CHAPTER 16 Clostridial Toxins and Endocrine Secretion: Their Use in Insulin-Secreting Cells J. LANG, R. REGAZZI and C. B. WOLLHEIM

16.1 Exocytosis of Insulin from the Pancreatic P-cell 16.1.1 Introduction Blood glucose concentrations are tightly regulated in the healthy individual. This is accomplished through the fine tuning of insulin secretion from the (3-cells in the pancreatic islets and the subsequent action of insulin on its target tissues. Failure of the (3-cells to release insulin promptly during food intake, as well as resistance to the action of the hormone, lead to various forms of diabetes mellitus (Polonsky, 1995, Turner et al., 1995). Insulin is stored in crystals in secretory vesicles (secretory granules), of which only a small proportion is liberated even under maximal stimulation. The secretory process, rather than the biosynthesis of the hormone, therefore constitutes the primary determinant of the concentration of insulin in the circulation. The secretion rate is the result of the dynamic interactions of a large number of stimulatory and inhibitory modulators. Glucose and leucine, as well as certain other nutrients, are initiators of insulin secretion, not requiring the presence of other stimuli (Prentki and Matschinsky, 1987). The metabolism of the nutrient secretagogues generates signals that trigger insulin secretion. In contrast, receptor agonists such as acetylcholine and gastrointestinal hormones (GLP-1 and GIP) potentiate insulin secretion. The secretion is potently inhibited by sympathetic neuronal activity, by epinephrine and by other neurohormones (Prentki and Matschinsky, 1987, Wollheim and Sharp, 1981).The p-cell contains a large number of insulin-containing secretory granules, averaging 10000 per cell (Orci, 1985).These granules correspond to the peptide-carrying large dense core vesicles (LDCV)of chromaffin cells and of neurons (De-Camilli and Rahn, 1990).Like other endocrine cells, the B-cell and derived cell lines also possess smaller, synaptic-like microvesicles (SLMV). The latter have been shown to contain y-aminobutyric acid (GABA). Their localization differs from that of the secretory granules in that the SLMVs are mostly, but not exclusively, found close to the Golgi complex (Reetz et al., 1991). The role of the SLMVs in the islet cell is unknown, although a paracrine function for GABA has been proposed through which

control of blood glucose by insulin

K. Aktories (Ed.),Bacterial Toxins. 0Chapman & Hall, Weinheim, 1997. ISBN 3-8261-0080-8

glucagon secretion from the a-cells in the islet could be inhibited (Sorenson et al., 1991).

16.1.2 Regulated Exocytosis All mammalian cells display constitutive secretion, which is thought to

stimulation of insulin secretion

be regulated at the stage of the budding of microvesicles from the trans-Golgi network (Burgess and Kelly, 1987). In addition, the specialized secretory cells exhibit regulated secretion. The docking of secretory vesicles on, and their fusion with, the plasma membrane are processes that appear to share many features with intracellular vesicle fusion (Scheller, 1995). The control over regulated exocytosis is exerted through second messenger pathways, in particular those implicating Ca2+and CAMP. Glucose and leucine stimulate insulin secretion by raising the concentration of cytosolic Ca2+ ([Ca'+],) in the b-cell and by providing metabolic coupling factors (Gembal et al., 1993)that may amplify the action of Ca2+on insulin exocytosis. The [Ca'+], rise is due to Ca2+ influx through voltage-sensitive Ca2+channels, mainly of the L-type, which open as a consequence of membrane depolarization (Bokvist et al., 1995).Glucose and leucine cause membrane depolarization by closure of ATP-sensitive K+ channels subsequent to the stimulation of oxidative metabolism and an increase in the ATP/ADP ratio in the cytosol (Ashcroft and Rorsman, 1989). The precise nature of the coupling factors that enhance the action of Ca2+in nutrient-evoked exocytosis remains obscure. Of the potentiating agents of insulin secretion, acetylcholine and cholecystokinin stimulate phospholipase C, thereby promoting Ca2+mobilization (and influx) and activation of protein kinase C (Wollheim and Regazzi, 1990).GLP-1 and GIP, on the other hand, generate cAMP and activate protein kinase A, as a consequence of the stimulation of adenylyl cyclase (Widmann et al., 1994). In electrically permeabilized insulin-secreting cells, the influence of soluble second messengers on exocytosis can be studied directly, since it is possible to dialyze such cells with respect to nucleotides and ions while cytosolic proteins are retained. In the presence of ATP, Ca2+stimulates insulin exocytosis with an ECS0of approximately 1.6 pM (Vallar etal., 1987, Ullrich etal., 1990).This is in close agreement with the value for Ca2+-stimulated exocytosis in patch-clamped mouse p-cells obtained using the capacitance method (Bokvist et al., 1995). In contrast to, cAMP is unable to stimulate exocytosis on its own, but potentiates Ca2+-inducedexocytosis (Vallar et al., 1987).This is consistent with the role of CAMP-generating hormones as potentiators of insulin secretion. The mechanism by which epinephrine and other neurohormones inhibit insulin secretion involves pertussis toxin-sensitive G-proteins. These hormones exert multiple actions, all contributing to the marked reduction of stimulated insulin secretion (Ullrich and Wollheim, 1988, Lang et al., 1993).They thus inhibit adenylyl cyclase activity, hyperpolarize the membrane potential by increasing K+ conductance, and

J. LANG, R. REGAZZI and C. B. WOLLHEIM

promote closure of voltage-sensitive channels. However, we have clearly demonstrated by various approaches in permeabilized cells that an overriding inhibitory influence on exocytosis is directly exerted by the activation of Gi and Goproteins (Lang et al., 1995). It has been suggested that stimulators of exocytosis cause a remodeling of the microfilamentous cell web, which could act as a barrier, interfering with the access of the secretory granules to the plasma membrane (Burgoyne, 1990). There is, however, no evidence in insulin-secreting cells that Ca2+and/or CAMPare capable of changing the arrangement of actin filaments. We found that drastic reduction of F-actin following treatment of HIT-T15 cells or rat pancreatic islets with C. botulinum C2 exotoxin mainly affects the recruitment of secretory granules to the plasma membrane (Li et al., 1994). It is unlikely, therefore, that stimulators or inhibitors of insulin exocytosis act primarily on the composition of the cytoskeleton.

16.1.3 The SNARE Hypothesis of Exocytosis As already mentioned, exocytosis has many features in common with intracellular vesicle fusion. Rothman and associates formulated the

SNARE hypothesis (Sollner et al., 1993) for the regulation of neurotransmitter exocytosis, based on the ubiquitous role in fusion of the N-ethylmaleimide-sensitive factor (NSF), and of the soluble NSF attachment proteins (SNAPS).The model suggests that proteins on the surface of secretory vesicles, vesicle SNAP receptors (v-SNARES) pair with proteins at the plasma (target) membrane t-SNARES for the docking of the vesicle. Fusion occurs when the cytosolic proteins aSNAP and NSF bind to the so-called core particle. NSF is an ATPase, and it is thought that the hydrolysis of ATP provides the energy for dissolution of the fusion particle, thereby promoting fusion of the vesicle and plasma membranes. The vesicle associated membrane proteins VAMP-1 and VAMP-2 are the main V-SNARES,while synaptosomal protein of molecular weight 25 kDa (SNAP-25) and members of the syntaxin family function as t-SNARES (Sollner et al., 1993, Scheller, 1995). The definition of the v- and t-SNARES was greatly facilitated by the use of the various clostridial neurotoxins which cleave the SNARE proteins with great selectivity (Niemann et al., 1994).The model further proposes that syntaxin triggers the fusion of the secretory vesicles by binding to the Ca2+-and phospholipidbinding protein synaptotagmin. This protein is a synaptic vesicle membrane protein, of which to date nine isoforms with different binding characteristics for phospholipids and syntaxin have been reported (Sudhof, 1995).The role of synaptotagmin as a Ca2+sensor has been proposed on the basis, among other evidence, of the impairment of the rapid phase of neurotransmitter release in hippocampal neurons of synaptotagmin-1 knock-out mice (Sudhof, 1995). The binding of Ca2+ to synaptotagmin may indeed uncover the receptor for SNAP/NSF on the docked vesicle to promote fusion. This, however, remains to be established. Clostridial Toxins and Endocrine Secretion: Their Use in Insulin-Secreting Cells

Other proteins that regulate exocytosis include n-secl (munc-18) and members of the Rab3 family, a low molecular weight G-protein. In particular, Rab3A and Rab3C have been shown to play a role in neurosecretion (Fischer von Mollard et al., 1994), while Rab3B appears to modulate exocytosis in pituitary cells (Lledo et al., 1993). The Rub3 proteins cycle between a soluble and a vesicle membranebound form, determined by the complex formation with either GDP or GTP which, in turn, is under the influence of several regulatory proteins (Fischer-vonMollard et al., 1994).

16.1.4 Application of the SNARE Hypothesis to Insulin Exocytosis Several of the proteins which, it has been suggested, regulate neurotransmitter release have recently been identified in the (3-cel1, and for a number of them a function in insulin exocytosis has been established. The ubiquitous fusion factor NSF is present in the insulinsecreting cell line HIT-T15. In addition, aSNAP, also present in these cells, appears to be one of the factors in cytosol required for Ca2+triggered insulin exocytosis (Kiraly-Borri et al., 1996). The V-SNARE VAMP-2 was localized to insulin secretory granules (Jacobson et al., 1994). It is also expressed on the SLMVs in the (3-cel1, as demonstrated by its colocalization with synaptophysin, a synaptic vesicle membrane protein (Regazzi et al., 1995).Clostridial neurotoxins have been used to investigate the functional importance of several of the SNARE proteins. As insulin-secreting cells do not express the ganglioside receptors for the clostridial neurotoxins on the cell surface, the toxins were introduced into the cells following cell permeabilization (Regazziet al., 1995, Boyd et al., 1995). Short treatment of the permeabilized cells with tetanus toxin (TeTx) light chain resulted in a dose-related cleavage of VAMP-2 and of cellubrevin which is also expressed both on secretory granules and SLMVs. Both TeTx and preactivated botulinum toxin B (BoNT/B) caused inhibition of Ca2+-stimulatedexocytosis in insulin-secreting cell lines and rat islet cells (Regazzi et al., 1995).At high concentrations of the toxins (50 nM), stimulated exocytosis was abolished. This suggests that VAMP-2 and/or cellubrevin are required for the docking/fusion process of the secretory granules. The t-SNARE SNAP-25 is also expressed in the 0-cell and has been localized mainly to the plasma membrane (Sadoul etal., 1995).SNAP25 was cleaved by treatment of SLO-permeabilized cells with botulinum toxins (BoNT)A or E. The two neurotoxins inhibited Ca2+-induced insulin exocytosis but failed to abolish the process completely (Sadoul et al., 1995). This could be due to the failure of the toxins to cleave SNAP-25 already complexed to other fusion proteins (escaping detection by Western blotting) or to the requirement for SNAP25 at a penultimate step in insulin exocytosis. The other t-SNARE candidate protein, syntaxin, is also expressed in islet cells (Jacobson et a/., 1994). Treatment of digitoninJ. LANG, R. REGAZZI and C. B. WOLLHEIM

permeabilized mouse islets with an antibody against syntaxin has been reported to attenuate Ca2+-stimulatedexocytosis (Martin et al., 1995). In recent studies, we observed complete inhibition of Ca2+mediated insulin exocytosis with BoNTKl, associated with syntaxin cleavage (Lang, Niemann and Wollheim, unpublished observations). Taken together, these results suggest that at least some of the proteins implicated in the regulation of synaptic vesicle fusion in nerve terminals also regulate exocytosis of secretory granules in the @-cell.

16.1.5 Role of Ca2+and Rab3A in Insulin Exocytosis The Ca2+-and phospholipid-binding protein synaptotagmin is also expressed in insulin-secreting cells (Lang and Wollheim, unpublished, Jacobson et al., 1994). It should be noted that the affinity for Ca2+of neurotransmitter exocytosis (Sudhof, 1995)is two orders of magnitude lower than the 1-2 pM Ca2+affinity reported for insulin exocytosis (Bokvist et al., 1995, Vallar et al., 1987, Ullrich et al., 1990).The latter process is similar to the slow secretion of peptide neurotransmitters stored in LDCVs. As with LDCVs, insulin secretory granules are not clustered at active zones, as is the case for synaptic vesicles. The short distance between the N-type Ca2+channels and the synaptic vesicles is thought to explain the high Ca2+concentrations required for the fast synaptic release. The greater distance between L-type Ca2+channels and the LDCVs, as well as insulin secretory granules, allows for buffering of Ca2+following channel gating, and would explain the higher affinity of the slow, endocrine type exocytotic process (Neher and Augustine, 1992). Nonetheless, because of the difference in Ca2+ affinity of its various isoforms (Sudhof, 1995), synaptotagmin could function as Ca2+sensor both in synaptic vesicle and endocrine-type exocytosis. Finally, the involvement of Rab3A in the regulation of insulin exocytosis is suggested by its localization in insulin-containing secretory granules. In addition, transient expression of mutants of Rab3A, which are either GTPase deficient or unable to bind guanine nucleotides, strongly inhibits stimulated insulin exocytosis (Regazzi et al., 1996). This observation agrees well with previous findings in chromaffin cells (Johannes et al., 1994, Holz et al., 1994). The precise interaction between Rab3A and the previously mentioned SNARE proteins remains to be clarified.

Clostridial Toxins and Endocrine Secretion: Their Use in Insulin-Secreting Cells

16.2 Permeabilization of Insulin-secreting Cells 16.2.1 General Remarks

assay of permeabilization

use pre-binding of toxin

In contrast to neuronal cells, a number of toxins, which provide interesting tools for the investigation of cell function, do not cross the cell membrane. Controlled cell permeabilization allows the intracellular application of these molecules in a system which retains the exocytotic response to stimulating or inhibiting agents. This can be achieved by the use either of detergents or of pore-forming toxins (Holz et al., 1992, Ahnert-Hilger et al., 1993). The detergent digitonin solubilizes membranes according to their cholesterol content and is therefore in general suitable for the creation of pores, mainly in the plasma membrane. Although this approach has often been used successfully in chromaffin cells (Holz et al., 1994, Holz et al., 1992), we did not find it a reliable method for insulin-secreting cells (Wollheim and Ullrich, unpublished). An alternative approach has been described, i.e., intracellular application of toxins by electrophoresis (Boyd et al., 1995).This method is feasible for toxins endowed with high biological activity and long half-life, as is the case for botulinum neurotoxins and tetanus toxin. In our hands, pore-forming toxins offer a far more reliable tool than detergents and are relatively easy to handle (Lang et al., 1995, KiralyBorri et al., 1996, Regazzi et al., 1995, Sadoul et al., 1995). As described in detail elsewhere in this volume, the hemolysin streptolysin 0 (SLO) from streptococci (commercially available from List, Sigma, Gibco,) binds as a monomer to cell membranes even at 4"C, and induces pores only at higher temperatures by forming oligomers. The effectiveness of the resulting permeabilization can easily be ascertained by observing dye-uptake by the cells. The major danger of this tool lies in over-permeabilization. Indeed, the streptolysin 0 monomer can enter the cytosol through the plasma membrane pores created by the oligomer and consequently permeabilize intracellular organelles. Theoretically, permeabilization is therefore achieved in the safest way by prebinding the toxin at 4"C, and washing off the unbound toxin. Subsequent pore-formation is achieved by shifting the cells to 37°C. However, this approach requires a relatively high amount of toxin, which may not be freely available. In addition, this procedure prolongs the experiment and can lead to the loss of cytosolic proteins essential for various cellular functions. Indeed, insulin-secreting cells demonstrate a run-down of their exocytotic response to Ca2+over time (Kiraly-Borri et al., 1996). We therefore prefer to perform the entire experiment at 37"C, thereby limiting the use of toxin. The activity of the toxin can be standardized by measuring the hemolysis of rabbit erythrocytes. In practice this is not required, as the permeabilization of the cell under study constitutes the critical parameter. In addition, the susceptibility of cell lines to the action of SLO may vary according to the passage number.

J. LANG, R. REGAZZI and C. B. WOLLHEIM

Cell permeabilization can be performed on cells in suspension or on attached cells. The use of attached cells avoids the need for cell detachment by agents which may alter cellular functions. This is of importance when studying receptor-mediated processes. Furthermore, attached cells do not require a recovery period as is necessary after trypsinization. Unfortunately, only a limited number of cell lines permit cell permeabilization when seeded on normal plastic. As far as the insulin-secreting cell lines are concerned, only the HIT-T15 cells (derived from hamster (3-cells) resist this procedure, whereas others such as RINmSF, INS-1 or primary islet cells tend to detach. Among the various procedures tested to improve cell adherence, only substrata coated with the extracellular matrix from bovine cornea gave good results. These specially coated sterile plastic wells or glass coverslips are commercially available in different forms and sizes (Eldan). Cells should be allowed to reach about 80% confluence, since cell contact generally favours adherence during subsequent manipulations. Another important issue concerns the intracellular buffers used. We have obtained good results with a potassium glutamate/HEPES buffer (see below).Whenever it might be important, the concentrations of free calcium ions have to be carefully controlled by the use of chelators. As the active concentration of free calcium for exocytosis in insulin-secreting cells ranges from 0.1 p M (basal) to 10 p M (maximal stimulatory levels) with an EC50at 2 pM, EGTA is a suitable chelator (Vallar et a/., 1987). The actual concentration of free Ca2+can be measured either by the use of an ion-specific Ca2+-electrode, or can be computed using an appropriate programme. The preparation and use of these electrodes have been described in detail (Baudet eta/., 1994). For obvious reasons they offer the most direct and reliable method to determine levels of free calcium. Although refined programs are available (Fohr et a/., 1993, Brooks and Storey, 1992), (e.g., WINMAXC) the computed values do not completely coincide with those measured by a calcium-specific electrode. However, they often allow calculation of free magnesium and nucleotides. Fluorescent Ca2+-indicatorssuch as Fura-2 are also commonly used to assess levels of free Ca2+.In practice the variations in computed values of free calcium are of minor importance when using only low (0.1pM) and high (10pM) levels of free calcium in insulin-secreting cells, but a considerable error can be introduced at intermediate free calcium concentrations. In addition, certain agents added to the system may in fact behave as calcium chelators and this can obviously only be detected by a calciumspecific electrode. In any case, calcium buffers have to be prepared extremely carefully using standard calcium solutions and paying particular attention to the pH-value of the solution, as the chelating action of EGTA is strongly dependent on the pH.

control of calcium concentration

Clostridial Toxins and Endocrine Secretion: Their Use in Insulin-Secreting Cells

16.2.2 Material Required 0

0

0 0 0

0

Cell culture facility Flat-bottomed 96 microwell plates (specially coated where appropriate) Waterbath at 37°C (equipped with a thermometer) Reliable and controlable water pump for aspiration Microcentrifuge (cooled or placed in a cold-room) Smoothly working pipettes.

16.2.3 Solutions All solutions should be carefully adjusted to the correct pH. For intracellular solutions use ultrapure KOH (Merck) to minimize further addition of calcium.

Krebs-Ringer/HEPES buffer (KRH)without added calcium: NaCl 125 mM; KCI 5 mM; M g S 0 4 2 mM; KH2P04 1.2 mM; HEPES 25 mM; EGTA 0.4 mM; adjust the pH to 74 with NaOH. Store in 20 ml aliquots at -20°C and warm to 37°C before use. This buffer is used to prewash the cells. The actual concentration of free calcium is about 10 ELM. 2x concentrated intracellular buffer (KG): potassium-glutamate 255 mM; NaCl 10 mM; M g S 0 4 14 mM; HEPES 40 mM; adjust the pH to 7.0 with ultrapure KOH (Merck).Store in 20ml aliquots at -20°C. Calcium buffers: 40 m M EGTA; adjust the pH to 7.0 with ultrapure KOH (Merck). CaCI2 100 mM stock solution: use a commercially available standard solution (Merck Ca-Titrisol). CapK5:EGTA 40 mM, CaCI2 40 mM; adjust pH to 7.0 with ultrapure

KOH. CapK7:mix 8.35 volumes of 40 m M EGTA with 1.65 volumes of CapK5; adjust pH to 7.0 with ultrapure KOH.

ATP: Exocytosis is greatly enhanced by the addition of ATP (Na2ATP, Boehringer). Generally 2 to 5 m M of ATP is sufficient. This nucleotide should be added in twice the amount required, just before the experiment, to the 2x concentrated intracellular buffer (KG).ATP will change the pH considerably. Therefore adjust the solution to pH 7.00 with ultrapure KOH (MERCK).ATP is stable in aqueous solution for a few hours when kept on ice. Remember that other nucleotides, like GTP, may also change the pH and the amount of free calcium. Take this into account by carefully adjusting the pH and, if required, introducing the corresponding concentration when computing the concentration of free calcium.

J. LANG, R. REGAZZI and C. 6. WOLLHEIM

16.2.4 Final Working Buffers low Ca2+buffer (LC; approx. 0.1 pM free Ca2+in the presence of 5 m M ATP): 1 volume CapK7+ 2 volumes 2 x KG + 1 volume double distilled water; adjust pH to 7.00 if necessary with ultrapure KOH (Merck).

High Ca2+buffer (HC; approx. 10pM free Ca2+in the presence of 5 m M ATP): 1 volume CapK5+ 2 volumes 2 x KG + 1 volume double distilled water; adjust pH to ZOO if necessary with ultrapure KOH (Merck).

Trypan Blue. Stock solution: 1 % trypan blue in distilled water; filter and store at 4°C. After 2 to 3 weeks precipitates will form which may greatly falsify the interpretation of dye-uptake into the cells. Working solution: 9 volumes LC + 1 volume trypan blue stock solution; prepare just before the experiment and keep at 37°C.

SlO solution. The appropriate amount of SLO (see below) should be dissolved in LC. Take into consideration that some preparations of SLO must be reduced with DTT before use. This is best achieved in a small volume of concentrated SLO solution by adding the appropriate amount of 1 M DTT to produce a final concentration of 50 m M DTT. Incubate the sample for 5 min at 37°C and dilute to the final concentration of SLO. 1 M stock solutions of DTT can be kept in small aliquots at -20°C.

Acid-ethanol for extraction of insulin: This solution is used to extract insulin from cells to determine the total cellular content: 750 ml ethanol + 235 ml H 2 0+ 15 ml concentrated HCI (37%). Keep solution at 4°C. Incubate cells overnight at 4" or -20°C before assaying insulin.

16.2.5 Experimental Outline

76.2.5.7 General Procedure Cells are seeded in a microwell plate 2 to 3 days before the experiment to reach about 80 % confluency on the day of the experiment to favour adherence during subsequent manipulations. Remember that certain cell lines have to be cultured on a special matrix. O n the day of the experiment cells in a microwell plate are transferred to a 37°C waterbath. The temperature should be checked with a thermometer since variations may influence exocytosis. Cells are first washed with KRH, than permeabilized with SLO in 0.1 pM free Ca2+.The cells are exposed to SLO for 7min. Subsequently, cells are preincubated (if necessary) at 0.1 p M Ca2+and then shifted to 0.1 y M or 10pM Ca2+. Finally, the supernatant is transferred to microcentrifuge tubes, centrifuged at 10 000 g for 2min to sediment any contaminating cells; thereafter aliquots are used for determination of the secretory product.

permeabilization Procedure

Clostridial Toxins and Endocrine Secretion: Their Use in Insulin-Secreting Cells

keep exact time protocol

During the experiment it is extremely important to keep an exact time protocol, since the action of calcium is very rapid, and minor variations may change the amount of secreted insulin. When employing 96-well plates, we generally work in rows of 6 wells, using the same conditions for 3 wells. For each row we allocate 50seconds to change the buffers, which allows us to manipulate 8 rows (48 wells in total or 16 different conditions in triplicate). To start with, it may be advisable to use a more relaxed schedule.

76.2.5.2 Determination of SlO Concentrations

assay of permeabilization

The dose-dependence of pore-forming activity for SLO is rather steep. Therefore prepare a series of 1 : 1 dilutions of SLO in LC. As outlined below, wash the cells with KRH, add 50 pI of your SLO dilutions to the wells for 7 min, aspirate and add trypan blue working solution. Leave the solution for 10 min (for reproducibility, the time of incubation with SLO and with trypan blue is important). Check the degree of permeabilization under the light microscope. The correct dilution of SLO is characterized by 100 % of blue cells and minimal loss of cells. Remember that precipitates in the trypan blue stock solution will falsify the result, since the final concentration of trypan blue in your working solution will be considerably lower.

use fresh trypan blue solution

76.2.5.3 Cell Culture Cells are seeded in a flat-bottomed 96-microwell plate 2 to 3 days before the experiment, at a density sufficient to reach about 80% confluence before further manipulation (lo5cells/well for HIT-T15, INS1 or RINm5F).Seed them in 6 to 8 rows, each row having 4 to 6 wells. The methods to culture insulin-secreting cells have been described in detail elsewhere (Asfari eta/., 1992, Wollheim et a/., 1990, Wollheim et a/., 1990).

16.2.6 Cell Permeabilization and Exocytosis Assay

make sure everything is ready

Examine the cells under the microscope. Be sure that all buffers are ready and equilibrated to 37°C. Test the waterpump; aspiration should be as gentle as possible. It is good practice to test the aspiration on a microwell filled with water (without cells). The end of the tubing should be fitted with a yellow pipette tip. Aspiration and addition of solutions should always be done at the same point of the well, for example at “12 o’clock”. If cells are well attached you may briefly touch the bottom with the aspiration tip and pipette tip. We use 100 pI for washes, 50 pl for all subsequent steps. If an agent to be tested is

J. LANG, R. REGAZZI and C. B. WOLLHEIM

very scarce, you may reduce the volume to 40~1,but be aware of evaporation at 37°C. Take care to aspirate the solutions completely: you must see the rectangle between the bottom and the wall of the well. Make sure that you have good lighting conditions.

1. Set the timer and have the protocol in a place where you can easily see it. You will have little time to rectify any mistakes !

2. Aspirate the medium carefully. Wash the cells with KRH, 100 pI/ well. If done in a regular manner, this does not require timing.

cell permeabilization assay

3. Set the timer and start permeabilization by aspirating the KRH from the wells and adding 50 pI of SLO solution/well. 4. After 7min, aspirate the supernatant gently and add 50pI of preincubation or incubation solution.

5. If preincubation is performed it should preferably last less than 10min as considerable rundown of the Ca2+ response may result.

6. Aspirate the preincubation solution, and add 50 pI incubation solution.

7. Aspirate the supernatant gently, and add 50 pl incubation sohtion.

8. Take out 50pI of supernatant and transfer it into microtubes placed in ice/waterbath.

9. Add 100 pl acid-ethanol (for total cellular content of insulin) or 1OOpI of trypan blue working solution in a number of wells; leave the microwell at 37°C for 10 min.

10. In the meantime centrifuge the supernatants for 2 min at 10 000 g at 4°C.

11. Take aliquots from the centrifuged microtubes (without touching the bottom of the tube) and place in an adequate amount buffer for determination of the secretion product.

12. Take the microwell plate to a light microscope. Carefully remove the trypan blue working solution till a thin film is left on the cells. Permeabilized cells will appear blue. Check for detachment of cells. In a good experiment, only a small empty spot should be visible at the place of aspiration and pipetting, viz.at “12 o’clock”. Be aware that most cells detach when taking out the supernatant with the pipette-tip at the end of the experiment. In fact, the experiment may have been more successful than it would appear! After a number of experiments you will be able to judge the success of permeabilization and degree of cell detachment simply by holding the microwell against a light source.

check for detachment of cells

Clostridial Toxins and Endocrine Secretion: Their Use in Insulin-Secreting Cells

16.2.7 Troubleshooting 76.2.17 Cell Detachment

cell detachment

Is the SLO concentration adequate? Different cell passages may exhibit different sensitivities to SLO. Retest the dose-dependence. Is the plastic support adequate ? Repeat with cells grown on extracellular matrix (ELDAN). Are aspiration and pipetting adequate? Ensure gentle aspiration and a smoothly working pipette.

76.2.12 Insufficient or No Response to Ca2+

poor response to Ca2+

Check the amount of free calcium in the buffers, if possible by the use of a Ca2+electrode. Check the pH of the buffers. Reduce preincubation time to minimize loss of cytosolic proteins. Test the response of cells directly after SLO permeabilization. In contrast to Ca2+,the response to GTP[S] should not be altered by rundown (Kiraly-Borri et a/., 1996). Supernatants have only been partially aspirated leading to dilution of incubation buffers and contamination with insulin secreted during the previous steps.

16.3 Transient Cotransfection Assay for Exocytosis 16.3.1 General Remarks The use of permeabilized cells in the study of exocytosis is not restricted to the investigation of agents which do not cross normally the plasma membrane, such as the clostridial neurotoxins, peptides or recombinant proteins. This approach can be combined with the transient overexpression of proteins of interest, e.g., components of the fusion machinery. The method described below applies to insulinsecreting HIT-T15 cells (Lang et a/., 1995). The cloning of stable cell lines expressing the protein under study is time consuming, especially when a number of different mutations are being investigated. In contrast, transient transfection is a rapid approach but will lead to (over)expression only in a limited percentage of cells. Therefore a reporter gene for secretion/exocytosis must be cotransfected. A first description using growth hormone in chromaffin cells to monitor secretion and exocytosis of large dense core vesicles demonstrated the feasibility of this approach (Holz et a/., 1994, Selden et a/., 1986), using a commercially available kit to determine the release of growth hormone (Nicholls). Each cell line may, however, handle a given secretory product in a distinct fashion. J. LANG, R. REGAZZI and C. 6. WOLLHEIM

Indeed, the use of growth hormone as a reporter signal is not very efficient in insulin-secreting cells (Lang, 199513).This is most probably due to differences in postranslational modifications, precursor processing, packing or sorting of the hormone executed by cell-specific enzymes. However, insulin of different origin from that of the cell line used can be measured reliably by taking advantage of the difference between species in the connecting peptide (C-peptide) of proinsulin. After transient transfection with human proinsulin cDNA, at least in HIT-T15 cells the human insulin C-peptide is cosecreted with mature insulin, and can be measured with commercially available RlAs (e.g. Euro-Diagnostica, Incsater)and ELlSAs (e.g. Dako, DRG).The ELlSAs from two distributors (Dako; DRG) have been tested, offer sufficient sensitivity, and discriminate well between C-peptide of hamster (as found normally in the insulin-secreting HIT-T15 cells) and human origin. In practice the choice of the reporter gene is given by the specific characteristics of the cell line or primary cells used, and by the availability of appropriate analytical methods, whose costs may vary considerably. An alternative approach has been the use of a fusion protein between secretogranin, a secretory product of chromaffin cells, and alkaline phosphatase (Parmer et al., 1993). However, its direct use in the study of exocytosis has not been reported to our knowledge. The choice of the transfecting agent depends on its efficiency and the level of expression required (Keown et al., 1990). Whereas the cheaper calcium phosphate precipitation is adequate for experiments using growth hormone (Holz et al., 1990), the use of human C-peptide as marker required more expensive commercially prepared formulas (Lang et al., 1995).Transfectam (Promega) is easy to handle (Barthel et al., 1993) and gives good results in insulin-secreting cells. A number of other lipofecting agents are commercially available and may be suitable (e.g. Lipofectamine, Gibco). The plasmids used do not require CsCI2gradients for preparation. Although this method yields preparations of high purity in large quantities, the obligatory use of considerable amounts of ethidium bromide is cumbersome. In general, the commercially available purification kits (e.9. Quiagen, Promega) will provide plasmid purification of good quality. The concentrations of plasmids used and the relative amount of reporter gene versus transgene under study may be varied according to the actual needs. Too high a degree of overexpression of the protein under study may in fact be undesirable especially when studying stoichiometric processes. In addition to (0ver)expressionof a protein, specific inhibition of protein expression should be feasible in this system by cotransfecting the reporter gene with plasmids carrying the appropriate antisense construct.

