Endotoxins: Structure, Function and Recognition (eBook)

Frontispiece 7
Preface 8
Contents 10
Contributors 12
About the Editors 15
Part I Structure and Properties of Endotoxins 16
1 Endotoxins: Lipopolysaccharides of Gram-Negative Bacteria 17
1.1 Introduction 18
1.2 Biosynthesize of LPS on the Surfaces of Inner Membrane 19
1.2.1 Beginning in the Cytoplasm to Form Kdo2-Lipid A 19
1.2.2 Connecting the Core Oligosaccharides to Lipid A 22
1.2.3 Synthesizing the O-antigen at the Cytoplasmic Surface of the Inner Membrane 23
1.2.4 Crossing the Inner Membrane 23
1.2.5 Assembling LPS at the Periplasmic Surface of the Inner Membrane 24
1.3 Export of LPS to the Surface of Bacteria 25
1.4 Structural Modification of LPS 26
1.4.1 Regulation of LPS Modification 26
1.4.2 Modifications in the Hydrophobic Region of LPS 29
1.4.3 Modifications in the Hydrophilic Region of LPS 30
1.5 Conclusion 32
References 32
2 Purification and Characterization of Lipopolysaccharides 40
2.1 Introduction 41
2.2 Extraction of Lipopolysaccharides 41
2.2.1 Large Scale Extraction of Lipopolysaccharides 41
2.2.1.1 Phenol--Water Extraction 42
2.2.1.2 Ether Extraction 43
2.2.1.3 EDTA Promoted Extraction 44
2.2.2 Micro-extraction of Lipopolysaccharides 45
2.2.2.1 Phenol--Water Micro-extraction 45
2.2.2.2 Phenol--Chloroform Micro-extraction 46
2.2.3 Extraction of Lipid A 46
2.2.3.1 Extraction of Lipid A from LPS 46
2.2.3.2 Extraction of Lipid A from Bacteria 47
2.2.3.3 Micro-extraction of Lipid A 47
2.2.3.4 Extraction of Free Lipid A 49
2.3 Purification of Lipopolysaccharides 49
2.3.1 Removal of Contaminants 50
2.3.2 Gel Filtration Chromatography of LPS 51
2.3.3 Ion-Exchange Chromatography of LPS 52
2.3.4 Capillary Electrophoresis of LPS 52
2.3.5 Purification of Lipid A 52
2.3.6 Micro-purification Method 54
2.4 Analysis of Lipopolysaccharides 54
2.4.1 Electrophoresis of LPS 55
2.4.2 Staining Methods 56
2.4.2.1 Silver Stain 56
2.4.2.2 Ethidium Bromide Stain 57
2.4.2.3 Zinc-Imidazole Stain 57
2.4.3 Immunoblotting Method 58
2.4.4 Analysis of Lipid A 58
2.5 Conclusions 58
References 59
3 Endotoxins: Relationship Between Structure, Function,INTbreak and Activity
3.1 Introduction 66
3.2 Physicochemical Characterisation of Endotoxins 66
3.3 Morphology and Size Distribution of Endotoxin Aggregates 69
3.4 Intramolecular Conformation 70
3.5 Biophysical Mechanisms of Agonism and Antagonism 72
3.6 Endotoxically Active Unit 74
References 76
4 The Diversity of the Core Oligosaccharide in Lipopolysaccharides 80
4.1 Introduction 81
4.2 Core Structures 86
4.2.1 Enterobacteria 86
4.2.2 Pasteurellaceae 90
4.2.3 Pseudomonas 92
4.2.4 Acinetobacter 94
4.2.5 Xanthomonas 95
4.2.6 Burkholderia 96
4.2.7 Ralstonia 98
4.2.8 Shewanella 98
4.2.9 Alteromonadaceae 99
4.2.10 Arenibacter Certesii 100
4.2.11 Psychromonas Arctica 101
4.2.12 Vibrionaceae 102
4.2.13 Rizhobiaceae 102
4.2.14 Loktanella Rosea 102
4.2.15 Halomonas Pantellerensis 104
4.3 Conclusion 105
References 105
5 Salmonella-Regulated Lipopolysaccharide Modifications 111
5.1 Introduction 112
5.1.1 Clinical Features and Relevance 112
5.1.2 Salmonella Pathogenesis 113
5.1.3 Host Defenses Against Salmonella Infection 115
5.1.4 Bacterial Modification of LPS and Other Surface Moieties 116
5.2 Two-Component Regulatory Systems 117
5.2.1 PhoP--PhoQ Regulatory System 119
5.2.2 PmrA--PmrB Regulatory System 120
5.2.3 RcsC--RcsD--RcsB Regulatory System 122
5.3 Additional Bacteria Capable of Modifying LPS 124
5.4 Summary and Significance 124
References 125
6 The Variation of O Antigens in Gram-Negative Bacteria 133
6.1 Introduction 134
