ISBN: 3-540-66125-5
TITLE: Bacterial Protein Toxins
AUTHOR: Aktories, K.; Just, I. (Eds.)
TOC:

CHAPTER 1
Uptake of Protein Toxins Acting Inside Cells
S. Olsnes, J. Wesche, and P.. Falnes. With 3 Figures 1
A. Introduction and Brief Description of Relevant Toxins 1
B. Binding to Cell-Surface Receptors 4
C. Endocytosis 5
D. Retrograde Vesicular Transport 7
I. Transport to the Golgi Apparatus 7
II. Transport to the Endoplasmic Reticulum 7
E. Translocation to the Cytosol 8
I. From the Surface 8
II. From Endosomes 9
III. From the ER 9
F. Stability of Toxins in the Cytosol 11
G. Translocation of Fusion Proteins 12
References 14
CHAPTER 2
Common Features of ADP-Ribosyltransferases
V. Masignani, M. Pizza, and R. Rappuoli. With 5 Figures 21
A. Introduction 21
B. The Well-Characterized Toxins 21
C. A Common Structure for the Catalytic Site 26
I. Region 1 27
II. Region 3 29
III. Region 2 30
D. Other Bacterial Toxins with ADPRibosylating Activity 33
E. Eukaryotic Mono-ADPRibosyltransferases 35
F. Practical Applications 37
References 39
CHAPTER 3
Diphtheria Toxin and the Diphtheria-Toxin Receptor
T. Umata, K.D. Sharma, and E. Mekada. With 4 Figures 45
A. Introduction 45
B. Diphtheria Toxin 45
I. Synthesis of Diphtheria Toxin 45
II. Toxicity of Diphtheria Toxin 46
III. Structure and Function of Diphtheria Toxin 48
1. The Catalytic Domain 48
2. The T Domain 48
3. The R Domain 49
IV. Sensitivity to Diphtheria Toxin 50
C. The Diphtheria-Toxin Receptor 51
I. Identification of the Diphtheria-Toxin-Receptor Protein 51
II. Cloning of the Diphtheria-Toxin-Receptor Gene 52
III. The Structure and Function of the Diphtheria-Toxin Receptor 53
IV. Molecules Associated with the Diphtheria-Toxin Receptor 55
1. DRAP27/CD9 55
2. Heparin-Like Molecules 57
V. Receptor and Toxin Entry Process 58
VI. Physiological Role of the Diphtheria-Toxin Receptor 59
1. EGF-Family Growth Factor 59
2. Juxtacrine Growth Regulator 59
3. Conversion of the Membrane-anchored Form to the Soluble Form 60
References 61
CHAPTER 4
Pseudomonas aeruginosa Exotoxin A: Structure/Function, Production, and Intoxication of Eukaryotic Cells
S.E.H. West. With 3 Figures 67
A. Introduction 67
I. Basic Structure 68
II. Role of ETA in Disease 70
B. Production of ETA by the Bacterial Cell 71
I. Characterization of the toxA Structural Gene 71
II. Environmental and Temporal Signals Affecting ETA Production 71
III. Regulation of ETA Production 72
IV. Secretion from the Bacterial Cell 75
C. Intoxication of Eukaryotic Cells 76
I. Binding to a Specific Receptor on the Eukaryotic Cell Surface and Internalization by Receptor-Mediated Endocytosis 77
II. Activation by Proteolytic Cleavage and/or a Conformational Change 79
III. Removal of a Terminal Lysine Residue and Translocation into the Cytosol 80
IV. ADPRibosylation of Elongation Factor 2 80
References 82
CHAPTER 5
Diphtheria-Toxin-Based Fusion-Protein Toxins Targeted to the Interleukin-2 Receptor: Unique Probes for Cell Biology and a New Therapeutic Agent for the Treatment of Lymphoma
J.R. Murphy and J.C. Vanderspek 91
A. Introduction 91
B. Diphtheria-Toxin-Based Cytokine Fusion Proteins 91
C. DAB389IL-2 as a Novel Biological Probe for Cell Biology 95
D. Pre-Clinical Characterization of DAB486IL-2 and DAB389IL-2 97
E. Clinical Evaluation of DAB486IL-2 and DAB389IL-2 98
I. Rheumatoid Arthritis 99
II. Psoriasis 100
III. Non-Hodgkin's Lymphoma 101
IV. Cutaneous T-Cell Lymphoma 102
References 104
CHAPTER 6
Structure and Function of Cholera Toxin and Related Enterotoxins
F. van den Akker, E. Merritt, and W.G.J. Hol. With 4 Figures 109
