Bacterial Toxins

Name: Bacterial Toxins | Tools in Cell Biology and Pharmacology
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Tools in Cell Biology and Pharmacology

Klaus Aktories(Herausgeber*in)

Wiley-Blackwell (Verlag)

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Erschienen am 26. September 2008

XXVI, 308 Seiten

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978-3-527-61460-8 (ISBN)

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Bacterial Toxins
Contents
CHAPTER 1 . Cholera Toxin: Mechanism of Action and Potential Use in Vaccine Development
1.1 Introduction
1.2 Molecular Aspects of Cholera Toxin Action
1.2.1 Structure and Relationship to Other Toxins
1.2.2 Toxin Entry into Cells and Events Leading to Pathogenesis
1.2.3 Enzymology of Cholera Toxin
1.2.4 In Vitro Stimulation of Cholera Toxin Activity by ARF
1.3 Practical Aspects of Cholera Toxin Use
1.3.1 Vaccine and Vaccine Development
1.3.2 Cholera Toxin as a Molecular Tool
1.4 Summary
CHAPTER 2 . Cholera Toxin and Escherichia coli Heat-labile Enterotoxin: Biochemical Methods for Assessing Enzymatic Activities
2.1 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 Gsa 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
2.5.1 Additional Reagents and Materials Required
2.5.2 Protocol
2.6 Assay 4: NAD Glycohydrolase Assay
2.6.1 Additional Reagents and Materials Required
2.6.2 Protocol
2.7 Comments and Considerations
2.7.1 Appropriate Controls and Analysis of Data
2.7.1.1 Controls
2.7.1.2 Data analysis
2.7.2 Optimization Interfering Substances, Troubleshooting, and Assay
2.7.2.1 Interfering substances
2.7.2.2 Troubleshooting
2.7.2.3 Assay optimization
2.7.3 Consideration for the Use of ARF
2.7.3.1 Lipid/Detergent and Nucleotide Requirements
2.7.3.2 Development of other Assay Conditions
CHAPTER 3 . Pertussis Toxin
3.1 Introduction
3.2 Genetic Regulation of Pertussis Toxin Production
3.3 Biogenesis of Pertussis Toxin
3.4 Receptor-binding and Translocation
3.5 ADP-ribosyltransferase Activity and Enzyme Mechanism
3.6 Biological Activities and Role of Pertussis Toxin in Pathogenesis
CHAPTER 4 . Pertussis Toxin as a Cell Biology Tool
4.1 Introduction
4.2 Pertussis Toxin as a Tool to Modify Cellular Functions
4.2.1 Cell Culture of Bordetella pertussis
4.2.2 Source of Pertussis Toxin and Preparation of Solution
4.2.3 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 ADP- ribosylation
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
4.4 SDS-Gel Electrophoresis
4.5 Reagents and Chemicals
CHAPTER 5 . Clostridium botulinum ADP-ribosyltransferase C3
5.1 Introduction
5.2 The Family of C3-like Transferases
5.3 Modification of Rho Proteins by C3-like Transferases
5.4 Characterization of the C . botulinum C3 ADP-Ribosyltransferase Reaction
5.5 Factors Affecting ADP-Ribosylation by C3
5.6 C3-Transferases in Cell Biology and Pharmacology
CHAPTER 6 . Clostridium botulinum C3 Exoenzyme and Studies on Rho Proteins
6.1 Introduction
6.2 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
6.3 Preparation of Swiss 3T3 Cells
6.4 Microinjection of C3 Transferase
6.5 Fixation and Staining of Cells
