\\. \ qÎ i MOLECULAR CHARACTERIZATION OF VARIANT SHIGA-L¡KE TOXIN GENES OF ESCHERICHIA COLI Adrienne Webster Paton, B.Sc.(Hons.)(FIinders) t I A thesis submitted for the degree of Doctor of Philosophy Department of Microbiology and lmmunology University of Adelaide August, 1993 , l\ \ tlr\ f'r\^¡, .ri¡ r:\ I i CONTENTS ABSTRACT I DECLARATION IV ACKNOWLEDGEMENTS V LIST OF ABBREVIATIONS V¡ CHAPTER 1: INTRODUCTION 1 1 .1 Diarrhoeagenic Escheríchia colí 1 1.1.1 Enterotoxigenic E. coli (ETEC) 2 1.1.2 Enteroinvasive E. colí (EIEC) 2 1.1.3 Enteropathogenic E. coli (EPEC) 3 1.1.4 Enteroaggregative E. coli (EAggEC) 3 1 .1 .5 Enterohaemorrhagic E. coli (EHEC) 4 1.2 Discovery of Shiga-like Toxins 5 1.3 Epidemiology 7 1.4 Disease States Associated with SLTs 9 1.4.1 Haemorrhagic Colitis (HC) 9 1.4.2 Haemolytic Uraemic Syndrome (HUS) 9 1.4.3 Thrombotic Thrombocytopoenic Purpura (TTP) 10 1.4.4 Oedema Disease of Piglets 10 1.5 Structure and Mode of Action 12 1.6 The Role of Toxin-Converting Bacteriophages 13 1.7 Evidence for the Existence of Multiple Forms of SLT 15 1.8 Gtoning and Sequence Analysis of SLT Genes 17 1.8.1 SLT-I 17 1.8.2 SHT 18 1.8.3 SLT-ll 19 1.8.4 SLT-ll Variants 20 1.9 Regulation of SLT Genes 20 1.1O Structure-Function Analysis of SLTs 24 1.10.1 Structure and Function of the A (Catalytic) Subunit 24 1.1O.2 Structure and Function of the B (Binding) Subunit 27 1.1O.2a Receptor Studies 28 1.1O.2b Functional Domains 29 1 .1 1 The Role of SLTs in the Pathogenesis of Disease 31 1.11.1 Clinical and Pathological Features 31 1.11.2 ln vitro Effects on Endothelial Cells 33 1.11.3 Role of Endotoxin and Cytokines in Pathogenesis 34 1.11.4 Animal Models for SLTEC Disease 36 1 .1 1 .5 Vaccination Against SLTEC Disease 39 1.12 Diagnosis of SLTEC lnfection 42 1.12.1 Detection of Faecal SLT and SLTEC 42 1.12.2 Sorbitol-MacConkey Agar for Detection of EHEC 43 1.12.3 Rapid Biochemical Test 44 1.12.4 Serological Methods of Detection 44 1.12.5 ELISAs for the Direct Detection of SLTs 45 1.12.6 Molecular Biological Diagnosis 45 1.13 Aims of the Work in this Thesis 47 CHAPTER 2: MATERIALS AND METHODS 49 2.1 Bacterial Strains 49 2.2 Bacteriophages 50 2.3 Gell Lines 50 2.4 Bacterial Growth Media 50 2.5 Cell Culture Media 51 2.6 Monoclonal Antibodies 52 2.7 Cloning Vectors 52 2.8 Routine Chemicals and Reagents 52 2.9 Solutions and Buffers 53 2.10 Restriction Endonucleases and Other Enzymes 53 2.11 Hybridization Membrane 54 2.12 lsolation of Bacterial Strains 54 2.13 Preservation of Bacterial Strains 54 2.14 Serotyping of E coli lsolates 55 2.15 Cell Culture 55 2.16 Storage of Cells and Recovery from Liquid Nitrogen 56 2.17 Cytotoxicity Assays 56 2.18 SLT Neutralization Assays 57 2.19 UV lnduction and lsolation of Bacteriophages 57 2.20 Preparation of E coli C6OO Lysogens 59 2.21 Bacteriophage