A. Introduction 109
B. Three-Dimensional Structures of Holotoxins 110
C. Toxin Assembly and Secretion 112
I. Design of Assembly Antagonists 113
D. Cell-Surface-Receptor Recognition 113
I. Design of Receptor Antagonists 115
E. Toxin Internalization 115
F. Enzymatic Mechanism 117
I. Substrates, Artificial Substrates, and Inhibitors 117
II. NAD-Binding Site 119
G. The LT-II Family 120
H. Perspectives 125
References 125
CHAPTER 7
Mechanism of Cholera Toxin Action: ADP-Ribosylation Factors as Stimulators of Cholera Toxin-Catalyzed ADP-Ribosylation and Effectors in Intracellular Vesicular Trafficking Events
W.A. Patton, N. Vitale, J. Moss, and M. Vaughan. With 4 Figures 133
A. Introduction 133
B. Cholera Toxin 135
I. Structure 135
II. Biochemistry 135
III. Toxin Internalization 136
C. ADP-Ribosylation Factors 137
I. Discovery of ARFs 137
II. Biochemical Characterization of ARFs 138
III. ARF Structure 139
1. The Primary Structures of ARFs 139
2. The Tertiary Structures of ARFs 140
IV. Other ARF Family Members 141
1. ARF-Related Proteins 141
2. ARF-Domain Protein 1 143
V. Molecules that Regulate ARF Function: GEPs and GAPs 145
1. ARF Guanine Nucleotide-Exchange Proteins 145
2. ARF GTPase-Activating Proteins 147
VI. Other ARF-Interacting Molecules 149
1. Phospholipase D 149
2. Arfaptins 150
VII. ARF in Cells 150
1. ARFs' Role in Vesicular Trafficking Events 150
2. Subcellular Localization of ARF 151
D. Summary 152
References 152
CHAPTER 8
Pertussis Toxin: StructureFunction Relationship
C. Locht, R. Antoine, A. Veithen, and D. Raze. With 5 Figures 167
A. Introduction 167
B. The Receptor-Binding Activity of PTX 168
C. Membrane Translocation of PTX 170
D. The Enzymatic Activity of S1 175
E. The Enzyme Mechanism of S1-Catalyzed ADPRibosylation 176
F. The Catalytic Residues of PTX 178
G. Substrate Binding by PTX 180
H. Conclusions 182
References 182
CHAPTER 9
Pertussis Toxin as a Pharmacological Tool
B. Nrnberg. With 3 Figures 187
A. Introduction 187
B. Molecular Aspects of PT Activity on G Proteins 189
I. General Considerations 189
II. PT-Sensitive G Proteins 190
1. Mechanism of PT Action 190
2. G-Protein Specificity 191
III. PT as a Tool with which to Study G-Protein-Subunit Composition 193
C. Functional Consequences of PT Activity 195
I. PT-Affecting ReceptorG-ProteinEffector Coupling 195
II. PT-Affecting Receptor-Independent Activation of G Proteins 197
III. Use of PT in Studying Cellular Signal Transduction 198
Appendix: Experimental Protocols for Using PT 199
I. Source of PT and Preparation of Solutions 199
II. Treatment of Mammalian Cell Cultures with PT 199
III. Activation of PT for in Vitro ADPRibosylation 199
IV. ADPRibosylation of Cell-Membrane Proteins by PT 200
V. ADPRibosylation of Isolated Proteins by PT 200
VI. Preparation of Samples for Sodium Dodecyl Sulfate Polyacrylamide-Gel Electrophoresis 200
VII. Cleavage of ADPRibose from Ga Subunits 201
References 201
CHAPTER 10
Clostridium Botulinum C3 Exoenzyme and C3-Like Transferases
K. Aktories, H. Barth, and I. Just. With 7 Figures 207
A. Introduction 207
B. Origin and Purification of C3 Exoenzymes 207
I. Origin of C3 Exoenzymes 207
II. Purification of C3 Exoenzymes 208
C. Genetics of C3 and C3-Like Exoenzymes 209
D. StructureFunction Analysis of C3 Exoenzymes 210
E. ADPRibosyltransferase Activity 212
I. Basic Properties 212
II. Regulation by Detergents and Divalent Cations 212
III. Rho Proteins as Substrates for C3 213
IV. Functional Consequences of the ADPRibosylation of Rho 215
F. Application of C3-Like Exoenzymes as Tools 218
