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i MOLECULAR CHARACTERIZATION OF VARIANT SHIGA-L¡KE TOXIN GENES OF ESCHERICHIA COLI
Adrienne Webster Paton, B.Sc.(Hons.)(FIinders)
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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. The other two SLT producers were an OX3:H21 strain isolated from the small bowel contents of a case of Sudden lnfant Death Syndrome and an O111:H- strain isolated from the faeces of a baby with haemolytic uraemic syndrome. Southern hybridization analysis of genomic DNA indicated that both these strains contained two SLT-related genes. None of these genes appeared to be phage- encoded. One of the SLT genes from the OX3:H21 strain was cloned as a 4.6 kb Ps¿l fragment into E. coli JM1O9 using the vector pUC19. JM1O9 cells harbouring the recombinant plasmid produced SLT, as judged by cytotoxicity for Vero cells. Nucleotide sequence analysis revealed that the SLT gene was related to, but distinct from previously reported variants of Shiga-like toxin type ll, produced bV E. coli from both human and animal sources. The A subunit of the SLT gene from OX3:H21 exhibited 95.9% homology (at both the DNA and derived amino acid sequence level) to the A subunit of the most closely related SLT-Il variant. The B subunit was less similar, exhibiting 88.6% and 88.8% homology to the related gene at the DNA and amino acid level, respectively. II
Clones containing the second SlT-related gene were not isolated from OX3:H21 gene banks. PCR was therefore used to amplify a 1,5 kb segment of DNA containing this operon from OX3:H21 genomic DNA, which was also cloned and subjected to DNA sequence analysis. This identified further variations compared with previously published SLT-ll sequences.
Southern hybridization analysis of chromosomal DNA from the O1 1 1:H- strain revealed that SLT-I- and SLT-ll-related genes were located on 8.S-kb and 4.5-kb EcoRl fragments, respectively. The larger fragment was cloned into E coti JM109 and the SLT-I gene was further localized to within a 3.O-kb Sphl-EcoRl fragment. Nucleotide sequence analysis revealed that the SLT-I A subunit gene from E coli 01 1 1:H- differed from the previously published sequences for SLT-I by 5 bp [resulting in two amino acid (aa) changesl. lt was more closely related to the gene encoding the A subunit of the Shiga toxin from Shigetta dysenteriae type 1, from which it differed by 3 bp (resulting in one aa change). The DNA sequence of the B subunit-encoding gene was identical to that of the other two toxins. The region of DNA upstream from the SLT-I gene of E. coli O1 1 1:H- contained an lS element, as well as a region with strong homology to a portion of the genome of bacteriophage lambda. Stable clones containing the SLT-Il-related gene were not detected in the 0111:H- gene bank, but the operon was cloned after PCR amplification and sequenced. The derived amino acid sequence of this gene differed by only one residue from that for one of the SlT-ll-related genes from the OX3:H21 strain. This difference, however, was associated with reduced cytotox¡city of the gene Product. The low isolation rate of SLT-producing E coli in this study may have been due to very low numbers of such organisms in patient samples, or to in vitro instability of SLT genes. To overcome this, a procedure for PCR- amplificatíon of SLT genes in crude DNA extracts of primary faecal cultures m was developed. A single pair of oligodeoxynucleotide primers was used for amplification of a 212- or 215-bp region of the A subunit gene. Genes were typed by hybridization of the PCR products to SLT-l- or SLT-ll-specific oligonucleotide probes. The procedure was capable of detecting less than 10 SlT-producing E. coli per ml culture against a background of greater than 109 other organisms per ml, and provided a rapid and sensitive means of screening primary faecal cultures for the presence of such strains. When this methodology was used to test primary cultures from gut contents or faeces from various patient groups, including apparently healthy infants, approximately half of all samples yielded positive results for SLT-I and/or SLT- ll sequences. IV
DECLARATION
This thesis contains no material which has been accepted for the award of any other degree or diploma in any University or other tertiary institution and, to the best of my knowledge and belief, contains no material previously published by any other person, except where due reference is made in the text. I consent to this copy of my thesis, when deposited in the University Library, being available for photocopying and loan, if accepted for the award of the degree.