lmmunity Studies 59 2.22 Extract¡on of Bacteriophage DNA 60 2.23 Extraction of Chromosomal DNA 61 2.24 Plasmid DNA Extraction 61 2.25 Restriction Endonuclease Digestion of DNA 62 2.26 Agarose Gel Electrophoresis 62 2.27 Band lsolation of DNA Fragments 63 2.28 Ligation of DNA Fragments 64 2.29 Preparation of Competent E. coli JM109 Cells 64 2.30 Transformation of E. coli JM109 Cells 65 2.31 Synthesis of Oligodeoxynucleotides 65 2.32 Ammonia Gleavage and Deprotection of Oligos 65 2.33 Labelling of Oligos with Digoxigenin 66 2.34 Random Primer Labelling of DNA Fragments with Digoxigenin 66 2.35 Preparation of Digoxigenin-Labelled SLT Probes by PGR 67 2.36 Preparation of Filters for Dot Blot Hybridization Analysis of Bacterial Lysates 67 2.37 Preparation of Filters for Southern Hybridization Analysis 68 234 Hybridization of Membranes with Oligo Probes 68 2.39 Hybridization of Membranes with DNA Probes 69 2.40 Development of DIG-Labelled Membranes 69 2.41 Stripping of Filters for Re-Hybridization 70 2.42 Rapid DNA Extraction for PCR Analysis 71 2.43 PGR-Amplification 71 2.44 Construction of Nested Deletion Derivatives 72 2.45 DNA Sequencing 73 2.46 Analysis of Sequence Data 74 2.47 Preparation of RNA 74 2.48 Gel Electrophoresis of RNA 75 2.49 Northern Hybridization Analysis of RNA 76 2.50 Reverse Transcription PCR 76 2.51 Protein Assay 76 CHAPTER THREE: ISOLATION AND PRELIMINARY ANALYSIS OF SLT-PRODUCING ESCHERICHIA COLI 77 3.1 Introduction 77 3.2 Results 77 3.2.1 Source of Bacterial Strains Tested 77 3.2.2 Screening for SLT Genes 78 3.2.3 Serotyping of SLT-Positive Strains 80 3.2.4 lnduction of Temperate Bacteriophages from SLT-Positive Strains 80 3.2.5 Restriction Analysis of Bacteriophage DNA 81 3.2.6 Superinfection Analysis 81 ir 3.2.7 Cytotoxicity of C600 Lysogens 81 3.2.8 Southern Hybridization Analysis of Bacteriophages 82 3.2.9 Cloning and Sequence Analysis of @031 DNA 83 3.2.10 Further Attempts to lsolate SLT-Converting Phages from E. coli Strains O31 and PH 84 3.3 Discussion 85 3.3.1 lsolation of SLTEC from Adelaide Children 85 3.3.2 Analysis of Bacteriophages 87 CHAPTER FOUR: CLONING AND NUCLEOTIDE SEOUENCE OF VARIANT SHIGA-LIKE TOXIN II GENES FROM ESCHERICHIA COLI OX3:H21 STRAIN 031 89 4.1 lntroduction 89 Ð 4.2 Results 89 fl 4.2.1 Further PCR Analysis 89 4.2.2 Southern Hybridization Analysis 90 4.2.3 DNA Sequence of the 031 PCR Product 91 4.2.4 Cloning of the SlT-Related Gene 91 4.2.5 DNA Sequence Analysis 92 4.2.6 Cloning of a Second SlT-ll-Related Gene from Strain 031 93 4.2.7 Sequencing of the Second SLT Gene from Strain 031 95 4.2.8 Expression of SLT-OX3 and SLT-OX3/2 Genes in E. coli Strain 031 96 4.3 Discussion 96 4.3.1 Presence of Two SlT-Related Genes in E. coli Strain 031 98 4.3.2 SLT-ll Sequence Variation 99 j I ,{ Þ- 4.3.3 Sequence Analysis of