G. Cellular Effects of C3 Exoenzymes 219
I. Effects of C3 on Cell Morphology and Actin Structure 219
II. Effects of C3 on CellCell Contacts 222
III. Effects of C3 on Endocytosis and Phagocytosis 223
IV. Effects of C3 on Cell Signalling not Directly Involving the Actin Cytoskeleton 223
1. Phospholipase D and PIP5 Kinase 223
2. Signalling to the Nucleus and Gene Transcription 224
H. Concluding Remarks 224
References 225
CHAPTER 11
Pseudomonas aeruginosa Exoenzyme S, a Bifunctional Cytotoxin Secreted by a Type-III Pathway
J.T. Barbieri and D.W. Frank. With 6 Figures 235
A. Introduction 235
B. Initial Biochemical Characterization of ExoS 236
C. Genetic Analysis of the Structural Genes Encoding ExoS 236
D. ExoS Requires FAS to Express ADPRibosyltransferase Activity 237
E. Molecular Properties of ExoS 237
F. Secretion of ExoS via a Type-III Secretion Pathway 239
G. Regulation of exoS Regulon Expression 240
H. The Carboxyl Terminus of ExoS Comprises the ADPRibosyltransferase Domain 240
I. Functional Mapping of ExoS 240
II. ExoS is a Biglutamic-Acid Transferase 240
III. ExoS can ADPRibosylate Numerous Proteins 242
IV. ExoS ADPRibosylates Ras at Multiple Arginine Residues 243
I. ExoS is a Bifunctional Cytotoxin 244
I. Cytotoxic Properties of ExoS 245
II. The Amino Terminus of ExoS Stimulates Rho-Dependent Depolymerization of Actin 245
III. The Carboxyl Terminus of ExoS is an ADPRibosyltransferase that is Cytotoxic to Cultured Cells 246
J. Mechanism for the Inhibition of Ras-Mediated Signal Transduction by ExoS 246
K. Functional and Sequence Relationship Between ExoS and the Vertebrate ADPRibosyltransferases 247
L. Conclusion 248
References 248
CHAPTER 12
Structure and Function of ActinAdenosine-Diphosphate-Ribosylating Toxins
I. Ohishi. With 2 Figures 253
A. Introduction 253
B. Clostridium Botulinum C_2 Toxin 253
I. ActinADP-Ribosylating Toxin of C. Botulinum Types C and D 253
II. Molecular Structure of Botulinum C_2 Toxin 254
III. Molecular Functions of Two Components of C_2 Toxin 256
IV. ADP-Ribosylation of Actin by C_2 Toxin 257
1. ADP-Ribosylation of Intracellular Actin of Cultured Cells 258
2. ADP-Ribosylation of Purified Actin 260
C. C. Perfringens Iota Toxin 262
I. ActinADP-Ribosylating Toxin of C. Perfringens Type E 262
II. Molecular Structure and Function 263
D. C. Spiroforme Toxin 265
I. ActinADP-Ribosylating Toxin of C. Spiroforme 265
II. Molecular Structure and Function 265
E. C. Difficile Toxin 266
I. ActinADP-Ribosylating Toxin of C. Difficile 266
II. Molecular Structure and Function 266
F. Concluding Remarks 268
References 269
CHAPTER 13
Molecular Biology of Actin-ADPRibosylating Toxins
M.R. Popoff. With 9 Figures 275
A. Introduction 275
B. Bacteria Producing ActinADP-Ribosylating Toxins 276
I. C. Botulinum 276
II. C. Perfringens 276
III. C. Spiroforme 277
IV. C. Difficile 277
C. Families of Actin-ADPRibosylating Toxins 277
I. C2-Toxin Family 278
II. Iota-Toxin Family 279
III. Relatedness Between C2-Toxin and Iota-Toxin Families 279
D. Actin-ADPRibosylating-Toxin Genes and Predicted Molecules 282
I. Iota-Toxin Genes and Iota-Toxin Proteins 282
II. C. Spiroforme Toxin and CDT Genes 283
III. C. Spiroforme Toxin and CDT Proteins 283
IV. C2-Toxin Genes and C2 Proteins 284
E. Relatedness of Actin-ADPRibosylating Toxins with Other Toxins 284
I. Relatedness with ADPRibosylating Toxins 284
II. Relatedness with Bacillus anthracis Toxins 288
1. Sequence Homology 288
2. Immunological Relatedness 289
3. Functional Comparison 289
III. Relatedness with Other Binary Toxins 290
1. Bacillus Binary Toxins 290