Clostridial Toxins and Endocrine Secretion: Their Use in Insulin-SecretingCells

16.3.2 Material Required 0 0 0

0 0

Cell culture facility Flat-bottomed 24-microwell plates (eventually specially coated) Plasmid encoding for human preproinsulin or another suitable secretory product Plasmid encoding for the protein to be studied Human C-peptide ELISA or RIA

16.3.3 Solutions 0 0 0

0 0

0 0 0

Complete cell culture medium. Sterile phosphate-buffered saline (PBS). Double concentration of Dulbecco's Mimimal Essential Medium (2 x DMEM). The commercially available powder DMEM is dissolved in half the amount of sterile bidistilled water indicated by the manufacturer (e.g. Gibco). The solution should be stored in aliquots at -20°C. Repetitive freeze-thawing should be avoided. Sterile double distilled water. Transfecting solution: Transfectam ( Promega), reconstituted with ethanol as indicated by the manufacturer. The resuspended solution can be kept for at least one month at 4°C. PBS (non-sterile) containing 8 mM EDTA, pH 7.4 (PBS-EDTA). Electrophoresis sample buffer without SDS. 20 % SDS solution in bidistilled water.

16.3.4 Procedure

lo6cells/well in a 24-well plate one day before transfection. The methods to culture insulinsecreting cells have been described in detail elsewhere (Asfari et al., 1992, Wollheim ef al., 1990, Wollheim et al., 1990).

1. Cells (HIT-T15)are plated at 0.75 x

2. O n the day of transfection, aspirate medium and wash the cells twice with PBS; leave the cells in PBS.

3. Preparation of the transfection solution: We use 5pg plasmid (pcDNAl or pcDNA3 from lnvitrogen containing the appropriate insert) and 1 pl of Transfectam per well in a final volume of 400 pI. The following amounts are given for one well:

4. Mix 2.5pg of the plasmid encoding the secretory product with 100p.1of 2 x DMEM.

5. Add the plasmid encoding the protein under study to this 100 pl. If less than 2.5 pg are used, fill to 2.5 pg with the appropriate vector. 6. Mix 1 pl of Transfectam with 100 pI 2 x DMEM, vortex vigorously, and leave for 10min. J. LANG, R. REGAZZI and C. B. WOLLHEIM

7. Mix the solution from 5. (plasmids) with solution from 6. (Transfectam) by vigorous vortexing, and leave the solution for 10 min.

8. Add 200 yI double distilled sterile water, vortex vigorously, leave for 10 min. 9. Aspirate PBS from the cells and add the transfecting solution.

10. After 4 to 8 hours aspirate the transfecting solution and add normal complete medium to the cells.

11. After 48 to 72 hours perform the exocytosis experiment on permeabilized cells as outlined above. The time schedule and volumes should be adapted where necessary. 350-400 yI of buffer will cover the cells adequately in a 24-well plate. Take into account that the buffers used during the exocytosis experiment do not contain BSA.

If the secretory product is rather lipophilic, add a small amount of BSA-containing buffer to the Eppendorf tubes to which the supernatants are transferred at the end of the experiment. 12. Perform the appropriate analytical method (RIA, ELISA, etc.) to measure the secretory product.

lipophilic secretory product

13. Detach the cells from some wells of the 24-well plate to verify overexpression of the protein under study. To this end, add 400yl PBS containing 8 m M EDTA, pH Z4 (PBS-EDTA),to the well, and leave at 37°C for 5 to 10 min. Detach the cells by rinsing the well several times with the PBS-EDTA. Transfer the cells in PBS-EDTA to a microtube, and centrifuge for 10 min at 2000 g. Resuspend the cells in 500yl electrophoresis sample buffer without SDS, sonicate, add 60 yI of 20 % SDS and boil the sample for 2 min before electrophoresis.

16.3.5 Troubleshooting

76.3.5.7 Insufficient Gene Expression Test for transfection efficiency by using a plasmid encoding forbgalactosidase (if you cannot find the plasmid in the lab next door, it is commercially available from several suppliers). About 10 % of cells should be positive using standard assays. If transfection efficiency is unsatisfactory, try changing the ratio of plasmid to Transfectam concentration as described (Barthel et al., 1993). Check the quality of the plasmids used, by electrophoresis on agarose gels. Try to express another secretory product. Use a different lipofection/transfection method. This may be particularly important when signs of cytotoxicity appear.

transfection efficiency

Clostridial Toxins and Endocrine Secretion: Their Use in lnsulin-Secreting Cells

76.3.5.2 Considerable Variability in the Expression Levels between the same or Different Plasmids

excessive variability

Measure the total cellular content of secretory products to check for variability in your transfection procedure. Redetermine the ODs of your plasmid preparations to ensure that equal amounts are used.

16.4 Tetanus Toxin as a Tool for Studying the Role of VAMPs in Exocytosis 16.4.1 Introduction Tetanus toxin (TeTx) is a zinc-dependent protease that has been shown to cleave VAMP-1, VAMP-2 and cellubrevin (Niemann et al., 1994). The toxin usually cleaves between a glutamine and a phenylalanine residue. The cleavage site of rat VAMP-1 is slightly different as the glutamine residue is replaced by valine. For this reason rat VAMP-1 is a poor substrate for TeTx (Niemann et al., 1994). Since the toxin is highly specific for VAMPs, it is a powerful tool to investigate the role of these proteins in different cellular processes.

16.4.2 General Considerations

A

comprehensive safety Precautions for neurotoxins

TeTx is a zinc-dependent enzyme and, therefore, care should be taken to avoid the presence of EDTA or any other substance capable of binding the cation in the incubation buffer. In our hands, the addition of zinc during the incubation period was not required. As a general rule, and for safety reasons, only well experienced personnel should be allowed to work with neurotoxins. For the experiments with neurotoxins the investigator should wear gloves and avoid using any material that could cause injuries such as needles, glassware etc. Any material that has been in contact with the solutions containing the neurotoxins is rinsed in concentrated sodium hypochlorite and collected in a plastic container. At the end of the experiment the container is autoclaved for 20 min at 120°C to inactivate completely the toxin that might possibly remain on the material. In principle, the secretion experiments described below can be performed by one person but, because of the precautions that should be taken with neurotoxins, we usually prefer to have the help of a second person. The first person removes the medium at given time points while the second adds the appropriate buffers.

J. LANG, R. REGAZZI and C. 6. WOLLHEIM

16.4.3 Specific Considerations for the Study of Insulin-Secreting Cells Insulin-secreting cells d o not express the receptors required for entry of the neurotoxins. Consequently, the cells need to be permeabilized before the addition of the neurotoxins. SLO has been shown to produce holes in the plasma membrane of several cell types that allow the passage of molecules up to 150 kDa. The permeabilization procedure with SLO described above yields reproducible results in insulin-secreting cells and is, therefore, the method of choice to test the effect of neurotoxins in these cells. Like the other clostridial neurotoxins, TeTx is composed of two subunits (Niemann et al., 1994).The heavy chain is required for crossing the plasma membrane while the light chain contains the catalytic activity. This means that the heavy chain is not needed for the action of neurotoxins in SLO permeabilized cells. Thus, the experiments can be performed using the light chain alone, which is much less dangerous than the whole toxin. If the whole toxin is used, the heavy and light chains need to be separated by reducing the cysteine bridge linking the two polypeptides before addition to the cells (see detailed protocol below). After permeabilization, the cells are incubated with TeTx to achieve cleavage of VAMPs in the cytosolic portion of the proteins. The toxin is added to the medium in the presence of resting free Ca2+concentrations (0.1pM). We have shown that, if the cells are not stimulated immediately after permeabilization, Ca2+-inducedexocytosis displays rapid rundown (Kiraly-Borri et a/., 1996). Thus, if the stimulation of the cells is delayed by 30min, Ca2+induced exocytosis is greatly reduced. Therefore it is necessary to find a compromise to ensure adequate cleavage of VAMPs while preserving appropriate exocytotic responses of the cells. For our experiments we chose an 8 min TeTx pretreatment of the cells. In insulin-secreting cells, 8min was found to be sufficient for 50 n M TeTx to digest between 80 and 90% of VAMP-2 and cellubrevin (VAMP-1 is not expressed at detectable levels in insulin-secreting cells) (Regazzi et al., 1995).After 8min the rundown of the exocytotic response is not very pronounced, and by increasing the free Ca2+concentration from 0.1 p M to 10 pM stimulations, of 4- to 10-fold can be obtained.

A

light chain safer than whole toxin

stimulate cells immediately

16.4.4 Practical Approach 76.4.4.7 Material Required Clostridial toxins are commercially available from Calbiochem and from Sigma. Antibodies against VAMPs, SNAP-25 and syntaxins can be obtained from Chemicon and from Sigma. No special material is required in addition to that given for cell permeabilization, except for a plastic container that should resist a temperature of 120°C. This conClostridial Toxins and Endocrine Secretion: Their Use in lnsulin-SecretingCells

tainer is used to collect the material that has come in contact with the neurotoxins and is autoclaved at the end of the experiment.

76.4.4.2 Solutions Required Prepare all buffers required for cell permeabilization and for secretion measurements (see Section 16.2.3). In addition to the buffers needed for cell permeabilization prepare: Solution LC supplemented with the required amount of TeTx. If the whole toxin (heavy plus light chains) is used, the catalytic subunit must be separated from the heavy chain by dithiothreitol (DTT) treatment. We usually incubate a 200-fold concentrated toxin in LC supplemented with DTT (final concentration 10 mM) for 30 min at 37°C. If the light chain is used this activation step is unnecessary. If the cleavage of VAMPs is to be verified prepare the following sobtions: Solubilization solution (SS): Tris, 10 mM; EDTA, 1 mM; Sodium dodecyl sulfate, 3 %; glycerol, 13 %; p-mercaptoethanol, 7 %. Homogenization solution (HS): Tris, 20 mM, pH 7.5; EDTA, 10 mM; Aprotinin, 2 pg/ml; Leupeptin, 10 pg/ml.

76.4.4.3 Preparation of Neurotoxins

control of Caz+ concentration

When using permeabilized cells care should be taken to avoid any addition to the buffers that could change the free Ca2+concentration. In particular, since a given free Ca2+concentration is obtained with Ca2+/EGTAbuffers, the pH of the solutions should be precisely controlled. In fact, the binding affinity of EGTA for Ca2+is extremely pHdependent. For this reason, we have used a TeTx dialyzed overnight at 4°C against KG buffer (see above) diluted 1 :2 in H 2 0 .We found that if not more than one freezehhaw cycle is used, aliquots of 5-10 p M TeTx are stable in this solution for several months at -80°C. Alternatively, concentrated solutions of TeTx can be used that will be diluted at least lOOx in the incubation buffer. A schematic representation of the protocol we currently use to analyse the role of VAMPs in insulin-secreting cells is given in Figure 1.

J. LANG, R. REGAZZI and C. 6. WOLLHEIM

1 1 1 1 Western blotting

Clostridial toxins

Stimulation

~

substrate cleavage

exocytosis

secretion

7 min

8 rnin

7 min

Fig. 1. Schematic representation of the protocol used to study the effect of clostridial toxins on insulin secretion. Insulin-secreting cells grown in multiwells are permeabilized at 37°C with streptolysin 0 (SLO)for 7 min. The SLO is then removed and the cells are incubated for 8min at 37°C in the presence or in the absence of the clostridial toxins under study. At the end of this treatment a fraction of the cells is processed for analysis by Western blotting of the cleavage of the toxin substrates (VAMPs, SNAP-25 or syntaxins). The remaining cells are stimulated for 7 min by increasing the concentration of free Ca2+, by adding poorly hydrolyzable GTP analogues or by adding CAMPThe amount of insulin released into the medium during the stimulation period is measured by radioimmunoassay

16.4.4.4 Detailed Procedure 1. Permeabilize the cells with SLO using the protocol described in 2.6.

2. Replace the medium with solution LC with or without TeTx. For 96-microwells we add 50 pI solution while for 24-multiwells the volume is 300p.1.

W

3. Incubate for 8 min at 37°C. 4. TeTx treatment is terminated by removing the medium. The cells are then stimulated to secrete by adding LC or HC buffer for an additional 7 min period. At the end of the TeTx incubation it is possible to test the degree of cleavage of VAMPs. In the case of primary P-cells and in INS-1 cells, where VAMPs can be detected in homogenates, the cells can be directly lysed in 300 yl solubilization solution (SS) after removing the medium. We usually scrape the cells off using the pipette tip and transfer the lysate into an Eppendorf tube. Because of the presence of large amounts of DNA, the cell solution is very viscous. Therefore, the lysate is sonicated briefly ( 3 x 3 s) to disrupt the DNA, and is then boiled for 3 min. In RINm5F and HIT-T15 cells VAMPs are not readily detectable in homogenates because of the limited number of secretory granules (Boyd etal., 1995, Wollheim et al., 1990).To test the extent of cleavage after the toxin pretreatment it is necessary to seed 2 x lo6cells in a dish of 21 cm2. After permeabilization and TeTx treatment the cells are immediately scraped in 800 pI ice-cold homogenization solution (HS). A crude membrane fraction is then prepared by briefly sonicating ( 3 x 3 s) the cells and centrifuging the homogenate at 4°C in an Clostridial Toxins and Endocrine Secretion: Their Use in Insulin-SecretingCells

Eppendorf centrifuge for 30 min at maximum speed (14 000 r.p.m). The crude membrane fraction (pellet) is resuspended by brief sonication in 150 PI HS supplemented with 1 % Triton XlOO and centrifuged again at 4°C for 30min at 14000 r.p.m. The proteins (including VAMPs) extracted from the membranes during the detergent treatment are found in the supernatant. The extent of VAMP cleavage can be assessed by Western blotting after separation of the proteins on SDS-PAGE. We found that the best results are obtained using a 12-18 % acrylamide gradient but a 13 % acrylamide gel also gives a satisfactory separation of VAMP-2 (18 kDa) and cellubrevin (14 kDa). Figure 2 shows the results of one experiment where the effect of TeTx on insulin secretion and the cleavage of VAMP-2 and cellubrevin have been analyzed in parallel in the rat @-cellline INS-1.

16.4.5 Troubleshooting 76.4.5.7 TeTx Does not Inhibit Exocytosis

no exocytosis

Verify the permeabilization of the cells after SLO treatment. Make sure that none of your buffers complexes Zn”. Verifv the cleavage of VAMPs as described above.

L

c ‘ 250 a,

3 & 200

5 .c a) t C

v

2

0-TeTx +TeTx

=

150 100

I

2011 0

0.1

10 0.1 Ca2+(pM)

10

VAMP9

Cb

VAMP-2

Cb

Fig. 2. Effect of TeTx pretreatment on insulin secretion and on VAMP expression in INS-1 cells. The figure shows the results of one experiment where the effect of TeTx on insulin secretion (left panel) and on VAMP-2 and cellubrevin cleavage (right panel) have been analyzed in parallel. INS-1 cells were seeded in 96-microwells (for the measurement of insulin secretion) and on 24-multiwells (to assess the cleavage of VAMPs). Two days later, the cells were permeabilized with SLO and treated with 50 n M TeTx using the protocol described in the text. After the treatment with TeTx, the cells in the 96-microwells were incubated at low (0.1yM) or high (10pM) free Ca2+for 7 min. The amount of insulin released into the medium during this period was assessed by radioimmunoassay. The results are given as mean f SEM (n = 3). The cells in the 24-multiwells were immediately disrupted in homogenization buffer as described in the text. The amount of VAMP-2 and cellubrevin (CB) present in the membranes of the cells at the end of the TeTx treatment was determined by Western blotting. The results are expressed in arbitrary units after densitometric scanning of the films J. LANb, K. KtbALLI and L. b. W U L L H t l M

16.4.5.2 No Cleavage of VAMPs Verify the activity of the toxin in vitro. This can be done using different procedures according to the tools available in the laboratory. The easiest method is to prepare a crude membrane fraction of the cells under study. The membrane fraction of a cell homogenate prepared in LC can easily be obtained by centrifugation (30 min) in an Eppendorf tube at 4°C and 14000 r.p.m. Eventually a crude brain membrane fraction prepared using the same protocol could be used as an additional control. An aliquot of the pellet (100pg for insulin-secreting cells, 20pg for brain) is then incubated in LC with TeTx at 37°C. The cleavage of VAMPs is verified by Western blotting. If recombinant VAMPs are available, the activity of the toxin in the incubation buffer can be tested using purified proteins and verifying the cleavage by protein staining (i.e. Coomassie blue or Silver staining) after SDS-PAGE.

no cleavage

16.5 The Use of Other Clostridial Neurotoxins for the Study of Exocytosis The protocol described above for TeTx has been applied successfully in our laboratory to study the effect of other clostridial neurotoxins on the exocytosis of insulin. Thus we have shown that pretreatment of insulin-secreting cells with BoNT/B, which cleaves VAMP-2 and cellubrevin between the same amino acid residues as TeTx (Niemann et al., 1994), blocks Ca2+-inducedsecretion (Regazzi et al., 1995). The same protocol was also used to investigate the effect on insulin secretion of BoNT/A and BoNT/E, which cleave the t-SNARE SNAP-25 (Sadoul et al., 1995), and of BoNT/Cl which cuts syntaxin (Lang, Niemann and Wollheim, unpublished).

16.6 Reagents and Chemicals Materials

Supplier

Cat-No.

Antibody anti-SNAP-25

SEROTEC CHEMICON Sternberger SIGMA CHEMICON CHEMICON Boehringer Eldan Merck Fluka DAKO SIGMA

MCA1308 ( # ) MAB331 SM181 SO664 MAB325lMAB329 ( # ) MAB335lMAB333 ( # ) 126 888 E-TCMT-F 1.09943.0001 03778 K 6218 G 5101

Antibody anti-syntaxin Antibody anti-VAMP ATP (Na2ATP) BCEM-plates CaCI2Titrisol EGTA Elisa C-peptide K+-glutamate

ClostridialToxins and Endocrine Secretion: Their Use in Insulin-SecretingCells

Materials KOH Suprapur Neurotoxin BoNT A Neurotoxin BoNT C Neurotoxin BoNT E Neurotoxin TeTx pCDNA3 Plasmid Maxi Kit Streptolysin-0 Transfectam

Supplier Merck Calbiochem Calbiochem Calbiochem Calbiochem Invitrogen Qiagen SIGMA Promega

Cat-No. 1.05002.0100 203650-Q ( # ) 203676 ( # ) 203673-Q ( # ) 582235-Q ( # ) V790-20 12262 S5265 (#) E 1232

Note that we have not tested commercial preparations indicated

by

(#I.

Distributors DAKO A/S : Produktionsvei 42, DK-2600 Glostrup, Denmark; phone +4544920044; fax +45-42841822 BOEHRINGER: MANNHEIM AG, lndustriestrasse 7, CH-6343 Rotkreuz, Switzerland; phone +41-42-654242; fax +41-42-644145; web address: http:// biochem.boehringer-mannheim.com CHEMICON Int. Inc. 28835 Single Oak Drive, Temecula, CA, 92590, USA; phone: +1-909-676-8080; fax +1-909-676-9209; web address: http://biochem.chemicon.com. ELDAN Tech Ltd., 28 Pierre Koenig St. Talpiot Industrial Area, Jerusalem 93469, Israel; phone: 972-2-781883; fax: 972-2-781852 FLUKA Chemie AG, PO6 260, CH-9471 Buchs, Switzerland; phone: +41-817552511; fax: +41-81-756-5449; web address: http://www.sigma.sial.com/fluka INVITROGEN BV, De Schelp 26, 9351 NV Leek, The Netherlands; phone: +315945-15175; fax: +31-5945-15312; e-mail: service gopher://gopher.electriciti.com:70/11Anvitrogen LIST: 501-6 Vandell Way, Campbell, CA 95008-6967; phone: +1-408-866-6363; fax: +1-408-866-6364 MERCK -E. Merck AG, Ruechlingstr. 20, CH-8953 Dietikon, Switzerland, phone +41-1-7451440, fax +41-1-7451420, e-mail: service address: http://www.merck.de/ NICHOLLS Institute Diagnostics B.V., Nieuweweg 172, NL-6603 BT Wiichen, The Netherlands; phone +31-8894-11424, fax +31-8894-23010 PROGEMA: 2800 Woods Hollow Road, Madison WI 53711 ;U.S.A.; phone +1-608274-4330; fax +1-608-277-2601; web address: http://www.promega.com QIAGEN: Max Volmer Str. 4, D-40724 Hilden, FRG; phone: +49-2103-892230; fax: +49--2103-892-240 SEROTEC Ltd, 22 Bankside, Kidlington, Oxford OX5 lJE, England; phone: +441865-379941; fax: +44-1865-373899; e-mail: 100116.3413SlGMA Chemie, PO6 260, CH-9470 BUCHS, Switzerland; phone: +41-81-7552721. STERNBERGER Monoclonals Inc., Hopkins Bayview Research Campus, 5210 Eastern Avenue, Baltimore, Md, 21224-2736, USA; phone +1-410-50-2644; fax: +1410-550-2643 WINMAXC, Chris Patton, Stanford University, Hopkins Marine Station, Pacific Grove, CA 93950-3094; e-mail: cpatton @ leland.stanford.edu.

References Ahnert-Hilger G, Stecher 6, Beyer C, et al. (1993):Exocytotic membrane fusion as studied in toxin-permeabilizedcells. In Methods Enzymol. 221: 139-49. Asfari M, Janiic D, Meda P, et a/. (1992): Establishment of 2-mercaptoethanoldependent differentiated insulin-secreting cell lines. In Endocrinology 130: 167-78. J. LANG, R. REGAZZI and C. B. WOLLHEIM

Ashcroft FM, Rorsman P (1989):Electrophysiology of the pancreatic p-cell. In Prog. Biophys. Molec. Biol. 54: 87-143. Barthel F, Remy JS, Loeffler JP, et a/. (1993): Gene transfer optimization with lipospermine-coated DNA. In DNA Cell. Biol. 12: 553-60. Boudet S, Hove-Madsen L, Bers DM (1994):How to make and use calcium-specific mini- and microelectrodes. In Methods Cell. Biol. 40: 93-113. Bokvist K, Eliasson L, Ammala C, et a/. (1995):Co-localization of L-type Ca2' channels and insulin-containing secretory granules and its significance for the initiation of exocytosis in mouse pancreatic B-cells. In E M 6 0 J. 14: 50-57. Boyd RS, Duggan MJ, Shone CC, et a/. (1995):The effect of botulinum neurotoxins on the release of insulin from the insulinoma cell lines HIT-T15 and RINm5F. In J. Biol. Chem. 270: 18216- 18. Brooks SP, Storey KB (1992):Bound and determined: a computer program for making buffers of defined ion concentrations. In Anal Biochem. 201: 119-26. Burgess TL, Kelly RB (1987): Constitutive and regulated secretion of proteins. In Annu. Rev. Cell Biol. 3: 243-93. Burgoyne RD (1990): Secretory vesicle-associated proteins and their role in exocytosis. In Annu. Rev. Physiol. 52: 647-59. De-Camilli P, Jahn R (1990):Pathways to regulated exocytosis in neurons. In Annu. Rev. Physiol. 52: 625-45. Fischer-von Mollard G, Stahl B, Li C, etal. (1994): Rub proteins in regulated exocytosis. In TlBS 19: 164-68. Fohr KJ, Warchol W, Gratzl M (1993): Calculation and control of free divalent cations in solutions used for membrane fusion studies. In Methods Enzymol. 221: 49-57. Gembal M, Detimary P, Gilon P, et a/. (1993):Mechanisms by which glucose can control insulin release independently from its action on ATP-sensitive K' channels in mouse B cells. In J. Clin. Invest. 91 : 871 -80. Holz RW, Bittner MA, Senter RA (1992):Regulated exocytotic fusion I: Chromaffin cells and PC12 cells. In Methods Enzymol. 219: 165-78. Holz RW, Brond k WH, Senter RA, et al. (1994): Evidence for the involvement of Rab3A in Ca -dependent exocytosis from adrenal chromaffin cells. In J. Biol.

x

Chem. 269: 10229-34. Jacobsson G, Bean AJ, Scheller RH, etal. (1994):Identification of synaptic proteins and their isoform mRNAs in compartments of pancreatic endocrine cells. In Proc. Natl. Acad. Sci. USA 91 : 12487-91. Johannes L, Lledo PM, Roa M, et al. (1994):The GTPase Rab3a negatively controls calcium-dependent exocytosis in neuroendocrine cells. In EM60 J. 13: 2029-37. Keown WA, Campbell CR, Kucherlapati RS (1990):Methods for introducing DNA into mammalian cells. In Methods Enzymol. 185: 527-37. Kiraly-Borri CE, Morgan A, Burgoyne RD, et a/. (1996): a-SNAP and NEMinsensitive factors are required for calcium-stimulated exocytosis of insulin. In Biochem. J. 314: 199-203. Lung J, Boulay F, Li GD, et al. (1993):Conserved transducer coupling but different effector linkage upon expression of the myeloid fMet-Leu-Phe receptor in insulin secreting cells. In E M 6 0 J. 12: 2671 -79. Lung J, Nishimoto I, OkamotoT, et al. (1995): Direct control of exocytosis by receptor activation of the heterotrimeric GTPases G, and Goor by the expression of their active G,-subunits. In EM60 J. 14: 3635-44. Lung J. (1995b):Application of human growth hormone transient gene expression system in insulin secreting cells. In Breakthrough Newsletter 3: 4-5. Li G, Rungger-Brandle E, Just I, etal. (1994):Effect of disruption of actin filaments by Clostridium botulinum C2 toxin on insulin secretion in HIT-T15 cells and pancreatic islets. In Mol. 6iol. Cell 5: 1199-1213. Lledo P-M, Vernier P, Vincent J-D, etal. (1993):Inhibition of Rab3B expression attenuates Ca2+-dependent exocytosis in rat anterior pituitary cells. In Nature 364: 540-44. Martin E, Moya F, Gutierrez LM, et al. (1995): Role of syntaxin in mouse pancreatic beta cells. In Diabetologia 38: 860-63. Clostridial Toxins and Endocrine Secretion:Their Use in Insulin-SecretingCells

Neher E, Augustine GJ (1992):Calcium gradients and buffers in bovine chromaffin cells. In J. Physiol. Lond. 450: 273-301. Niemann H, Blasi J, Jahn R (1994):Clostridial neurotoxins: new tools for dissecting exocytosis. In Trends Cell Biol. 4: 179-85. Orci L (1985):The insulin factory: a tour of the plant surroundings and a visit to the assembly line. In Diabetologia 28: 528-46. Parmer RJ, Xi XP, Wu HJ, et a/. (1993):Secretory protein traffic. Chrornogranin A contains a dominant targeting signal for the regulated pathway. In J. Clin. Invest. 92: 1042-54. Polonsky KS (1995): The p-cell in diabetes: from molecular genetics to clinical research. In Diabetes 44: 705-17. Prentki M, Matschinsky FM (1987):Ca2+,CAMPand phospholipid derived messengers in the coupling mechanisms of insulin secretion. In Physiol. Rev. 67: 1185-1248. Reetz A, Solimena M, Matteoli M, et a/. (1991):GABA and pancreatic beta-cells: colocalization of glutamic acid decarboxylase (GAD)and GABA with synapticlike microvesicles suggests their role in GABA storage and secretion. In EMBOJ. 10: 1275-84. Regazzi R, Wollheim CB, Lang J, et a/. (1995): VAMP-2 and cellubrevin are expressed in pancreatic p-cells and are essential for Ca2+-but not for GTPySinduced insulin secretion. In EM60 J. 14: 2723-30. Regazzi R, Ravazolla M, lezzi M, et a/. (1996): Expression, localization and functional role of small GTPases of the Rub3 family in insulin-secreting cells. In J. Cell Sci. 109: 2265-73. Sadoul K, Lang J, Montecucco C, et a/. (1995):SNAP-25 is expressed in islets of Langerhans and is involved in insulin release. In J. Cell Biol. 128: 1019-28. Scheller RH (1995):Membrane trafficking in the presynaptic nerve terminal. In Neuron 14: 893-97. Selden RF, Howie KB, Rowe ME, et a/. (1986):Human growth hormone as a reporter gene in regulation studies employing transient gene expression. In Mol. Cell Biol. 6: 3173-79. Sollner T, Whiteheart SW, Brunner M, et a/. (1993): SNAP receptors implicated in vesicle targeting and fusion. In Nature 362: 318-24. Sorenson RL, Garry DG, Breljee TC (1991):Structural and functional considerations of GABA in islets of Langerhans. In Diabetes 40: 1365-74. Sudhof TC (1995):The synaptic vesicle cycle: In Nature 375: 645-53. Turner RC, Hattersley AT, Shaw JTE, et a/. (1995):Type II diabetes: clinical aspects of molecular biological studies. In Diabetes 44: 1-10, Ullrich S, Wollheim CB (1988):GTP-dependent inhibition of insulin secretion by epinephrine in permeabilized RINm5F cells. In J. Biol. Chem. 263:8615-20. Ullrich S, Prentki M, Wollheim CB (1990): Somatostatin inhibition of Ca2+-induced insulin secretion in permeabilized HIT-T15 cells. In Biochem. J. 270: 273-76. Vallar L, Biden TJ, Wollheim CB (1987): Guanine nucleotides induce Ca2+independent insulin secretion from permeabilized RINm5F cells. In J. Biol. Chem. 262: 5049-56. Widmann C, Burki E, Dolci W, et al. (1994): Signal transduction by the cloned glucagon-like peptide-1 receptor: comparison with signaling by the endogenous receptors of beta cell lines. In Mol. Pharmacol. 45: 1029-35. Wollheim CB, Sharp GW (1981):Regulation of insulin release by calcium. In Physiol. Rev. 61 : 914-73. Wollheim CB, Meda P, Halban PA (1990):Isolation of pancreatic islets and primary culture of the intact microorgans or of dispersed islet cells. In Methods Enzymol. 192: 188-223. Wollheim CB, Meda P, Halban PA (1990): Establishment and culture of insulinsecreting beta cell lines. In Methods Enzymol. 192: 223-35. Wollheim CB, Regazzi R (1990):Protein kinase C in insulin releasing cells. Putative role in stimulus secretion coupling. In FEBS Lett. 268: 376-80.