SLT-OX3 from E. coli I Strain 031 100 I 4.3.4 Sequence Analysis of the Second SLT-ll-Related li Gene (SLT-OX3l2l from Strain 031 102 'I I 4.3.5 Functional Significance of Amino Acid Sequence i Variation 104 I 4.3.6 Expression of SLT-OX3 and SLT-OX3/2 107 i 4.3.7 Clinical Significance 107 CHAPTER FIVE: CLONING AND NUCLEOTIDE SEOUENCE OF VARIANT SHIGA-LIKE TOXIN GENES FROM ESCHERICHIA COLI O111:H- STRAIN PH 109 5.1 lntroduction 109 5.2 Results 109 þ 5.2.1 Cytotoxicity of SLT Produced by Strain PH and Neutralization with Monoclonal Antibodies 109 5.2.2 Southern Hybridization Analysis 110 5.2.3 Cloning of the SlT-l-Related Gene from E. coli Strain PH 111 5.2.4 Nucleotide and Amino Acid Sequences of the SlT-l-Related Operon in Strain PH 112 5,2.5 Cloning and Sequencing of a Second SlT-Related Gene from Strain PH 114 5.2.6 Detection of SLT Transcripts in Strain PH 115 5.3 Discussion 116 5.3.1 Sequence Analysis of a Variant SLT-I Operon in Strain PH 116 5.3.2 Analysis of Sequences Downstream from the PH SLT-I Operon 118 I / I il Þ^. 5.3.3 Analysis of Sequences Upstream of the PH ¡ SLT-I Operon 118 { 5.3.4 Sequence Analysis of SLT-ll/O111 from Strain PH 121 t .l 123 'I 5.3.5 Expression of SLT Genes in Strain PH I t 5.3.6 Relative lmportance of SLT-I versus SLT-Il I I in Pathogenesis 123 CHAPTER SIX: DEVELOPMENT OF A PCR ASSAY FOR DIRECT DETECTION OF SLT-RELATED GENES IN PRIMARY CULTURES OF FAECES AND GUT CONTENTS 128 6.1 lntroduct¡on 128 6.2 Results 129 6.2.1 Development of a Novel SLT-I- and SLT-Il-Specific PCR Assay 129 6.2.2 Preparation of Samples for PCR Analysis 131 6.2.3 Sensitivity of Direct Detection of SLT-Related Genes in Faecal Cultures 131 6.2.4 Detection of SLT Genes in Clinical Samples 132 6.4.5 Amplification of Complete SLT-ll-Related Operons from Faecal DNA Extracts 133 6.3 Discussion 134 6.3.1 Direct Detection of SLT Genes in Faecal Samples 134 6.3.2 Direct Cloning of PCR-Amplified SLT Genes from Extracts of Faecal Cultures 138 CHAPTER SEVEN: FINAL DISCUSSION 140 BIBLIOGRAPHY 144 APPENDIX l: PUBLICATIONS 175 APPENDIX ll: SEOUENCE DATABASE ACCESSION NUMBERS 176 I ABSTRACT f n recent years strains of Escherichia colí producing Shiga-like toxins (SLTs) have been associated with serious human disease. However, the incidence and nature of such strains in Adelaide children has not previously been examined. ln this study, 1,475 E. coli isolates from this population were screened for the presence of SLT genes using the polymerase chain reaction (PCR) and by hybridization with specific DNA and oligodeoxynucleotide probes. Four SLT-producing strains were isolated. Two of these were lysogenized with toxin-converting bacteriophages, which were indistinguishable by restriction analysis from the reference SLT-l-encoding bacteriophage H198, and were not further characterized.