Cholera toxins

1 An Introduction to Cholera ........................................................................ 1 1.1 Nature of the Disease ............................................................................ 1 1.2 Epidemics and Pandemics .................................................................... 1 1.3 Causation of the Disease ...................................................................... 3 1.4 Treatment Basics .................................................................................. 3 2 Bacterial Toxins: A Brief Overview ........................................................... 5 2.1 Introduction .......................................................................................... 5 2.2 Different Types of Toxins ..................................................................... 5 2.3 Bacterial Endotoxins: A General Introduction ..................................... 6 2.4 Bacterial Exotoxins: A General Introduction ....................................... 8 2.4.1 Extracellularly Acting Toxins ................................................... 8 2.4.2 Intracellularly Acting Toxins .................................................... 9 2.4.3 Toxins Whose Mechanisms of Action Are Not Well Defined ............................................................................. 10 2.5 Toxins of Vibrio cholerae ..................................................................... 11 3 Vibrio cholerae, the Causative Organism ................................................... 13 3.1 Introduction .......................................................................................... 13 3.2 Classification and Identification ........................................................... 13 3.3 Subtyping .............................................................................................. 15 3.3.1 Serogroups ................................................................................ 15 3.3.2 Serotypes ................................................................................... 15 3.3.3 Biotypes .................................................................................... 16 3.3.4 Phage Types .............................................................................. 17 3.4 Molecular Subtyping ............................................................................ 17 3.4.1 Plasmid Profile Analysis ........................................................... 17 3.4.2 RFLP Analysis of ctx Genes ..................................................... 17 3.4.3 RFLP Analysis of the rRNA Genes .......................................... 18 3.4.4 DNA or Gene Probes ................................................................ 18 3.4.5 Multilocus Enzyme Electrophoresis (MEE) Types .................. 19 3.4.6 Pulsed Field Gel Electrophoresis (PFGE) ................................ 20 3.5 Growth Requirements and Characteristics ........................................... 20 x Contents 3.6 Ultrastructure of the Cell .................................................................. 21 3.6.1 Pili (Fimbriae) ...................................................................... 22 3.7 Cholera Bacteriophages .................................................................... 24 3.8 Genetics of V. cholerae: An Outline of Some Relevant Areas ......... 27 4 Endotoxin of Vibrio cholerae: Physical and Chemical Characterization ...................................................................................... 33 4.1 Introduction ...................................................................................... 33 4.2 LPS of V. cholerae: Site of Occurrence ............................................ 34 4.3 Extracellular LPS .............................................................................. 34 4.4 Chemical Composition of Lipid A ................................................... 37 4.5 Chemical Structure of Lipid A ......................................................... 38 4.6 3-Deoxy-D-Manno-Octolusonic Acid (Kdo) ................................... 39 4.7 Core-PS: Chemical Constituents ...................................................... 41 4.7.1 O1 Vibrios ............................................................................ 41 4.7.2 Non-O1 Vibrios .................................................................... 41 4.8 Core-PS: Chemical Structure ........................................................... 42 4.9 O-PS: Chemical Constituents ........................................................... 44 4.9.1 O1 Vibrios ............................................................................ 44 4.9.2 Non-O1 Vibrios .................................................................... 44 4.10 O-PS: Chemical Structure ................................................................ 46 4.11 The Capsular Polysaccharide (CPS) ................................................. 49 4.11.1 Colony Morphology and CPS............................................. 49 4.11.2 CPS: Site of Occurrence ..................................................... 50 4.11.3 CPS: Sugar Composition .................................................... 51 4.11.4 CPS: Chemical Structure .................................................... 51 4.12 Emerging Research Trends and Future Possibilities ........................ 53 5 Endotoxin of Vibrio cholerae: Genetics of Biosynthesis ......................... 55 5.1 Introduction ........................................................................................ 55 5.2 Lipid A ................................................................................................ 56 5.3 Core-PS ............................................................................................... 56 5.4 O-Antigen Polysaccharide (O-PS) of V. cholerae .............................. 60 5.4.1 V. cholerae O1 ......................................................................... 60 5.4.2 V. cholerae O139 ..................................................................... 64 5.4.3 V. cholerae of Serogroups Non-O1 Non-O139 ....................... 69 5.5 Progenitor of V. cholerae O139 .......................................................... 75 5.6 Genesis of O139 O-PS Gene Cluster .................................................. 76 5.7 Concluding Remarks .......................................................................... 77 6 Endotoxin of Vibrio cholerae: Biological Functions ............................... 81 6.1 Introduction. ....................................................................................... 81 6.2 Endotoxic Activities ........................................................................... 82 6.2.1 Role of Lipid A ....................................................................... 82 6.2.2 Roles of Particular Constituent Chemical Groups .................. 82 6.2.3 Effect on Cell Morphology ..................................................... 83 Contents xi 6.2.4 Effect on Neutrophil Chemotaxis ......................................... 84 6.2.5 Effect on Hemagglutinating Activity of Bacterial Cells ....... 