J. LANG, R. REGAZZI and C. 6. WOLLHEIM

Bacterial Toxins

Klaus Aktories Copyright 0 2002 WILEY-VCH Verlag GmbH & Co. KGaA

CHAPTER 17

Pore-forming Toxins S. BHAKDI, A. VALEVA, I. WALEV, U. WELLER, and M. PALMER

17.1 Origin of Pore-forming Toxins (PFTs) Pore-forming toxins (PFTs) are produced as water-soluble proteins that undergo a hydrophilic-amphiphilic transition as they insert into target lipid bilayers to produce transmembrane pores. Most bacterial pathogens produce one or several PFTs whose contribution to microbial virulence has in some cases been demonstrated experimentally. Reviews on various aspects of PFTs are available in the recent literature (Bhakdi et al., 1996; Bhakdi et al., 1994; Parker et al., 1996; Van der Goot et a/., 1994).Amongst the best studied PFTs are the staphylococcal alpha-toxin (Bhakdi and Tranum-Jensen, 1991), streptolysin 0, pneumolysin, perfringolysin (the last three of which are members of the family of cholesterol-binding toxins) (Alouf and Geoffroy, 1991), aerolysin of A. hydrophila (Parker et a/., 1996), and E. coli hemolysin (HlyA) (the prototype of the RTX family of toxins) (Welch, 1991; Welch et al., 1995). Most PFTs can be categorized within families of related proteins. The two largest known families are the cholesterol-binding toxins (Alouf and Geoffroy, 1991) and the RTX family (for Repeats in Toxin) (Welch, 1991; Welch et al., 1995).An emerging family is the group of toxins produced by Vibrio species. The PFTs of S. aureus (alpha-toxin, gamma-toxin, and leukocidin) (Bhakdi and Tranum-Jensen, 1991; Prevost et a/., 1995) exhibit a low degree of relatedness. A large group of PFTs is represented by the larval insecticidal toxins of Bacillus thuringiensis (Gazit and Shai, 1993; Li et al., 1991; Schwartz et al., 1993), which are potentially useful tools to permeabilize insect cells.

families of PFTs

17.2 Evolution of the PFT Field The concept of membrane damage as an effector mechanism of bacterial exotoxins arose during the 1970s, mainly through the work on alpha-toxin by Freer, Arbuthnott and Bernheimer (Bernheimer, 1974; Freer et a/., 1968) and, subsequently, Thelestam and Mollby (1975).These studies gave rise to the idea that staphylococcal alphatoxin produced permeability defects in membranes. However, it was K. Aktories (Ed.),Bacterial Toxins. 0Chapman & Hall, Weinheim, 1997. ISBN 3-8261-0080-8

nature of PFTs

difficult at that time to envisage how a soluble protein might generate discrete functional lesions in lipid bilayers, and the molecular basis for the membrane damaging effects of alpha-toxin remained obscure. In 1978, papers appeared describing the isolation and ultrastructural characterization of pore-forming C5b-9 complement complexes, which were shown to be amphiphilic, lipidbinding molecules (reviewed in Bhakdi and Tranum-Jensen, 1987). These studies essentially proved the validity of Mayer’s doughnut hypothesis, which in 1972 had proposed that C5-C9 complement proteins assembled into pore-forming protein complexes in lipid bilayers (Mayer, 1972). Using approaches that had been employed in the complement work, alphatoxin pores were then purified and characterized. In 1981, alpha-toxin became the first bacterial toxin recognized as forming pores in membranes of mammalian cells (Fussle et al., 1981). Conceptually, this formed a counterpart to membrane-damaging bacterioicins (in particular, to certain colicins), which create small channels in the plasma membrane of target microorganisms (Cramer et al., 1995). Descriptions followed of aerolysin (Howard and Buckley, 1982), streptolysin-0 (Bhakdi et a/., 1985), E. coli hemolysin (Bhakdi et al., 1986) and the larval insecticidal toxins (see e.g. Li et al., 1991) as pore-forming toxins. Since then, the field of PFTs has been expanding steadily, and pore-formers are today known to be produced by the majority of bacterial pathogens. Major advances have been the identification of various toxin families; the elucidation of the crystal structure of the water-soluble form of aerolysin (Parker et al., 1994); the development of molecular approaches to study the mode of pore-formation, particularly with the use of alpha-toxin as a model (Valeva et al., 1996); the pending elucidation of the three-dimensional structure of the alpha-toxin pore; the discovery of secondary cellular reactions provoked by PFTs (reviewed in Bhakdi et al., 1994); the use of genetically engineered bacterial strains to demonstrate the biological relevance of several PFTs (e.g. Bramley et al., 1989; O’Reilly et al., 1986; Paton et al., 1993; Welch et al., 1981); and the introduction of PFTs as tools in cell biology (Ahnert-Hilger et al., 1985; Ahnert-Hilger et al., 1988; Bhakdi et a/., 1993).

17.3 Relationship to Other Toxins Structural homologies between PFTs and other toxins have not been identified. However, the process of membrane permeabilization may be operative in many cases where proteins have to escape from an intracellular compartment. Well known examples are diphtheria toxin, the neurotoxins and anthrax toxin. Specific domains in many intracellularly active toxins have in fact been shown to produce pores in artificial lipid bilayers, and membrane permeabilization is thought to form the basis for translocation of the active moieties from the late endosome to the cytoplasm (reviewed in Montecucco et al., 1994). The molecular mechanism of this translocation remains obscure. In the S. BHAKDI et al

case of anthrax toxin, it has recently been shown that one component (the protective antigen) self-associates to form ring-shaped structures in endosomal membranes (Milne et al., 1994), a phenomenon that is reminiscent of the formation of pores by alpha-toxin. Lethal factor (LF) of anthrax toxin, once released into the cytoplasm, causes K' efflux and release of interleukin-1 (Hanna et al., 1993), processes that again remind one of the pore-forming action of alpha-toxin (Walev et al., 1995). Thus, LF may in fact be a pore-former that permeabilizes the plasma membrane from within the cells. Overall, convergent tendencies in different areas of microbial toxin research are becoming apparent. The search for structural homologies amongst toxins may soon receive a new impetus when it becomes possible to screen explicitly for sequences that are functionally relevant. As a first example, the maior and perhaps sole membrane-inserting sequence in alpha-toxin is confined to a continuous stretch of only 15 amino acid residues (Valeva et al., 1996). A directed search (Bayley, 1994a) revealed that this sequence bears remarkable homology to the putative transmembrane domain of the y-subunit of Na+-K+-ATPase,and with a sequence in phospholemman, a putative activator of channel proteins; both are proteins containing putative membrane-insertion domains, but otherwise with no homologies to alpha-toxin. It can be anticipated that analogous discoveries will follow in the wake of this example.

17.4 Molecular Mechanism of Action PFTs from gram-positive organisms are produced and secreted as water-soluble monomers, and generally d o not seem to require further processing to acquire functional activity. In contrast, PFTs from gram-negative bacteria generally must be post-translationally modified to acquire functionality. Two types of modifications have been identified: first, acylation with fatty acids, as exemplified by the RTXtoxins (Hackett et al., 1994; Issartel et al., 1991;Stanley et al., 1994); and second, proteolytic activation as found, for example, for aerolysin (Parker et al., 1996; Van der Goot et al., 1992; Van der Goot et al., 1993) and Vibrio cholerae hemolysin (Hall and Drasar, 1990). The process of pore-formation comprises a coordinated series of molecular events that often can be distinguished and dissociated from each other by using functionally defective mutant proteins (see e.g. Bayley, 199413; Valeva et al., 1996; Walker and Bayley (1994; Walker et al., 1995).

17.4.1 Binding Binding studies have only been undertaken with alpha-toxin (Cassidy and Harshman, 1976; Hildebrand et al., 1991), aerolysin (Howard and Buckley, 1982), streptolysin 0 (Palmer et al., 1993) and perfringolysin Pore-forming Toxins

Table 1. Specific binding site

Specific features

Alpha-toxin

Present on rabbit erythrocytes, but not identified; not absolutely required for toxin binding to lipid bilayers

Streptolysin 0 and Perfringolysin

Yes: membrane cholesterol; primary binding reversible

Binding site on toxin molecule probably conformational and may involve C- and N-terminal halves of the molecule; maximal binding at 20-25°C; no dependency on metal ions Hydrophobic C-terminal domain essential for binding; binding occurs at 0-37°C; no dependency on metal ions

Aerolysin

Apparently present, but not absolutely required for binding to lipid bilayers Not detected; binding non-saturable and irreversible

E. coli Hemolysin

Binding site on toxin molecule unknown; conformation of binding site dependent on Ca”; bindability not dependent on fatty acid acylation

(lwamoto et al., 1990; Ohno-lwashita et al., 1988), and most recently with E. coli hemolysin (Bhakdi et al., unpublished). These studies collectively demonstrate the diversity in binding behavior, salient features of which are summarized in Table 1.

17.4.2 Oligomerization The majority of PFTs that have been studied in sufficient detail have been found to self-associate in membranes to form non-covalently bonded oligomers. Examples are alpha-toxin, the cholesterol-binding toxins, aerolysin and the hemolysins of Vibrio species. Oligomerization occurs as membrane-bound toxin protomers collide with each other via lateral diffusion in the bilayer. Probably, membrane binding serves to orient the molecules uniformly, so that the critical interacting surfaces can contact each other when collision occurs. Interlocking of toxin protomers with each other involves tight molecular interactions that probably provide the energy to drive the pore-forming domains into the bilayer. Pores may represent a homogeneous population of oligomers, or they may be heterogenous in composition and size. Alpha-toxin is the classical example of a homogeneous pore, which has been identified as a heptamer by low-resolution X-ray crystallography (Gouaux et al., 1994).Aerolysin and the Vibrio hemolysin family probably also form homogeneous oligomeric pores. In contrast, the cholesterol-binding toxins produce large, heterogeneous pores (reviewed in Bhakdi et al., 1996; Parker et al., 1996). Two features render detection of toxin oligomers easy. The first is the presence of electron-microscopically visible structures (arcs and rings) on membranes. The second is the resistance of oligomers S. BHAKDI et al.

towards dissociation by detergents. Such resistance can often be observed even in SDS when samples are not heated (e.g. alphatoxin, Vibrio hemolysins, aerolysin). Oligomers formed by the cholesterol-binding toxins dissociate in SDS but persist in nondenaturing detergents (e.g., deoxycholate). E. coli hemolysin displays neither of these two characteristics. Here, data from functional studies appear to indicate formation of small oligomers (Benz et al., 1989), but stringent proof for their existence has not yet been obtained.

17.4.3 Pore Formation A salient feature of all known pores is their irreversible anchorage in the lipid bilayer (Bhakdi and Tranum-Jensen, 1987). Hence, membrane-bound PFTs must be amphiphilic, possessing a lipid binding surface and a hydrophilic face that lines the aqueous channel. The pore-forming domains themselves are unlikely to harbor extended stretches of hydrophobic amino acid residues. Rather, they would be expected to possess amphipathic secondary structure. Rules to identify or predict pore-forming sequences do not exist, and even the solution of the structure of a water-soluble protomer form will not permit conclusions to be drawn on the structure of the pore. This situation is given in the case of aerolysin (Parker et al., 1996). The first and at present the only pore structure that is on the verge of resolution is that of alpha-toxin. Here, molecular studies using cysteine scanning mutagenesis and spectrofluorometric analyses led to an identification of 15 residues in the center of the molecule that are the major, if not only pore-forming domain (Valeva et al., 1996).The pore-forming sequence in alpha-toxin displays no remarkable features that would have permitted its identification on the basis of any theoretical considerations of primary structure. It has the overall conformation of an amphipathic p-barrel, and seven polypeptide loops participate in creating a cylindrical molecule with a hydrophilic channel running through its center. X-ray crystallographic studies are refining this model,at atomic resolution. In sum, pores may be formed by the insertion of quite short amino acid sequences into the bilayer. These sequences should assume an amphipathic conformation. If oligomerization occurs, closed ring structures can be formed. In some cases, incomplete oligomerization leads to formation of heterogeneously sized arcs. It is also conceivable, although not yet proven, that some pore-formers act as monomers with corresponding variations in functional pore size. In the same context, such slit-opening polypeptide sequences may render possible the translocation of other protein domains across the bilayer, as is required for release of certain toxins from intracellular compartments.

structure of al~ha-toxinpores

Pore-forming Toxins

17.5 Cell-biological Effects PFTs evoke a broad spectrum of cell-biological effects that may be broadly differentiated into two categories. The first comprise effects that are the direct or indirect consequences of transmembrane ion fluxes. The second category encompasses effects that are not recognizably due to deregulated ion fluxes. This differentiation may prove useful especially when PFTs are employed to permeabilize cell membranes: the first effects are dependent on ion movement and can be manipulated at will. In contrast, when an effect is not recognizably dependent on ion flux, it can turn out to be difficult to suppress or control.

17.5.1 Cell-biological Effects Resulting from Ion Fluxes Certain cellular processes are specifically influenced by gradients of monovalent ions across the plasma membrane. It appears that stimulation of some such processes are actually counteracted by concomitant Ca2+flux. Ca2+-dependentprocesses in turn may be abrogated when pores are large enough to permit rapid egress of cytoplasmic proteins. Therefore, it is useful to differentiate between three types of pores: (a) those that are selectively permissive for monovalent ions (e.g. staphylococcal alpha-toxin); (b) those that are permissive for Ca2+and small molecules, but not for proteins (e.g. E. coli hemolysin); and (c) large pores that allow passage of macromolecules (e.g. streptolysin 0).

715.7.7 Reactions Governed by Flux of Monovalent Ions Two possibly related phenomena have been found to be dependent on the flux of monovalent ions. The hypothetical common link is represented by a newly discovered family of intracellular proteases whose activity may be influenced by K+ concentrations. lnterleukin converting enzyme (ICE) is the best studied member of this family. Efflux of K' from monocytes leads to activation of ICE, so that the cells rapidly process and export 11-18 (Walev etal., 1995).An ICE-related protease is involved in regulating programmed cell death, which may be the reason why formation of K+-permissivepores by alpha-toxin in human T-lymphoctes causes apoptosis (Jonas et al., 1994). Both apoptosis and ICE-activation are inhibited when alpha-toxin treated cells are suspended in K+-richmedium. It is of interest that simultaneous flooding of cells with Ca2+,such as occurs when larger pores are formed in lymphocytes (e.g. at high alpha-toxin concentrations or with E. coli hemolysin) counteracts the apoptosis-promoting effect of K+-efflux (Jonas et al., 1994).

S. BHAKDI et al.

7Z5.7.2 Reactions Triggered by Ca2+Influx Many of these reactions are compositely triggered by a flux of extracellular Ca2+into the cells, combined with liberation of Ca2+from intracellular storage pools. The present discussion is oversimplified and will not take the latter event into account.

Secretion. Exocytotic liberation of granular constituents has been demonstrated in leukocytes, platelets and neurological cells. Platelets attacked by alpha-toxin secrete platelet factor 4 and factor 5 (Arvand et al., 1990; Bhakdi et al., 1988). Release of the latter leads to assembly of platelet-bound prothrombinase complexes that generate thrombin. Alpha-toxin can thus promote coagulation via its permeabilizing action on platelets. Release of granule constituents from leukocytes has been observed after permeabilization of these cells by HIyA. For example, large amounts of elastase are secreted into the extracellular medium (Bhakdi et al., 1989). Generation of lipid mediators. Ca2+-dependentphospholipase activation has been shown to stimulate synthesis of lipid mediators derived from arachidonic acid. The production of these lipid mediators can provoke both short- and long-range effects on bystander cells (e.g. (Bhakdi etal., 1994; Suttorp etal., 1985; Suttorp etal., 1992). Cytoskeletal dysfunction. Ca2+-dependentcytoskeletal dysfunction has been observed in endothelial cells under attack by PFTs; cellular contraction leads to formation of intercellular gaps causing leakage of macromolecules across a confluent monolayer (Suttorp et al., 1990; Suttorp et al., 1988). Stimulation of constitutive NO-synthase. Both S. aureus alpha-toxin and HlyA increase synthesis of NO by the constitutive NO synthase in endothelial cells (Suttorp et al., 1993). NO in turn can provoke an array of further reactions in bystander cells.

17.5.2 Reactions not Recognizably Governed by Flux of Ions through Toxin Pores

7Z5.2.7 Short Circuiting of G-protein-dependent Signalling Pathways This property is characteristic of HlyA and probably extends to other members of the RTX family. The initial interaction of HlyA with the target membrane triggers a G-protein-dependent pathway, and causes generation of diacylglycerol and inositol triphosphate (Grimminger et al., 1991). HlyA has indeed been identified as the most potent proPore-forming Toxins

use high HIYA concentrations

teinaceous inducer of phosphoinositide hydrolysis. The mechanism underlying this stimulatory process has not been delineated. When HlyA is used to permeabilize cells, the use of high toxin concentrations is recommended because rapid pore-formation counteracts the Gprotein dependent signalling pathways.

725.2.2 Proteolytic Shedding of Membrane Proteins Many membrane-associated molecules can be released from cells by a proteolytic cleavage process called shedding. The shed molecules often retain their ligand-binding capacity and can cause a spectrum of secondary reactions including, for example, trans-signalling. This term denotes the transfer of sensitivity towards a ligand onto cells that primarily lack the respective receptor. Trans-signalling can occur when the soluble receptor binds to a bystander cell (Bazil, 1995; Mackiewicz et al., 1992). Pore-forming toxins have been found to induce rapid and massive shedding of CD14 and lL-6R, and cleaved soluble IL-6R was biologically active in trans-signalling. Current evidence indicates that membrane permeabilization leads to activation of the shedding proteases present in the host cell membranes, but the underlying mechanism remains unknown (Walev et al., 1996). The possibility that pore-forming proteins can indirectly activate cellderived proteases may attain significance when these toxins are used as tools in cell biology.

17.6 Pathogenic Aspects

cytocidal action

inflammation

s. BHAKDI eta/

Secondary reactions provoked by pore-forming toxins are very diverse, and are probably responsible for short- and long-range effects in the host organism (for review, see Bhakdi et al., 1994). Locally, PFTs can counteract the host defense system directly by killing phagocytes and lymphocytes, and they promote spread of infections by their cytocidal action on tissue cells (see e.g. Bhakdi et al., 1994; Paton et al., 1993; Trifillis et al., 1994; Welch, 1991; Welch et al., 1995; Welch et al., 1981). Mediators, derived for example from arachidonic acid, will provoke inflammation. These processes can be complemented and enhanced by the liberation of biologically active cellular mediators from intracellular compartments or from the cell membrane. Thus, it is understandable that PFTs provoke inflammatory lesions and acute organ dysfunction in vitro and in vivo, and are lethal in experimental animals. When perfused through an isolated lung, PFTs provoke profound pathophysiological alterations in the pulmonary microvasculature and cause irreversible pulmonary edema. The underlying mechanisms are complex, but include a direct toxic action on endothelial cells, and the pro-

duction of lipid mediators that in turn provoke arterial hypertension and edema formation. PFTs thus have the capacity to provoke acute pulmonary failure such as is typically found in patients presenting with respiratory distress syndrome during severe bacterial infections (reviewed in Bhakdi et a/., 1994). In experimental animals, roles for PFTs in the pathogenesis of mastitis (Bramley eta/., 1989), suppurative skin lesions (O’Reilly et a/., 1986), pyelonephritis (O’Hanley et a/., 1991; Welch et a/., 198l), and bacterial pneumonia (Paton et a/., 1993) have been established through the use of isogenic bacterial strains expressing or lacking a given PFT. Moreover, antibodies against alpha-toxin confer significant protection upon animals against various infections with S. aureus (Hungerer et a/., unpublished). Altogether, it has become clear that many PFTs do represent significant determinants of microbial pathogenicity.

17.7 Purification Protocols In the following, we will describe our current protocols for isolating staphylococcal alpha-toxin and streptolysin-0 from bacterial culture supernatants. The protocol for isolating recombinant streptolysin-0 from E. coli has been published (Weller et a/., 1996) and will not be repeated here.

17.7.1 Purification of Staphylococcal Alpha-Toxin The strain most widely used for toxin production is S. aureus Wood 46. Note that the presence of proteases may give rise to proteolytic cleavage of alpha-toxin, which results in altered pore-forming activity (Palmer et a/., 1993).Moreover, proteolytic activity contaminating the final toxin preparation may interfere with cell-biological experiments. Per liter of culture, the following protocol typically yields 20mg of alpha-toxin suitable for cell-biological applications with a strain available from this laboratory. The protocol given below requires a suitable membrane filtration device (molecular weight cut-off below 30 kDa; effective surface ca. 0.5m2 for 2-31 of culture) as available from Filtron or Millipore for concentration of culture supernatants.

interference by proteolysis

Materials

2 x TY medium: dissolve 20g/l Bacto-Tryptone (Difco), l o g yeast extract (Difco), and l o g NaCl in 1 I water, and autoclave Column buffer A: ammonium acetate 20 mM, pH 6.2 Column buffer B: ammonium acetate 500 mM, pH 6.2

Pore-forming Toxins

Procedure 1. 2 xTY medium is used. A starter culture of 30 ml is inoculated, shaken overnight and diluted 1 :lo0 with 2 x TY to yield the production culture. To achieve good aeration, the production culture is divided into portions of 500 ml contained in 2 l Erlenmeyer flasks, which are incubated in an orbitary shaker for 18 h (37"C, 250 r.p.m in an orbitary shaker (New Brunswick Scientific G25).

2. The culture is then transferred into centrifuge bottles, the bacterial cells are pelleted by centrifugation (lOOOOg, 20 min), and the supernatant is collected.

3. Since high concentration promotes membrane-independent oligomerization of alpha-toxin, the following steps should be carried out at 4°C. The culture supernatant is cycled through a suitable membrane concentration device until the volume has been reduced by about 90 %.

4. To the concentrate, 5 volumes of buffer A (26mM ammonium acetate, pH 6.2) are added, and the filtration is continued. This step is repeated twice.

5. The final concentrated solution is cleared by centrifugation (15000g, 20 min) and applied to a column of S-Sepharose FF (Pharmacia; bed volume 10 ml/l I of culture) pre-equilibrated with buffer A at a flow rate of 2 ml/min. The column is rinsed with buffer A until the absorbance at 280 nm (A280)of the eluate has reached a stable baseline, and then developed with a linear gradient to 100 % buffer B (500mM ammonium acetate, pH 6.2; 90 min, flow rate 2 ml/min). The monomeric toxin elutes at about 200 mM ammonium acetate.

6. The fractions recovered from the column are assayed for purity by SDS-PAGE (the monomeric toxin of 33kDa is inevitably accompanied by heptamers which are detected in the gel if the samples are not boiled).The protein concentration is determined by measuring A280 (which is 0.19 for 0.1 mg toxin/ml) or by the Bradford method (A5g5= 0.6 for 10 pg toxin/ml assay mixture).

7. The hemolytic activity can be assayed as follows: the sample is initially diluted 1 :100. Serial two-fold dilutions are then prepared in a microtiter plate with PBVO.1 % BSA. Rabbit erythroytes are added to 1.25% ( 1 . 2 5 108cells/ml) ~ and plates incubated at room temperature for 30 min before visual reading of the hemolytic titer. 1 mg toxin/ml should have a titer of > 1 :20000. 8. Fractions containing the toxin are pooled, dialyzed against 10 m M ammonium acetate, and aliquots are lyophilized and stored at -70°C. Alternatively, the dialysis can be performed with PBS, and aliquots of the solutions are frozen directly. If repeated freeze-thaw-cycles are avoided, the toxin activity is stable for years. S. BHAKDI eta1

17.7.2 Purification of Streptolysin 0 The protocol is applicable to bacterial cultures of from 2 to 20 I, and yields 0.2 to 0.6 mg toxin per liter of culture. Preparation of culture medium and production of culture supernatants containing SLO. Seeding cultures of Streptococcus pyogenes, Richards strain, are prepared in Brain-Heart-Infusion medium by overnight incubation at 37°C. For long term storage of the strain, the culture is supplemented to 50% (by volume) with sterile glycerol and kept at -70°C.

1. Toxin production medium is prepared from Bacto Proteose Peptone No. 3 (Difco) 20g, and Yeast Extract 20 g (Difco),and flasks of the appropriate volume (up to 5 I ) are filled with distilled water to bottom of the neck.

toxin production

2. After autoclaving at 110°C for 30min, 25 mI of sterile 10 % N a H C 0 3 and 20 ml of sterile 50 % glucose are added. 3. The culture is started with 20ml inoculum per liter. Streptococci are cultured at 37°C and after 4 h the addition of N a H C 0 3and glucose is repeated. The pH of the medium is monitored every

30 min and adjusted to 7.5 with 1 M NaOH, while stirring with a magnetic bar. 4. After 8 h the culture is harvested by centrifugation, 10000 g for 15 min, 4°C.

5. Fractionation of culture supernatant: The subsequent steps, excluding the hydrophobic interaction chromatography (HIC), are carried out at 4°C. The supernatant is concentrated by membrane filtration, preferably by employing a tangential flow ultraconcentrator to 1/40 of the culture volume.

6. The concentrate is supplemented with 10mM EDTA, 10mM benzamidine and 0.5 m M phenylmethylsulfonyl fluoride, precipitated by addition of saturated (NH4)2S04 buffered to pH 6.5 to 60 % saturation, kept overnight and recovered by centrifugation.

7. The (NHJ2S04precipitate is dissolved in PBS (150mM NaCI, 10 m M Na2HP04/NaH2P04,pH 6.8), about 2 ml/litre of culture, centrifuged at 50000 g, 30 min and sterile filtered. Covalent chromatography: The method described by Alouf and Geoffrey (1988)is followed with some modifications. A set of columns is used in tandem. The first is a Thiopropyl-Sepharose 6B@ column (about 1 ml/per I of culture) in the free thiol form. The second (about 2 ml/per I of culture) is activated with thiopyridine (if necessary, the gel is activated according to the manufacturerS instruction).The gel of the first column is converted to the reduced form by washing with Pore-forming Toxins

0.1 M sodium borate, 1 m M EDTA, 100mM DTT (3 column volumes), followed by PBS + 2 m M EDTA (5 column volumes) and connected to the second column filled with the thiopyridine-activated gel. The dissolved precipitate is applied with a flow of 0.1 ml/min, and the column set is washed with column buffer, about 10 column volumes overnight. The reducing column is disconnected and the second column is eluted with the latter buffer supplemented with 10mM DTT, at a flow of 0.1 ml/min. Fractions of about 1/5 column volume are collected and assayed by SDS-PAGE and the hemolytic activity determined. Hydrophobic interaction chromatography: Fractions containing hemolytic activity from covalent chromatography column are subjected to hydrophobic-interaction chromatography on AlkylSuperoseB (Pinkney et a/., 1995). Prior to application to the column, the fractions are mixed with an equal volume of 2M (NH4)2S04 in 50 m M Na2P04(brought to pH 6.5 with N a O H ) and centrifuged at 10000 g for 5 min. The sample is applied to the column equilibrated with buffer A (1 M (NH&S04, 5 0 m M Na2P04, p H 6.5) and the column is washed with 4 column volumes of the latter buffer. Streptolysin 0 is eluted by a linear gradient of 5 column volumes to buffer B (50mM NaH2P04, pH6.5) at a flow rate of 0.5mVmin. Fractions (1 ml) are assayed by SDS-PAGE, and hemolytic activity determined. Streptolysin 0 containing fractions are pooled and stored at -70°C.

storage conditions

Storage of purified streptolysin 0.The eluate from the hydrophobic chromatography column may be aliquoted and kept frozen at -70°C. Freeze drying of purified streptolysin 0, without loss of activity (even of dilute samples), is possible if 0.1 % (w/v) bovine serum albumin is added.