6.2 O Serotyping Schemes 135
6.2.1 Conventional O Serotyping Methods 135
6.2.2 Molecular Typing Methods 136
6.2.2.1 PCR-RFLP 136
6.2.2.2 Gene-Specific PCR 137
6.2.2.3 DNA Microarray 137
6.3 O antigen Diversity in Gram-Negative Bacteria 138
6.3.1 Enterobacteriaceae 138
6.3.1.1 Escherichia and Shigella 138
6.3.1.2 Salmonella 139
6.3.1.3 Yersinia 139
6.3.1.4 Citrobacter 140
6.3.1.5 Klebsiella 140
6.3.1.6 Serratia 140
6.3.1.7 Hafnia 141
6.3.1.8 Proteus 141
6.3.2 Pseudomonadaceae 141
6.3.2.1 P. aeruginosa 142
6.3.2.2 P. syringae 142
6.3.3 Vibrionaceae 142
6.4 General Properties of O Antigen Gene Clusters 142
6.4.1 O Antigen Biosynthesis 143
6.4.2 Examples of O Antigen Gene Clusters 145
6.4.2.1 E. coli, S. enterica and Shigella spp 145
6.4.2.2 Enterobacter Sakazakii 146
6.4.2.3 P. aeruginosa 146
6.4.2.4 V. cholerae 146
6.4.2.5 Yersinia 147
6.4.2.6 Legionella Pneumophila 148
6.4.2.7 C. freundii 148
6.5 Formation and Distribution of O Antigen Gene Clusters 148
6.5.1 Lateral Transfer of O Antigen Genes by Homologous Recombination 149
6.5.2 Insertion and Deletion of O Antigen Genes Mediated by IS Elements 150
6.5.3 Role of Plasmids in Evolution of O Antigen gene Clusters 150
6.5.4 O Antigen Modification Mediated by Phages 151
6.6 O antigen and Virulence 153
6.6.1 The Complete Loss of O Antigen Leads to Severe Attenuation of Virulence 153
6.6.2 The O Antigen Differences Can Account for Differences in the Nature of Pathogenicity 153
6.6.3 The Effect of the Chemical Composition and Structure of an O Side Branch on Virulence 154
6.6.4 The O Antigen Chain Lengths is Important for the Full Virulence 154
6.7 Conclusions 155
References 155
7 Regulators of TLR4 Signaling by Endotoxins 163
7.1 Introduction 164
7.2 TLR4 Signaling Pathway 165
7.2.1 MyD88-Dependent Pathway 166
7.2.2 MyD88-Independent Pathway 168
7.3 Localisation of TLR4 and its Adaptor Molecules 168
7.4 Chaperones 169
7.5 Negative Regulators of TLR4 Signaling 170
7.5.1 Cell Surface Receptors 170
7.5.2 Splice Variants 171
7.5.3 Inhibitory Molecules 172
7.5.3.1 IRAK-M 172
7.5.3.2 TRAF1 and TRAF4 172
7.5.3.3 TANK 173
7.5.3.4 RIP3 173
7.5.3.5 A20 173
7.5.3.6 SOCS-1 174
7.5.3.7 Rab7b 175
7.6 Conclusion 175
References 175
8 Membrane Partitioning: Is Location Everything When It Comes to Endotoxin Recognition? 182
8.1 Introduction 183
8.2 The Innate Immune System 183
8.2.1 The Toll Like Receptor Family 184
8.2.2 Innate Immune Recognition of Bacterial Endotoxin or Lipopolysaccharide 185
8.2.3 Protein--Protein Interactions in Innate Immunity: PRRs Are Part of Multi-component Sensor Apparatuses 186
8.2.4 Is TLR4 Recruited in Membrane Microdomains Upon Ligand Engagement? 187
8.2.5 Does Membrane-Partitioning Play a Major Role in Protein Uptake and Intracellular Routing? 188
8.2.6 Concluding Remarks 189
References 190
Part II Infection, Treatment and Immunity 194
9 Endotoxin Detection from Limulus Amebocyte Lysate to Recombinant Factor C 195
9.1 General Introduction 196
9.2 Gram-Negative Bacterial Membrane A Wall of Fire 197
9.3 Lipopolysaccharide: A Mediator of Septic Shock Pathophysiological Properties 197
9.4 The Structure of LPS 198
9.4.1 The O-Specific Chain 198
9.4.2 The Core Oligosaccharide Domain 199
9.4.3 The Lipid A 200
9.5 Plasma LPS-Binding Proteins Protect and Provoke Septic Shock The Achilles Heel? 201
9.6 Overcoming the LPS Problem The Horseshoe Crab, a Creature Small and Great 204
9.7 Drawbacks with LAL 206
9.7.1 Differential Endotoxin Reactivities and Lack of Specificity 206
9.7.2 Problems with Sample and Specimen Preparations 207