Adrienne W. Paton
August 5, 1 993 V
ACKNOWLEDGEMENTS
I would like to thank my supervisors, Professor Paul Manning, Dr. James Paton and Dr. Paul Goldwater, for guidance and many helpful discussions during the course of this study, and for assistance with the compilation of this thesis. I would also like to thank Dr. Paul Goldwater for obtaining clinical specimens for analysis. In addition, I am grateful to Dr. David Hansman for permission to undertake this work in the Department of Microbiology, Adelaide Children's Hospital. I am indebted to Dr. Uwe Stroeher for assistance with the DNA sequencing and to Rachel Combe and Rebekah Miller for technical assistance. This work was supported by grants from the Sudden lnfant Death Research Foundation of South Australia and the Adelaide Children's Hospital Research Foundation. The financial assistance of an Australian Postgraduate Research Award is also acknowledged. VI
LIST OF ABBREVIAT¡ONS
aa Amino acid Aooo Absorbance 600 nm
ATCC American Type Culture Collection
bp Base pair
BSA Bovine serum albumin
cDso 5Oo/o cytotoxicity dose
CFA Colonization factor antigen cfu Colony forming units
CNS Central nervous system
dATP Deoxyadenosine 5'-triphosphate
dCTP Deoxycytosine 5'-triPhosPhate
dGTP Deoxyguanosine 5'-tri phosphate
DIG Digoxigenin
DMEM Dulbecco's Modified Eagles Medium
DMF Dimethyl formamide
DNA Deoxyribonucleic acid
DNase Deoxyribonuclease
dNTP Deoxyribonucleoside triphosphate
dTTP Deoxythymid ine 5'-triphosphate
dUTP Deoxyurid ine 5'-triphosphate
EAF Enteropathogenic Escherichia coli adherence factor
EAggEC Enteroaggregative Escherichia coli
EDTA Ethylene-diamine-tetra-acetic acid
EHEC Enterohaemorrhagic Escherichia coli
EIEC Enteroinvasive Escherichia coli
ELISA Enzyme-linked immunosorbent assays
EPEC Enteropathogenic Escherichia coli VII
ETEC Enterotoxigenic Escheríchía coli
FCS Foetal calf serum g Unit gravitational force
Gbg G lobotriaosyl ceramide
Gb+ Globotetraosyl ceramide
GIS Gastroi ntestinal system
GM Growth medium
HC Haemorrhagic colitis
HeLa Human cervical carcínoma cells
HEp-2 Human laryngeal epithelioma cells
HEPES 4- (2-hV droxyethyl ) - 1 -pe pe razi neetha nes u lfo n ic acid
HSVECS Human saphenous vein endothelial cells
HUS Haemolytic uraemic syndrome
HUVECS Human umbilical vein endothelial cells rL-1ß lnterleukin 1 beta i.p. lntraperitoneal
IPTG lsopropyl-ß-D-thiogalactopyra noside
IS lnsertion sequence kb Kilobase pairs kcal Kilocalorie kDa Kilodalton
LB Luria-Bertani broth LDso 50% lethal dose
LPS Lipopolysaccharide
LT Heat-labile enterotoxin
MDa Megadalton
MM Maintenance medium
MOPS 4-morpholinepropanesulfonic acid Ml Relative molecular mass VIII
MRNA Messenger ribonucleic acid
MUG 4-methylumbelliferyl-ß-D-gl ucu ronide
NBT Nitroblue tetrazolium salt
No.(s) Number(s)
NP4O Nonidet P4O oligo(s) Oligodeoxynucleotide (s )
ORF Open reading frame
PBS Phosphate buffered saline
PCR Polymerase chain reaction pfu Plaque forming units psi Pounds per square inch
RBS Ribosome binding site
RNA Ribonucleic acid
RNase Ribonuclease rpm Revolutions per minute rRNA Ribosomal ribonucleic acid
SDS sodium dodecyl sulphate
SHT Shiga toxin
SIDS Sudden lnfant Death Syndrome
SLT Shiga-like toxin
SLTEC Shiga-like toxin producing Escherichia coli
SSC Standard saline citrate
ST Heat stable enterotoxin
TBE Tris-borate-EDTA buffer
TE Tris-EDTA buffer
TET Tris-HCl-EDTA-Triton buffer
TLC Thin layer chromotography
TNF-ø Tumour necrosis factor alpha
Tris Tris ( hyd roxymethyl )aminomethane IX
tRNA Transfer ribonucleic acid
TTP Thrombotic thrombocytopoenic purpura
UV Ultraviolet VT Verocytotoxin
VTEC Verocytotoxigenic E. coli vlv Volume per volume
W/V Weight per volume
X-gal 5-Bromo-4-chloro-3-indolyl-ß-D-ga lacto-pyranoside X-phosphate 5-bromo-4-chloro-3-indolyl phosphate Él'I Y
1
CHAPTER ONE
INTRODUCTION
1.1 Diarrhoeagenic Escherichia coli