17.8 Application of PFTs as Tools in Research permeabilization of plasma membranes

advantages of PFTs

S. BHAKDI etal.

The first paper describing the use of PFTs as tools to permeabilize plasma membranes was published in 1985 (Ahnert-Hilger et a/., 1985). In that study, chromaffin cells permeabilized with alpha-toxin and streptolysin 0 were shown to retain their capacity to secrete granule constituents following stimulation with low concentrations of Ca”. Since then, an ever increasing number of investigators are confirming that PFTs are better tools for membrane permeabilization than digitonin or saponin. As a consequence, alpha-toxin and, especially, streptolysin 0 are now being employed by over 100 groups as tools in cell biology, and this number is steadily growing. The advantages of PFTs are manifold. The effects of digitonin or saponin are normally difficult to predict, control or monitor. In contrast, the actions of PFTs are well characterized and easy to control. The toxins are easy to handle. The two agents currently used by most groups, alpha-toxin and streptolysin 0, are stable and can be stored in lyophilized form for years. No special procedures are required to

activate the toxins: pores are generated within minutes after the application to target cells, without the requirement for divalent ions and over a wide pH range. The processes of binding and pore-formation can generally be dissociated by simple temperature shifts (AhnertHilger et al., 1988). Binding can be achieved at low temperature without pore-formation; the cells may then be washed free of unbound toxin, and permeabilization can be achieved by transferring the cells to 37°C. In this way it can be guaranteed that pore-formation occurs exclusively in the plasma membrane. Pores formed by PFTs are usually fairly well defined in size, so that judicious choice of toxin and of experimental conditions will generally enable one to roduce stable membrane lesions ranging from approximately 8 to 300 8, in functional diameter. It becomes possible to manipulate the intracellular ionic milieu, to introduce small molecules such as nucleotides into the cells, or to apply large molecules such as antibodies to the cytoplasm. Since pure preparations of PFT are devoid of enzymatic (proteolytic) activity, they cause no direct alterations of cell constituents. In the following, simple strategies for utilization of PFTs will be outlined. When very small pores that are permissive for monovalent but not for divalent ions are required, alpha-toxin should be used in a concentration range of 0.5-5 yglml. Most cells will become permeabilized.Note, however, that certain cells exhibit a natural resistance towards alpha-toxin and pore formation will not occur. A simple means to discern whether permeabilization has taken place is to observe whether the cells swell; increases in cell volume are the consequence of an uncontrolled flux of monovalent ions and water, and can be observed microscopically or by flow cytometry. Another simple method is measurement of cellular ATP One hour after toxin application, cells are lysed with Triton X-100, and ATP is quantified using the luciferase assay (method described in (Bhakdi et al., 1989)). ATP depletion will always be found in cells that have been permeabilized. When larger pores that allow fluxes of Ca2+ and ATP/GTP are required, several possibilities arise. The first is the use of high concentrations (>20 yg/ml) of alpha-toxin: this leads to generation of Ca2+-permissivepores in most cells. A further possibility is the use of E. coli hemolysin (HIyA), but two points should be heeded. First, the RTX- toxins require Ca2+in order to bind to target cells. It is therefore more difficult to construct experiments in which Ca2+-dependentcellular processes are to be studied. Second, low concentrations of HlyA provoke short-circuiting of G-protein-dependent signaling pathways. Such reactions may superimpose themselves on the cellular processes under study. HlyA has not yet been used in membrane permeabilization experiments, but this toxin harbors potential advantages since it will permeabilize most mammalian cells to create pores that permit flux of Ca2+and small molecules such as nucleotides, without allowing efflux of cytosolic proteins (reviewed in Bhakdi e t al., 1993).

well defined pore size

1

control of pore size

induction of very small pores

induction of larger pores

use of different toxins

Pore-forming Toxins

production of very large pores

future developments

When very large pores are needed, streptolysin 0 is presently the most convenient toxin to apply. Streptolysin 0 pores are so large that cytoplasmic proteins rapidly egress from the cells; however, intracellular organelles and the cytoskeleton remain, so that key cellular processes such as vesicle trafficking and movement of peptides amongst cellular compartments can be analyzed, e.g. with the use of antibodies. A promising permeabilizing agent is tetanolysin, because this PFT appears to create pores covering a wide range of sizes dependent simply on the concentration applied (Weller et al., unpublished). Finally, engineered Streptolysin 0 toxin mutants are currently being produced in our laboratory that may permit pores of controllable size to be created. A series of alpha-toxin mutants are also becoming available that contain built-in switches and triggers, thus permitting yet more subtle control over the permeabilization event (Bayley, 1994b; Chang et al., 1995; Valeva et a/., 1996; Walker and Bayley, 1994).

17.9 Reagents and Chemicals Materials

Supplier

Cat-No.

Pall-Filtron Pall-Filtron

Prova- F P OSOlOC72

Difco Difco Pharmacia

0123-07-5 0127-07-1 17-0729-01

Merck Difco Pall-Filtron Pharmacia Pharmacia

10493 0122-01 OS030C72 17-0420-01 17-0587-01

Alpha toxin: Tangetial flow concentrator Provario 3 Ultrafiltration membrane Ultrasette Omega (cut off 10 kDa) Bacto Tryptone Bacto Yeast Extract SP-Sepharose fast flow

Streptolysin 0: Brain heart infusion Bacto Profuse-Peptone No. 3 Ultrafiltration membrane (cut off 30 kDa) Thiopropyl-Sepharose 68 Alkyl-Superose HR 10/110 column

Acknowledgements Our studies have received the continued support of the DFG (SFB 311) and the Verband der Chemischen Industrie.

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Grimminger F, Sibelius U, Bhakdi S et al. (1991):Escherichia coli hemolysin is a POtent inductor of phosphoinositide hydrolysis and related metabolic responses in human neutrophils. In J Clin Invest 88: 1531-1539. Hackett M, Guo L, Shabanowitz J et al. (1994): Internal lysine palmitoylation in adenylate cyclase toxin from Bordetella pertussis. In Science 266: 433-435. Hall RH, Drasar BS (1990):Vibrio cholerae hemolysin is processed by proteolysis. In Infect lrnrnun 58: 3375-3379. Hanna PC, Acosta D, Collier RJ (1993):O n the role of macrophages in anthrax. In Proc Natl Acad Sci USA 90: 10198- 10201. Hildebrand A, Pohl M, Bhakdi S (1991):Staphylococcus aureus alpha-toxin. Dual mechanisms of binding to target cells. In J Biol Chem 266: 17195-17200. Howard SP, Buckley JT (1982):Membrane glycoprotein receptor and hole-forming properties of a cytolytic protein toxin. In Biochemistry 21: 1662-1667. lssartel J-P, Koronakis V, Hughes C (1991):Activation of Escherichia coli prohaemolysin to the mature toxin by acyl carrier protein-dependent fatty acylation. In Nature 351 :759-761. lwamoto M, Ohno-lwashita V, Ando S (1990): Effect of isolated C-terminal fragment of @toxin (perfringolysin0)on toxin assembly and membrane lysis. In Eur J Biochern 194: 25-31. Jonas D, Walev I, Berger T et al. (1994):Small transmembrane pores created by staphylococcal alpha-toxin in T lymphocytes evoke internucleosomal DNAdegradation. In Infect lrnrnun 62: 1304-1312. Li JD, Carroll J, Ellar DJ (1991):Crystal structure of insecticidal delta-endotoxin from Bacillus thuringiensis at 2.5 A resolution. In Nature 353: 815-821. Mackiewicz A, Schooltink H, Heinrich PC et al. (1992):Complex of soluble human IL-6-receptor/lL-6up-regulates expression of acute-phase proteins. In J lmrnunol 149: 2021 -2027. Mayer MM (1972): Mechanism of cytolysis by complement. In Proc Natl Acad Sci USA 69: 2954-2959. Milne JC, Furlong D, Hannah PC etal. (1994):Anthrax protective antigen forms oligomers during intoxication of mammalian cells. In J Biol Chem 269: 20607-20612. Montecucco C, Papini E, Schiavo G (1994):Bacterial protein toxins penetrate cells via a four-step mechanism. In FEBS Lett 346: 92-98. O’Hanley P, Lalonde G, Ji G (1991):Alpha-hemolysin contributes to the pathogenicity of piliated digalactoside-binding Escherichia coli in the kidney: efficacy of an alpha-hemolysin vaccine in preventing renal injury in the BALB/c mouse model of pyelonephritis. In Infect lrnrnun 59: 1153-1161. O’Reilly M, Azavedo JCS, Kennedy S et al. (1986): Inactivation of the alphahaemolysin gene of Staphylococus aureus 8325-4 by site-directed mutagenesis and studies on the expression of its haemolysins. In Microb Pathog 1: 125-138. Ohno-lwashita V, Ywamoto M, Mitsui K et al. (1988): Protease nicked @-toxin of Clostridium perfringens a new membrane probe with no catalytic effect reveals two classes of cholesterol as toxin-binding sites on sheep erythrocytes. In EurJ Biochem 176: 95-101. Palmer M, Valeva A, Kehoe M et al. (1995):Kinetics of streptolysin 0 assembly. In Eur J Biochern 231 : 388-395. Palmer M, Weller U, MeOner M et al. (1993):Altered pore-forming properties of proteolytically nicked staphylococcal a-toxin. In J Biol Chern 268: 11963-11967. Parker MW, Buckley JT, Postma JPM et al. (1994):Structure of the Aerornonas toxin proaerolysin in its water-soluble and membrane-channelstates. In Nature 367: 292-295. Parker MW, van der Goot FG, Buckley JT (1996):Aerolysin -the ins and outs of a model channel-forming toxin. In Mol Microbiol 19: 205-212. Paton JC, Andrew PW, Boulnois GJ et al. (1993):Molecular analysis of the pathogenicity of Streptococcus pneumoniae: The role of pneumococal proteins. In Annu Rev Microbiol47: 89- 115. Pinkney M, Kapur V, Smith J et al. (1995): Different forms of streptolysin 0 produced by Streptococcus pyogenes and by Escherichia coli expressing recombinant toxin: cleavage by streptococcal cystein protease. In Infect lrnrnun 63: 2776-2779. S. BHAKDI et al

Prevost G, Cribier B, Couppie P et al. (1995): Panton-Valentine leucocidin and gamma-hemolysin from Staphylococcus aureus ATCC 49775 are encoded by distinct genetic loci and have different biological activities. In Infect lmmun 63: 4121 -4129. Schwartz JL, Garneau L, Savaria D et al. (1993): Lepidopteran-specific crystal toxins from Bacillus thurnigiensis form cation- and anion-selective channels in planar lipid bilayers. In J Membr Biol 132: 53-62. Stanley P, Packman LC, Koronakis V et al. (1994): Fatty acylation of two internal lysine residues required for the toxic activity of Escherichia coli hemolysin. In: Science 266: 1992-1996. Suttorp N, Seeger W, Dewein E et al. (1985):Staphylococcal alpha-toxin-induced PG12 production in endothelial cells: role of calcium. In Am J Physiol248: C127C135. Suttorp N, Buerke M, Tannert-Otto S et al. (1992):Stimulation of PAF-synthesis in pulmonary artery endothelial cells by Staphylococcus aureus alpha-toxin. In Thrornb Res 67: 243-252. Suttorp N, Fuhrmann M, Tannert-Otto S et al. (1993):Pore-forming bacterial toxins potently induce release of nitric oxide in porcine endothelial cells. In J Exp Med 178:337-341. Suttorp N, Floer B, Seeger W et al. (1990):Effects of E coli hemolysin on endothelial cell function. In Infect lmmun 58: 3796-3801. Suttorp N, Hessz T, Seeger W et al. (1988):Bacterial exotoxins and endothelial permeability for water and albumin. In Am J Physiol255: C369-C376. Thelestam M, Mollby R (1975):Sensitive assay for detection of toxin-induced damage to the cytoplasmic membrane of human diploid fibroblasts. In Infect lmmun 12: 225-232. Trifillis AL, Donnenberg MS, Cui X etal. (1994):Binding to and killing of human renal epithelial cells by hemolytic P-fimbriated E. coli. In Kidney.Int 46: 1083- 1091. Valeva A, Weisser A, Walker B et al. (1996): Molecular architecture of a toxin pore: a 15-residue sequence lines the transmembrane channel of staphylococal atoxin. In EM60 J 15: 15: 1857-1864 Van der Goot FG, Lakey J, Pattus F et al. (1992):Spectroscopic study of the activation and oligomerization of the channel-forming toxin aerolysin: identification of the site of proteolytic activation. In Biochemistry 31 : 8566-8570. Van der Goot FG, Ausio J, Wong KR et al. (1993):Dimerization stabilizes the poreforming toxin aerolysin in solution. In J Biol Chem 268: 18272-18279. Van der Goot FG, Pattus F, Parker MW etal. (1994):Aerolysin: from the soluble form to the transmembrane channel. In Toxic01 87: 19-28. Walev I, Reske K, Palmer M etal. (1995):Potassium-inhibitedprocessing of 11-18 in human monocytes. In EM60 J 14: 1607-1614. Walev I, Vollmer P, Palmer M et al. (1996):Pore-forming toxins trigger shedding of receptors for interleukin 6 and lipopolysaccharide. In Proc Natl Acad Sci USA93: 7882- 7887. Walker B, Bayley H (1994):A pore-forming protein with a protease-activated trigger. In Protein Engineering 7: 91 -97. Walker B, Braha 0, Cheley S et al. (1995):An intermediate in the assembly of a pore-forming protein trapped with a genetically-engineered switch. In Chemistry & Biology 2: 99- 105. Welch RA (1991):Pore-formingcytolysins of gram-negative bacteria. In Mol Microbiol5: 521 -529. Welch RA, Bauer ME, Kent AD et al. (1995):Battling against host phagocytes: the wherefore of the RTX family of toxins? In Infect. Agents and Disease 4: 254-272. Welch RA, Dellinger EP, Minshew B et al. (1981): Haemolysin contributes to virulence of extra-intestinal E. coli infections. In Nature 294: 665-667. Weller, U, Muller L, Messner M et al. (1996): Expression of active streptolysin-0 in E. coli as MBP-SLO fusion protein. The N-terminal 70 amino acids are not required for hemolytic activity. In EurJ Biochem, 236: 34-39.

Pore-forming Toxins

Bacterial Toxins

Klaus Aktories Copyright 0 2002 WILEY-VCH Verlag GmbH & Co. KGaA

CHAPTER 18

Application of Alpha-Toxin and Streptolysin 0 as Tools in Cell Biological Research G. AHNERT-HILGER and U. WELLER

18.1 Permeabilized Cells: an Approach to Study lntracellular Processes Permeabilized cells allow the study of intracellular processes in situ under conditions which are believed to be close to the physiological situation in intact cells. Permeabilization by bacterial pore-forming toxins, alpha-toxin and streptolysin 0 (SLO)is now a widely accepted approach in the functional analysis of intracellular organelles. The native forms of the toxins assemble into amphiphilic polymers in the target lipid bilayers where they generate stable transmembrane pores. Alpha-toxin pores comprise a homogeneous population of ring-structured heptamers that allow the free passage of low molecular mass solutes (for review see Bhakdi and Tranum-Jensen, 1987). SLO pores are heterogeneous with a larger diameter which permits the exchange of proteins (Bhakdi and Tranum-Jensen, 1987; AhnertHilger et al., 1989a,b; Ahnert-Hilger et a/., 1993; Bhakdi et al., 1993). The intracellular processes studied after permeabilization with bacterial pore-forming toxins comprise:

1. exocytotic membrane fusion in a growing variety of secretory cells and preparations (Ahnert-Hilger et a/., 1985, 1987; 1989c; 1992; Bader et al., 1986; Howell et al., 1987; Schrezenmeier et al., 1988; van der Merwe etal., 1989; Dekker etal., 1989; Stecher et al., 1989; Stecher et al., 1991, Galli etal., 1994; Sadoul et al., 1995; Lung et al., 1995),

structure of pore-forming toxins

applications

2. intracellular calcium regulation (Fohr et al., 1989; 1991),

3. glucose (McEwen and Arion, 1985; Baldini et al., 1991) and arachidonic acid (Suttorp et al., 1985) metabolism, 4. stimulus-contraction coupling (Kitazawa et al., 1989),

5. intracellular membrane trafficking (Pimplikar and Simon, 1993).

K. Aktories (Ed.),Bacterial Toxins. 0 Chapman & Hall, Weinheim, 1997. ISBN 3-8261-0080-8

18.2 Alpha-Toxin and Streptolysin 0 as Tools in the Study of Secretory Processes Alpha-toxin permeabilizes cells for small molecules ( 53 kDa) whereas SLO renders cells permeable for both small and large molecules (? 150 kDa). In this respect SLO resembles digitonin (AhnertHilger et a/., 1989a,b; 1993; Bhakdi et al., 1993).

18.2.1 Biological Activity Alpha-toxin from Staphylococcus aureus (strain Wood 46, ATCC 10832, DSM 20491) was kindly provided by S. Bhakdi, lnstitut fur Medizinische Mikrobiologie und Hygiene, Johannes Gutenberg Universitat, Mainz, Germany. The toxin can be easily purified from the culture supernatant (Lind et al., 1987; Palmer et al., 1994). The purified toxin can be lyophilized or if necessary dialyzed against an intracellular buffer (see below). Dissolved alpha-toxin can be stored in aliquots at -20°C for months without loss of activity. Streptolysin 0 was purified from the culture supernatant of group A (3-hemolytic streptococci (S. pyogenes) (Pinkney et al., 1995). The excellent final product can be stored in aliquots at -20 "C for years. Wildtype SLO is only active in the reduced form, thus addition of 1 m M dithiothreitol is recommended. The yield of SLO-production from S. pyogenes is low. To overcome this problem a translational fusion between the genes of E. coli maltose binding protein (MBP) and SLO was constructed using the commercially available plasmid pMAL-c2. After transfection into Ecoli high levels of the soluble fusion protein MBP-SLO can be produced. After purification on amylose resin, free active SLO can be obtained by cleaving with factor Xa or trypsin, followed by ion exchange chromatography on hydroxyapatite (Weller et al., 1996). Uncleaved MBP-SLO exhibits similar biological activity to native SLO. This approach allows the production of large amounts of pure SLO for permeabilization. Introduction of a mutant in which cysteine 530 was replaced by alanine provides a SLO with similar biological activity but without the necessity for reduction by DTT (Pinkey et al., 1989; 1995) (see Fig. 1). Both toxins are also commercially available. An alpha-toxin preparation from lnstitut Pasteur has a relatively low titre, but can be concentrated by two successive ammonium sulfate precipitations (55 % and 65 %), followed by dialysis against an intracellular buffer (Schrezenmeier et al., 1988). SLO can also be obtained from Wellcome Diagnostics (Dartford, United Kingdom). The international units (I.U.) given by the company referred to its application to determine the antistreptolysin 0-titre and cannot be compared directly to the hemolytic units mentioned above. However, the material is rather impure (freeze-dried preparation of a partially purified culture filtrate) containing large amounts of reducing cysteine which, when used without further purification, may impair the processes under study. G. AHNERT-HILGERandU. WELLER

8o w

u

1

0-

m+

7J

p

I

20

2~g/rnl SLO

5pg/ml IKg/ml SLO fusion protein

5pg/rnl lbg/ml SLO-mutant

Fig. 1. Comparison of various SLO preparations using Ca2+-stimulatedexocytosis from PC 12 cells. The experiment was performed as described in section 2.2 of this chapter using either SLO purified from the culture supernatant, SLO fusion protein, or the fusion protein of the alanine mutant. In this assay the SLO fusion protein was slightly more effective than the mutant protein

The biological activity of various preparations of both toxins can be easily checked using rabbit red blood cells. Normally, a red blood cell suspension (2.5 Yofinal concentration) in PBS (50 mM phosphate, pH 7, supplemented with 4 % sodium citrate), which can be stored for 3-4 days at 4 "C, was mixed with various toxin dilutions (final volume 55 PI). After 40 min at 37 "C hemolysis is monitored spectrophotometrically at 412 nm in 30 PI of the supernatant, diluted with 1 ml of distilled water. Total hemolysis is determined after the addition of SDS (0.2 %) which gives an extinction of about 1.2. The dilution of toxin hemolyzing 50 % of the red blood cells is taken as the number of hemolytic units per milliliter of the undiluted toxin solution (Lind et al., 1987; AhnertHilger et al., 1989a,b).

78.2.7.7 Assay to Compare Biological Activity of Various PoreForming Toxins Using Rabbit Erythrocytes Solutions

4 Yo sodium citrate:To make 100 ml, dissolve 4g of sodium citrate in distilled water and adjust to a total volume of 100 ml. Phosphate buffered saline (PBS): To make 1 I dissolve 6.9 g NaH2P04x 2 H 2 0 and Z6 g NaCl in about 600ml of distilled water, titrate to pH 7.2 with N a O H and adjust to a total volume of 1 I. Store at 4 "C. 2 % SDS: To make 100 ml dissolve 2 g of sodium dodecylsulfate in 100 ml of distilled water.

Application of Alpha-Toxin and Streptolysin 0 as Tools in Cell Biological Research

Protocol

1. Mix 9 volumes of fresh rabbit blood with 1 volume of 4 % sodium citrate. assay of pore-forming ability

2. Wash 3 times with PBS by a 5 min centrifugation at 3000 g. 3. Dilute the erythrocyte pellet 1 :40 in PBS, which gives a suspension containing about 2.5 % erythrocytes. 4. Mix 5yl of toxin (diluted in PBS, supplemented with 1 m M dithiothreitol in the case of SLO in PBS), 5 pl PBS alone for control, or 5 yI 2 % SDS with 50 yI of the 2.5 70erythrocyte suspension.

5. Incubate for 40min at 37 "C with constant shaking in a water bath.

6. Centrifuge at 12000 g for 2 min. 7. Remove 30 pI of the supernatant and dilute with 1 ml of water. 8. Estimate hemoglobin content spectrophotometrically at 412 nm. The reciprocal dilution of the toxin hemolyzing 50% of the erythrocyte suspension at 37 "C within 40min is taken as the number of hemolytic units per milliliter of the undiluted toxin solution.

checking membrane permeability

An increased permeability of cells can easily be checked using membrane-impermeable dyes which stain either various components of the cell body (trypan blue or eosin) or the nucleus (azure A). Toxintreated cells and the dye, both in an isotonic medium, are mixed to yield a final dye concentration of 0.2%. The percentage of stained cells should be determined immediately in the light microscope, since prolonged incubation leads to staining of intact cells as well. Release of other low molecular weight substances such as Rb+,ATP, or Ca2+is also suitable as a first check of permeability. The permeability of alpha-toxin-treated cells to all small molecules was the same as after treatment with SLO or digitonin (Ahnert-Hilger et al., 1989). The release of cytoplasmic enzymes such as lactate dehydrogenase (mol. wt. 135 kDa) can be used to monitor the permeability for proteins after application of SLO or digitonin (Ahnert-Hilger et a/., 1985; Howell et a/., 1987).Another approach involves the accumulation of antibodies to intracellular proteins (Ahnert-Hilger et al., 1989a) or the application of membrane-impermeable clostridial neurotoxins (Ahnert-Hilger et al., 1989a,c; Stecher et al., 1989; 1991) after permeabilization with SLO or digitonin. The permeability due to alpha-toxin is restricted to the plasma membrane, as the alpha-toxin monomers (34 kDa) are too large to pass through the pores generated by the heptamer (Bhakdi and Tranum-Jensen, 1987; Bhakdi et al., 1993). Accordingly, proteins are not lost during permeabilization. The large pores generated by the SLO monomers after oligomerization also allow proteins to pass. To avoid damage to intracellular membranes by SLO monomers, the cells were incubated very briefly, or at 0 "C. Under the latter conditions the SLO monomers only bind to the plasma membrane. After

G. AHNERT-HILGER and U. WELLER

washing, pore-formation is initiated by warming up the preparation. In contrast to SLO, membrane permeabilization with digitonin is insensitive to the temperature and therefore more difficult to control (Ahnert-Hilger et al., 1989a,b).

avoiding damage to intracellular membranes

18.2.2 Assay for Exocytosis in Permeabilized Cells Permeabilized secretory cells are widely used to study the final events during secretion by exocytosis. Convenient cellular models are bovine adrenal chromaffin cells in short term culture and the rat pheochromocytoma cell line PC 12. Both cell types take up labeled catecholamines and store them in secretory vesicles, from which they can be released upon stimulation. The released catecholamines can be detected in the supernatant. After permeabilization of the plasma membrane, release of catecholamines can be triggered by micromolar concentrations of Ca2+. In most of the studies dealing with exocytosis by permeabilized cells an "intracellular medium" is applied, containing potassium as the main cation, and glutamate as an anion (Ahnert-Hilger et al., 1989b; 1993).Since the free Ca2+concentration within the cells under resting conditions as well as during stimulation is in the micromolar range, this ion must be carefully controlled in the buffers used. A combination of chelators for divalent cations is suitable to buffer the free Ca2+concentration from 0.1-100 pM under experimental conditions. Added Mg2+and ATP as well as the pH of the medium must be considered, because they alter the equilibrium between Ca2+and the chelators present. The free Ca2+and Mg2+concentrations are calculated by a computer program and controlled by Ca2+and Mg2+ specific electrodes (Fohr et a/., 1993). Each Ca2+buffer is prepared separately from stock solutions, with a final check of pH, pCa, or pMg. If no Ca2+electrode is available, the calculated total amount of Ca2+(as CaCI2)and Mg2+(as M g ( C H 3 C 0 0 ) 2must ) be added before the pH adjustment. Buffers can be stored at -20°C but should be thawed only once, mainly because of decomposition of ATE The experimental procedures for both PC 12 and adrenal chromaffin cells, using alpha-toxin or SLO to permeabilize the plasma membrane, are given below. The secretory response decreases with time after permeabilization. The amount of the decrease differs between different cells and preparations. In SLO-permeabilized PC 12 cells, exocytosis can be restored using cytosolic fractions. The free Ca2+concentration necessary to elicit exocytosis varies between 1 and 10 p M depending on the cell preparations, but is always 3-5 fold higher in alpha-toxinpermeabilized cells. Since the permeability to Ca2+is the same in cells treated with alpha-toxin, SLO or digitonin (Fohr et al., 1989, 1991) buffering by intracellular proteins may be the reason for this variability.

control of CaZ+ concentration

Application of Alpha-Toxin and Streptolysin 0 as Tools in Cell Biological Research

Solutions for the assay of exocytosis

Examples of KG-buffers used in the analysis of exocytosis from permeabilized chromaffin or PC 12 cells: 1 M M g ( C H 3 C 0 0 ) 2stock solution: prepare by dissolving 2.145 g M g ( C H 3 C 0 0 ) 2x 4 H 2 0 in 10 mI of distilled water.

1 M CaCI2stock solution: prepare by dissolving 1.47 g CaCI2x 2 H 2 0 in 10 ml of distilled water. KG-buffer I with Mg2+/ATP for permeabilization and incubation before the stimulation (150m M K+-glutamate, 2 mM EGTA, 2 m M EDTA, 20 m M PIPES, 2 m M Na+/ATP,1 m M free Mg2+):For 500 ml dissolve 13.9 g K+-glutamate, 0.38 g EGTA (free acid), 0.2 g EDTA (free acid), 3.02 g PIPES (free acid) and 0.61 g Na/ATP in about 300ml of water, add KOH to give a pH of 7.0 and stir at room temperature. Add 2.4 ml of 1 M M g ( C H 3 C 0 0 ) 2 to give a final free Mg2+concentration of 1 m M and adjust pH exactly to 7.0 with KOH. Make up to 500ml. KG-buffer II with Mg2'/ATP and Ca2+(15 p M free) for stimulation: For 100ml dissolve one fifth of the amount of salts for KG buffer I in about 60ml. Add 370pl of 1 M Mg(CH3C00)2and 310y.1of 1 M Ca(C1)2,to give a final free Mg2+concentration of 1 m M and a final free Ca2+concentration of 15 pM. Adjust pH exactly to 7.0 with KOH. Make up to 100ml. In some experiments (i.e. in permeabilized PC 12 cells) ATP may be omitted during permeabilization and/or stimulation (Ahnert-Hilger et a/., 1985, 1989a,b). KG-buffer 111 without ATP and Ca2+:For 500 ml the procedure is the same except that Na/ATP is left out. Add 1.55ml of 1 M M g ( C H 3 C 0 0 ) to 2 give a final free Mg2+concentration of 1 mM, and adjust pH exactly to 70 with KOH. Make up to 500 ml. KG-buffer IV without ATP but with Ca2+(15 y M free): For 100 ml the procedure is the same as for KG-buffer II except that Na/ATP is left out. Add 1901.11of 1 M Mg(CH3C00)2and 3101.11 of 1 M Ca(Q2 to give a final free Mg2+concentration of 1 m M and a final free Ca2+ concentration of 15 pM. Adjust pH to ZO and make up to 100 ml. Ca2+-freeKrebs-buffer (140mM NaCI, 4.7 m M KCI, 1.2 m M KH2P04, 1.2mM MgS04, 20mM PIPES, 11 mM glucose, pH 7.0): For 1 I weigh

the following substances: 8.2 g NaCI, 0.35 g KCI, 0.16 g KH2P04,0.3 g MgS04 x 7H20, 4.8 g HEPES (free acid), dissolve in 800ml of distilled water, adjust pH to 7.0 with NaOH and make up to 1 I. Store in appropriate aliquots at -20 "C. Add glucose (0.218 g per 100ml) immediately before use. 100mM ascorbic acid: For lOml, weigh 0.176 g and dissolve in distilled water. Store in aliquots at -20 "C. G. AHNERT-HILGERand U. WELLER

l-[7,8-3H] Noradrenaline, ( [3H]NE, 15 Ci/mmol): Cat No. TRA.584, Am ers ha m BuchIer, Braunsc hwe ig , Germa ny. To load one 100 mm culture dish mix 100 pI 100 m M ascorbic acid with 10 pI [3H]NEand 10 ml of DMEM. For one 24 multiwell mix 66 pI of 100 m M ascorbic acid with 7 p.1 [3H]NEand 6.6 ml DMEM. Assay of exocytosis for cells in suspension, e.g. PC 12 cells

1. Label one 100mm dish with 10 ml [3H]NE-solutionfor 2 h in the cell incubator

2. Aspirate the labelling medium and chase cells for 1-2 h in DMEM.

exocy+osisof cells in suspension

3. Aspirate DMEM and wash cells with KR-buffer four times for 5 min.

4. Wash cells once with 10ml KG buffer I or Ill.

5. Suspend cells in 0.6-0.9ml KG buffer I or Ill and divide among 12-18 vials (50pl each, corresponding to about 2-3 x lo5cells/ sample) already containing 50 pI of 100 HU/ml alpha-toxin or SLO, corresponding to about 300-500 HU/107 cells. Incubate for 20-30 min at 25 "C, 30 "C or 37 "C with alpha-toxin or 5 min on ice with SLO. 6. Centrifuge each sample at 30009 for 20 sec and remove supernatant.

7. Resuspend pellet in 1OOp.I of KG-buffer I or Ill containing the substances to be tested and incubate for 5 up to 40min at 25 "C, 30 "C or 37 "C. 8. Repeat step 6. 9. Stimulation: Resuspend in loop1 KG-buffer II or Wand incubate for 3- 10 min at 25 "C, 30 "C or 37 "C. 10. Remove supernatant and determine released catecholamines by liquid scintillation counting.

11. Add 200 pl/well of 0.2 % SDS to solubilize the cells and to determine the remaining catecholamines. Assay of exocytosis for cells attached to culture plates

1.

Label cells (250 pl/well with a diameter of 20 mm) with radioactive noradrenaline for 2 h in the cell incubator.