6.6 C3 Protein Inhibits LPA-stimulated Stress Fibre Assembly and Focal Adhesion Clustering
6.7 Timecourse of Focal Adhesion Breakdown
6.8 Alternative Delivery of C3 Transferase
6.9 Conclusion
6.10 Reagents and Chemicals
CHAPTER 7 . Preparation of Clostridium botulinum C3 Exoenzyme and Application of ADP-ribosylation of Rho Proteins in Biological Systems
7.1 Introduction
7.2 C3 Exoenzyme Expression and Purification of Recombinant
7.3 ADP-ribosylation Reaction
7.4 Application of C3 Exoenzyme in Biological Systems
CHAPTER 8 . Actin-ADP-ribosylating Toxins: Cytotoxic Mechanisms of Clostridium botulinum C2 Toxin and Clostridium perfringens Iota Toxin
8.1 Introduction
8.2 Clostridium botulinum C2 Toxin
8.3 Other actin-ADP-ribosylating Toxins
8.4 Actin as the Substrate for ADP-ribosylation
8.5 Model for the Cytopathic Effects of Actin- ADP-ribosylating Toxins
CHAPTER 9 . Purification. Activation and Endocytosis of Botulinum C2 Toxin
9.1 Introduction
9.2 Assay Method for the Toxin
9.3 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
9.5 Endocytosis of Two Nonlinked Protein Components in 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 II
9.6 Reagents and Chemicals
CHAPTER 10. The Role of the Clostridium botulinum C2 Toxin as a Research Tool to Study Eucaryotic Cell Biology
10.1 Introduction
10.2 Identifying Potential Uses
10.3 Isolation and Purification
10.4 C2 Toxin as a Research Tool
10.4.1 Binding Step
10.4.2 Internalization Step
10.4.3 lntracellular Step
CHAPTER 11 . Probing the Actin Cytoskeleton by Clostridium botulinum C2 Toxin and Clostridium perfringes Iota Toxin
11.1 Introduction
11.2 Effects of Clostridium botulinum C2 Toxin in Intact Cells
11.2.1 Treatment of Intact Cells with Clostridium botulinum C2 Toxin
11.2.2 Effects of C2 on the Microfilament System
11.2.2.1 Visualization of the Microfilament System with FITC-phalloidin
11.2.2.2 Quantitative Determination of F-actin Depolymerization
11.2.3 Detection of C2-catalyzed ADP-ribosylation in Intact Cells
11.2.3.1 Direct Method by Metabolical Labeling
11.2.3.2 Quantification of ADP-ribosylated Actin
11.2.4 Controls of the in vivo Effects of C2 Toxin
11.3 ADP-ribosylation of Actin
11.3.1 ADP-ribosylation of Actin in Cell Lysates
11.3.2 ADP-ribosylation of Skeletal Muscle a-Actin to Study Actin Functions
11.3.3 ADP-ribosylation of Skeletal Muscle a-Actin for Microinjection
11.3.4 Troubleshooting
11.4 Reagents and Chemicals
CHAPTER 12 . Clostridium difficile Toxins
12.1 Introduction
12.2 Molecular Characteristics of the Toxins
12.3 Cellular Effects of the Toxins
12.3.1 Actin Cytoskeleton is the Toxin Target
12.3.2 The Receptor for ToxA Contains Galactose
12.3.3 The Toxins Act lntracellularly after Endocytosis
12.4 Molecular Mode of Action of the Toxins
12.4.1 C . difficile Toxins Modify Rho Proteins
12.4.2 C . difficile Toxins are Glucosyltransferases
12.5 Biological Consequences of Toxin Action on Cells
12.5.1 In Cultured Cells
12.5.2 In Animal Models
12.6 Relationship of the C . difficile Toxins to Other Large Clostridial Cytotoxins
12.7 Conclusions and Perspectives for the Future
CHAPTER 13 . Clostridium difficile Toxin B as a Probe for Rho GTPases
13.1 Introduction