The gastrointestinal tracts of humans and many animal species have been successfully colonized by the facultative anaerobe Escherichía coli. Members of this extremely diverse species usually live in a symbiotic relationship with their host (Levine, 1987). However, there are specific groups within the species which are capable of perturbing this relationship and causing potentially life-threatening disease. Five major classes of E. have been identified\ enterotoxigenic E. coli lETECl, X diarrhoeagenic coli \ enteroinvasive E. coli (EIEC), enteropathogenic E. coli (EPEC), enteroaggregat¡ve E. coli (EAggEC) and enterohaemorrhagic E coli (EHEC) (Levine, 1987). This classification system is based on various characteristics such as ability to invade or adhere to the intestinal mucosal surface, O:H serogrouping, production of toxins, and clinical syndromes which are associated with specific groups (Levine, 1987). Particular O:H serogroups are often associated with a given class of diarrhoeagenic E. coli and serotyping has been used as a guide in diagnosis. However, whilst the O:H serogroup may be a useful clonal marker, it is not a determinant of virulence per se and definitive classification must be based on the other molecular features referred to above. Comparative features and pathogenic mechanisms of these diarrhoeagenic E. colíhave been extensively reviewed in recent years (Levine, 1987; Robins-Browne, 1987 and 199o; Sansonetti, 19921 and so for the purposes of this thesis only a brief overview of the different classes will be presented. 2
1.1.1 Enterotoxigenic E. colí (ETEC) ETEC are a major cause of bacterial diarrhoea in developing countries and are often the aetiological agent of the diarrhoea which plagues international travellers. ETEC produce fimbrial or fibrillar colonization factor antigens (CFA) or putative colonization factors (PCF) which permit attachment of the organism to enterocytes of the small intestine. More than a dozen antigenically distinct fimbriae or fibrillae have been identified and these have been assigned different CS (coli surface) numbers (Levine, 1987), ETEC are also distinguished from the other E. coli groups by their ability to produce either or both of two plasmid-encoded toxins; heat-labile enterotoxin (LT) and heat-stable enterotoxin (ST). The mode of action of these two toxins on enterocytes involves the stimulation of adenylate cyclase by LT and guanylate cyclase by ST, which results in secretion of fluid by the cell into the gut lumen and hence development of diarrhoea (Levine,1987l Karmali, 1989).
1.1.2 Enteroinvasíve E. coli (EIEC) The EIEC group produce a dysentery-like diarrhoeal disease similar to that produced by Shigellae and there is substantial O-antigen cross-reactivity between these two groups (Levine, 1987). Both organisms have the capacity to invade epithelial cells, part¡cularly those of the colonic mucosa. They multiply inside the cell and eventually cause its death, thereby disrupting the mucosal barrier and permitting the release of erythrocytes and polymorphonuclear leukocytes into the gut lumen (Robins-Browne, 1990). The invasive capacity of both species is believed to be mediated by several plasmid-encoded outer membrane proteins, which are also closely related antigenically between EIEC and Shigella spp. (Levine, 1987). Indeed the virulence plasmids are interchangeable between EIEC and Shigella (Sansonetti et al., 1983). ln addition, chromosomally-encoded determinants are required for full virulence (Robins-Browne, 1990). 3
1.1.3 Enteropathogeníc E. colí (EPEC) EPEC are known to be associated with outbreaks of diarrhoea, particularly in infants. EPEC strains exhibit an attaching and effacing adherence to the membrane of enterocytes, which results in the bacterium becoming partially surrounded in a cup-shaped protrusion of the host cell membrane. This intimate adherence is thought to account for the destruction of the microvilli of the gut seen on histopathological examination (Levine, 1987). The adhesin which mediates attaching and effacing adherence was thought to be encoded by genes located in the chromosome of EPEC (Levine, 1987; Karmali, 1989; Robins-Browne, 1990). lndeed, Jerse et al' (1990) identified a chromosomal gene, denoted eae, which induced attaching and effacing lesions on cultured cells. These authors reported that the deduced amino acid sequence of the eae gene had significant homology to the invasin gene of Yersinia pseudotuberculosis (Jerse et al., 1990). A later study by Jerse and Kaper (1991) showed that the eae gene encodes a94- kDa protein.