2. Aspirate the labeling medium and chase cells for 1-2h in DMEM. 3. Aspirate DMEM and wash cells with KR-buffer four times for 5 min.

W

exocytosisof attached cells

Application of Alpha-Toxin and Streptolysin 0 as Tools in Cell Biological Research

4. Wash cells once with 200yl KG buffer I or Ill 5. Aspirate supernatant and add 200yl of alpha-toxin or SLO dissolved in KG-buffer I or Ill. Incubate with pore forming toxins: alpha-toxin 20-30 min at 25 "C, 30 "C or 37 "C, SLO 5 min at 0 "C or between 1 and 2 min at 25 "C, 30 "C or 37 "C. About 30 HU/ml alpha-toxin or SLO, corresponding to 300 HU/107 cells are used.

6. Aspirate solution. 7. Incubate with substances to be tested, dissolved in KG buffer I or Ill, for 5 to 40min at 25 "C, 30 "C or 37 "C. 8. Same procedure as in step 6. 9. Stimulation: Add 200 pl KG-buffer II and incubate for 3-10 min at 25 "C, 30 "C or 37 "C. 10. Remove supernatant and determine released catecholamines by liquid scintillation counting.

11. Add 200yllwell of 0.2% SDS to solubilize the cells and to determine the remaining catecholamines. Fig. 1 shows the result of a representative experiment using Ca2+induced exocytosis from PC 12 cells. Here, native SLO, the fusion protein, and the fusion protein of the alanine mutant, were compared.

18.3 Regulation of Vesicular Transmitter Transporters in Permeabilized Cells and Synaptosomes Transmitter uptake into secretory vesicles is an ATP-dependent process. So far these studies have been restricted to isolated secretory vesicles, where intracellular substances necessary for regulation may be lost during purification. Neuroendocrine cells or synaptosomes permeabilized with both pore-forming toxins can be used to study the regulation of transmitter storage without the necessity of purifying secretory vesicles.

18.3.1 Catecholamine Uptake into SLO-Permeabilized PC 12 Cells Catecholamine uptake is driven by the pH gradient across the vesicle membrane (Schuldiner et al., 1979).

G. AHNERT-HILGER and U. WELLER

Solutions for transmitter uptake into secretory vesicles in permeabilized cells Buffer I: For 500 ml dissolve 34.23 g sucrose (200mM), 1.86 g KCI (50mM), 3.024 g PIPES (20mM), 0.7608 g EGTA (4 mM) and 0.1017 g MgCI2 (1 mM) adjust to pH 7.0 using KOH Buffer II: use the same constitutents as for buffer I, add 2.87 g Mg/ ATP (2 mM) for 50 ml, and adjust to pH 7.0 with KOH. Assay of ATP-dependent uptake of catecholamines or amino acid transmitters The assay is performed in suspension. 1. Remove medium from cells, and resuspend them in KrebsRinger-HEPES and wash them twice with Krebs-Ringer-HEPES buffer by centrifugation.

2. Resuspend pellet in ice cold buffer I containing SLO or alphatoxin.

assay of catecholamine

uptake

3. Incubate samples with SLO for 5min on ice, alpha-toxinsamples for 10 min at 32 "C (incubation time may be increased). 4. Centrifuge at 4 "C and resuspend in ice-cold buffer I.

5. Incubate for 20min at 36 "C to remove soluble cytosolic components.

6. Centrifuge at 4 "C and aspirate supernatant.

7. Resuspend in buffer II containing 3H labeled transmitters such as GABA or noradrenaline and incubate for 20 min at 36 "C. 8. Stop the reaction by diluting with 1 ml ice-cold buffer I followed by centrifugation.

9. Remove supernatant and dissolve pellet in 0.2 % SDS. 10. Use one aliquot to count radioactivity, and the other one for the determination of protein content. Results of a representative experiment are given in Table 1. As expected the ATP-dependent uptake is inhibited by reserpine. A similar experimental protocol can be applied to bovine adrenal chromaffin cells attached to culture plates.

18.3.2 Acidification of Small Synaptic Vesicles in SLO-Permeabilized Synaptosomes The uptake of aminoacid neurotransmitter has been characterized by bioenergetic studies (Hell et al., 1992; Hartinger and Jahn, 1993). In contrast to catecholamine and GABA uptake, glutamate uptake does not depend on a pH gradient over the vesicle membrane. Application of Alpha-Toxin and Streptolysin 0 as Tools in Cell Biological Research

Table 1. Catecholamine uptake into permeabilized PC 12 cells

3H noradrenaline uptake (pmoles/mg protein) no ATP ATP ATP + reserpine 2 pM ATP + reserpine 0.5 pM

11.0 f 0.8 30.6 f 3.2 9.9 f 1.8 12.3 f 0.7

PC 12 cells were permeabilized with purified SLO as given in section 18.31 of this chapter. Protein content was measured using the BCA-method. Values are the mean of three samples k SD. Similar results were obtained using either wild-type SLO fusion protein or its alanine mutant. The assay can also be performed using alpha-toxin for permeabilization.

uptake of glutamate

Uptake of glutamate results in an acidification of the intravesicular lumen. This acidification can be indicated by the fluorescent dye acridine orange, which accumulates in acid compartments. Synaptosomes were prepared (McMahon etal., 1992) and stored as pellets of about 1 mg overlaid with 200 pl Krebs-Ringer-HEPES buffer on ice. Acidification was performed with either glutamate or chloride (Hell et al., 1992; Hartinger and Jahn, 1993).To get rid of the non-vesicular glutamate extensive washing of the permeabilized synaptosomes is required. Measurement is performed under constant stirring at 492 nm using 530 nm as a reference with the Aminco dual beam spectrophotometer. Acidification in permeabilized synaptosomes Solutions: Acidification buffer: To prepare 500 ml dissolve 51.35 g sucrose (300mM); 246.5mg M g S 0 4 (2mM); 203mg MgC& (2mM); 1.046 g MOPS (10mM) and adjust to pH Z4 with KOH.

EGTA K+-salt100 mM: dissolve 3.8 g free acid in 100 ml and adjust to pH 7.0 with KOH. Oligomycin B stock solution 805.1 pg in 1 ml ethanol (1 mM); store in aliquots of 100-200 pI. Ouabain 7.288 mg in 1 ml water (10mM), store in aliquots of 200 pl. NaVanadate 550 pg in 1 ml water (5 mM), prepare immediately before use ATP-generating system: Creatine kinase (Boehringer Mannheim) 4 mg/ml in glyceroVwater (1 + 1) store in aliquots of 200 PI; creatine phosphate (800mM) 261.76 mg/ml in water, store in aliquots of 200 pl; mix 1 part of CP and one part of CK.

G. AHNERT-HILGERand U. WELLER

ATP disodium salt: dissolve 73.2 mg in 1 ml water (120mM) adjust to about pH 6 with KOH. 1 M K-glutamate: dissolve 185,2 mg glutamic acid, monopotassium salt in 1 ml distilled water. 3 M KCI: dissolve 223,7 mg in 1 ml distilled water. 3 M (NH4)2S04: dissolve 396,3 mg in 1 ml distilled water. Acridine orange 1 mM: dissolve 2,654 mg/lO ml 30 % ethanol, store at -20 "Cin the dark. Permeabilization:

1. Remove the Ringer solution from the synaptosomal pellet, resuspend the pellet in 200 pI acidification buffer containing EGTA and SLO (5p1/200pl, dilution depends on batch) or alpha-toxin (12-20 pV200 pl, dilution depends on batch). 2. Incubate SLO-samples for 5min on ice, a-toxin-samples for 10 min at 32 "C (incubation time may be increased).

permeabilization of synaptosomes

3. Spin down for 2min at 5500 rpm, remove supernatant and resuspend pellet in 200 pl permeabilization buffer. 4. Incubate for 5 min at 32 "C dilute with 1 ml of ice cold permeabilization buffer (acidification buffer plus EGTA).

5. Spin down for 2 min at 5500 rpm. 6. Wash three times with ice cold buffer. 7. Resuspend pellet in 1 ml acidification buffer, incubate at 32 "C for 5 min, and spin down. 8. Store the permeabilized synaptosomes as pellets on ice until use.

Glutamate- and chloride-induced acidification: 1. Resuspend permeabilized synaptosomes in 1 ml acidification buffer containing 200 p M ouabain, 100 p M vanadate, 100 p M oligomycin B pre-warmed to 32 "C,

2. Add 34 pl of CK/CP plus 20 pl 1 mM acridine orange.

assay of permeabilization

3. Start measurement. 4. Add subsequently 40 pl 120 m M ATP, 10 pI 1 M K-glutamate, 15 pl 3 M KCI, and 101.113 M (NH4)2 SO4(see Figure 2). Figure 2 illustrates representative experiments using rat brain synaptosomes. Acidification can be measured only in permeabilized synaptosomes.

Application of Alpha-Toxin and Streptolysin 0 os Tools in Cell Biological Research

3

i

min -

Glut

KCI

NH,'

-1

-1

-1

k

Fig. 2. Acidification of small synaptic vesicles by glutamate and chloride in synaptosomes. The acidification assay was performed as described in section 3.2 of this chapter. Two representative experiments with intact (upper trace) or SLOpermeabilized (lower trace) synaptosomes are shown. The ordinate gives the changes of absorbance obtained ( A 492-530). Final concentrations of potassium glutamate (Glut), KCI, and ammonium sulfate (NH,') were lOmM, 45mM and 30 mM, respectively.The uptake of glutamate and chloride result in an acidification of the lumen of small synaptic vesicles, which increases the vesicular uptake of acridine orange, resulting in a decrease in the amount of extravesicular dye. This acidification can be only observed when the plasma membrane is permeabilized

18.4 Reagents and Chemicals Materials

Supplier

Cat-No.

Acridine orange ATP (sodium salt) Creatine kinase Creatine phosphate 3H Noradrenaline Oligomycin B Ouabain Reserpine Vanadate (sodium salt)

Sigma Sigma Boehringer Sigma Amersham Sigma Sigma Sigma Fluka

A 4921 A 3377 127566 P 7936 TRA.584 0 5126 0 3125

R 0875 72060

Acknowledgements The author would like to thank B. Wiedenmann and S. Bebenroth, Medizinische Klinik und Poliklinik Abteilung Gastroenterologie, Universitatsklinikurn Benjamin Franklin, Freie Universitat Berlin, S. Bhakdi Medizinische Mikrobiologie und Hygiene, Johannes Gutenberg Universitat, Mainz, Germany and R. Jahn, Howard Hughes Medical School Department of Pharmacology, Yale University, New Haven USA. Work was supported by the DFG (SFB 515) and by a short term grant of the Boehringer lngelheirn Fonds to G.A.H. G. AHNERT-HILGERand U. WELLER

References Ahnert-Hilger G, Bhakdi S, Gratzl M (1985):Minimal requirements for exocytosis.A study using PC12 cells permeabilized with staphylococcal alphatoxin. J. Biol. Chem. 260: 12730-12734. Ahnert-Hilger G, Brautigam M, Gratzl M (1987): Ca2+-stimulatedcatecholamine release from permeabilized PC 12 cells: Biochemical evidence for exocytosis and its modulation by protein kinase C and G-proteins. Biochemistry 26: 7842- 7848. Ahnert-Hilger G, Bader M-F, Bhakdi S, et al. (19890): Introduction of macromolecules into bovine adrenal medullary chromaffin cells and rat pheochromocytoma cells (PC12) by permeabilization with streptolysin 0: Inhibitory effect of tetanus toxin on catecholamine secretion. J. Neurochem. 52: 1751-1758. Ahnert-Hilger G, Mach W, Fohr KJ etal. (l989b): Poration by alphatoxin and streptolysin 0:An approach to analyze intracellular processes. Methods in Cell Biol. 31: 63-90. The tetanus toxin light Ahnert-Hilger G, Weller U, Dauzenroth ME, et 01. (1989~): chain inhibits exocytosis. FEBS Lett. 242: 245-248. Ahnert-Hilger G, Wegenhorst U, Stecher B et al. (1992): Exocytosis from permeabilized bovine adrenal chromaffin cells is differently modulated by GTPyS and GMPPNHF! Evidence for the involvement of various guanine nucleotide-binding proteins. Biochem. J. 284: 321 -326. Ahnert-Hilger G, Stecher B, Beyer C et al. (1993): Exocytotic membrane fusion as studied in toxin-permeabilized cells. Methods in Enzymology, 221 : 139- 149. Bader MF, Thierse D, Aunis D etal. (1986):Characterization of hormone and protein release from alphatoxin-permeabilized chromaffin cells in primary culture. J. Biol. Chem. 261 : 5777-5783. Baldini G, Hohman R, Charron MJ et al. (1991): Insulin and nonhydrolyzable GTP analogs induce translocation of GLUT4 to the plasma membrane in alphatoxinpermeabilized rat adipose cells. J. Biol. Chem. 7: 4037-4040. Bhakdi S, Tranum-Jensen J (1987): Damage to mammalian cells by proteins that form transmembrane pores. Rev. Physiol. Biochem. Pharmacol. 107: 147-223. Bhakdi S, Weller U, Walev I et al. (1993):A guide to the use of pore-forming toxins for the controlled permeabilization of cell membranes. Med. Microbiol. Immunol. 183: 167-175. Dekker LV, De Graan PNE, Oestreicher AB etal. (1989):Inhibition of noradrenaline release by antibodies to B-50 (GAP-43).Nature 342: 74-76. Fohr KJ, Warchol W, Gratzl M. (1991): Calculation and control of free divalent cations in solutions used for membrane fusion studies. Methods in Enzymology, 221 : 149- 153. Fohr KJ, Scott J, Ahnert-Hilger G et al. (1989):Characterizationof the inositol 1,4,5,trisphosphate-induced calcium release from permeabilized endocrine cells and its inhibition by decavanadate and p-hydroxymercuribenzoate. Biochem. J. 262: 83-89. Fohr KJ, Ahnert-Hilger G, Stecher B etal. (1991): GTP and Ca2+modulate the inosito1 1,4,5,-trisphosphate-dependentCa2+release in streptolysin 0-permeabilized bovine adrenal chromaffin cells. J. Neurochem. 56: 665-670. Galli T, Chilcote T, Mundigl 0 et a/. (1994):Tetanus toxin-mediated cleavage of cellubrevin impairs exocytosis of transferrin receptor-containing vesicles in CHO cells. J. Cell Biol. 125: 1015-1024. Hartinger J, Jahn R (1993):An anion binding site that regulates the glutamate transporter of synaptic vesicles. J. Biol. Chem. 268: 23122-23127. Hell JW, Maycox PR, Jahn R (1990):Energy dependence and functional reconstitution of the y-aminobutyric acid carrier from synaptic vesicles. J. Biol. Chem. 265: 2111-211z Howell Tw, Cockcroft S, Gomperts BD (1987):Essential synergy between Ca2+and guanine nucleotides in exocytotic secretion from permeabilized mast cells. J. Cell Biol. 105: 191- 197. Kitazawa T, Kobayashi S, Horiuti K et a/. (1989):Receptor coupled, permeabilized smooth muscle: Role of the phosphatidylinositol cascade, G-proteins and Application of Alpha-Toxin and Streptolysin 0 as Tools in Cell Biological Research

modulation of the contractile response to Ca”. J. Biol. Chem. 264: 5339-5342. Lung J, Nishimoto I, Okamoto T, et a/. (1995): Direct control of exocytosis by receptor-mediatedactivation of the heterotrimericGTPases G, and Goor by the expression of their active G, subunits. EMBO J. 14: 3635-3644. Lind I, Ahnert-Hilger G, Fuchs G et a/. (1987):Purification of alpha-toxin from staphylococcus aureus and application to cell permeabilization. Analyt. Biochem. 164: 84-89. McEwen BF, Arion WJ (1985): Permeabilization of rat hepatocytes with Staphylococcus aureus alphatoxin. J. Cell Biol. 100: 1922-1929. McMahon HT, Foran P, Dolly JO et al. (1992):Tetanus toxin and botulinum toxins type A and B inhibit glutamate, y-aminobutyric acid, aspartate, and metenkephalin release from synaptosomes. J. Biol. Chem. 276: 21338-21343. Palmer M, Jursch R, Weller U et a/. (1993):Staphylococcus aureus a-toxin. Production of functionally intact, site-specifically modifiable protein by intoduction of cysteine at positions 69, 130, and 186. J. Biol. Chem. 68: 119-11962. Palmer M, Valeva A, Kehoe M etal. (1995):Kinetics of streptolysin 0 self-assembly. Eur. J. Biochem. 231 : 388-395. Pimplikar S, Simons K (1993): Role of heterotrimeric G-proteins in polarized membrane transport. J. Cell Sci. 17: 27-32. Pinkney M, Beachey E, Kehoe M (1989):The thiol-activated toxin streptolysin 0 does not require a thiol group for activity. Infect. Immun. 57: 2553-2558. Pinkney M, Kapur V, Smith J etal. (1995):Different forms of streptolysin 0 produced by Streptococcus pyogenes and by Escherichia coli expressing recombinant toxin: cleavage by streptococcal protease. Infect. Immun. 63: 2776-2779. Sadoul K, Lung J, Montecucco C etal. (1995):SNAP-25 is expressed in islets of Langerhans and is involed in insuline release. J. Cell Biol. 128: 1019-1025. Schrezenmeier H, Ahnert-Hilger G, Fleischer B (1988):AT cell receptor-associated GTP-binding protein triggers T cell receptor-mediatedgranule exocytosis in cytotoxic T lymphocytes. J. Immunol. 141: 3785-3790. Schuldiner S, Fishkes H, Kanner BI (1978):Role of a transmembrane pH gradient in epinephrine transport by chromaffin granule membrane vesicles. Proc. Natl. Acad. Sci. 75: 3713-3716. v Stecher B, Weller U, Habermann E et a/. (1989):The light chain but not the heavy chain of botulinum A toxin inhibits exocytosis from permeabilized adrenal chromaffin cells. FEBS Lett. 255: 391 -394. Stecher B, Ahnert-Hilger G, Weller U etal. (1992):Amylase release from streptolysin 0-permeabilized pancreatic acinar cells: effects of Ca2+,GTPyS, CAMP,tetanus toxin and botulinum A toxin. Biochem. J. 283: 899-904. Suttorp N, Seeger W, Dewein E et a/. (1985):Staphylococcal alpha-toxin-induced PGI, production in endothelial cells: Role of calcium. Am. J. Physiol. 248: C127C134. van der Merwe PA, Millar RP, Wakefield IK et a/. (1989):Mechanism of luteinizinghormone exocytosis in Staphylococcus aureus-alphatoxin-permeabilized sheep gonadotropes. Biochem. J. 264: 901 -908. Weller U, Muller L, MeOner M et al. (1996):Expression of active streptolysin-0 in E. coli as maltose-binding protein-streptolysin-0 fusion protein: The N-terminal 70 amino acids are not required for hemolytic activity. Eur. J. Biochem. 236: 34-39

G. AHNERT-HILGER and U. WELLER

Bacterial Toxins

Klaus Aktories Copyright 0 2002 WILEY-VCH Verlag GmbH & Co. KGaA

CHAPTER 19

Toxins as Transporting Tools S. OLSNES, 0. KLINGENBERG, R. MUNOZ, I? 0. FALNES, and A. WIEDLOCHA

19.1 Introduction It is being recognized that an increasing number of protein toxins from bacteria and plants act on targets located in the cytosol. Recently it has been shown that such toxins can be used to carry passenger proteins across cellular membranes into the cytosol. A common feature of the toxins in question is that they consist of two functionally different parts, A and B. The B-moiety binds the toxin to cell surface receptors, a binding which is required to produce the toxic effect at low concentrations of the toxin. The A-moiety is an enzyme that enters the cytosol and damages intracellular components. The B-moiety may be a separate part of the same protein as the A-moiety, or it may consist of one or several separate polypeptides. Toxins of this group include diphtheria toxin, Pseudomonas aeruginosa exotoxin A, cholera toxin, E. coli heat labile toxin, Shigella toxin, Shiga-like toxin, tetanus and botulinum toxins, Clostridium difficile toxin, anthrax toxin, invasive adenylate cyclase from Bordetella pertussis, and possibly Pasteurella multocida toxin. A number of plant cytotoxins, such as ricin, abrin, modeccin, viscumin and volkensin also act on targets in the cytosol (Olsnes and Sandvig, 1985).All these toxins are potential carriers for peptides and proteins targeted to the cytosol. So far, however, only diphtheria toxin and, to a lesser extent Pseudomonas toxin and anthrax toxin, have been exploited for this purpose.

toxins that act in cytosol

19.2 Diphtheria Toxin Diphtheria toxin is the main pathogenicity factor in diphtheria (Pappenheimer, 1977).The toxin gene is carried by a bacteriophage, fir which is lysogenic in Corynebacterium diphtheriae. Toxigenic strains of the bacteria cause local infection of the throat. After recovery from the acute phase of the disease, life-threatening organ complications often occur, mainly in the heart, which are due to toxin produced by the bacteria in the throat and released into the circulation. Due to mass vaccination, the disease is now almost extinct in developed countries. K. Aktories (Ed.), Bacterial Toxins. 0 Chapman & Hall, Weinheim, 1997. ISBN 3-8261-0080-8

-

a

A-fragment(21 kD)

w

b

Passenger protein A-fragment

B-fragment(38 kD)

&fragment

1

Trypsln

Catalytic domain

d

u

Binding domaln

Transmembrane domain

Fig. 1. Schematic structure of intact and nicked diphtheria toxin (a) and of a fusion protein where a passenger protein has been fused to the N-terminus of the Afragment (b)

structure

Diphtheria toxin is produced as a single polypeptide chain (Fig. 1a) which is easily cleaved ("nicked") by trypsin and trypsin-like proteases into two disulfide-linked fragments, A and B (Pappenheimer, 1977).The structure of the nicked toxin resembles that of the plant toxins ricin, abrin, modeccin, viscumin and others (Olsnes and Sandvig,

1985). Diphtheria toxin inactivates elongation factor 2, an enzyme required for protein synthesis (Pappenheimer, 1977) through catalyzing its ADP-ribosylation, thereby inhibiting protein synthesis and inducing cell death. Elongation factor 2 contains a unique amino acid, diphthamide, which is formed by posttranslational modification of a histidine residue (Van Ness et al., 1980).The ADP-ribose binds covalently to this unusual amino acid.

19.2.1 Toxin-resistant Cell Mutants toxin-resistant mutants

S. OLSNES et 01.

It is important that toxin-resistant mutants can be obtained easily. Mutations in the coding region of the elongation factor 2 gene close to the histidine residues that normally becomes modified to form diphthamide, may render the protein unrecognizable by the modifying enzymes, so that diphthamide is not formed (Moehring et al., 1980; Omura et a/., 1989, Foley et al., 1995).Mutations in the gene for one of the modifying enzymes may also result in the absence of diphthamide. Surprisingly, elongation factor lacking diphthamide is still fully active and the cells may exhibit normal growth, but they are completely resistant to diphtheria toxin (and to Pseudomonas toxin).

19.2.2 Receptor for Diphtheria Toxin The entry of diphtheria toxin to reach its intracellular target is initiated by binding to cell surface receptors (Boquet and Pappenheimer, 1976). The receptor has been identified as the uncleaved precursor of heparin-binding epidermal growth factor-like growth factor (Naglich etal., 1992).This precursor is easily cleaved close to the membrane to release the growth factor. Such cleavage is induced by phorbol esters, which also renders the cell highly resistant to diphtheria toxin (Olsnes et al., 1986; Dluz et al., 1993; Lanzrein et al., 1995).The cleaved receptor remains to a large extent bound at the cell surface due to interaction with surface heparans (Lanzreinet al., 1995),so that the ability of the cells to bind toxin is therefore not much reduced. However, in the presence of soluble heparin the cleaved receptor is released from the cells. Cells vary greatly in the number of toxin-binding sites. The most sensitive cell lines are certain strains obtained from the kidney of the African green monkey; such as Vero, COS and CV-1 (Middlebrook et al., 1978).These cells contain of the order lo5 binding sites per cell. The human osteosarcoma cell line, U2 OS, also contains a high number of receptors. Other cell lines, such as HeLa, exhibit an order of magnitude fewer binding sites, but the cells are still sensitive to the toxin (Middlebrok et al., 1978; Sundan et al., 1982). Mouse and rat cells are highly resistant to the toxin, and they show little or no specific binding due to minor differences in the amino acid sequence of the growth factor precursor (Hooper and Eidels, 1995). Insect cells are also resistant to the toxin (Valdizan et al., 1995). In cells containing low amounts of surface heparans, the toxin does not bind well unless soluble heparin is added (Valdizan et al., 1995). Apparently, the heparin-binding site of the toxin receptor covers the binding site for the toxin until heparin is bound. In normal animal cells surface heparan proteoglycans carry out this function. Murine or insect cells transfected with human or simian diphtheria toxin receptor become highly sensitive to the toxin (Naglich et al., 1992; Valdizan et al., 1995). The transmembrane domain of the receptor is apparently not required for activity, since cells transfected with a mutant receptor in which the transmembrane and intracellular domains have been replaced by a sequence signalling the addition of a glycophosphoinositol (GPI)anchor are highly sensitive to the toxin (Lanzrein et al., 1996).The finding that a GPI anchor can replace the transmembrane domain to yield functional receptor, whereas the cleaved but cell-associated receptor is not functional, indicates that the main function of the receptor is to position the toxin in the correct orientation and the correct distance with respect to the membrane, so that the toxin is ready to insert once the pH is reduced, which is the event inducing translocation of the A-fragment of diphtheria toxin (see below).

number of binding sites

Toxins os Transporting Tools

19.2.3 Translocation of the Toxin The next step in the entry process is endocytic uptake of the bound toxin. The uptake is comparatively slow and appears to occur from clathrin-coated pits (Morris et a/., 1985). Once in endosomes, the toxin is exposed to acidic conditions: a pH below 5.3 is necessary for the penetration to the cytosol (Sandvig and Olsnes, 1981).If acidification of the endosomes is prevented by exposure of the cells to NH&I or monensin, the cells are protected from poisoning. However, the protection can be overcome if the cells are exposed to buffer with acidic pH (Sandvig and Olsnes, 1981). Under these conditions toxin bound at the cell surface penetrates directly through the surface membrane (Moskaug et a/., 1988). Low pH induces a conformational change in the B-fragment to expose hydrophobic regions that are normally hidden (Sandvig and Olsnes, 1981, Zhao and London, 1990). As a result, the B-fragment inserts itself into the membrane and apparently becomes an integral membrane protein. Simultaneously, the A-fragment is translocated to the cytosol (Moskaug et al., 1988). If, at this stage, the cells are treated with proteolytic enzymes, such as pronase, the translocated A-fragment and a 25 kDa piece of the B-fragment are protected against proteolytic digestion. The protected part of the B-fragment which represents its C-terminal region, is inserted into the membrane or translocated into the cell (Moskaug et al., 1991). If the cells are subsequently permeabilized with low concentrations of saponin, the Afragment is released into the medium, whereas the B-fragment remains associated with the membranes. This indicates that only the A-fragment is free in the cytosol. Extensive unfolding of the A-fragment appears to be required for translocation to occur. The A-fragment does not contain an internal disulfide bond, but when such a bond was introduced, translocation did not take place (Falnes etal., 1994).In fact, experiments where one disulfide bond was introduced at four different locations in the Afragment showed that translocation was prevented in each case. This indicates that if the protein is not able to unfold, translocation does not take place (Fig. 2). When the intrafragment disulfide was placed close to the N-terminal end of the A-fragment, the interfragment disulfide was reduced upon exposure to low pH and the A-fragment became stuck half-way during translocation (Falnes and Olsnes, 1995).When the disulfide was C-terminal, the inter-fragment disulfide was also reduced, but the A-fragment was released into the medium. This indicates that the translocation is initiated from the C-terminal end of the A-fragment.

19.2.4 Further Requirements for Translocation When translocation is induced at the surface membrane, by exposure to low pH, only a fraction (10-50 %) of the bound toxin is translocated (Falnes et a/., 1994; Moskaug et a/., 1987).A number of requirements S. OLSNES etal.

@I B

AA

inhibitors

4 --I++ low pH

7 Mutants

'S

Fig. 2. Mechanism for the low pH-induced translocation of the diphtheria toxin Afragment across the plasma membrane. The first step in the translocation process is the cell-mediated reduction of the interfragment disulfide bond. This step is dependent on low pH and can be inhibited by various inhibitors, such as the alkyluting agent N-ethylmaleimide, the anion transport inhibitor DIDS, and acidification of the cytosol. The second step is the complete translocation of the A-fragment to the cytosol, and various mutant A-fragments containing internal disulfide bonds are blocked at this step, leading either to partial translocation of the A-fragment or to a release of the A-fragment into the medium surrounding the cells

must be fulfilled for translocation to take place. In addition to low external pH, it is necessary that the internal pH is not too low. Thus, an inwardly directed pH-gradient is required (Moskaug et a/., 1988). A further requirement is the presence of permeant anions in the external medium, so that there is no outwardly directed anion gradient (Sandvig and Olsnes, 1986; Moskaug etal., 1989).If these requirements are not fulfilled, insertion of the B-fragment still takes place upon exposure to low pH, but the translocation of the A-fragment to the cytosol is prevented. An inwardly directed proton gradient and permeant anions are therfore somehow involved in the penetration process. Inwardly directed proton gradient is required to unfold the A-fragment at low pH, and to refold it at the neutral pH in the cytosol. The pHgradient may therfore constitute the driving force in the translocation process. The role of permeant anions is not understood.

19.2.5 Formation of Cation Channels Concomitant with translocation, cation channels are formed in the membrane (Sandvig and Olsnes, 1988; Papini et a/., 1988) which are selective for monovalent cations. Small ions penetrate more easily Toxins as Transporting Tools

than larger ones, but even comparatively large cations such as choline and glucosamine penetrate to some extent. The ability to form cation channels resides in the B-fragment, and the B-fragment alone is more efficient in forming channels than the whole toxin (Stenmark et al., 1989). Binding to the specific toxin receptors is a requirement for channels to be formed. It is possible that the inserted B-fragment, alone or together with the toxin receptor, forms the channel. Certain mutations of the highly a-helical T-domain of the toxin B-fragment strongly reduce the ability of the toxin to form channels (Falnes et al., 1992; Silverman et al., 1994). In addition, when the transmembrane domain of the receptor was replaced by a GPI anchor, channel formation was not observed (Lanzrein etal., 1996).In spite of this, poisoning occurred in all cases, albeit to a somewhat reduced extent. This indicates that the channels are not directly involved in the translocation process. In fact, the number of active channels is much lower than that of translocated molecules (Lanzrein et al., 1996),further indicating that there is no close link between channels and translocation.