2. Leukocidins and gamma-Lysins 291
F. Genetics of the Actin-ADPRibosylating Toxins 292
I. Genomic Localization 292
II. Gene Transfer 293
G. Gene Expression 294
I. Genes of Enzymatic and Binding-Component Genes are Organized in an Operon 294
II. Gene Regulation 295
H. Identification of Actin-ADPRibosylating-Toxin-Producing Clostridia by Genetic Methods 297
I. Functional Domains 297
I. Enzymatic-Component Domains 297
1. Enzymatic Site 297
2. Enzymatic-Component Domain which Interacts with the Binding Component 299
3. Actin-Binding Site 300
II. Binding-Component Domains 300
K. Concluding Remarks 302
References 302
CHAPTER 14
Molecular Mechanisms of Action of the Large Clostridial Cytotoxins
I. Just, F. Hofmann, and K. Aktories. With 6 Figures 307
A. Introduction 307
B. Structure of the Toxins 308
C. Cell Entry 309
D. Molecular Mode of Action 315
I. Elucidation of the Molecular Mechanism of Action 315
II. Enzymatic Activity 316
1. Co-Substrates 316
2. Catalytic Domain and Requirements for Catalysis 318
3. Recognition of the Protein Substrates 320
III. Cellular Targets of the Cytotoxins 321
1. Rho and Ras Proteins as Substrates 321
2. Site of Modification 321
3. Cellular Functions of Rho Proteins 322
IV. Functional Consequences of Glucosylation 324
1. Consequences on the GTPase Cycle 324
2. Biological Consequences 325
E. Concluding Remarks 327
References 327
CHAPTER 15
Molecular Biology of Large Clostridial Toxins
J.S. Moncrief, D.M. Lyerly, and T.D. Wilkins. With 2 Figures 333
A. Introduction 333
B. Purification and Characterization of Large Clostridial Toxins 335
I. Toxin Production 335
II. Purification and Physicochemical Properties 336
1. C. Difficile Toxins 336
2. C. Sordellii and C. Novyi Toxins 337
III. Biological Properties 337
1. C. Difficile Toxins 337
2. C. Sordellii and C. Novyi Toxins 339
3. Receptors 340
C. Mechanism of Action 341
D. Molecular Genetics of the Toxins 342
I. C. Difficile Toxin A and B Genes 342
II. C. Difficile Toxigenic Element 344
III. Atypical Strains of C. Difficile 344
IV. C. Sordellii and C. Novyi Genes 345
V. Sequence Identity and Conserved Features of the Toxins 346
1. N-Terminal Glucosyltransferase Domain 346
2. Repeating Units 347
3. Additional Conserved Features 348
VI. Gene Transfer in C. Difficile 349
E. Regulation of C. Difficile Toxins 349
F. Conclusions 351
References 351
CHAPTER 16
The Cytotoxic Necrotizing Factor 1 from Escherichia Coli
P. Boquet and C. Fiorentini. With 5 Figures 361
A. Introduction 361
B. The CNF1 Gene and the Prevalence of CNF1-Producing Strains among Uropathogenic E. coli 362
I. The CNF1 Gene 362
II. Prevalence of CNF1-Producing Strains among Uropathogenic E. coli 363
C. Production, Purification and Cellular Effects of E. coli CNF1 364
I. Production and Purification of E. coli CNF1 364
II. Cellular Effects of E. coli CNF1 365
D. CNF1 Molecular Mechanism of Action 365
I. Intracellular Enzymatic Activity of CNF1 365
II. Consequences of CNF1 Activity on Rho GTP-Binding Proteins 368
E. StructureFunction Relationships of CNF1 and the Family of Dermonecrotic Toxins 373
I. The C-Terminal Part of CNF1 Contains its Enzymatic Activity 373
II. The N-Terminal Part of CNF1 Contains its Cell-Binding Activity 375
F. Possible Roles for CNF1 as a Virulence Factor 375
I. CNF1 and Induction of Phagocytosis 375
II. CNF1 and Cell Apoptosis 377
III. CNF1: Epithelial Cell Permeability and PMN Trans-Epithelial Migration 378
G. Conclusions 379
References 379
CHAPTER 17
Shiga Toxins of Shigella dysenteriae and Escherichia coli
A.R. Melton-Celsa and A.D. O'Brien. With 1 Figure 385