EPEC strains can also be classified on the basis of in vitro adherence to HEp-2 (human laryngeal epithelioma) cells. The factor responsible for this is referred to as EPEC adherence factor (EAF) and is plasmid-encoded. Two subclasses of EPEC have been identified; Class I includes those strains of classical EPEC serotypes that exhibit localized adherence to HEp-2 cells and that are usually EAF+. Class ll includes those strains of classical EPEC serotypes which exhibit diffuse or no adherence to HEp-2 cells and that are usually EAF- (Levine, 1987).
1.1.4 Enteroaggregatíve E. coli (E,aggEC) The EAggEC, as their name suggests, are classified on the basis of their distinctive in vitro adherence pattern to HeLa and HEp-2 cells. These 4 bacteria form a network of clusters as they adhere to each other and to the cultured cells, but in a pattern distinct from the localized adherence described for classical EPEC stra¡ns (Levine, 1987; Robins-Browne, 1990). This aggregative adherence is mediated by plasmid-encoded fimbriae which are different again from the fimbriae associated with ETEC and EHEC groups.
Although very little is known about this group, it is thought that they are associated with diarrhoeal disease in children and travellers' diarrhoea (Levine, 1987 ; Robins-Browne, 199O).
1.1.5 Enterohaemorrhagíc E. coli (EHEC)
The EHEC group is known to be associated with specific disease states in humans such as bloody diarrhoea, haemorrhagic colitis, thrombotic thrombocytopoenic purpura and the haemolytic uraemic syndrome (Karmali ef al., 1983; O'Brien ef al., 1983b; Riley et al., 1983). Strains from this group have been identified as high producers of one or more potent cytotoxins, which were distinct from the ETEC toxins LT and ST. These cytotoxins were referred to as Verocytotoxins or (more commonly) Verotoxins, because of their cytotoxicity for cultured Vero cells (African green monkey kidney cells) and were also referred to as Shiga-like toxins (SLTs) because of their structural similarity to Shiga toxin produced by Shigella dysenteríae (Levine, 1987). EHEC strains of serotype O157 H7 have been shown to carry a 60- MDa plasmid which encodes fimbriae that are involved in the in vitro attachment to Henle 4O7 cells (gut derived epithelial cells) (Tzipori et al., 1987).
A chromosomally-encoded adhesin has also been identified, which is thought to mediate attabhment and effacement of enterocytes, resulting in damage to microvilli. These lesions closely resemble the attachment and effacement lesions associated with EPEC infections (Levine, 1987; Tzipori et al., 1989). An EHEC eae gene thought to be responsible for these lesions 5 was cloned by Yu and Kaper (1992). The nucleotide sequence homology between the EPEC and EHEC eae genes was reported to be 86%, Some strains of classical EPEC serotypes also have the capacity to produce SLTs but generally they have been identified as low-producers.
However, some EPEC high producers do occur; for example strains belonging to EPEC serotypes 0111 and 026 are often high SLT producers and have been isolated from individuals with bloody diarrhoea, haemorrhagic colitis or haemolytic uraemic syndrome. These strains therefore, ate more appropriately classified as EHECs. The SLTs have been of particular interest to researchers in recent years as their precise role in the pathogenesis of E. coli-related gastrointestinal disease in humans and animals has yet to be elucidated. For this reason, they have been chosen as the subject of this thesis.