19.3 Pseudomonas Exotoxin A In contrast to diphtheria toxin, fseudomonas aeruginosa exotoxin A does not play a prominent role in a maior infectious disease. The bacterium is mainly pathogenic in immuno compromised patients with severe diseases. Like diphtheria toxin, also Pseudomonas aeruginosa exotoxin A inactivates elongation factor 2 by ADP-ribosylating diphthamide (Wick et al., 1990; Pastan et a/., 1989). In spite of the identical enzymatic activity, there is little resemblance in the primary structure of the two toxins. Some similarity can, however, be detected at the enzymatically active site. The structure/function relationship is less clear than in the case of diphtheria toxin. The toxin has been sequenced and analyzed by X-ray crystallography at 3 8, resolution (Alured et al., 1986). It was found to consist of three domains, one of which (domain Ill) carries the enzymatic activity, whereas another one (domain I) apparently binds to cell-surface receptors. The remaining domain (domain II) is believed to be involved in the translocation of domain Ill to the cytosol. Endocytosed toxin is cleaved near Arg297to yield two disulfide-linked fragments of 28 and 37 kDa (Ogata et al., 1990).Elimination of this disulfide was found to strongly reduce the toxicity (Madshus and Collier, 1989).The 37 kDa fragment appears to be translocated to the cytosol (Ogata et al., 1990). Like diphtheria toxin, Pseudomonas aeruginosa exotoxin A requires low pH to act (FitzGerald et al., 1980). In spite of this, it has not been possible to induce translocation of fseudomonas toxin across the surface membrane by exposure to low pH. It appears that the toxin must be transported beyond the endosomes, possibly to the trans-Golgi network or even to the endoplasmic reticulum to find conditions required for translocation (Chaudhary et al., 1990). In fact domain Ill ends with an amino acid sequence that (after removal of a terminal S. OLSNES et al

arginine residue) resembles the KDEL sequence which is a recognition sequence for luminal proteins that are retained in the endoplasmic reticuIum. The plant toxins ricin, abrin, modeccin, volkensin and viscumin (van Deurs et al., 1989), as well as Shigella toxin, may also require retrograde transport to the Golgi apparatus or to the endoplasmic reticulum (Sandvig et al., 1992) to find conditions suitable for translocation. In the case of Shigella toxin it has been shown that it is in fact transported back to the endoplasmic reticulum and that transport to this location is correlated with the toxic effect. Some of the plant toxins require low pH for penetration, but others do not (Sandvig and Olsnes, 1982).The requirement of low pH in the case of some of these toxins is possibly related to correct routing or processing of the toxin, rather than to the translocation process as such.

19.4 Non-cytocidal Toxins with lntracellular Sites of Action The toxins mentioned above have the disadvantage as targeting vehicles that they can only be used on toxin-resistant cells as they will kill normal cells. It is possible to make mutant toxins with highly reduced toxicity, but so far such mutants have proved to be less active in translocation than the wild-type toxins. In future work it may therefore be necessary to look for other toxins that do not kill the cells. Cholera toxin and E. coli heat-labile toxin ADP-ribosylate the asubunit of trimeric G-proteins (G,) so that they are unable to hydrolyze GTP (Casey and Gilman, 1988; Neer and Claphan, 1988). As a result, the adenylate cyclase stays in a persistently active state. The toxin is taken up from the apical pole of the enterocytes, whereas the adenylate cyclase is located at the basolateral side. Since membrane glycolipids such as the toxin receptor (ganglioside GM,) tend not to cross tight junctions, it is unlikely that the toxin reaches the basolateral side by lateral diffusion. Most likely it enters the cytosol and diffuses to the target which is also located at the cytosolic side of the membrane. Pertussis toxin ADP-ribosylates a cysteine residue of a-subunits of G-proteins involved in the regulation of adenylate cyclase (G,,Go) and of transducin, with the result that the G-protein is unable to interact with the receptors and the signal is therefore not transmitted. This toxin consists of 5 subunits that are linked by noncovalent bonds to the enzymatically active S1-subunit(Casey and Gilman, 1988; Neer and Claphan, 1988). The Sl-subunit is activated by reduction of an internal disulfide bond. A number of toxins that act on intracellular targets have a somewhat different organization, being reconstituted at the cell surface from two different proteins (bipartite toxins).This type of organization was first discovered in the toxin from Bacillus anthracis (Leppla, 1982). This toxin has a three-component structure, consisting of two subunits

bipartite toxins

Toxins as Transporting Tools

toxins that ADP-ribosylate actin

that confer toxicity (the lethal factor (83 kDa) and the edema factor (89 kDa)) and one subunit called protective antigen (82.7 kDa) because it is used in vaccines. The protective antigen acts as a receptor for both the lethal factor and the edema factor. It binds to cellsurface receptors and is then cleaved and a fragment is released. The remaining fragment (63 kDa) becomes a binding site for the two toxic subunits which bind in a mutually exclusive manner. The lethal factor confers cytotoxicity by a mechanism not yet known, whereas the edema factor has adenylate cyclase activity (Leppla, 1982; Singh et a/., 1989).The adenylate cyclase enters the cytosol, where it binds to calmodulin and thereby becomes activated. A number of toxins that act by ADP-ribosylating actin have a similar molecular organization to that of anthrax toxin. Botulinum C2 toxin (not the neurotoxin) from Clostridium botulinum types C and D consists of two components, I and II (Aktories and Wegner, 1989; Aktories etal., 1986).Component II (- 100 kDa) binds to cell surface receptors and is cleaved by trypsin-like enzymes to generate a fragment (- 72 kDa) to form a binding site for the 50 kD component I. The toxin ADP-riboslyates non-muscle, monomeric G-actin at Arg-177. The modified actin is unpolymerizable, and acts as a capping agent inhibit ing f urther polymerization of microfilaments . Act in is therefore trapped in its monomeric G-form with the result that stress fibres disappear and the cells round up. The iota toxin from Clostridium perfringens, the Clostridium spiroforme toxin, and an ADP-ribosyl-transferase from Clostridium difficile have a similar structure and mechanism of action (Aktories and Wegner, 1989; Popoff et a/., 1989; Henrique et al., 1987). Endocytosis may not be required for the entry of an invasive adenylate cyclase from Bordella pertussis (Hanski and Ferfel, 1985; Donovan and Storm, 1990).This is a single chain protein (mol. wt. approx. 200 kDa) which resembles the edema factor from anthrax toxin in that it must interact with calmodulin to become active. In contrast to anthrax toxin, it consists of only one polypeptide which is, however, easily cleaved by proteases and thereby activated. An enzymatically active 45 kDa fragment is not active on whole cells, but it could in conjunction with the rest of the molecule enter the cytosol. The facts that this toxin acts much more rapidly than anthrax toxin, and that it is active even at 4 "C and on erythrocytes that have little, if any, endocytosis, suggest that the toxin is able to penetrate directly through the cell surface membrane.

19.5 Methods to Assess Translocation In a number of experimental situations it is desirable to translocate proteins from the exterior into the cytosol, and this may also be desirable for vaccination purposes. Since many proteins retain their function in fusion proteins, cDNA for the protein in question may be linked to the cDNA of a toxin A-moiety and, after expression and reconstituS. OLSNES et al.

tion with the appropriate B-moiety (Fig. 1 b), it may be translocated across cell membranes to reach the cytosol. In experiments on transmembrane translocation of fusion proteins it is necessary to have a reliable and convenient way of monitoring translocation of the fusion protein. Measuring the toxic effect of the fusion protein provides an indication of translocation, but is not sufficient, since it only shows that the enzymatic A-fragment has been translocated. Thus, the passenger peptide or protein could have been removed proteolytically before translocation takes place. It should also be noted that monitoring toxicity is not very accurate in assessing how much A-fragment (or fusion protein) is translocated. A number of more direct methods can be used to demonstrate that the fusion protein has indeed been translocated:

importance of reliable method for monitoring translocation

1. The fact that diphtheria toxin A-fragment is able to penetrate into cells from the surface membrane upon lowering the pH, opens the possibility of direct assay of the translocated material. Thus, by binding radioactively labeled toxin or reconstituted fusion protein to the cell surface under conditions where endocytosis is reduced, and then exposing the cells to a pulse at low pH, toxin or fusion protein may be translocated across the plasma membrane. Untranslocated material remaining at the cell surface can then be removed with pronase. In this way one can determine directly how much radioactive protein is translocated, and in which form it is translocated. Only if the protected molecule migrates in SDS-PAGE like the intact fusion protein, is it likely that it has been translocated in the intact form (Moskaug et al., 1988; Stenmark et al., 1991; Wiedlocha et al., 1992).

alternative methods for assessing tanslocation

2. The fusion protein can be supplied with a signal recognized by enzymes or other molecules exclusively present in the cytosol. (a) The protein can be supplied with a C-terminal prenylation signal, a CaaX (C, cysteine; a, an aliphatic amino acid; X, any out of several possible amino acids)-box (Fig. 3). If the four Cterminal amino acids of a cytosolic protein form a CaaX-box, enzymes in the cytosol will link a prenyl group (farnesyl or geranylgeranyl) onto the cysteine residue with subsequent removal of the three terminal amino acids and carboxylmethylation of the exposed terminal cysteine residue. We have used the CaaX-box Cys-Val-lle-Met (from Ki-Ras 4B), which signals the addition of a farnesyl group (Falnes et al., 1995; Wiedlocha et al., 1995). E. coli does not have farnesyl transferase, making it easy to produce unmodified protein. Farnesylating enzymes are present in the cytosol of eukaryotic cells and the modification can be assessed in different ways. (i) Labelling the cells with mevalonic acid, a precursor of the farnesyl group, will label all cytosolic proteins containing a CaaX box. The translocated protein can then be visualized by subsequent solubilisation of the cells and immunoprecipitation with specific antibody, followed by SDS-PAGE. Toxins as Transporting Tools

'

PP

(FPP)

V l CaaX

1

+

ra tFarnesyl n>

sy:;:>

m

EC-Methyl

Fig. 3. Farnesylation and further processing of proteins containing a C-terminal CaaX-motif. Farnesyl transferase catalyses the transfer of a farnesyl moiety from farnesyl pyrophosphate FPP) to the cysteine residue in the CaaX-motif, where C = Cys, a = usually aliphatic amino acids, and X = Met, Ser; Cys, Ala, Gln. The three C-terminal amino acids (aaX) are then cleaved off, and a methyl group is transferred from S-adenosyl methionine to the now C-terminal cysteine residue

(ii) The modified protein usually migrates slightly more rapidly in SDS-PAGE than the unmodified molecule (Fig. 4). (iii) The modified protein tends to partition into Triton X-114, even if the unmodified molecule does not.

Fig. 4. In vivo farnesylation of diphtheria toxin A-fragment with a C-terminal CaaXmotif. The mutant A-fragment was translated in vitro in a rabbit reticulocyte lysate in the presence of [35S]methionine, and full-length toxin was formed by adding a translation mixture containing unlabelled B-fragment, and then dialysing overnight. To this mixture was added 1 mM unlabelled methionine, and the complete mixture was incubated 1 h at 0 "C with Vero cells. The cells were subsequently washed, briefly exposed to pH 4.8 to induce translocation of the A-fragment to the cytosol, and then incubated for various time periods at 37 "C in the presence of 10 FM monensin. Finally, the cells were lysed, and the TCA-precipitable radioactivity from the post-nuclear supernatant was analysed by non-reducing SDS-PAGE and fluorography S. OLSNES et al.

(b) A nuclear localization signal can be added to the protein. After being translocated to the cytosol, the protein may then be transported to the nucleus (Wiedlocha et al., 1994; Wiedlocha et al., 1996). Cell fractionation followed by SDS-PAGE can be used to assess how much is present in the nucleus and, by inference, how much has been translocated into the cytosol. A caveat here is that many labelled proteins bind unspecifically to the plastic of the cell culture vessel and can be released when the cells are solubilized with a non-ionic detergent. In the subsequent centrifugation to separate the nuclear and cytosolic fractions they will often precipitate with the nuclei (R. Muiioz, unpublished observations). To avoid this problem, treatment of the cells with trypsin or pronase is recommended, followed by addition of a protease inhibitor, before the cells are solubilized. (c) A signal for degradation by the proteasome system can be added (I? Falnes, unpublished results). A control lacking this signal will also be required. If the protein is rapidly degraded after translocation has been induced, this indicates a cytosolic localization, if the control without the degradation signal is stable in the cytosol. The simplest modification is to place a destabilizing amino acid at the N-terminal end according to the N-end rule (Varshavsky, 1992).This can be done by expressing the protein as a fusion protein, e.g. with maltose binding protein behind a cleavage site specific for a rare-cutting protease, such as factor X,. In this case the N-terminal amino acid can be any amino acid except proline.

(d) Tyrosine phosphorylation (but not phosphorylation of serine and threonine) appears to occur selectively in the cytosol and in the nuclei. If an externally added protein carrying a tyrosine phosphorylation site becomes phosphorylated at this site, it may be taken as evidence that the protein has crossed the membrane to gain access to the cytosol or the nucleoplasm.

19.6 Examples of Translocated Peptides and Proteins It has been shown that a number of peptides linked to the N-terminal end of the diphtheria toxin A-fragment are translocated to the cytosol (Stenmark et al., 1991, Stenmark et al., 1992; Ariansen et al., 1993).It is an interesting question to what extent such peptides can subsequently be presented by class I major histocompatibility antigens. If this is the case, fusion proteins of viral or cancer-related peptides and enzymatically inactive mutants of diphtheria toxin could be used for vaccination purposes to expand desired populations of cytotoxic CD 8' T- Iy mphocytes. Toxins as Transporting Tools

The Clostridium botulinum exoenzyme C3 is able to ADP-ribosylate the small G-protein, rho, in a cell free system, but is unable to enter cells (except at very high concentrations) because it lacks a B-moiety. Constructs where exoenzyme C3 was linked to the N-terminal end of diphtheria toxin or to toxin where the N-terminal part of the Afragment had been deleted, were able to translocate the exoenzymecontaining fusion protein into the cytosol and interfere with the organization of actin filaments (Aullo et al., 1993). Externally added aFGF (acidic fibroblast growth factor) contained in a fusion protein with diphtheria toxin A-fragment is able to stimulate DNA synthesis in serum-starved cells without a measurable increase in tyrosine phosphorylation (Wiedlocha et al., 1994; Wiedlocha et al., 1996). The DNA synthesis is only stimulated when the fusion protein is translocated to the cytosol (and subsequently to the nucleus), but not when the translocation is prevented by the presence of heparin or by introduction of disulfide bridges into the toxin A-fragment to prevent unfolding of the protein. A deletion mutant of the growth factor lacking an N-terminal putative nuclear localization sequence was also unable to stimulate DNA synthesis, even though it was efficiently translocated to the cytosol. It is a tempting interpretation that accumulation of the growth factor in the nucleus is required to stimulate DNA synthesis (Imamura et al., 1990), but the possibility must also be kept in mind that deletion of the N-terminal region could induce a structural perturbation. Thus, the sensitivity to trypsin and pronase of the deletion mutant measured in the presence of heparin was clearly higher than that of full length aFGF. Once translocated into the cells, fusion protein with growth factor containing the nuclear localization signal was still detectable after 48 h. O n the other hand, AaFGF disappeared completely from the cells upon incubation for 24 h after removal of AaFGF from the medium. It is not clear if it is degraded in the cells, or if growth factor that is not retained in the nucleus is transported out of the cells using the same mechanism by which aFGF is exported from cells that produce this growth factor. Dihydrofolate reductase has been used extensively in translocation experiments. A fusion protein with diphtheria toxin A-fragment was shown to be translocated to the cytosol (Klingenberg and Olsnes, 1996). The translocation was inhibited by methotrexate, which induces a tight folding of the protein. A fusion with a mutated dihydrofolate reductase that does not bind methotrexate tightly was also translocated, and in this case methotrexate was not able to prevent the translocation. This indicates that not only must the toxin Afragment be unfolded, but the passenger protein must also be able to unfold for translocation to occur. The bacterial RNase, barnase, linked to Pseudomonas aeruginosa exotoxin A or to a non-toxic deletion mutant of the toxin lacking the enzymatic domain, was found to be toxic to cells to a greater extent than either component alone (Prior et al., 1991; 1992). A C-terminal KDEL sequence was required for toxicity. Cells resistant to the intracellular action of the toxin were also sensitive to the fusion protein. This S. OLSNES etal.

suggests that the fusion protein is translocated to the cytosol, although direct evidence for this was not provided. Fusion proteins have been constructed from peptide epitopes from influenza A antigens and the binding and translocation domains of Pseudomonas exotoxin A (Donelly et al., 1993).When target cells were incubated with these fusion proteins, and subsequently exposed to cytotoxic T lymphocytes (CTLs) specific for the relevant epitopes, a CTL mediate lysis of the target cells was observed. These experiments suggest that the translocation machinery supplied by protein toxins may be useful tools for bringing peptides into cells for presentation via the maior histocompatibility class I (MHC I) system. It should be noted that no direct evidence was provided that the translocation occurred by the toxin pathway, and it cannot be excluded that the toxin was only instrumental in accumulating the peptide on the surface of the cells and in the endocytic pathway. The N-terminal part (residues 1-254) of the lethal factor (LF) component of anthrax toxin is nontoxic, but its binding to the protective antigen (PA) is retained (Arora and Leppla, 1993). When the enzymatic domains of diphtheria toxin (Arora and Leppla, 1994; Milne et al., 1995), Pseudomonas exotoxin A (Arora and Leppla, 1994), or shiga toxin (Arora and Leppla, 1994) were fused to this fragment of LF, the resulting fusion proteins were highly toxic to cells in the presence of PA. These results suggest that the anthrax toxin translocation machinery may be useful for bringing foreign peptides and proteins into cells, but more work remains to be done to establish the efficiency of the translocation process.

19.7 limitations and Possibilities of the System The main limitations of the translocation system described here are that (a) many passenger proteins interfere with the structure of the toxin so that it cannot recognize the toxin receptor, and that (b) the passenger protein may not be able to unfold. We have tested a large number of proteins, and most of them are not translocated due to one of these problems (Klingenberg and Olsnes, 1996).This may also be a problem with smaller peptides, but usually less than with protein passengers. Conceivably, modification of the fusion proteins with polypeptide linkers and by mutations in the passenger protein may overcome the problem, but this will require a considerable amount of work in each case. The question whether toxins can be used to translocate nucleic acids into cells is very interesting, but this has not been tested seriously up to now. Toxins are interesting candidates for delivery of antisense RNA, ribozymes and genes for transformation and gene therapy. So far there is not convincing evidence that toxins have been effective for such purposes. From what is now known about the translocation of diphtheria toxin, it is likely that the nucleic acid would have to be linked end-to-end to the N-terminus of the A-fragment of this

factors affecting tr~nslocation

Toxins as Transporting Tools

toxin. This would have to be done chemically and with high specificity. It is more difficult to predict what would be the requirements for other toxins. Once the conditions for linkage were worked out, there is no obvious reason why toxins should not be useful for this purpose.

19.8 Methods 19.8.1 In vitro Transcription and Translation The cDNA encoding the fusion protein is cloned into a plasmid downstream of a SP6, T7 or T3 RNA polymerase promoter. The plasmid is linearized downstream of the inserts by digesting with an appropriate restriction enzyme, and transcribed in vitro with the appropriate RNA polymerase (75).

transcription and translation of plasmid

1. Mix in an Eppendorf tube: 20 pI 5 x transcription buffer (200 m M Tris-HCI, pH 7.9, 30 m M MgCI2, 10 m M spermidine, 50 m M NaCI) 10 pI 100 m M dithiothreitol 4 pI RNasin (Promega) 20 pl of a mixture containing 2.5 m M each of ATP, CTP, UTP and GTP 2 p1 of linearized plasmid 10-20 U of either T3, T7 or SP6 RNA polymerase H 2 0to a total volume of 100 pI

2. Incubate at 37 for 60 min O

3. The mRNA is then precipitated with 2.5 volumes ethanol and dissolved in 10 pI H 2 0containing 10 m M dithiothreitol and 0.1 U/ pI RNasin

4. The transcripts is translated in a rabbit reticulocyte lysate system (Promega, WI, USA), as follows: To 35 pl nuclease-treated reticulocyte lysate, add: 7 PI H20 1 pl 1 m M amino acide mixture (19 amino acids without methionine)

2 pI mRNA 5 pL [35S]methionine(1200 Ci/mmole, 10 mCi/ml, Amersham) Total volume 50 pl.

5. Incubate at 30 "C for 60 min to obtain labeled fusion protein. To obtain unlabeled fusion protein, the incubation is carried out in the presence of 25 p M unlabelled methionine instead of [35S]methionine to obtain maximal yield of fusion protein. synthesis of unlabeled protein S. OLSNES et al.

To estimate the amount of translation product in this case, an aliquot (5 PI) is incubated in parallel in the presence of 5 pM [35S]methionine in addition to the unlabelled methionine. The radioactive translation product is analyzed by SDS-PAGE and fluorography. The amount of 35S-labelled protein is calculated by incubating the isolated gel band with Opti-Fluor (Amersham) for 30 min and then measuring the radioactivity by scintillation counting. After translation, the whole cell-free translation mixture (referred to as lysate below) is dialyzed at 4 "C first for 16 h against PBS and then for 4 h against HEPES medium, to remove free [35S]methionineand the reducing agent, allowing disulfide bridges to be formed (Fig. 5). An aliquot of the lysate is then analyzed by SDS-PAGE under reducing and non-reducing conditions as indicated, followed by fluorography. Translation mixtures can either be used undiluted or diluted with HEPES medium, pH 7.2.

estimation of amount of Protein

19.8.2 SDS-PAGE SDS-PAGE is carried out in 0.75 mm thick slab gels as described by Leammli (1970). In most cases 10-12% acrylamide and 0.27% N,N'-methylenebisacrylamide gels were used. The gels are subsequently fixed in 4 % acetic acid/27 % methanol for 30 min and then treated with 1 M Na-salicylate, pH 5.8., in 2 % glycerol for 30 min, to visualize proteins labeled with [35S] methionine. Dried gels are exposed to Kodak XAR-5 films in the absence of intensifying screens at -80 "C for fluorography. When '251-labeledproteins were analyzed, the gels were not amplified and the dried gels were layered on Kodak XARS films in the presence of an intensifying screen at -

SDS-PAGE of fusion proteins

80 "C.

Dialysis membrane

F&+g&+ +e Add sample

Dialyse

Fig. 5. Convenient method for dialysing small sample volumes. The bottom part of an Eppendorf tube is cut away, and a hole is cut in the cap. A dialysis membrane which has been pre-soaked in water is streched across the inner cylinder of the cap, and the sample (typically 40-80 PI) is added through the whole in the cap. Finally, the cap containing the sample is gently placed floating (e.g. using tweezers) in the desired buffer, and gentle stirring can be applied. It is advisable to check that the cap is not leaky by floating it on the buffer before the sample is added

Toxins as Transporting Tools

19.8.3 Cell Culture Cells are maintained and propagated under standard conditions (5 % C 0 2 in EagleS minimal essential medium containing 5 % FCS, 2 m M glutamic acid and 50 pg gentamycin). Two days prior to the experiments, the cells are seeded into 12 or 24 well Costar (Cambridge, MA) plates at a density of lo5 and 5 x lo4 cells/well, respectively in the same medium at 37 "C in a humidified atmosphere with 5 % c0;I.

19.8.4 Cell Binding and Translocation Assay To measure binding, dialyzed translation mixture (diluted 5 times in HEPES medium) is added to Vero cells growing as monolayers in 12 well microtiter plates and kept at 24 "C for 20 min in the presence of 1 mM unlabelled methionine and 10 p M monensin (Moskaug et al., 1988).To measure translocation, undiluted, dialyzed lysates are added to cells and kept for 20 min at 24 "C in the presence of 1 mM methionine (to reduce incorporation of any labeled methionine that had not been completely removed during dialysis) and 10 p M monensin. The cells are then washed 3 times with ice cold HEPES medium and exposed to pH 4.5 for 3-5 min at 37 "C, treated with pronase and analyzed by SDS-PAGE and fluorography as described above.

19.8.5 Expression in Bacteria and Purification of Recombinant Proteins A CaaX motif (Cys-Val-lle-Met) can be introduced C-terminally into the protein in question by standard techniques. The cDNA obtained can be cloned into an expression vector for E. coli, such as pTrc-99A (Pharmacia), and then transfected into E. coli DH5 a and induced with 5 m M isopropyl-(3-D-thioglactopyranoside (IPTG).The protein is isolated from the bacterial pellet. For purification of aFGF-CaaX, the bacterial pellet was suspended in 20 mM Tris-HCI, pH 7.5, 0.5 M NaCI, 1 m M DTT, 1 m M EDTA and sonicated. The clear supernatant, after centrifugation, was applied to a heparin cartridge (Bio-Rad), and the bound material was eluted with a linear NaCl gradient (0.5-2 M) in the above buffer. For purification of dtA-CaaX, the bacteria were harvested, washed with PBS, resuspended in PBS and sonicated. After centrifugation, the supernatant was chromatographed on a Sephacryl S-100HR column equilibrated with 20 m M Tris-HCI, pH 7.5, 0.5 M NaCI, 1 m M DTT. Fractions containing dtA-CaaX were collected, dialyzed against 20 mM phosphate buffer, pH 6.0, 1 m M DTT, and chromatographed on a Q-cartridge (Bio-Rad)with a linear NaCl gradient (0-1 M) in the same buffer. S. OLSNES efal.

19.8.6 In vitro Farnesylation For in vitro labeling, 0.2 pCi [3H]farnesyl pyrophosphate (Du Pont New England Nuclear) and 2 ng of the CaaX-tagged protein is added to 20 1-11 of reticulocyte lysate (Promega) as source of farnesyl transferase. The mixture is adjusted to contain 5 mM MgCI2.To carry out the full modification, 1 pl of dog pancreatic microsomes (Promega), must also be added. The mixture is then incubated for 30 min at 37 "C. Labeled proteins can be recovered from the reaction mixture by immunoprecipitation with an appropriate antibody adsorbed to protein A-Sepharose, and analyzed by SDS-PAGE and fluorography (Wiedlocha et al., 1992). 1. Mix in an Eppendorf tube: 0.2 pl farnesyl pyrophosphate (0.2 pCi) (Du Pont) 20 pl rabbit reticulocyte lysate (Promega) 2-20 ng CaaX-tagged protein MgCI2to a final concentration of 5 m M 1 pl dog pancreatic microscomes (Promega). (Optional, to carry out the full modification, but not required to obtain labelling)

farnesylation procedure

2. Incubate for 30 min at 37 "C.

3. Analyse by immunoprecipitation, SDS-PAGE and fluorography,

19.8.7 Analysis of in vivo Farnesylation Cells growing in 25 cm2 flasks are serum-starved for 36 hr and then preincubated overnight in serum free medium containing 1 pCi/ml [3H]mevalonic acid (Du Pont), 1 pCi/ml ["C] mevalonic acid and 4 yg/ml of lovastatin (to prevent formation of endogenous mevalonic acid). Protein with a CaaX-box is added and the cells are incubated overnight. In some cases the protein is added at the same time as the labeled mevalonic acid. The cells are then washed twice with HEPES medium, lysed (Wiedlocha et al., 1994) and centrifuged for 5 min at 14000 r.p.m. at 4 "C. The supernatant is centrifuged once more and the second supernatant (the cytoplasmic fraction) is mixed by rotation for 2 h at 4 "C with protein A-Sepharose C-4B that had previously been treated with 2 pI of antibody against the protein.

1. Cells, growing in one well of a 6-well plate - a 25 cm2 flask, serum-starved if needed.

Toxins as Transporting Tools

2. Add an appropriate volume of serum-free medium containing: 1-6 pCi/ml [3H]Mevalonic acid (Du Pont) (Dry in a spin-vac) 1 pCi/ml ['4C]Mevalonic acid (Du Pont) (Dry in a spin-vac) 4-10 pg/ml Lovastatin

3. Preincubate at 37 "C for 0-12 h, add CaaX-tagged protein. 4. Incubate at 37 "C for 8-24 h.

5. Wash twice with HEPES medium 6. Lyse cells in the presence of protease inhibitors.

7. Centrifuge for 5 min at 12000 r.p.m. in an Eppendorf centrifuge (to remove nuclei).

8. Collect CaaX-tagged protein from the supernatant by immunoprecipitation, TCA-precipitation, Heparin-sepharose-precipitation or by another suitable method.

9. Analyse by SDS-PAGE and fluorography.

19.8.8 Triton X-114 Partitioning of Farnesylated Proteins 1. Triton X-114 equilibrated with PBS is diluted to a 20 % stock solution (Madshus et a/., 1984). 2. PBS (300 pl) and 100 pl of the Triton X-114 stock solution are mixed and kept on ice.

3. Protein is added, and the mixture is kept at 0°C for further 15 min. 4. To separate the phases, the solution is incubated for 15 min at 37 "C, and then centrifuged for 2 min at room temperature in an Eppendorf centrifuge.

5. The upper (water) phase is transferred to a new tube, and the lower (Triton) phase is washed at 37 "C with 500 pI PBS.

6. Both phases are then diluted to 1 ml with PBS (0 "C), followed by immunoprecipitation with antibodies adsorbed to protein ASepharose. The precipitated material is analyzed with SDSPAGE and fluorography.

J\

remOve detergents

S. OLSNES etal.

To study partitioning of proteins from cells, the cells are lysed directly in the Triton/PBS mixture in the presence of 1 m M PMSF, and, after removal of the nuclei by centrifugation, the procedure for phase separation described above is followed. It should be noted that many antibodies do not bind well to their antigens in the presence of high concentrations of detergent. It is therefore advisable to dilute the Triton X-114 phase with water at 0 "C before immunoabsorbtion.

Acknowledgements This work was supported by grants from The Norwegian Cancer Society, The Norwegian Research Council, Rake1 og Kristian Bruun’s legat, Torsteds legat, The Family Blix legat, The Jahre Foundation and The Novo Nordisk Foundation.