9.7.3 LAL Production Endangers the Horseshoe Crab 207
9.8 Factor C: A Horseshoe Crab Serine Protease with Multiple High Affinity LPS-Binding Sites LPS Detection and Prevention Strategies Towards Non-LAL Based LPS Detection 208
9.9 Genetic Engineering and Production of Recombinant Factor C (rFC) Necessity Spawns Innovation: Cloning and Subcloning the Factor C cDNA into Bacterial, Yeast, Insect and Mammalian Cells 208
9.10 Development of a Quantitative Endotoxin Assay 209
9.11 Commercialization of the Endotoxin Detection Kit The Route to PyroGene and Pyrosense 210
References 211
10 The Role of Endotoxin in Infection: Helicobacter pylori and Campylobacter jejuni 217
10.1 Introduction 218
10.1.1 Helicobacter pylori and Campylobacter jejuni Infections 218
10.1.2 Nature of H. pylori and C. jejuni Endotoxins 219
10.2 Structure and Properties of H. pylori and C. jejuni Lipid A Moieties 221
10.2.1 Structural Analysis of H. pylori Lipid A 221
10.2.2 Structural Analysis of C. jejuni Lipid A 223
10.2.3 Molecular and Supramolecular Basis for the Contrasting Immunological Activities of C. jejuni and H. pylori Lipid A Moieties 224
10.3 Relevance of Low Endotoxin and Lipid A Immuno-Activities to Chronic Infection: H. pylori as a Comparative Model 225
10.4 Molecular Mimicry in H. pylori Endotoxin 227
10.4.1 H. pylori Expression of Lewis (Le) and Blood Group Antigen Mimicry 227
10.4.2 Anti-Le Antibodies in Autoimmune Pathogenesis 229
10.4.2.1 Anti-Le Antibodies and the Inflammatory Response 229
10.4.2.2 Anti-Le Antibodies in Gastric Atrophy 230
10.5 Molecular Mimicry in C. jejuni Endotoxin 232
10.5.1 Expression of Gangliosiode Mimicry by C. jejuni 232
10.5.2 Pathogenic Anti-Ganglioside Antibodies and C. jejuni Ganglioside Mimicry 235
10.5.2.1 Pathogenic Anti-Ganglioside Antibodies in GBS 235
10.5.2.2 T-Cells in GBS Development 236
10.5.2.3 C. jejuni Ganglioside Mimicry and Anti-Ganglioside Antibodies 236
10.5.3 Relevance of Molecular Mimicry in the Pathogenesis of GBS 237
10.5.3.1 Galway Postulates 237
10.5.3.2 Experimental Models 238
10.6 Conclusions and Future Perspective 239
References 240
11 The Role of Pseudomonas Lipopolysaccharide in Cystic Fibrosis Airway Infection 249
11.1 Introduction 250
11.2 Cystic Fibrosis is an Important Disease of Children and Young Adults 250
11.2.1 Persistent Inflammation and Chronic Infection are the Hallmarks of CF Pulmonary Disease 251
11.2.2 P. aeruginosa Is an Important Pathogen in the CF Airway 252
11.2.3 Adaptation of P. aeruginosa to the Airway Is Important for CF Lung Disease 252
11.2.4 P. aeruginosa Lipid A Structures in CF Clinical Isolates are Distinct from Those Seen in Acute Clinical Infections and Isolates from the Environment 252
11.2.5 Synthesis of Cystic Fibrosis-Specific Lipid A Modifications in P. aeruginosa 255
11.2.6 P. aeruginosa Lipid A Modifications Promote CAP Resistance 256
11.2.7 P. aeruginosa Lipid A Modifications Modulate Host Inflammatory Responses 258
11.3 Conclusions 259
References 259
12 Development of Small-Molecule Endotoxin Sequestering Agents 262
12.1 Introduction 263
12.2 Endotoxin, the Trigger in Gram-Negative Sepsis 264
12.2.1 Host Responses to Endotoxin 264
12.2.2 Complexation of LPS by Macromolecules: A Failed Therapeutic Strategy? 267
12.2.3 The Paradigm of Non-Immunologic Sequestration of LPS by Small Molecules: A More Accessible Strategy? 267
12.3 Lipopolyamines as Endotoxin Sequestrants 269
12.3.1 Further SAR Lessons Learned En Route to DS-96, an N-Alkylhomospermine Lipopolyamine 275
12.3.2 Activity of DS-96 in Human Blood 279