1.2 Discovery of Shiga-like Toxins
During the late 197Os in Canada, Konowalchuk and co-workers¡ tested X the suitability of Vero cells for the detection of heat labile enterotoxin (LT) produced bV E.coli isolates (Konowalchuk et al., 19771. Of the 136 isolates tested, filtrates from 1O caused profound and irreversible cytotoxicity in Vero cells. These same filtrates had no effect on Chinese hamster ovary cells and Y1 mouse adrenal cells, and therefore it was considered that a toxin other than LT was responsible (Konowalchuk et. al., 1977; Smith and Scotland, 1988; Karmali, 1989). This new toxin was designated Verocytotoxín or Verotoxin (VT) and E. coli strains capable of producing it became known as Verotoxic E. coli (VTEC). lsolates tested by Konowalchuk included a number of different E. coli serolypes including representatives of the EPEC group. Seven of the 1O VT* strains had been isolated from infants with diarrhoea, and one other was isolated from a piglet with diarrhoea; the other two 6
cytotoxic strains were isolated from cheese (Konowalchuk et al., 19771. Clearly, the association of VTEC isolates with diarrhoeal cases was worthy of further investigation. One of Konowalchuk's VT-producing isolates, strain H3O (serotype 026:H11), was chosen for toxin purification and characterization studies (O'Brien et al., 1983a). The purified toxin was shown to have strikingly similar structure and biological activity to Shiga toxin (SHT) produced by Shígella dysenteríae type 1, resulting in the new nomenclature of Shiga-like toxin (SLT). lt was also demonstrated that this SLT could be neutralized by anti-SHT (O'Brien et al., 1982; O'Brien and LaVeck, '1983). Both SLT and VT nomenclature systems persist in the literature today. However, for the purposes of this thesis, the toxins will be referred to only as SLT and organisms producing these toxins will be referred to as "Shiga-like tox¡n- producing E. coli" (SLTEC) rather than Verotoxigenic E. coli (VTEC). Of the SLT isolates described by Konowalchuk ef a/. (19771, it was observed that the SLT from one of the human isolates and the SLT from the piglet ísolate could not be neutralized by anti-serum raised against the H30 . SLT. Subsequent work by a number of groups showed that there were indeed two types of SLT; the original referred to as SLT-I and another related toxin group referred to as SLT-ll, which could not be neutralized by antibodies to either Shiga toxin or SLT-I (Scotland et al., 1985; Strockbine et al., 1986). Further work has also shown that the SLT-I¡ group includes variant toxins (SLT-Ilv) which have been associated with oedema disease in piglets (Marques et al., 1987) and human variants of SLT-ll have also been described (Gannon et al., 1990; lto ef al., 1990). The genetic characteristics of the SLT family of toxins will be discussed in Section 1.8.
During the early 198Os, clinical surveys testing for the incidence of SLTEC in diarrhoea cases were conducted in England and lndia, but a causal 7
relationship between SLTEC and diarrhoeal disease was difficult to establish (Karmali, 1989). However, in 1983, work performed in the United States and Canada suggested that SLTEC were implicated in serious human disease associated with diarrhoea' An uncommon serotype of E' coli (0157:H7l which was SlT-positive, was isolated from outbreaks of haemorrhagic colitis (HC) in both countries. Furthermore, in 1983 and 1985 SLTEC were isolated from cases of the haemolytic uraemic syndrome (HUS) (Karmali et al., 1983; Karmali et al., 1985). The precise role of the SLTs in the pathogenesis of diarrhoea, HC and HUS was not understood at the time, but a number of observations from the cases of HUS pointed directly to the involvement of SLT. These cases invariably had SLTEC isolated from their stools, and more importantly, faecal filtrates tested positive for SLT. Also, serum from HUS patients taken during the course of the illness, showed increasing antibody titres to SLT (Karmali et al., 1983; Karmali et al., 1985; Karmali, 1989).