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FitzGerald D, Morris RE, Saelinger CB (1980):Receptor-mediated internalization of pseudomonas toxin by mouse fibroblasts. Cell 21 : 867-873. Foley T, Moehring JM, Moehring TJ (1995): Mutations in the elongation factor 2 gene which confer resistance to diphteria toxin and Pseudomonas exotoxin A. J Biol Chem 270: 23218-23225. Hanski H, Farfel Z (1985): Bordetella pertussis invasive adenylate cyclase. J Biol Chem 260: 5526-5532. Henriques B, Florin I, Thelestam M (1987):Cellular internalizationof Clostridiurn difficile toxin A. Microb Pathogen 2: 455-563. Hooper KP, Eidels L (1995): Localization of a critical diphtheria toxin-binding domain to the C-terminus of the mature heparin-binding EGF-like growth factor region of the diphtheria toxin receptor. Biochem Biophys Res Cornrnun 206: 710-717. lmamura T, Engleka K, Zhan X, Tokita Y, Forough R, Roeder D, Jackson M, Maier JAM, Hla, T, Maciag T (1990):Recovery of mitogenic activity of a growth factor mutant with a nuclear translocation sequence. Science 249: 1567-1570. Just I, Selzer J, Wilm M, von Eichel-Streiber C, Mann M, Aktories K (1995):Glucosylation of Rho proteins by Clostridium difficile toxin B Nature 375: 500-503. Just I, Wilm M, Selzer J, Rex G, von Eichel-Streiber C, Mann M and Aktories K (1995):The enterotoxin from Clostridiurn difficile (ToxA) monoglucosylates the Rho protein. J Biol Chem 270: 13932-13936. Klingenberg 0, Olsnes S (1996):Ability of methotrexate to inhibit translocation to the cytosol of dihydrofolate reductase linked to diphtheria toxin. Biochem J 313: 647-653. Laemmli UK (1970): Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685. Lanzrein M, Garred 0, Olsnes S, Sandvig K (1995): Diphtheria toxin endocytosis and membrane translocation is dependent on the intact membrane anchored receptor (HB-EGF precursor): Studies on the cell-associated receptor cleaved by a metalloprotease in TPA-treated cells. Biochem J 310: 285-289. Lanzrein M, Sand 0, Olsnes S (1996): GPI-anchored diphtheria toxin receptor allows membrane translocation of the toxin without ion channel activity. EM60 J 15: 725-734. Leppla SH (1982): Anthrax toxin edema factor: a bacterial adenylate cyclase that increases CAMP concentrations in eucaryotic cells. Proc Nut1Acad Sci USA 79: 3162-3166. Link E, Edelmann L, Chou JH, Binz T, Yamasaki S, Eisel U, Baumert M, Sudhof T C, Niemann H, Jahn R (1992):Tetanus toxin action: Inhibition of neurotransmitter release linked to synaptobrevin proteolysis. Biochem Biophys Res Comrnun 189: 1017-1023. Madshus I H, Collier R J (1989): Effect of eliminating a disulfide bridge within domain II of Pseudomonas aeruginosa exotoxin A. Infect lmrnun 57: 1873-1878. Madshus I H, Olsnes S, Sandvig K (1984):Requirements for entry of poliovirus RNA into cells at low pH. EMBOJ. 3: 1945-1950. McGill S, Stenmark H, Sandvig K, Olsnes S (1989):Membrane interactions of diphtheria toxin analysed using in vitro synthesised mutants. EM60 J 8: 2843-2848. Middlebrook JL, Dorland R B, Leppla SH (1978):Association of diphtheria toxin with Vero cells: Demonstration of a receptor. J Biol Chern 253: 7325-7330. Milne JC, Blanke SR, Hanna PC, Collier RJ (1995): Protective antigen-binding domain of anthrax lethal factor mediates translocation of a heterologous protein fused to its amino- or carboxy-terminus.Mol Microbioll5: 661-666. Moehring JM, Moehring TJ, Danley DE (1980): Posttranslational modification of elongation factor 2 in diphtheria-toxin-resistantmutants of CHO-K1 cells. Proc Natl Acad Sci USA 77: 1010- 1014.

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Morris RE, Gerstein AS, Bonventre PF, Saelinger CB (1985): Receptor-mediated entry of diphtheria toxin into monkey (Vero) cells: Electron microscopic evaluation. Infect lmmun 50: 721-727. Moskaug J 0 , Sandvig K, Olsnes S (1989):Role of anions in low pH-induced translocation of diphtheria toxin. J Biol Chern 264: 11367-11372. Moskaug J 0 , Sandvig K, Olsnes S (1988): Low pH-induced release of diphtheria toxin A-fragment in Vero cells. Biochemical evidence for transfer to the cytosol. J Biol Chern 263: 2518-2525. Moskaug J 0 , Stenmark H, Olsnes S (1991): Insertion of diphtheria toxin into the plasma membrane at low pH. Characterization and topology of inserted regions. J Biol Chern 266: 2652-2659. Moskaug J 0 , Sandvig K, Olsnes S (1987): Cell-mediated reduction of the interfragment disulfide in nicked diphtheria toxin. A new system to study toxin entry at low pH. J Biol Chern 262: 10339-10345. Naglich JG, Metherall JE, Russell DW, Eidels L (1992):Expression cloning of a diphtheria toxin receptor: Identity with a heparin-binding EGF-like growth factor precursor. Cell 69: 1051-1061. Neer EJ, Claphan DE (1988):Roles of G protein subunits in transmembrane signalling. Nature 333: 129-134. Ogata M, Chaudhary VK, Pastan I, FitzGerald DJ (1990):Processing of Pseudomonas exotoxin by a cellular protease results in the generation of a 37,000-Da toxin fragment that is translocated to the cytosol. J Biol Chem 265: 20678-20685. Olsnes S, Sandvig K (1985):Entry of polypeptidetoxins into animal cells. In Endocytosis (Pastan I, Willingham MC, eds), pp 195-234, Plenum pub1 corp, NY Olsnes S, Carvaial E, Sandvig K (1986):Interactions between diphtheria toxin entry and anion transport in Vero cells. 111. Effect on toxin binding and anion transport of tumor-promoting phorbol esters, vanadate, fluoride and salicylate. J Biol Chem 261 : 1562-1569. Omura F, Kohno K, Uchida T (1989):The histidine residue of codon 715 is essential for function of elongation factor 2. EurJ Biochem 180: 1-8. Papini E, Sandona D, Rappuoli R, Montecucco C (1988):On the membrane translocation of diphtheria toxin: at low pH the toxin induces ion channels on cells. EMBO J 7: 3353-3359. Pappenheimer AM Jr (1977):Diphtheria toxin. Ann Rev Biochem 46: 69-94. Pastan I, FitzGerald D (1989) Pseudomonas exotoxin: Chimeric toxins. J Biol Chern 264: 15157-15160. Popoff MR, Milward FW, Bancillon B, Boquet P (1989):Purificationof the Clostridium spiroforrne binary toxin and activity of the toxin on HEp-2 cells. Infect lrnrnun 57: 2462-2469. Prior TI, FitzGerald DJ, Pastan I (1992):Translocation mediated by domain II of Pseudornonas exotoxin A: Transport of barnase into the cytosol. Biochemistry 31 : 3555-3559. Prior TI, FitzGerald DJ, Pastan I (1991):Barnase toxin: A new chimeric toxin composed of Pseudomonas exotoxin A and barnase. Cell 64: 1017-1023. Sandvig K, Olsnes S (1981): Rapid entry of nicked diphtheria toxin into cells at low pH. Characterization of the entry process and effects of low pH on the toxin molecule. J Biol Chem 256: 9068-9076. Sandvig K, Olsnes S (1986):Interactions between diphtheria toxin and anion transport in Vero cells. IV. Evidence that entry of diphtheria toxin is dependent on efficient anion transport. J Biol Chem 261 : 1570-1575. Sandvig K, Olsnes S (1988): Diphtheria toxin-induced channels in Vero cells selective for monovalent cations. J Biol Chem 263: 12352-12359. Sandvig K, Olsnes S (1982): Entry of the toxic proteins abrin, modeccin, ricin and diphtheria toxin into cells. II. Effect of pH, metabolic inhibitors and ionophores and evidence for penetration from endocytotic vesicles. J Biol Chern 257: 7504-7513. Sandvig K, Garred 0, Prydz K, Kozlov JV, Hansen SH, Deurs B (1992):Retrograde transport of endocytosed Shiga toxin to the endoplasmic reticulum. Nature 358: 510-512.

Toxins as Transporting Tools

Schiavo G, Rossetto 0, Catsicas S, de Laureto PP, DasGupta BR, Benfenati F, Montecucco C (1993):Identification of the nerve terminal targets of botulinum neurotoxin serotypes A, D, E. J Biol Chem 268: 23784-23787. Schiavo G, Benfenati F, Poulain B, Rossetto 0, de Laureto PP, DasGupta BR, Montecucco C (1992):Tetanus and botulinum-B neurotoxins block neutransmitter release by proteolytic cleavage of synaptobrevin. Nature 359: 832-835. Silverman JA, Mindell JA, Finkelstein A, Shen WH, Collier RJ (1994): Mutational analysis of the helical hairpin of diphtheria toxin transmembrane domain. J Biol Chem 269: 22524-22532. Singh Y, Chaudhary VK, Leppla SH (1989):A deleted variant of Bacillus anthracis protective antigen is non-toxic and blocks anthrax toxin action in vitro. J Biol Chem 264: 19103-19107. Stenmark H, Afanasiev BN, Ariansen SA, Olsnes S (1992):Reconstitution of active diphtheria toxin and its fusion proteins from separate A- and B-fragments. Biochem J 281 : 619-625. Stenmark H, McGill S, Sandvig K, Olsnes S (1989):Permeabilisationof the plasma membrane by deletion mutants of diphtheria toxin. EMBO, J 8: 2849-2853. Stenmark H, Moskaug J 0 , Madshus IH, Sandvig K, Olsnes S (1991):Peptides fused to the amino-terminal end of diphtheria toxin are translocated to the cytosol. J Cell Biolll3: 1025-1032. Sundan A, Olsnes S, Sandvig K, Pihl A (1982): Preparation and properties of chimaeric toxins prepared from the constituent polypeptides of diphtheria toxin and ricin. Evidence for the entry of ricin A-chain via the diphtheria toxin pathway. J Biol Chem 257: 9733-9739. Valdizan EM, Loukianov EV, Olsnes S (1995): Induction of toxin sensitivity in insect cells by infection with baculovirus encoding diphtheria toxin receptor. J 6\01 Chem 270: 16879-16885. van Deurs B, Petersen OW, Olsnes S, Sandvig K (1989):The ways of endocytosis. Int Rev Cytol 117: 131-177. Van Ness BG, Howard JB, Bodley JW (1980):ADP-ribosylation of elongation factor 2 by diphtheria toxin. NMR spectra and proposed structure of ribosyldiphthamide and its hydrolysis products. J Biol Chem 255: 10710-10716. Varshavsky A (1992):The N-end rule. Cell 69: 725-735. Wick MJ, Hamood AN, lglewski BH (1990):Analysis of the structure-function relationship of Pseudomonas aeruginosa exotoxin. A. Mol Microbiol4: 527-535. Wiedlocha A, Falnes PO, Madshus IH, Sandvig K, Olsnes S (1994): Dual mode of signal transduction by externally added acidic fibroblast growth factor. Cell, 76: 1039-1051. Wiedlocha A, Madshus IH, Mach H, Middaugh CR, Olsnes S (1992):Tight folding of acidic fibroblast growth factor prevents its translocation to the cytosol with diphtheria toxin as vector. EM60 J. 11 : 4835-4842. Wiedlocha A, Falnes PO, Rapak A, Klingenberg 0, Mutioz R, Olsnes S (1995): Translocation to cytosol of exogenous, CaaX-tagged acidic fibroblast growth factor. J Biol Chem 270: 30680-30685. Wiedlocha A, Falnes P 0 , Rapak A, Mufioz R, Klingenberg 0, Olsnes S (1996): Stimulation of proliferation of a human osteosarcoma cell line by exogenous acidic fibroblast growth factor requires both activation of receptor tyrosine kinase and growth factor internalization.Mol Cell Biol 16: 270-280. Zhao J-M, London E (1990): Conformation and model membrane interactions of diphtheria toxin fragment A. J Biol Chem 263: 15369-15377.

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Bacterial Toxins

Klaus Aktories Copyright 0 2002 WILEY-VCH Verlag GmbH & Co. KGaA

CHAPTER 20

A Brief Guide to the Safe Handling of BioIogica I Toxins A. B. MAKSYMOWYCH and L. L. SIMPSON

20.1 Introduction Generally speaking, the use of toxins as research tools is technically straightforward. One merely adds activated toxin to the cells of interest, then waits an appropriate length of time for the toxin to exert its effects. However, the technical ease of using toxins should not divert attention from the fact that materials must be handled in a way that does not pose a hazard to laboratory workers or to others in the vicinity. Fortunately, the safeguards that are needed to ensure the health and welfare of everyone can be implemented in a way that poses relatively little burden on investigators. The most important element in reducing the risk of toxins - or sources of toxins, such as microbiological cultures - is strict adherence to standard policies and procedures.’ Persons working with toxins or infectious materials must be aware of potential hazards and be trained and proficient in the safe handling of these materials. This is ordinarily accomplished by: (a) identifying each real or potential hazard, (b) identifying the circumstances under which hazardous materials will be encountered, (c) developing or adopting procedures for handling hazardous materials, and (d) preparing written guidelines that stipulate the responses to accidents and/or contamination.

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adherence to standard Policies and Procedures

20.2 Preventive Measures Policies and procedures that relate to safe handling of toxins and infectious material can be broken down into four maior categories: preventive measures, containment, disposal and decontamination. There are three maior steps that should be taken to safeguard the welfare of workers who handle toxins:

1. An institutional physician knowledgeable about human poisoning should be notified that the work is in progress, and this physician should be given the names of all persons who handle the toxin. Similarly, all laboratory persons should know the identity and whereabouts of the physician knowledgeable about poisoning.

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physician

’ Many of the guidelines enumerated in this chapter are extracted from the Thomas Jefferson University Guide to Laboratory Safety.

K. Aktories (Ed.), Bacterial Toxins. 0 Chapman & Hall, Weinheim, 1997. ISBN 3-8261-0080-8

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material safety data sheets

protective equipment

2. Material Safety Data Sheets describing the toxin should be prepared and made available to all laboratory workers.

3. Laboratory workers should wear appropriate personal protective equipment. At a minimum, this includes a laboratory coat and gloves.

20.3 Containment

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containment

20.3.1 Containment Procedures There are two broad dimensions to the concept of containment. Within the laboratory, one must use procedures that restrict exposure to the toxin and minimize the potential for causing immediate harm. Beyond the laboratory, one must adopt procedures that prevent spread of biologically active material. Both of these dimensions of containment must be viewed in the context of the type of laboratory that is used.

20.3.2 Containment Facilities The selection of an appropriate laboratory or biosafety level for a particular type of work hinges on a number of factors. Some of the more important of these factors are the potency and stability of the toxin being handled, the virulence and pathogenicity of the infectious agents being handled, and the availability of protective vaccines or therapeutic measures. The United States Department of Health, Education and Welfare has developed a system for classifying toxins and other etiological agents on the basis of hazard. Depending on the level of hazard, the etiological agent can be placed in one of four biosafety levels. A brief description of these biosafety levels is given in Table 1.

20.3.3 Containment within the laboratory and beyond the Laboratory The maior guidelines for minimizing risk within a laboratory are:

1. Doors to the laboratory should be clearly marked to indicate the presence of a biohazard. These doors should remain locked at all times, and only properly authorized personnel should have access.

2. The laboratory itself should conform to the requirements of a BL-2 or higher level facility, depending on the exact nature of the work. A detailed description of such facilities can be found in the CDC-NIH Biosafety in Microbiological and Biomedical Laboratories booklet. A. B. MAKSYMOWYCH and L. L. SIMPSON

Table 1. Description of Biosafety Levels Biosafety level 1 represents a basic level of containment that relies on standard microbiological practices with no special primary or secondary barriers recommended, other than a sink for washing hands. Special containment equipment or facility design is not required nor is it generally used. Biosafety level 2 applies to facilities in which work is done with the broad spectrum of indigenous moderate-risk agents present in the community and associated with human disease of varying severity. This level of containment is appropriate when work is done with any humanderived blood, body fluids, or tissues where the presence of an infectious agent may be unknown. Personnel working with human-derived materials should refer to the Occupational Exposure to Bloodborne Pathogen Standard (OSHA) for specific, required precautions. Biosafety level 3 applies where work is done with indigenous or exotic agents with potential for respiratory transmission, and which may cause serious and potential lethal infection. Biosafety level 4 applicable to work with dangerous and exotic agents which pose a high individual risk of life-threateningdisease, which may be transmitted via the aerosol route, and for which there is no available vaccine or therapy.

The descriptions given above are summaries of the information found in the third edition of the Department of Health and Human Services Publication (CDC) 93-8395, Biosafety in Microbiological and Biomedical Laboratories. This publication provides information relating to practices, facilities, and safety equipment, as well as recommendations for incorporating these items into four biosafety levels of laboratory operation.

3. Stock solutions of toxin should be stored in a refrigeratodfreezer that is marked to indicate the presence of a biohazard. Ideally, stock solutions should be diluted to create working solutions that have the lowest level of toxicity consistent with the scholarly work in progress.

4. Laboratory personnel should wear gloves and appropriate protective garments, as indicated above, and this personal protective equipment should be removed when leaving the laboratory. In addition, laboratory counters should be covered with absorbent material.

5. To the fullest extent practical, toxin should be transported, diluted and used in disposable pipettes and containers.

6. To the fullest extent practical, toxin should be handled by mechanical means (e.g., automatic pipettes). Care should be taken to avoid creation of aerosols, and the toxin must not be allowed to come into contact with the mouth or broken skin. The guidelines for ensuring that biologically active toxin does not go beyond the research laboratory are:

1. Toxin should be transported outside the research laboratory only when there is good justification (i.e., transport to another research facility). When toxin is shipped, it must be packaged and labeled according to government standards. A Brief Guide to the Safe Handling of Biological Toxins

2. Steps should be taken to ensure that toxin is contained within the research laboratory. The techniques for preventing escape are inherent in the design of BL-2 and BL-3 facilities (see above). For example, air from the laboratory should not be recirculated into non-laboratory areas.

3. As indicated above, no unauthorized persons should be allowed access to areas where they might accidentally come into contact with the toxin and inadvertently carry it out of the laboratory.

4. As indicated below, toxin and contaminated materials that are intended for disposal should be handled as a biohazard and, whenever practical, treated to abolish toxicity.

20.4 Disposal

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safe disposal

Techniques that have been widely adopted for disposing of protein toxins are:

1. Solutions of toxin are inactivated in an autoclave. Questions about residual toxicity can be resolved by performing a bioassay.

2. Potentially infectious waste, such as material from tissue cultures, should be autoclaved and prepared for disposal by incineration.

3. Animal carcasses, bedding and absorbent material from bench tops should be wrapped in biohazard containers and prepared for disposal by incineration.

4. Disposable labware, such as pipettes, should be autoclaved and placed in biohazard containers. Non-disposable labware should be retained in the facility until it has been decontaminated.

20.5 Decontamination

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decontamination

Decontamination typically occurs in two settings: as a general cleaning procedure that is part of good laboratory practice, and as an emergency intervention when there has been a significant spill or other accident. The decontamination procedures that are part of good laboratory practices can be enumerated as follows:

1. All biohazardous materials and items contaminated with infectious agents should be either decontaminated before being washed and stored, or discarded properly. (a) Whenever possible, contaminated items and biohazardous materials should be sterilized by autoclaving (saturated steam under pressure). (b) Each individual working with biohazardous material or contaminated items should be responsible for their decontamination or disinfection before disposal or reuse. A. B. MAKSYMOWYCHand L. L. SIMPSON

(c)When autoclaving, test tape or another suitable indicator must be used on each load placed in the autoclave. This will aid in determining which items have been sterilized.

2. All laboratories in which work with biohazardous materials is carried out must have labeled, leak-proof, covered containers for temporary holding of infectious materials awaiting disinfection or disposal.

3. All floors, laboratory benches, and other surfaces in areas where biohazardous materials are handled must be disinfected upon completion of operations involving plating, pipetting, centrifugation and similar procedures.

4. Floors should be wet mopped with disinfectant. Avoidance of dry sweeping and dusting will reduce the formation of aerosols. If sweeping is necessary, a push broom and floor sweeping compound should be used. Waxing and buffing should be done after mopping.

5. Floor drains must be flooded with water periodically in order to fill traps and prevent the backflow of sewer gases.

20.6 Emergency Intervention The following procedures should be followed in the event of a biohazard spill. The initial steps are the responsibility of the person(s) whose actions led to the spill.

1. Remove contaminated protective garments (including shoes)

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emergencies

immediately at the door before leaving the contaminated area.

2. Warn others of the spill, and isolate the area. 3. Notify the laboratory supervisor. 4. Wash hands and face or, if facilities are available, shower. Use germicidal soap.

5. Notify the institutional health and safety officer. After the above immediate actions are accomplished, decontamination and cleanup will be directed by the laboratory supervisor and/or a person knowledgeable about biohazard decontamination. This subsequent phase of cleanup should include the following elements.

1. The affected area should not be re-entered for a minimum of 30 minutes if there is a danger of airborne particles. 2. Personnel responsible for the cleanup should wear all appropriate personal protective equipment.

3. An appropriate disinfection solution (e.g., 10 % Chlorox) should be used to treat the spill area. The disinfection solution should be A Brief Guide to the Safe Handling of Biological Toxins

poured gently rather than sprayed, thus minimizing the risk of creating aerosols.

4. Transfer all materials, such as absorbent cloth from the spill area, to an autoclave container.

5. Wash and mop the spill area and adjacent areas with disinfectantdetergent solution.

6. Gas sterilize equipment that requires decontamination but cannot be subjected to liquids.

7. Before leaving the immediate area, the decontamination team should remove shoe covers and wipe shoes on pads soaked with disinfectant solution. Other personal protective equipment should be removed for appropriate decontamination or autoclaving. Personnel should then shower using germicidal soap.

8. The laboratory supervisor should assure that all waste, equipment, clothing, and respirators are properly decontaminated or disinfected and disposed in appropriate containers.

20.7 Resources Expanding access to computers and networks represents a resource which should not be overlooked. There are two Universal Resource Locators that represent a sound starting point for obtaining safety related resources available on the internet. First, the full text version of the Biosafety in Microbiological and Biomedical Laboratories, 3rd edition, March 1993 may be accessed at http://www.cdc.gov/od/ ohslbiosftylbmbl-l.htm. Another excellent starting point is the Occupational Safety and Health Administration home page at http:// www.osha.gov, which allows access to multiple sources of information, as well as a listing of Safety and Health Internet Sites.

20.8 Conclusion The various guidelines presented above should not be seen as allinclusive. They are general guidelines that are likely to apply to most research facilities. Additional precautionary steps may be needed, depending on the amount of toxin and type of work involved. However, all of the guidelines can be implemented in a way that safeguards personnel while still permitting vigorous and scholarly research.

Acknowledgements Supported in part by a grant (NS-22153) from NlNDS and a contract (DAMD17-95-C-5004) from USDOA. A. B. MAKSYMOWYCH and L. L. SIMPSON

Bacterial Toxins

Klaus Aktories Copyright 0 2002 WILEY-VCH Verlag GmbH & Co. KGaA

Subject Index

Abrin 279 Acetylcholine - Inhibition of release by clostridial neurotoxins 169, 194 Acidic fibroblast growth factor, Fusion protein 284 Actin - ADP-ribosylation 95-96, 134-138,280 Clostridium botulinum C2 toxin 94, 280 Effects on cytoskeleton 98-99 Microfilament network 98 Quantification 133-134 - ATPase activity 96 - Capping protein 96 - Complex with gelsolin 96-97 - Cytoskeleton Clostridium difficile toxins 144 Control by Rho GTPases 166 Depolymerization 167 Disruption by Clostridium difficile toxins 147 - Depolymerization Assay 131-132 C2 toxin 129 Cytochalasins 129 - F-actin 98 - Functions 95 - G-actin 98 - Identification by immunoprecipitation 133 - lsoforms 95 - Phalloidin labelling 77, 131 - Polymerization 95-96, 123, 125 - Purification 135 - Regulation C2 toxin (C2T) 123, 125 Rho proteins 62, 71, 159 - Stability 135 Actinomorphic response, Clostridium novyi alpha-toxin 150

Activation, Streptolysin 0 (SLO) 260 Active site - ADP-ribosyltransferase C3 62 - Diphtheria toxin 39 - E. coli heat-labile toxin 39 - Pertussis toxin 39 Adamalysin, Zinc endopeptidase 172 Adenylate cyclase 279 - Activation by cholera toxin 4 - Bordetella pertussis 280 ADP-ribosylation - Actin 95-96, 98-99, 134-138, 280 Effects on cytoskeleton 98-99 - Auto-ADP-ribosylation by cholera toxin 4 - Blockage by brefeldin A 37-38 - C2 toxin (C2T) 133-134 - Cholera toxin 279 - Effect on microfilament network 98-99 - Enhancement by ARFs 5 - G proteins 49-55,279, 284 E. coli heat-labile toxin 279 - Gasubunits 41 - Pertussis toxin 38,4751 -54 - Rho proteins 85,87-89, 165,284 ADP-ribosylation factors (ARFs) 15-16, 19-20, 22-25,27 - Activator of phospholipase D 5 - Activities 15-16 - Cellular locations 15-16 - Conservation 5 - Functions 5 - Properties 5 - Purification 17-18 - Sources 17 - Stimulation of ADP-ribosylation

5 ADP-ribosyltransferase - Clostridium difficile 280 - Mode of action 1-2 - Pertussis toxin 33-34, 38-41

- Pseudomonas aeruginosa 2 ADP-ribosyltransferaseC3 61-62, 145 - Active site 62 - ADP-ribosylation 165 - ADP-ribosylation of G proteins 284 - ADP-ribosylation of Rho proteins 284 - Assay of activity 74 - Determination of protein concentration 74 - Effect on cells 77-80, 89 - Endocytosis 81, 164 - Enzyme activity 64 - Factors affecting ribosylation 65 - Fusion protein 66, 81 - Inactivation of Rho proteins 71 - Microinjection 75-76, 89 - Modification of Rho proteins 62-64 - production by Clostridium botulinum 61, 85 - Purification 71-74, 86 - Rho GTPase inhibitor 81 - Rho proteins, assay 89-91 - Storage 73-74 - Structure 61-62, 85 - Toxicity 65 - Uptake 81 Aerolysin from A. hydrophila 241 -245 aFGF see Acidic fibroblast growth factor Ag mat ine - ADP-ribosylation by cholera toxin 4 - Assay 18-19,22-23 Agrobacterium tumefaciens, virB operon 36 Alpha-toxin 150, 241 - Actinomorphic response 150 - Assay 261-263 - Binding 243-244

K. Aktories (Ed.), Bacterial Toxins. 0 Chapman & Hall, Weinheim, 1997. ISBN 3-8261-0080-8

- Effect on interleukin converting enzyme (ICE) 246 - Induction of apoptosis 246 - Mutant 254 - Oligomerization 244-245 - Permeabilization 259-263 - Pore formation 242-243,245, 259 - Pore size 253 - Purification 249-250 - Storage 252,260 - Study of exocytosis 260-266 - Study of secretory processes 260-266 Anthrax toxin - Homology to iota toxin 94 - Permeabilization 243 - Pore formation 243 - Translocation 285 Antibiotic-associated pseudomembranous colitis, Clostridiurn difficile 141 Aplysia neurons, Assay for clostridial neurotoxins 198 Apoptosis 246 Assay - Alpha-toxin 261-263 - Botulinum neurotoxin (BoNT) 183-184 - C2 toxin (C2T) 103-105, 120 - Catecholamine uptake 266-267 - Cholera toxin 16-29 - Cleavage of synaptobrevin 235-237 - Clostridial neurotoxins 183-184, 197-198,204-206 - Exocytosis 228-232,263-266 - Glucosylation of Rho GTPases 164-166 - Glutamate uptake 267-270 - LD50 169, 197 - E. coli heat-labile toxin 16-29 - Permeability 262 - Permeabilization 222,263-266 - Pore-forming ability 261-263 - Rho proteins 87-91 - Streptolysin 0 261-263 - Tetanus neurotoxin 183-184 - ,,Time-to-death" 104 - Toxicity 183 - Translocation of fusion proteins 288 Assay systems - Aplysia neurons 198 - Autonomic nervous system 198 - Drosophila neurons 198 - Endocrine cells 199 - Invertebrate neurons 198 - Neuromuscular junction preparations 197- 198 Subject Index

- Primary cultures of CNS neurons 198 Astacin, Zinc endopeptidase 172 ATPase, Actin 96 Autonomic nervous system, Assay for clostridial neurotoxins 198

Bacillus anthracis toxin 279-280 Bacillus cereus exoenzyme 61, (see also ADP-ribosyltransferaseC3) Bacillus sphaericus, Mosquitocidal toxin 39 Bacillus thuringiensis, Pore-forming toxins 241 Bacterial toxins, Transporting tools 273 Bacteriocins, Pore-forming toxins 242 Balanced pathogenicity 173-174 Barnase, Fusion protein 284-285 Binary toxins 117-118, 279-280 BoNT see Botulinum neurotoxin Bordetella pertussis - Adenylate cyclase 280 - Cause of whooping cough 33 - Culture 48 - Production of pertussis toxin 33 - Toxin 2 Botulinum neurotoxin (BoNT) 120 - see also Clostridial neurotoxins - Action on neuromuscular junctions 169, 174-175, 194 - Action on peripheral nervous system 170 - Activation mechanism 170-171 - Assay 183-184 - Cleavage of SNAP-25 176-177, 181 - Cleavage of synaptobrevin 175-177 - Cleavage of syntaxin 175-178 - Different types 180, 193-194 - Formation of ion channels 175-176 - Inhibition of acetylcholine release 194 - Inhibition of neurotransmitter release 170, 193 - Insulin exocytosis 220 - Internalization 170, 175-176 - Mechanism of toxicity 170 - Mode of action 193-196 - Purification 182-183 - Receptor 175 - Recognition motifs in target proteins 180 - Serotypes 169 - Structure 170 - Therapeutic use 185