A. Profile of the Shiga-Toxin Family 385
I. Nomenclature and History 385
II. The Stx Family 386
1. Traits that Make the Members Part of a Family 386
2. Characteristics that Distinguish Stx Family Members 387
III. Role of Stxs in S. Dysenteriae Type 1 and STEC Disease 388
1. Pathogenesis of Infection Caused by Organisms that Produce Stxs 388
2. Associations Between Stxs and Development of HC and/or the HUS 388
3. Findings with Animal and Tissue Culture Models that Support a Primary Role for Stxs in Virulence of Shiga's Bacillus and STEC 389
B. Stx Genetics and Expression 389
I. Location, Organization, and Nucleotide and Deduced Amino Acid Sequences of Stx Family Member Operons 389
II. Regulation of Toxin Production 391
III. Toxin Purification 391
C. StructureFunction Analyses of Stx Family Members 392
I. Structure of Stx 392
II. Genetic Analyses of Stx Function 393
D. Intracellular Trafficking of the Shiga Toxins 396
E. Virulence/Toxicity Differences among the Stxs 397
F. Immune Response to Stxs, Passive Anti-Stx Therapy, and Vaccine Development 397
I. Anti-Toxin Responses during STEC Infection 397
II. Passive Therapy with Anti-Stx Antibodies 398
III. Vaccine Development 398
G. Summary 399
References 399
CHAPTER 18
Clostridial Neurotoxins
H. Bigalke and L.F. Shoer. With 8 Figures 407
A. Introduction 407
B. Tetanus and Botulism in Man and Animals 409
I. Modes of Poisoning 409
II. Clinical Manifestations 410
III. Pathophysiology 410
C. Structure of Clostridial Neurotoxins 412
I. Genetic Determination 412
II. Structure of Proteins 413
D. Toxicokinetics of Clostridial Neurotoxins 417
I. Receptor Binding and Internalization 417
II. Translocation from Endosomes into the Cytosol and Priming 419
III. Sorting, Routing and Axonal Transport 421
E. Toxicodynamics of Clostridial Neurotoxins 422
I. Mode of Action of Clostridial Neuroproteases 422
II. Function of Substrates 425
F. Clostridial Neurotoxins serve as Tools in Cell Biology and as Therapeutic Agents 427
References 431
CHAPTER 19
Anthrax Toxin
S.H. Leppla. With 1 Figure 445
A. Introduction 445
B. Toxin Genes 446
I. Gene Location and Organization 446
II. DNA Sequences and Transcriptional Regulation 446
C. Toxin-Component Proteins 447
I. Toxin Production, Purification, and Properties 447
II. PA Structure and Function 448
III. LF Structure and Function 452
IV. EF Structure and Function 454
V. PA Family Members 455
D. Cellular Uptake and Internalization 456
I. Cellular Receptor for PA 456
II. Proteolytic Activation of PA 456
III. LF and EF Binding to PA63 457
IV. Endocytic Uptake 458
V. Channel Formation 459
VI. Translocation and Cytosolic Trafficking 461
E. Intracellular Actions 462
I. EF Adenylate Cyclase 462
II. LF Metalloprotease 463
F. Therapeutic Applications of LF Fusion Proteins 464
G. Summary and Future Prospects 465
References 465
CHAPTER 20
Adenylyl-Cyclase Toxin from Bordetella pertussis
E.L. Hewlett and M.C. Gray. With 1 Figure 473
A. Introduction and Background 473
B. Gene and Protein Structure 474
C. Biological Activities of AC Toxin 475
I. Enzymatic Activity 475
II. Cell-Invasive Activity 476
III. Pore Formation and Hemolysis 477
IV. Summary 479
D. Possible Role/s for AC Toxin in Pathogenesis 480
E. AC toxin as a Protective Antigen 481
F. Uses of AC Toxin as a Novel Research Reagent 482
G. Future Directions 482
References 483
CHAPTER 21
Helicobacter Pylori Vacuolating Cytotoxin
W. Fischer and R. Haas. With 4 Figures 489
A. Introduction 489
B. Identification and Purification of the H. pylori Vacuolating Cytotoxin 490