84 6.3 Antigenic Properties .......................................................................... 84 6.4 Immunological Responses ................................................................ 86 6.4.1 Vibriocidal Antibody Level and Immunity ........................... 86 6.4.2 Monoclonal Antibodies ......................................................... 87 6.4.3 Immunoglobulin Subclasses ................................................. 89 6.4.4 Antibody Assay for Encapsulated Cells ............................... 90 6.4.5 Synthetic Oligosaccharides ................................................... 90 6.5 Role in the Intestinal Adhesion and Virulence of the Vibrios .......... 92 6.6 LPS as Phage Receptor ..................................................................... 94 6.7 Biofilm Formation and the Structure of LPS .................................... 96 6.8 Capsular Polysaccharide (CPS) ........................................................ 99 6.9 Concluding Remarks ......................................................................... 100 6.9.1 Recognition of LPS and Activation of the Innate Immunity of the Host ............................................................ 100 6.9.2 Serogroup Surveillance and Monitoring ............................... 100 6.9.3 LPS and Cholera Vaccine ...................................................... 102 7 Cholera Toxin (CT): Structure ............................................................... 105 7.1 Introduction ....................................................................................... 105 7.2 Isolation and Purification .................................................................. 106 7.3 Primary Structures of CT and LT ...................................................... 107 7.4 Structures of CT and LT from X-Ray Crystallography .................... 109 7.4.1 B Monomer and Pentamer .................................................... 112 7.4.2 A1 Fragment of the A Subunit .............................................. 114 7.4.3 A2 Fragment of the A Subunit .............................................. 115 7.5 Receptor Binding .............................................................................. 115 7.6 Structure at the Binding Site ............................................................. 117 7.7 Structural Basis of Toxicity............................................................... 118 7.8 Structure-Based Inhibitor (Drug) Design: Possible Approaches ...... 122 8 Cholera Toxin (CT): Organization and Function of the Relevant Genetic Elements ........................................................... 125 8.1 Introduction ....................................................................................... 125 8.2 The CTX Genetic Element ................................................................ 126 8.3 Cloning and Sequencing of the ctxAB Operon ................................. 127 8.4 CTX Genetic Element Belongs to a Filamentous Bacteriophage ................................................................................... 128 8.5 The CTX Genome .......................................................................... 129 8.5.1 The Core Region ................................................................... 129 8.5.2 RS2 Region ........................................................................... 130 8.6 RS1 Element: The Flanking Region ................................................. 130 8.7 TLC: Another Upstream Element ..................................................... 131 8.8 Variation of CTX Among Classical, El Tor, O139, and Non-O1 Non-O139 Strains ........................................................ 131 xii Contents 8.8.1 Variation in Copy Number and Location Within Chromosome ......................................................................... 131 8.8.2 Variation in Sequence of rstR and Ig-2 ................................. 132 8.8.3 Variation in the Integration Site ............................................ 132 8.8.4 Variation in the Flanking Region .......................................... 132 8.8.5 Production of Infectious Particles ......................................... 134 8.9 Overview of CTX Biology ............................................................. 134 8.9.1 Infection of CTX .............................................................. 134 8.9.2 Integration of CTX ......................................................... 135 8.9.3 Replication of the CTX Genome ........................................ 135 8.9.4 CTX Gene Expression ........................................................ 136 8.9.5 Assembly and Secretion of CTX ....................................... 136 8.10 The Vibrio Pathogenicity Island-1 (VPI-1) ....................................... 137 8.11 Toxin-Coregulated Pili (TCP) ........................................................... 139 8.12 Other Horizontally Acquired Gene Clusters ..................................... 142 8.12.1 VPI-2 ................................................................................... 142 8.12.2 VSP-I and VSP-II Islands ................................................... 143 9 Cholera Toxin (CT): Regulation of the Relevant Virulence Genes ..... 147 9.1 Introduction ....................................................................................... 147 9.2 Modulation of Cholera Toxin Expression by Environmental Factors ................................................................. 148 9.3 The Regulation of Virulence: An Overview ..................................... 149 9.4 The ToxR Regulon ............................................................................ 150 9.5 ToxT-Dependent Transcription ......................................................... 151 9.6 Regulation of ToxT Transcription ..................................................... 156 9.7 Transcriptional Regulation of tcpPH ................................................ 158 9.8 Direct Transcription of ompU and ompT by ToxR ........................... 159 9.9 Environmental Regulation of tcpPH Transcription .......................... 160 9.9.1 Osmolarity ............................................................................. 161 9.9.2 Temperature .......................................................................... 161 9.9.3 Quorum Sensing .................................................................... 161 9.9.4 Glucose Availability .............................................................. 164 10 Cholera Toxin (CT): Secretion by the Vibrios ....................................... 165 10.1 Introduction ..................................................................................... 165 10.2 Early Electron Microscopic Enquiries ............................................ 165 10.3 The Secretion Mechanism: An Overview ....................................... 167 10.4 Translocation Across the Inner Membrane ..................................... 168 10.5 Protein Folding in the Periplasm ..................................................... 169 10.6 Secretion Across the Outer Membrane ........................................... 