1.3 Epidemiology
There is now a very broad spectrum of disease thought to be associated with SLTs. This includes serious and indeed life threatening disorders in humans such as HC, HUS and rare cases of thrombotic thrombocytopoen¡c purpura (TTP) (Karmali et al., 1983; O'Brien et al., 1983b; Riley et al., 1983; Morrison et al., 1985; Riley, 1987). However, some individuals infected with SLTEC may only suffer a mild diarrhoea, whilst others may have a completely asymptomatic infection. Interestingly, it has been reported that infection with some strains of E. colí l'J^157 may be asymptomatic in spite of the presence of free toxin in the faeces (Edelman et al., 1988; Brian et al., 1992]'. Epidemiological surveys have revealed that SLTEC are prevalent in the gastrointestinal tracts of many domestic animals, (both symptomatic and 8 asymptomatic) but are particularly common in cattle. SlT-associated disease in the animal population includes oedema disease in piglets, as well as diarrhoea in calves and cows (Smith and Scotland, 1988; Gyles, 1992), lt has also been reported that SLTEC have been isolated f rom cats with diarrhoea (Abaas et al., 1989). SLTEC strains isolated from symptomatic animals have included serotypes often associated with SLTEC disease in humans (Karmali, 1989). SLTECs have also been isolated from fresh retail meats including pork, beef, poultry and lamb (Smith and Scotland, 1988; Karmali, 1989). From these epidemiological data it has been shown that the highest carriage rate of SLTECs is in the bovine population, but that the animal reservoir may well be much broader than first indicated. SLTEC-associated disease in humans often occurs in epidemic outbreaks affecting all age groups, but with the most serious disease occurring in the paediatric and geriatric populations (Bopp et al., 1987; Karmali, 1989). This is likely to be a consequence of a lack of acquired immunity to SLTEC in the very young and the waning of antibody levels in geriatric populations, as is the case for many infectious diseases (Bopp et al., 1987; Karmali, 1989). lnfection often occurs after consumption of inadequately cooked beef or unpasteurized milk products. There have been many reports in the literature of outbreaks occurring in institutional settings such as nursing homes or child care centres, where a common contaminated food source has been identified. There is, however, increasing evidence that person to person infection also occurs, such as in family contacts of index cases, or during institutional outbreaks in nursing staff who were known not to have consumed the contaminated food source (Karmali, 1989). The preferred treatment of patients with SLTEC is supportive, involving replacement of fluid and electrolytes, as well as intensive therapy where renal failure is involved. Antibiotic therapy has not been demonstrated to be beneficial. lndeed disturbances to gut flora by prior exposure to antibiotics 9 riI à may be a predispos¡ng factor for both SLTEC disease and bacillary dysentery, I therapy with such drugs may facilitate release of toxin from and subsequent ¡ t bacterial cells into the gut lumen (Karmali, 1989). I L
i 1.4 Disease States Associated with SLTs I
I
I 1.4.1 Haemorrhagic Colit¡s (HC) i HC is a serious illness, involving a presentation with severe abdominal cramps and watery diarrhoea, which progresses to a haemorrhagic discharge. lndividuals suffering HC are generally afebrile (Riley, 1987). A number of neurological sequelae have been described in individuals with HC; these include lethargy, severe headache, convulsions and encephalopathy (Tesh and O'Brien, 1991). N
ru
1.4.2 Haemolytic Uraemic Syndrome (HUS) Two forms of HUS (classical and atypical) have been described and these can be distinguished by differences in their prodromal phases. The prodrome of the classical syndrome is characterized by watery diarrhoea and severe abdominal cramps, followed by the development of bloody diarrhoea as in HC. The prodrome of atypical HUS does not involve diarrhoea, but does include vomiting and fever. Upper and lower respiratory tract symptoms are often evident as well. ln HUS, the illness progresses from either of the above, to acute renal failure, microangiopathic anaemia and thrombocytopoenia. Although HUS is reported in all age groups, its incidence is higher in infants, young children and the elderly. lndeed, it is a major cause of acute renal failure in the paediatric population (Smith and Scotland, 1988; Karmali, 1989; Kaplan et al., 1990).