- Toxicity 169 Species differences 169 - Toxin channels 176 - Vaccine 182 - Zinc endopeptidase 172 Botulism 169-170, 185, 194 - Flaccid paralysis 169, 194 Brefeldin A, Blockage of ADPribosylation 37-38

C3 exoenzyme see ADPribosyltransferase C3 Ca2+,Insulin exocytosis 221 Catecholamine uptake - Assay 266-267 - Permeabilized cells 266-268 Cation channels, Formation by diphtheria toxin 277-278 Cdc42, Regulation of filopodia 71, 159 Cell adhesion, Rho proteins 71 Cell biology, Use of Clostridiurn difficile ToxB 166-167 Cell motility, Effect of Clostridium botulinurn C2 toxin 125 Cellubrevin - Cleavage by clostridial neurotoxins 196 - lsoform of synaptobrevin 178 Central nervous system, Action of tetanus neurotoxin 169-70, 194 Chickens, Tetanus resistance 169, 179 Chimeric toxins see Fusion proteins Cholera 1 - Fluid and electrolyte loss 4 - Vaccine 5-6 Cholera toxin - Action on G protein 279 - Activation 16-17 - Activation of adenylate cyclase 4 - Adjuvant 7 - ADP-ribosylation 279 agmatine 4 - ADP-ribosyltransferase 1-2 - Antigen delivery system 7 - Assay 15-29 - Auto-ADP-ribosylation 4 - Binding site for ganglioside GMl 2 - Catalytic action 15 - Catalytic His residue 41 - Cellular substrate 3-4 - Endocytosis 3 - Entry into cells 3 - Hydrolysis of NAD' 4, 15 - Immune response 6,7 - Increase of calcium flux 3 - Mode of action 1 - Molecular tool 7

- NAD+-glycohydrolysis 39 - Purification 16 - Releaseof 5-HT 4 - Structure 2-3, 15-16 - Targeting molecules 7 - Targeting to G ,, ganglioside 7 - Uses 5-8 - Vaccine development 5-7 - X-ray crystallography 3 Cholesterol-bindingtoxins 241 - Oligomerization 244 Classification, Pore-formingtoxins 241 Clathrin, Endocytosis 276 Clostridia, Ecology 173-174 Clostridial neurotoxins - Amino acid sequence 170-172 - Apo-neurotoxin 172 - Assay 183-184, 197, 199-200, 204-206 - Binding to cells 174 - Binding to neuromuscular junctions 169, 174-175, 194 - Decontamination 182,201 - Diseases 169 - Function 173 - Inhibition of acetylcholine release 169 - Inhibition of exocytosis 237 - Inhibition of neurotransmitter release 170, 181-182, 193 - Internalization 169, 175-176, 194 - LDSo 183 - Mobile genetic elements 173 - Mode of action 193-196 - Neurospecificity 169 - Origin 169 - Precursors 185-186 - Purification 182-183 - Recognition sequences 180 - Research tool 117, 184, 196 - Safety 182 - Sources 201 - Specificity 180, 195-196 - Structure 170, 173, 194 - Synaptobrevin cleavage 176 - Therapeutic use 117, 193 - Therrnolysin-like proteases 173 - Toxicity 169, 183, 201 - Zinc endopeptidases 172-174, 176, 185-186 Clostridiurn barati, Cause of botulism 169 Clostridiurn botulinum - C3 exoenzyme see ADPribosyltransferaseC3 - Production of neurotoxins 193 Clostridium botulinurn C2 toxin (C2T) 93, 103, 117-118

- Action on cytoskeleton 119, 123-125 - Action on G-actin 166-167 - Activation 104, 109 - ADP-ribosylation 133-134 - ADP-ribosylation of actin 94, 280 - Antibody preparation 111-112 - Assay 103-105, 120 - Autoregulation of actin synthesis 125 - Binary structure 129-130 - Binary toxin 117-118 - Cell motility 125 - Channel formation in membranes 122-123 - Chemical mediators Release 123-124 Storage 124 - Depolymerization of actin 129 - Detoxification 104 - Effect on cytoskeleton 119, 123-125, 131 - Effect on microfilaments 131 - Endocytosis 93, 110, 119 - lmmunofluorescence 110, 112-114 - Internalization 118, 122 - Mechanism of action 118-119 - Microinjection 119 - Purification 104-108, 120-121 - Purification of antibodies 112 - Research tool 121, 123-126, 129 - Sensitivity of cells 130 - Specificity 94-5 - Storage 108-109 - Structure 93-4, 103, 110 - Toxicity 117-118, 122, 130 - Translocation 93 Clostridiurn botulinurntype C 103 - see also Clostridiurn botulinurn C2 toxin Clostridium bufyricum, Cause of botulism 169 Clostridium difficile - ADP-ribosyltransferase 280 - Cytotoxic activity 141 - Glucosyltransferase activity 159 - Molecular toxicity 151 - Pathogenicity 151 - Pseudomembranouscolitis 141, 151 - Transferase 93 Clostridiurn difficile cytotoxin see Clostridium difficile, ToxB Clostridiurn difficile enterotoxin see Clostridium difficile, ToxA Clostridium difficile ToxA - Antibodies 143 - Breakdown of tight junctions 149 - Cytoskeletal breakdown 149 - Cytotoxic activity 160 - Effects on nuclei 147

-

Glucosylation of Rho proteins 149 Homologies 142 Intestinal effects 149, 152 lntracellular calcium release 148 Mechanism of action 149 Molecular size 142, 159 Purification 142, 160-161 Receptor 144 - Stability 160 - Storage 160 - Structure 142-143, 152, 159 - Toxicity 141, 149 Clostridiurn difficile ToxB 141 - Antibodies 143 - Cell biology tool 166-167 - Cytoskeletal breakdown 159 - Cytotoxicity 160, 164 - Glucosyltransferase activity 161 - Homologies 142 - Inhibition of DNA synthesis 148 - Lytic effects 152 - Mechanism of action 149 - Molecular size 142, 159 - Probe for Rho GTPases 159-160 - Purification 142, 160-161 - Receptor-mediatedendocytosis 164 - Storage 160 - Structure 142, 152, 159 - Toxicity 141, 143, 149 Clostridium difficile toxins - see also Clostridiurn difficile, ToxA; Clostridiurn difficile Tox B - Cytokine release 148 - Cytopathogenic effect 144 - Disruption of actin cytoskeleton 147 - Disruption of epithelial barrier 148-149 - Effect on tight junctions 148 - Effects on animals 148-149 - Endocytosis 144 - Glucosylation of Rho proteins 146-147, 162-163 - Glucosyltransferases 146-147 - Internalization 145 - lntracellular processing 144-145 - Modification of Rho proteins 145 - Research tool 153 - Structure 170-172 - Tumor therapv 153 - UDP-glucose glycohydrolase 161-162 - Vaccination 153 Clostridium Limosum exoenzyme 61, (see also ADPribosyltransferase C3) Clostridium novyi alpha toxin see Alpha-toxin -

Subject Index

Clostridium perfringens iota toxin 93 - Binding component 94 - Effect on actin cytoskeleton 129 - Homology 94 - Mode of action 280 - Specificity 95 - Structure 94, 280 - Toxicity 94 Clostridium sordelli toxins 150 Clostridium spiroforme toxin 93 - Binding component 94 - Mode of action 280 - Structure 94, 280 Clostridium tetani 169 - see also Tetanus neurotoxin (TeTx) - Production of tetanus neurotoxin 193 CNS neurons, Assay for clostridial neurotoxins 198 Coated pits, Endocytosis 144 Colicins, Pore-formingtoxins 242 Collagenase, Zinc endopeptidase 172 Colon cancer therapy, Clostridium difficile toxins 153 Corynebacterium diphtheriae, Cause of diphtheria 2,273 Crotalus durissus phosphodiesterase 94 CTA (Cholera toxin subunit A) see Cholera toxin Culture - Bordetella pertussis 48 - Clostridium botulinum type C 106 - E. COI~ 72 - For immunofluorescence labelling 113 - Swiss 3T3 cells 74-75 - Vero cells 113 Cytochalasins - Actin depolymerization 129 - Cytopathogenic effect 144 Cytocidal action, Pore-forming toxins 248 Cytokine release, Clostridium difficile toxins 148 Cytopathogenic effect - Actinomorphic 144 - Clostridium difficile toxins 144 - Cytochalasins 144 Cytoskeleton - Action of Clostridium botulinum C2 toxin 119, 123-125 - Effect of ADP-ribosylationof actin 98-99 - Effect of Clostridium difficile ToxA 149 Subject Index

- Effect of Clostridium difficile ToxB 159 - Effect of pore-forming toxins 247 Decontamination 298-300 - Clostridial neurotoxins 182,201 - Clostridium botulinum C2 toxin 104 - Tetanus neurotoxin 232,234 Diabetes mellitus 217 Dihydrofolate reductase, Fusion protein 284 Diphthamide, Acceptor substrate for toxins 39, 41, 274 Diphtheria, Symptoms 273 Diphtheria toxin - Active site 39 - ADP-ribosylation of elongation factor 274 - ADP-ribosyltransferase 2 - Cause of diphtheria 273 - Differences in cellular sensitivity 275 - Effect of phorbol esters 275 - Endocytosis 276 - Formation of cation channels 277-278 - Inhibition of protein synthesis 274 - NAD+-glycohydrolysis 39 - Receptor 275 - Resistant cells 275 - Resistant mutations 274 - Structure 37, 274 - Translocation 276-277, 281 DNA synthesis, Inhibition by ToxB 148 DNase inhibition, Actin depolymerization assay 131-132 Drosophila neurons, Assay for clostridial neurotoxins 198 Dystonias, Treatment with botulinum neurotoxin 185-186

E. coli, Culture 72 E. coli heat-labile toxin (LT) 1 - Activation 16 - Active site 39 - ADP-ribosylation of G proteins 279 - ADP-ribosyltransferase 2 - Assay 15-29 - Purification 16 - Structure 2, 15 - Structure of holotoxin 3 - Subunits 15 - X-ray crystallography 3 E. coli hemolysin 241-42

- Binding 244 - Oligomerization 244-245 - Pore size 253 - Pore-forming toxin 241 EDlN 61 (see also ADPribosyltransferase C3) Elongation factor 2 (EF-2), ADPribosylation 2 Emergency procedures 299-300 Endocrine cells, Assay for clostridial neurotoxins 199 Endocytosis - ADP-ribosyltransferaseC3 81, 164 - Cholera toxin 3 - Clostridium botulinum C2 toxin 93, 110 - Clostridium difficile toxins 144 - Coated pits 144,276 - Diphtheria toxin 276 Enterochromaffincells, Release of 5-HT by cholera toxin 4 Epithelial barrier, Disruption by Clostridium difficile toxins 148 Exocytosis - Assay 228-232,263-266 - Inhibition by clostridial neurotoxins 237 - SNARE hypothesis 219-221 - Study using clostridial neurotoxins 196 - Study using pore-forming toxins 260-266 - Transient cotransfection assay 228-232 Exotoxin A see Pseudomonas aeruginosa exotoxin A F-actin 98 Farnesylation, Recombinant proteins 289-290 Filopodia, Cdc42 71, 159 Fixation of cells 76, 131 Flaccid paralysis, Botulism 169, 194 Focal adhesions - Control by Rho proteins 80, 145, 159 - Effect of ADP-ribosyltransferase C3 80 - FAK 76 -Paxillin 76 - Talin 76-77 - Vinculin 76-77 Fusion proteins 280-281 - Acidic fibroblast growth factor 284 - ADP-ribosyltransferaseC3 66,

81 - Barnase 284-285

-

Dihydrofolate reductase 284 Influenza A antigens 285 Limitations 285-286 Production 286-290 - Therapeutic potential 121 - Translocation 280-286 Fusion toxins see Fusion proteins

G proteins - Action of cholera toxin 279 - Action of pertussis toxin 38, 41, 47 - ADP-ribosylation 49-55, 279, 284 - Control of insulin secretion 218-219 - Effects of pore-forming toxins 247-248 - Removal of ADP-ribose 56 - Signal transduction cycle 42 Ga subunits, ADP-ribosylation 41 G-actin 98 - Effect of Clostridium botulinum C2 toxin 166-167 Ganglioside GM1, Binding to cholera toxin 2 Gelsolin, Complex with actin 96-97 Glucosylation - Recombinant GTPases 163-164 - Rho GTPase, assay 164-166 - Rho proteins 146-148, 161-63 Glucosyltransferases, Clostridium difficile toxins 146-147, 159, 161 Glutamate uptake, Assay 267-270 Glycophosphoinositol (GPI) anchor, Toxin receptor 275 GM1 ganglioside, Targeting by cholera toxin 7 Golgi apparatus, ARFs 16 GSCI - GTPase activity 4 - Structure 4 - Substrate for cholera toxin 3-4 GTPase - Activity of Gs, 4 - Recombinant, Glucosylation 163- 164 - Rho proteins 159 Hemorrhagic toxin, Clostridium sordelli 150 5-HT, Release by cholera toxin 4 lmmunolabelling 76-77 - Clostridium botulinum C2 toxin 110, 112-114

- Focal adhesions 76 Inflammation, Effects of poreforming toxins 248-249 Influenza A antigens, Fusion protein 285 Inhibitory synapses, Blockade by tetanus neurotoxin 170 Insect cells - Permeabilization 241 - Resistance to diphtheria toxin 275 Insecticidal toxins 241 Insulin secretion - Botulinum neurotoxin 220 - Ca2' 221 - Control 217-219 - Effect of pertussis toxin 41 - Rab3A 221 - SNARE hypothesis 220-221 - Synaptotagmin 221 - Syntaxin 220 - Tetanus neurotoxin (TeTx) 220 Insulin-secretingcells, Permeabilization 222-228 Interleukin converting enzyme (ICE) 246 Internalization - Botulinum neurotoxin 170, 175-176 - Clostridial neurotoxins 169, 175-176, 194 - Clostridium botulinum C2 toxin 118, 122 - Clostridium difficile toxins 145 - Tetanus neurotoxin 170, 175-176 Intestine, Effect of Clostridium difficile ToxA 149 Invertebrate neurons, Assay for clostridial neurotoxins 198 Ion channels, Formation by clostridial neurotoxins 175-176 Ion fluxes, Generation by poreforming toxins 246-247 Iota toxin see Clostridium perfringens iota toxin Islet-activating protein see Pertussis toxin Lamellipodia protrusion, Rac protein 71 Large clostridial cytotoxins 150-151 a-Latratoxin, Effect on synaptosomes 199 - Inhibition of botulinum neurotoxin/A 181 LD50 - Assay 197 - Botulinum neurotoxin 169 - Clostridial neurotoxins 183

- Tetanus neurotoxin 169 50 % lethal dose see LDW Lethal toxin, Clostridium sordelli 150 Lipid mediators, Generation by pore-forming toxins 247 Membrane protein shedding, Effect of pore-forming toxins 248 Membrane proteins, ADPribosylation 51-54 Membrane ruffling - Control by Rho proteins 145 - Rac protein 159 Metalloendoproteases see Zinc endopeptidases Methods - see also Assay; Culture; Purification - Actin labeling 131 - Activation of Clostridium botulinum C2 toxin 109 - Activation of pertussis toxin 50-51 - ADP-ribosylation Actin 134-138 G proteins 49-55 membrane proteins 51-54 Rho proteins 87-89 - ADP-ribosyltransferaseC3, expression 86-87 - ARFassay 20-28 - Assessing translocation 280-283 - Cell homogenates 51 - Cell permeabilization 222-228,235 - Cleavage of synaptobrevin (VAMP) by tetanus neurotoxin 233-237 - Clostridium botulinum C2 toxincatalysed ADP-ribosylation

133-134 - Clostridium botulinum type C, culture 106 - Fixation of cells 76 - Fusion proteins 286-290 - Glucosylation of Rho proteins 162- 163 - lmmunolabelling 76-77 - Microinjection of ADPribosyltransferase C3 75-76 - Micropipettes 75 - Permeabilizationof insulinsecreting cells 222-228 - Phalloidin labelling of actin 131 - Preparation of Clostridium botulinum C2 toxin antibodies 111-112 - Quantitative determination of actin depolymerization 131-132

Subject Index

- Recombinant ARFs 18 - Removal of ADP-ribose from G-proteins 56 - SDS-PAGE of G-proteins 55-57 - Subfractionation of synaptosomes 210-211 - Synaptosome poisoning 207-209 - Synaptosome preparation 202-204 - Toxicity testing 183 Microfilaments - Effect of ADP-ribosylation 98-99 - Effect of Clostridium botulinum C2 toxin 131 - Regulation by Rho proteins 166 Microinjection - ADP-ribosyltransferase C3 75-76 - Clostridium botulinum C2 toxin 119 Micropipettes, Preparation 75 Mitogenicity, Pertussis toxin 41 Mobile genetic elements, Clostridial neurotoxins 173 Modeccin 279 Mosquitocidal toxin 39, 41 Mycotoxins, Depolymerization of actin 129 NAD+-glycohydrolysis - Cholera toxin 39 - Diphtheria toxin 39 - Exotoxin A 39 - Pertussis toxin 38-39 Neuromuscular junctions - Action of neurotoxins 169, 174-175, 194 - Assay for clostridial neurotoxins 197 - Assay system 197-198 Neurospecificity, Clostridial neurotoxins 169 Neurotransmitter release - Blockade by clostridial neurotoxins 181-182 - Monitoring 204-206 NO-synthase, Stimulation by poreforming toxins 247 Paxillin 76 Perfringolysin - Binding 243-244 - Pore-formingtoxin 241 Peripheral nervous system, Action of botulinum neurotoxin 170 Permeability, Assay 262 Permeabilization Subject index

-

Alpha-toxin 259-263 Anthrax toxin 243 Assay 222,263-266 Insect cells 241 - Insulin-secretingcells 222-228 - Membranes 241 -243,259-266 - Pore-formingtoxins 222 - Saponin 276 - Streptolysin 0 233, 259-263 - Synaptosomes 268-270 Permeabilized cells - Assay for exocytosis 263-266 - Catecholamine uptake 266-268 Pertussis, vaccine 33 Pertussis toxin - Accessory genes (ptl) 35-36 - Action on G proteins 38, 41, 47 - Activation 47-48, 50-51 - Active site 39 - ADP-ribosylation of G proteins 38,279 - ADP-ribosylation of membrane proteins 51-54 - ADP-ribosyltransferase 2, 33-34,38-41 - Binding properties 37 - Biogenesis 35 - Biological activity 41-42 - Catalytic His residue 41 - Cause of whooping cough 33 - Effect on insulin secretion 41 - Mechanism of action 33 - Mitogenicity 41 - NAD+-glycohydrolysis 38-39 - Pertussis toxin liberation genes (ptl) 35-36 - Physiological effects 33 - Purification 33 - Receptors 36-37 - Secretion 35 - Sources 48 - Structural genes (ptx) 34-35 - Structure 33-35,3747 - Translocation 36-3747 - Virulence genes 35 PFTs see Pore-forming toxins (PFTs) Phalloidin - Actin labeling 77 - Labelling of stress fibers 164 Phospholipase D - Activation by ARFs 5, 16 - Regulation by Rho proteins 63 Phosphoramidon - Inhibition of tetanus neurotoxin activity 176 - Inhibition of zinc endopeptidases 176 Phosphorimager, Analysis of radiolabeled gels 165 Pinocytosis see Endocytosis

Plant toxins 279 Pneumolysin, Pore-forming toxin 241 Pore-forming toxins (PFTs) - Aerolysin from A. hydrophila 241-245 - Alpha-toxin 241-243, 245,253, 259 - Applications 259 - Bacillus thuringiensis toxins 241 - Bacteriocins 242 - Binding 243-244 - Cell-biological effects 246-248 - Cholesterol-binding toxins 241 - Classification 241 - Colicins 242 - Cytocidal action 248 - E. coli hemolysin 241 -242 - Effects on G proteins 247-248 - Effects on signaling pathways 247-248 - Inflammation 248-249 - Mode of action 243-245 - Pathogenic aspects 248-249 - Perfringolysin 241 - Permeabilization 222 - Pneumolysin 241 - Pore formation 245 - Pore size 253 Alpha-toxin 253, 260 Streptolysin 0 254, 260 Tetanolysin 0 254 - Purification 249-252 - Research tools 252-254 - RTX family 241 - Staphylococcus aureus 241 - Streptolysin 0 222,241 -242,259, 262-263 - Tetanolysin 254 - Vibrio toxins 241 Protein synthesis, Inhibition by diphtheria toxin 274 Pseudomembranous colitis, Clostridium difficile 151 Pseudomonas aeruginosa exotoxin A - ADP-ribosylation 2 - Inactivation of elongation factor 278 - NAD+-glycohydrolysis 39 - Structure 37, 278 - Translocation 278-279 Purification - Actin 135 - ADP-ribosylation factors 17-18 - ADP-ribosyltransferaseC3 71-74, 86 - Alpha-toxin 249-252 - Botulinum neurotoxin (BoNT) 182-183 - Cholera toxin 16

- Clostridial neurotoxins 182-183 - Clostridium botulinum C2 toxin 104-108, 120-121, 105-108, 112 - Clostridium difficile toxins 142, 160-161 - E. coli heat-labile toxin 16 - Pertussis toxin 33 - Pore-formingtoxins 249-252 - Recombinant proteins 288 - Streptolysin 0 251-252 - Synaptosomes 203-204 - Tetanus neurotoxin 182-183 Rac protein 63-64 Lamellipodia protrusion 71 - Membrane ruffling 159 Receptor-bindingregions - Botulinum neurotoxin 173 - Tetanus neurotoxin 173 Receptor-mediatedendocytosis, Clostridium botulinurn C2 toxin

-

110, 119 Receptors - Diphtheria toxin 275 - Pertussis toxin 35-36 Recognition motif - For clostridial neurotoxins 180 - SNAP-25 180 - Synaptobrevin (VAMP) 180 - Syntaxin 180 Recognition sequences - Botulinum neurotoxin 180 - Tetanus neurotoxin 180 Recombinant proteins - Cholera vaccine development 6 - Expression in bacteria 288 - Farnesylation 289-290 - In vitro farnesylation 289 Research tools, Pore-forming toxins 252-254 Retrograde axonal transport, Tetanus neurotoxin 194 Rho proteins (Rho GTPases) - Actin regulation 62, 71, 159 - Action of Clostridium difficile toxins 145 - Activation of transcription factors 159 - ADP-ribosylation 85,87-89, 165,284 - Assay 87-91 - Basal activity 77 - Control of focal adhesions 71, 80,89, 145, 159 - Control of membrane ruffling 145 - Control of stress fibers 145, 159 - Cytoskeleton regulation 166 - Enzyme regulation 62-63

- Function 89 - Glucosylation 146-148, 161-63 by Clostridium difficile toxins 162- 163 - Glucosylation assay 164-166 - GTPase activity 62, 159 - Inactivation by ADPribosyltransferaseC3 71 - Mammalian 85 - Phospholipase D regulation 63 - Regulation of tight junctions 148 - Regulation of transcription factors 166 - Stress fiber assembly 71, 89 - Structure 62, 85 - Substrate specificity 64 Ricin 279 RTX family, Pore-formingtoxins 241 Staphylococcus aureus, Pore-forming toxins 241 Safety 295-300 - Clostridial neurotoxins 182 - Containment 296-298 - Decontamination 298-300 - Disposal 298-300 - Emergencies 299-300 - Information resources 300 - Preventive measures 295-296 - Tetanus neurotoxin 201, 232-233 Saponin, Permeabilization 276 Shigella toxin 279 Sialoglycoproteins, Pertussis toxin receptors 36-37 Signal transduction cycle, G protein 42 Signaling pathways, Effects of pore-forming toxins 247-248 SLO see Streptolysin 0 SNAP-25 - Cleavage by botulinum neurotoxin 176-178, 195 - Cleavage by clostridial neurotoxins 181, 196 - Complex with syntaxin 181 - Conservation 196 - Function 179, 181 - Molecular size 179 - Recognition motif 180 - Structure 180 - t-SNARE 181 - Tissue distribution 179 SNARE complex 181-182 SNARE hypothesis - Exocytosis 219-221 - Insulin exocytosis 220-221 - Synaptobrevin (VAMP) 219

- Synaptotagmin 219 - Syntaxin 219 Staphylococcal alpha-toxin see Alpha-toxin Storage - Alpha-toxin 252, 260 - C2 toxin 108 - Streptolysin 0 252, 260 Strabismus, Treatment with botulinum neurotoxin 185 Streptococcal glucosyltransferases, Homology with Tox A and B 142 Streptolysin 0 (SLO) 242 - Activation 260 - Applications 252, 254 - Assay 261-263 - Binding 243-244 - Cell permeabilization 225-228, 233,259-263 - Fusion protein 260 - Large pore size 254, 260 - Pore formation 222, 241,259, 262-263 - Purification 251-252 - Storage 252,260 - Study of exocytosis 260-266 - Study of secretory processes 260-266 Stress fibers - Effect of Clostridium botulinum C2 toxin 131 - Effect of Rho proteins 71, 145, 159 - Phalloidin labelling 164 Structure - Botulinum neurotoxin 170 - Clostridial neurotoxins 170, 173, 194 - Clostridiurn botulinum C2 toxin 93. 103 - Diphtheria toxin 37 - Pertussis toxin 33,47 - Pseudomonas aeruginosa exotoxin A 37,278 - SNAP-25 180 - Synaptobrevin 178-180 - Syntaxin 180 - Tetanus neurotoxin 170, 233 Synaptobrevin (VAMP) - Action of tetanus neurotoxin 232 - Assay of cleavage 235-237 - Association with synaptophysin

178 - Association with VAP-33 178 - Cleavage by botulinum neurotoxin 175-178, 195 - Cleavage by clostridial neurotoxins 176-178, 196 - Cleavage by tetanus neurotoxin 195,233-237 Subject Index

- Conservation 196 - Function 181 - Recognition motif 180 - SNARE hypothesis 219 - Structure 178-180 - Vesicle receptor protein 181 Synaptophysin, Association with synaptobrevin 178 Synaptosomal-associatedprotein of 25 kDa see SNAP-25 Synaptosomes - Assay for clostridial neurotoxins 199-200 - Effect of a-latratoxin 199 - Permeabilization 268-270 - Poisoning 207-209 - Preparation 202-204 - Properties 199 - Purification 203-204 - Subfractionation 210-211 Synaptotagmin - Insulin exocytosis 221 - Receptor for botulinum neurotoxin 175 - SNARE hypothesis 219 Syntaxin - Cellular distribution 179 - Cleavage by botulinum neurotoxin 176-178, 195 - Cleavage by clostridial neurotoxins 196 - Complex with SNAP-25 181 - Conservation 196 - Function 181 - Insulin exocytosis 220 - lsoforms 179-180 - Recognition motif 180 - SNARE hypothesis 219 - Structure 180 - t-SNARE 181 t-SNARE 181 - SNAP-25 181 - Syntaxin 181 Talin 76-77 Tetanolysin, Pore-formingtoxins 254 Tetanus 169, 194 - Resistance of chickens 169, 179 - Resistance of rats 169, 179 Tetanus neurotoxin (TeTx) 169 - see also clostridial neurotoxins - Action on central nervous system 169-170, 194 - Activation mechanism 170-171 - Assay 183-184 - Binding to neuromuscular junction 169, 174-175

Subject Index

- Blockade of inhibitory synapses 170 - Blockade of neurotransmitter release 170, 176, 193 - Cellubrevin cleavage 232 - Decontamination 232, 234 - Formation of ion channels 175-176 - Insulin exocytosis 220 - Internalization 170, 175-176 - Mechanism of toxicity 170 - Migration to inhibitory interneurons 169 - Mode of action 193-196 - Purification 182-183 - Receptor-bindingregions 173 - Recognition sequences 180 - Retrograde axonal transport 169, 194 - Safety 201,232-233 - Specificity 232 - Structure 170, 233 - Synaptobrevin cleavage 176-178, 181-182, 232-7 - Syntaxin cleavage 175-176 - Toxicity 169 - Vaccine 182 - Zinc endopeptidase 172,232 TeTx see Tetanus neurotoxin Therapeutic use, Botulinum neurotoxin 185 Thermolysin-like proteases - Clostridial neurotoxins 173 - Zinc co-ordination 172 Tight junctions - Effect of Clostridiurn difficile toxins 148 - Regulation by Rho proteins 148 "Time-to-death" assay 104, 197 ToxA see Clostridiurn difficile, ToxA ToxB see Clostridium difficile, ToxB Toxicity - ADP-ribosyltransferaseC3 65 - Botulinum neurotoxin 169 - Clostridial neurotoxins 169, 183, 201 - Clostridium botulinurn C2 toxin 117-118, 122, 130 - Iota toxin 94 - Resistance 119 - Species differences 169 - Testing 183 - Tetanus neurotoxin 169 - Tox A from Clostridium difficile 141, 149 - Tox B from Clostridiurn difficile 141, 143, 149 Toxin channels see Ion channels Toxins, Chimeric see Fusion proteins

Transcription factors, Regulation by Rho proteins 159, 166 Transient cotransfection assay, Exocytosis 228-232 Translocation - Anthrax toxin 285 - Assay 288 - C2 toxin 93 - Diphtheria toxin 276-277, 281 - Fusion proteins 280-286 - Methods of assessment 280-283 - Pertussis toxin 36-37, 47 - Pseudornonas aeruginosa exotoxin A 278-279 Tumor therapy, Clostridiurn difficile toxins 153

UDP-gIucose g Iyco hyd rolase, Clostridiurn difficile toxins

161-162

V-SNARE see Vesicle receptor protein Vaccine - Botulinum neurotoxin 182 - Cholera 5-7 - Clostridium difficile 153 - Pertussis 33 - Tetanus neurotoxin 182 - Whooping cough 42 VAMP see Synaptobrevin VAP-33, Association with synaptobrevin 178 Vesicle receptor protein (v-SNARE) - Synaptobrevin (VAMP) 181 - Synaptotagmin 181 Vibrio cholerae - Cause of cholera 1 - Strains 6 Vibrio hemolysins, Oligomerization 244-245 Vibrio toxins, Pore-formingtoxins 241 Vinculin 76-77 Viscumin 279 Volkensin 279

Whooping cough - Caused by Bordetella pertussis 33 - Vaccine 42

Zinc endopeptidases 172 - Active site 172 - Clostridial neurotoxins 172-174, 176, 185-186 - Thermolysin-like enzymes 172