13.2 Purification of Toxin A and Toxin B
13.3 Glucosyltransferase Activity of Toxin B
13.3.1 Glucosylation in Cell Lysates
13.3.2 Glucosylation of Recombinant Rho Proteins or Membranous Fractions
13.3.3 Glucosylation of Recombinant GTPases for Microinjection
13.4 Toxin B Action on Intact Cells
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
CHAPTER 14 . Clostridial Neurotoxins
14.1 The Origin of Clostridial Neurotoxins
14.2 Structure of Clostridial Neurotoxins
14.3 Mechanism of Action of Clostridial Neurotoxins
14.3.1 Cell Binding
14.3.2 Internalization and Membrane Translocation
14.3.3 lntracellular Activity
14.3.3.1 VAMP/Synaptobrevin
14.3.3.2 SNAP-25
14.3.3.3 Syntaxin
14.3.3.4 Target Recognition
14.4 Clostridial Neurotoxins and the Blockade of Neurotransmitter Release
14.5 Biomedical Applications of Clostridial Neurotoxins
14.5.1 Safety Warnings
14.5.2 Purification of Clostridial Neurotoxins
14.5.3 Functional Assay of Clostridial Neurotoxins
14.5.3.1 In vitro Assay of Clostridial Neurotoxins
14.5.3.2 Use in Cell Biology
14.6 Therapeutic Uses of Botulinum Neurotoxins
14.7 Concluding Remarks
14.8 Chemicals
CHAPTER 15 . Isolated Nerve Terminals as a Model System for the Study of Botulinum and Tetanus Toxins
15.1 Introduction
15.2 Clostridial Neurotoxins: How Do They Work?
15.3 Model Systems for the Study of Toxin Action
15.3.1 Bioassay
15.3.2 Neuromuscular Junction Preparations
15.3.3 Synaptic Preparations Derived from the Autonomic Nervous System
15.3.4 Invertebrate Neurons
15.3.5 Primary Cultures of CNS Neurons
15.3.6 Endocrine Cells in Culture and Endocrine Cell Lines
15.3.7 Synaptosomes from Mammalian Brain
15.4 Practical Considerations
15.4.1 Sources for Clostridial Neurotoxins
15.4.2 Precautions
15.4.3 Preparation of Synaptosomes
15.4.4 Monitoring Release of Neurotransmitter
15.4.5 Poisoning of Synaptosomes
15.4.6 Subfractionation of Synaptosomes
15.5 Relevant ChemicaIs
CHAPTER 16 . Clostridial Toxins and Endocrine Secretion: Their Use in Insulin-Secreting Cells
16.1 Exocytosis of Insulin from. the Pancreatic ß-cell
16.1.1 Introduction
16.1.2 Regulated Exocytosis
16.1.3 The SNARE Hypothesis of Exocytosis
16.1.4 Application of the SNARE Hypothesis to Insulin Exocytosis
16.1.5 Role of Ca2+ and Rab3A in Insulin Exocytosis
16.2 Permeabilization of Insulin-secreting Cells
16.2.1 General Remarks
16.2.2. Material Required
16.2.3 Solutions
16.2.4 Final Working Buffers
16.2.5 Experimental Outline
16.2.5.1 General Procedure
16.2.5.2 Determination of SLO Concentrations
16.2.5.3 Cell Culture
16.2.6 Cell Permeabilization and Exocytosis Assay
16.2.7 Troubleshooting
16.2.7.1 Cell Detachment
16.2.7.2 Insufficient or No Response to Ca2+
16.3 Transient Cotransfection Assay for Exocytosis
16.3.1 General Remarks
16.3.2 Material Required
16.3.3 Solutions
16.3.4 Procedure
16.3.5 Troubleshooting
16.3.5.1 Insufficient Gene Expression
16.3.5.2 Considerable Variability in the Expression Levels between the same of Different Plasmids
16.4 Tetanus Toxin as a Tool for Studying the Role of VAMPs in Exocytosis
16.4.1 Introduction
16.4.2 General Considerations
16.4.3 Specific Considerations for the Study of Insulin-Secreting Cells