12.3.3 In Vivo Potency, Pharmacodynamics and Pharmacokinetics of DS-96 279
12.3.4 Toxicity of DS-96 279
12.3.5 Pharmacokinetics of DS-96 and Further Developments 283
12.4 Conclusions and Future Prospects 284
References 285
13 Development of an Anti-Endotoxin Vaccine for Sepsis 291
13.1 Introduction 292
13.2 Endotoxin as a Therapeutic Target 292
13.3 Endotoxin Structure and Immunity 293
13.4 Initial Studies of Anti-LPS Core Antibodies as Therapy for Gram-Negative Bacterial Infections 295
13.4.1 Anti-Core LPS Antibody Levels 297
13.4.2 Anti-Lipid A Antibodies 297
13.5 Anti Core Monoclonal Antibodies 298
13.6 Development of J5 Subunit Vaccine 298
13.6.1 Initial Studies with Unadjuvanted Vaccine 299
13.6.2 J5 dLPS/OMP Vaccine and Adjuvants 301
13.7 Other Anti-Endotoxin Vaccines 301
13.8 Immunization Strategies with Anti-Endotoxin Vaccines 303
13.9 Other Potential Applications of Anti-Endotoxin Vaccine 303
References 304
14 Synthetic and Natural TLR4 Agonists as Safe and Effective Vaccine Adjuvants 309
14.1 Introduction 310
14.2 Immune Recognition of Lipopolysaccharide (LPS) and Related Molecules 314
14.3 Lipid A Structure and Activity 315
14.4 Formulation Effects on TLR4 Agonist Activity 317
14.5 Conclusion 322
References 323
15 Targeting Endotoxin in the Treatment of Sepsis 328
15.1 Introduction 329
15.2 Endotoxin and the Initiation of the Sepsis Cascade 329
15.3 Rationale for Targeting Endotoxin in Sepsis 330
15.4 Potential for Targeting Endotoxin in Sepsis 331
15.5 Anti-Endotoxin Antibodies 331
15.6 Vaccines 333
15.7 Endotoxin-Binding Peptides 333
15.7.1 Bactericidal/Permeability Increasing Protein 333
15.7.2 Human Lactoferrin 334
15.8 Lipid A Analog 334
15.9 Phospholipid Emulsion 335
15.10 Hemoperfusion Through a Polymixin B Embedded Cartridge 335
15.10.1 Animal Studies 336
15.10.2 Human Studies 337
15.11 Conclusion 338
References 338
16 Lipopolysaccharides in Rhizobium-Legume Symbioses 344
16.1 Introduction 345
16.2 Rhizobial Lipopolysaccharide Structures 347
16.2.1 Lipid A (LA) Structures 348
16.2.2 Core Oligosaccharide (COS) Structures 350
16.2.3 O-Chain Polysaccharide (OPS) Structures 354
16.3 Rhizobial Lipopolysaccharide Biosynthesis 359
16.3.1 LA Biosynthesis 359
16.3.2 Core Biosynthesis 364
16.3.3 O-Chain Polysaccharide Synthesis 366
16.4 Structural Modifications to Rhizobial LPSs During Symbiosis 371
16.4.1 Modifications to the O-Chain Polysaccharide During Symbiosis 372
16.4.2 Modifications to the Core Oligosaccharide During Symbiosis 376
16.4.3 Modifications to the LA During Symbiosis 376
16.5 Rhizobial Lipopolysaccharide Function 377
16.5.1 The Symbiotic Function of the Unique Structural Features and Modifications of Rhizobial Lipopolysaccharides 377
16.5.2 Rhizobial Lipopolysaccharide, Symbiosis, and the Plant Defense Response 380
References 382
17 Lipopolysaccharides and Plant Innate Immunity 392
17.1 Introduction 393
17.2 LPS as a MAMP 394
17.2.1 LPS as a Direct Inducer of Basal Plant Defenses 394
17.2.2 LPS as a Primer of Plant Defense Response Induction 395
17.2.3 LPS can Modulate the Hypersensitive Response, a Programmed Cell Death Associated with Resistance 395
17.2.4 LPS Induces Systemic Effects in Plants 396
17.3 Different Sub-Structures Within LPS can Act as MAMPs 397
17.4 Structural Variations in LPS Influence its Activity in Plants 398
17.5 Subversion of LPS-Induced Effects 399
17.6 Perception of LPS by Plants 399
17.6.1 SNARE Proteins, Vesicle Trafficking and Plant Defense 400
17.6.2 A Role for the Syntaxin PEN1 in LPS Signalling in Plants? 401
17.7 Concluding Remarks 403
References 404
Index 409