HUS is also a well-recognized complication of S. dysenteriae type 1 infection. However, organisms producing SlT-related toxins are not the only 10 I I'ir à- causes of HUS and other infectious agents (e.g. streptococcus pneumoniae, Salmonella typhí, and Campylobacter jejunil have been implicated, especially in the atypical presentation (Kaplan et al., 199O). Also there are types of HUS which do not appear to have an infectious aetiology. These include an inherited form which occurs in children and an adult-associated HUS which occurs as a complication in pregnancy, with the use of oral contraceptives and other drugs, and various other illnesses (Smith and Scotland, 1988; Kaplan et al., 1990).
1.4.3 Thrombotic Thrombocytopoenic Purpura (TTP) TTP is similar to HUS in that individuals present with microangiopathic haemolytic anaemia, thrombocytopoenia, and renal dysfunction. The features of TTP which distinguish it from HUS include the presence of fever and the i development of neurological complications. The peak incidence is in persons aged 30 to 40 years. Generally, TTP cases do not involve any prodrome, although there have been reported cases of prodromal bloody diarrhoea from which E. coli O157:H7 was isolated (Karmali, 1989)'
'1.4.4 Oedema Disease of Piglets Oedema disease is a serious illness in weanling piglets, which is associated with low morbidity, but a mortality rate which is often as high as 9Oo/o. lt is associated with certain serotypes of E. colí (including O138:K81, O139:K82 and O141 :K85) which multiply rapidly in the digestive tract and colonize the small intestine. The high susceptibility of piglets immediately after weaning is thought to be due to a number of factors, including changes in the piglets' environment, changes in diet and the subsequent disturbance in gut flora, and the loss of maternal immunity imparted to the piglet during lactation, lt has also been suggested that there is a genetic susceptibility or pre-disposition involved in this disease (Morris and Sojka, 1985; lmberechts 11 et al., 1992Ì.. Oedema disease is characterized by neurological symptoms including ataxia, convulsions and paralysis. Oedema is typically present in the eyelids, brain, stomach, intestine and mesentery of the colon, Anorexia, diarrhoea, and fever may be observed as a prodromal phase in some animals, but usually the onset of disease is acute, and results in sudden and unexpected death in previously healthy and robust animals (Nielsen and Clugston, 1971; Morris and Sojka, 1985; lmberechts et al., 1992l,.
The above symptoms were recognized as being consistent with a toxaemia, and the role of putative toxins in the pathogenesis of this disease was examined in a number of animal experiments. Timoney (1950) induced the clinical hallmarks of this disease in healthy animals when they were injected intravenously with the supernatants of centrifuged gut contents of animals with oedema disease. Further experimental work conducted by a number of groups confirmed that the E. coli serotypes associated with this disease produced a toxin which was referred to as oedema disease principle
(fmberechts et al., 1992). Oedema disease principle (later identified as a variant SLT tslT-llvl) was absorbed via the gut, caused vascular damage to the tissues outlined above and resulted in the oedema and subsequent neurological dysfunction characteristic of this disease (Nielsen and Clugston, 1971; Methiyapun ef al., 1984). Thus, SLT-llv was directly implicated in the pathogenesis of this disease (lmberechts et al., 1992l'. Notwithstanding this, the ability of the SLTEC to colonize and persist in the gut is an important virulence trait. Although these strains are not considered invasive, they have been isolated from the mesenteric lymph nodes of affected piglets (Morris and Sojka, 1985; lmberechls et al., 19921. Also, it has been reported that scrapings of the mucosal surface of the intestine of affected animals, have much higher numbers of SLTEC than the lumen of the gut, implying a capacity to adhere, persist and proliferate (Bertschinger and Pohlenz, 1983; lmberechts ef al., 1992). 12
1.5 Structure and Mode of Action