16.4.4 Practical Approach
16.4.4.1 Material Required
16.4.4.2 Solutions Required
16.4.4.3 Preparation of Neurotoxins
16.4.4.4 Detailed Procedure
16.4.5 Troubleshooting
16.4.5.1 TeTx Does not Inhibit Exocytosis
16.4.5.2 No Cleavage of VAMPs
16.5 The Study of Exocytosis The Use of Other Clostridial Neurotoxins for the Study of Exocytosis
16.6 Reagents and Chemicals
CHAPTER 17 . Pore-forming Toxins
17.1 Origin of Pore-forming Toxins (PFTs)
17.2 Evolution of the PFT Field
17.3 Relationship to Other Toxins
17.4 Molecular Mechanism of Action
17.4.1 Binding
17.4.2 Oligomerization
17.4.3 Pore Formation
17.5 Cell-biological Effects
17.5.1 Cell-biological Effects Resulting from Ion Fluxes
17.5.1.1 Reactions Governed by Flux of Monovalent Ions
17.5.1.2 Reactions Triggered by Ca2+ Influx
17.5.2 Reactions not Recognizably Governed by Flux of Ions through Toxin Pores
1 7.5.2.1 Short Circuiting of G - protein - dependent SignaIIing Pathways
17.5.2.2 Proteolytic Shedding of Membrane Proteins
17.6 Pathogenic Aspects
17.7 Purification Protocols
17.7.1 Purification of Staphylococcal Alpha-Toxin
17.7.2 Purification of Streptolysin 0
17.8 Application of PFTs as Tools in Research
17.9 Reagents and Chemicals
CHAPTER 18 . Application of Alpha-toxin and Streptolysin O as Tools in Cell Biological
18.1 Permeabilized Cells: an Approach to Study In tracellular Processes
18.2 Alpha-Toxin and Streptolysin O as Tools in the Study of Secretory Processes
18.2.1 Biological Activity
18.2.1.1 Assay to Compare Biological Activity of Various Pore-Forming Toxins Using Rabbit Erythrocytes
18.2.2 Assay for Exocytosis in Permeabilized Cells
18.3 Regulation of Vesicular Transmitter Transporters in Permeabilized Cells and Synaptosomes
18.3.1 Catecholamine Uptake into SLO-Permeabilized PC 12 Cells
18.3.2 Acidification of Small Synaptic Vesicles in SLO-Permeabilized Synaptosomes
18.4 Reagents and Chemicals
CHAPTER 19 . Toxins as Transporting Tools
19.1 Introduction
19.2 Diphtheria Toxin
19.2.1 Toxin-resistant Cell Mutants
19.2.2 Receptor for Diphtheria Toxin
19.2.3 Translocation of the Toxin
19.2.4 Further Requirements for Translocation
19.2.5 Formation of Cation Channels
19.3 Pseudomonas Exotoxin A
19.4 Non-cytocidal Toxins with lntracellular Sites of Action
19.5 Methods to Assess Translocation
19.6 Example of Translocated Peptides and Proteins
19.7 Limitations and Possibilities of the System
19.8 Methods
19.8.1 In vitro Transcription and Translation
19.8.2 SDS-PAGE
19.8.3 Cell Culture
19.8.4 Cell Binding and Translocation Assay
19.8.5 Expression in Bacteria and Purification of Recombinant Proteins
19.8.6 In vitro Farnesylation
19.8.7 Analysis of in vivo Farnesylation
19.8.8 Triton X-114 Partitioning of Farnesylated Proteins
CHAPTER 20 . A Brief Guide to the Safe Handling of Biological Toxins
20.1 Introduction
20.2 Preventive Measures
20.3 Containment
20.3.1 Containment Procedures
20.3.2 Containment Facilities
20.3.3 Containment within the Laboratory and beyond the Laboratory
20.4 Disposal
20.5 Decontamination
20.6 Emergency Intervention
20.7 Resources
20.8 Conclusion
Subject Index

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