172 10.6.1 Genetics of the Extracellular Protein Secretion (Eps) Apparatus .......................................................................... 172 10.6.2 Molecular Architecture of the Secretory Machinery ........ 174 10.6.3 The Inner Membrane Complex ......................................... 176 Contents xiii 10.6.4 The Pseudopilus ................................................................ 177 10.6.5 The Outer Membrane Complex ........................................ 181 10.6.6 The T2SS System: A Summary ........................................ 182 10.6.7 Targeting Signals for the Translocation of CT Through the OM ............................................................... 183 11 Cholera Toxin (CT): Entry and Retrograde Trafficking into the Epithelial Cell ............................................................................. 185 11.1 Introduction ..................................................................................... 185 11.2 Attachment to the Cell Membrane .................................................. 186 11.3 Endocytosis ..................................................................................... 187 11.4 Retrograde Trafficking to the Golgi Complex and Endoplasmic Reticulum ........................................................... 189 11.5 Unfolding of CTA1 and Its Translocation to the Cytosol ............... 191 11.6 Reactions in the Cytosol Leading to the Activation of Adenylate Cyclase, Chloride Channel Outpouring, and Diarrhea .................................................................................... 193 12 Cholera Toxin (CT): Immune Response of the Host and Vaccine Production ........................................................................... 199 12.1 Introduction ..................................................................................... 199 12.2 Protective Immunity by Anti-CT .................................................... 199 12.3 Immunomodulation by Cholera Toxin (CT) ................................... 201 12.3.1 Adjuvant Properties of CT ................................................ 201 12.3.2 Adjuvant Properties of B Subunits of CT ......................... 203 12.3.3 Adjuvant Properties of Nontoxic Derivatives of CT .............................................................. 203 12.3.4 Mechanism of Adjuvant Activity of CT ........................... 204 12.4 Role of Cholera Toxin (CT) in the Preparation of Vaccines ........... 205 12.4.1 Background Knowledge .................................................... 205 12.4.2 The Vaccine Strains ........................................................... 206 12.5 Where Do We Stand Now? ............................................................. 211 13 Other Toxins of Vibrio cholerae .............................................................. 213 13.1 Introduction ..................................................................................... 213 13.2 Zonula Occludens Toxin (Zot) ........................................................ 214 13.2.1 Zonula Occludens (ZO) .................................................... 214 13.2.2 Discovery of Zot ............................................................... 215 13.2.3 Properties of Zot ............................................................... 216 13.2.4 Genetics ............................................................................. 218 13.2.5 The Zot Receptors ............................................................. 219 13.2.6 Applications of Zot ........................................................... 220 13.3 Accessory Cholera Enterotoxin (Ace) ............................................ 221 13.3.1 Discovery of Ace .............................................................. 221 13.3.2 The ace Gene and the Encoded Protein ............................ 222 xiv Contents 13.3.3 Purification of Ace ............................................................ 223 13.3.4 Mode of Action of Ace ..................................................... 224 13.4 Hemolysin ....................................................................................... 225 13.4.1 The Gene and the Encoded Protein ................................... 225 13.4.2 Protein Purification and the Crystal Structure .................. 226 13.4.3 Biological Activity ............................................................ 227 13.5 Repeats in Toxin (RTX) .................................................................. 229 13.5.1 Discovery of RTX ............................................................. 229 13.5.2 Organization of the RTX Locus ........................................ 229 13.5.3 Structural Features of RtxA .............................................. 230 13.5.4 Secretion of RTX Toxin from V. cholerae......................... 231 13.5.5 Mode of Action of RTX Toxin .......................................... 233 13.6 Chinese Hamster Ovary (CHO) Cell Elongation Factor (Cef) ..................................................................................... 236 13.6.1 Purification, Stability, and Biological Activity ................. 236 13.6.2 Cloning of the cef Gene and the Encoded Protein ............ 237 13.7 New Cholera Toxin (NCT).............................................................. 237 13.8 Shiga-Like Toxin (SLT) .................................................................. 238 13.9 Thermostable Direct Hemolysin (TDH) ......................................... 239 13.10 Heat-Stable Enterotoxin of Nonagglutinable Vibrios (NAG-ST) ........................................................................................ 239 13.10.1 Discovery ........................................................................ 239 13.10.2 Purification of NAG-ST .................................................. 240 13.10.3 Amino Acid Sequence of NAG-ST ................................. 240 13.10.4 Cloning and Sequencing of the stn Gene Encoding NAG-ST .......................................................................... 241 13.10.5 Mode of Action of NAG-ST ........................................... 242 13.11 Toxin WO7 ...................................................................................... 242 13.11.1 Discovery ........................................................................ 242 13.11.2 Purification of WO7 Toxin .............................................. 243 13.11.3 Biological Activity .......................................................... 243 14 Concluding Notes ..................................................................................... 245 14.1 An Introspection .............................................................................. 245 Appendix ........................................................................................................... 249 References ......................................................................................................... 267 Index .................................................................................................................. 311