C. Gene Structure and Mechanism of Secretion 491
I. Cloning and Molecular Characterisation of vacA Encoding the Vacuolating Cytotoxin 491
II. Autotransporter Organisation of the VacA Precursor 493
III. Mosaic Gene Structure of vacA Alleles in the H. pylori Population 493
IV. Consequences of the vacA Mosaic Gene Structure 494
V. Presence of vacA Homologues in the H. pylori Genome 494
D. Regulation of vacA Gene Expression 495
E. Extracellular Structure and Activation of the Vacuolating Cytotoxin 496
I. Processing and Quaternary Structure 496
II. Activation by Acid 496
F. Effects of VacA on Eucaryotic Cells 497
I. Binding to Target Cells and Mechanism of Uptake 497
II. Vacuole Formation 499
III. Other Effects of VacA 501
G. Clinical Relevance of the Vacuolating Cytotoxin 502
H. VacA as a Vaccine Candidate 503
I. Concluding Remarks 503
References 504
CHAPTER 22
Staphylococcal alpha Toxin
S. Bhakdi, I. Walev, M. Palmer, and A. Valeva. With 8 Figures 509
A. Occurrence and Biological Significance 509
B. Purification and Properties of Monomeric Toxin 509
C. Mechanism of Action 510
I. Binding 510
II. Oligomerization 511
III. Pore Formation 512
D. Structure of Oligomeric Pores 512
I. Structure of the Heptameric Pore Formed in Detergent Solution 512
II. Structure of the Membrane-Bound Oligomer 514
E. Biological Effects 517
I. Cytocidal Action 517
II. Secondary Cellular Reactions 517
1. Reactions Provoked by Transmembrane Flux of Monovalent Ions 517
2. Ca^{2+}-Dependent Reactions 518
3. Long-Range Effects of alpha Toxin 520
4. Synergism Between alpha Toxin and Other Toxins 520
F. Resistance and Repair Mechanism 520
G. Use of alpha Toxin in Cell Biology 522
H. Medical Relevance of alpha Toxin 523
References 524
CHAPTER 23
Bacterial Phospholipases
R.W. Titball and J.I. Rood. With 5 Figures 529
A. Introduction 529
B. Related Groups of Phospholipases 529
I. Zinc Metallophospholipase Cs 530
II. Gram-Negative PLCs 535
III. Phosphatidylinositol PLCs 535
IV. Phospholipase Ds 536
C. Functional and Biological Properties of Phospholipases 536
D. Modulation of Eukaryotic Cell Metabolism 538
I. Hydrolysis of Membrane Phospholipids 538
II. Hydrolysis of Membrane Phospholipids Modulates Cell Metabolism 541
E. Regulation 542
I. Regulation of the C. perfringens plc gene 542
II. Environmental Control of PLC Production in P. aeruginosa 542
III. Regulation of the Listeria Phospholipases by PrfA 543
F. Role in Disease 544
I. Gas Gangrene 544
II. P. aeruginosa Infections 546
III. The Pathogenesis of Listeriosis 547
IV. Caseous Lymphadenitis in Ruminants 548
G. Conclusions 548
References 549
CHAPTER 24
Pore-Forming Toxins as Cell-Biological and Pharmacological Tools
G. Ahnert-Hilger, I. Pahner, and M. Hltje. With 5 Figures 557
A. Permeabilized Cells: an Approach to the Study of Intracellular Processes 557
B. alpha-Toxin and SLO as Tools with which to Study Functional Aspects of Intracellular Organelles 559
I. Biological Activity and Cell Permeability 559
1. Protocol 1: Permeabilization of Attached Cells by alpha-Toxin or SLO 560
a) Alternate Protocol 1 560
2. Protocol 2: Permeabilization of Cells in Suspension 560
a) Commentary for Protocols 1 and 2 561
3. Protocol 3: Assay to Compare Biological Activity of Various Pore-Forming Toxins Using Rabbit Erythrocytes 561
a) Commentary for Protocol 3 562
4. Protocol 4: Trypan-Blue Exclusion Test 562
a) Commentary for Protocol 4 563
II. Introduction of Membrane Impermeable Proteins 563
1. Protocol 5: Introduction of Membrane-Impermeable Proteins. Immunofluorescence for Synaptophysin 565
a) Commentary for Protocol 5 565
2. Protocol 6: Introduction of Membrane-Impermeable Proteins. TeNT/LC 566
a) Commentary for Protocol 6 567
III. Assay for Exocytosis in Permeabilized Cells 567
1. Protocol 7: Measuring Exocytosis in Permeabilized Suspension Cells 568
2. Protocol 8: Measuring Exocytosis in Permeabilized Attached Cells 570
a) Commentary for Basic Protocols 7 and 8 570
IV. Regulation of Vesicular Transmitter Transporters in Permeabilized Cells 570
1. Protocol 9: Regulation of Vesicular Transmitter Transporters in Permeabilized Cells 571
a) Commentary for Basic Protocol 9 572
C. Chemicals Used in the Protocols 572
D. Concluding Remarks 573
References 573
CHAPTER 25
Heat-Stable Enterotoxin of Escherichia Coli 
T. Hirayama and A. Wada. With 3 Figures 577
A. Introduction 577
B. Heat-Stable Enterotoxin STa 578
I. Structure and Biological Properties of STa 578
II. Receptor for STa 580
C. STa-like Heat-Stable Enterotoxin 585
D. Heat-Stable Enterotoxin STb 585
I. Structure of STb 585
II. Biological Function of STb 586
E. Concluding Remarks 588
References 588
CHAPTER 26
Superantigenic Toxins
B. Fleischer. With 1 Figure 595
A. Summary 595
B. Introduction 595
C. PETs of S. Aureus and S. Pyogenes 596
I. Genes and Molecules 596
II. Molecular Mechanism of Action 600
1. Binding to MHC class-II Molecules 600
2. Non-MHC Receptors 601
3. Interaction with the TCR 602
III. Biological Significance of PETs 604
1. Role of PETs as Virulence Factors 604
2. Role in Pathogenesis 604
3. Association with Human Autoimmune Disease 605
4. The Enterotoxic Activity 606
D. Other Superantigens (or Pseudosuperantigens) of Gram-Positive Cocci? 607
I. The ETs of S. Aureus 607
II. M Proteins and SPE B of S. Pyogenes 607
III. The Mitogenic Factor of S. Pyogenes 608
E. The M. Arthritidis Superantigen 610
F. The Y. Pseudotuberculosis Mitogen 610
G. Concluding Remarks 611
References 611
CHAPTER 27
Structure and Activity of Endotoxins
S. Hauschildt, W. Brabetz, A.B. Schromm, L. Hamann, P. Zabel, E.T. Rietschel, and S. Mller-Loennies. With 10 Figures 619
A. Introduction 619
B. The Chemical Structure of LPS 624
I. Structural Characteristics of the O-Specific Chain 627
II. Structural Characteristics of the LPS Core 627
III. Structural Characteristics of Lipid A 629
C. Biosynthesis of LPS 632
I. Biosynthesis of Lipid A 632
II. Biosynthesis of the Core Region 632
III. Biosynthesis of the O-Specific Chain 633
D. StructureActivity Relationships of LPS and Lipid A 634
E. Cellular and Humoral Responses to LPS in Mammals 636
F. Strategies for the Treatment of Gram-Negative Infections 644
I. Antibacterial Agents 645
II. Antagonists of Endotoxic Effects 647
III. Neutralizing Antibodies Against Endotoxin 649
G. Final Remarks 651
References 652
CHAPTER 28
Translocated Toxins and Modulins of Yersinia
M. Aepfelbacher, R. Zumbihl, K. Ruckdeschel, B. Rouot, and J. Heesemann. With 5 Figures 669
A. Introduction 669
B. Yersinia Protein Type-III Secretion/Translocation System 670
I. Virulence Plasmid pYV 670
II. Regulation of Yop Expression, Secretion and Translocation 671
C. Translocated Toxins and Modulins of Yersinia (Effector Yops) 674
I. YopH, a Highly Active Tyrosine Phosphatase 674
II. YopE, an Actin-Disrupting Cytotoxin 677
III. YopP, Modulator of Multiple Signal Pathways Leading to Apoptosis and Cytokine Suppression 678
IV. YopT, Another Actin-Disrupting Cytotoxin 681
V. YopM  So Far, no Evidence for an Intracellular Function 681
VI. YpkA, a Putative Serine/Threonine Kinase Affecting Cell Shape 682
D. Perspectives 683
References 685
Subject Index 691
END