ln 1983 O'Brien and LaVeck successfully purified SLT from E. coli strain H30, an EPEC strain originally identified as being capable of inducing cytotoxicity in Vero cells (Konowalchuk et. al., 1977; O'Brien and LaVeck, 1983). This permitted molecular characterization of the toxin, which indicated that SLT-I was structurally and biologically indistinguishable from SHT produced by S. dystenteriae type 1. They found that it was cytotoxic for HeLa and Vero cells, enterotoxic for ligated rabbit ileal loops and lethal for mice (O'Brien and LaVeck, 1983). The M, of the holotoxin is approximately
70,OOO, comprising a single catalytic A subunit of Mr 32,2OO, and a multimeric B subunit (each monomer Mt 7,7OO) involved in the binding of the toxin to specific glycolipid receptors on the surface of target cells. Biochemical crosslinking analysis suggested that the holotoxins of both SHT and SLT include 5 B subunit monomers (Donohue-Rolfe et al., 1 984). However, x-ray crystallographic analysis of purified SHT and SLT-l B subunit has produced conflicting data. Hart et al. (1991), suggested a tetrameric arrangement for the B multimer, whereas Stein et al. (1992) repclrted a pentameric arrangement, consistent with the crosslinking studies. To clarify this point, further x-ray crystallography needs to be carried out on the holotoxins (O'Brien et al., 1992). The process of B subunit binding and cell receptor specificity will be discussed in detail in Section 1.1O.2. Once bound to a target cell membrane, toxin molecules are thought to be internalized by a process of receptor-mediated endocytosis, as shown in Figure 1.1. Briefly, this involves formation of a clathrin-coated pit within the cell membrane, which subsequently pinches off to form a sealed coated vesicle, with toxin bound to the internal surface. The toxin-bound vesicles may undergo fusion with cellular lysosomes and transportation via the Golg¡ Figure 1 .1 Entry of SLTs into mammalian cells
Model for the endocytic entry and processing of SLT as proposed by O'Brien and Holmes (1gg7). sLT enters the cell by receptor-mediated endocytosis. The B subunit of the toxin binds to the mammalian cell receptor. The clathrin-coated pit is pinched off and the coated vesicle is formed. The vesicle is acidified and it may fuse with lysosomes. The mechanism by which the enzymatically active A1 fragment of the toxin is generated and reaches the cytosol is not known, but is presumed to involve proteolytic nicking and reduction of disulphide bonds in the A subunit. The A1 fragment w¡thin the cytosol binds to the 605 ribosomal subunit, leading to an inhibition of protein synthesis (O'Brien and Holmes, 1987). EXTERNAL TOXIN
A A CLATHRIN COATEO PIT
A
COATEO VES¡CLE
OO
FUSIOH ()F TYSOSOMES WITH COATEO VESICLE
D PRocESsrNc oF Toxtl tt{ cYTosot D D AlBIN0S T0 + I 60s Rl8osoMEs
POLYSOHES I II{H¡BINOil (lF PRÍITEII{ SYIITHESIS: CEtL DEATH 13 before being translocated to the cytosol (Sandvig et al., 1989; Tesh and O'Brien, 1991; Sandvig et al., 1992!.. The mechanism for toxin translocation from the endocytic vesicles to the cytosol is not understood. However, it has been suggested that an entry domain might exist which could facilitate translocation (Keusch, 1981). Moreover, hydropathy profiles of both the B subunit and a port¡on of the A subunit referred to as the A2 fragment, show areas of hydrophobicity that could be important ¡n this process (Seidah et al., 1986; Jackson et al., 1987a1. The A2 fragment is a consequence of post- translational processing of the A subunit, presumably by an E. coli protease. This generates a catalytically active 27-kDa N-terminal A1 fragment and the 4-kDa C-terminal A2 fragment, which remain linked by a disulphide bond. However, after translocation into the cytosol, this di-sulphide bond is reduced, thereby releasing the active A1 component (Tesh and O'Brien, 1991), The SLT family of toxins are all potent inhibitors of eukaryotic protein synthesis. They are RNA N-glycosidases capable of cleaving a specific N- glycosidic bond in the 28S ribosomal RNA (Endo et al., 1988). This causes the loss of a single adenosine residue (at position 43241 and prevents elongation factor-1-dependent binding of the aminoacyl-tRNA to the 605 ribosomal subunit (lgarashi et al., 1987¡ Endo et al', 1988; Ogasawara et al', 1988), thereby inhibiting peptide chain elongation and ultimately causing cell
death.
1.6 The Role of Toxin-Gonverting Bacteriophages
Smith and Linggood (1971) demonstrated that EPEC strain H19 (serotype O26:H11) produced, at that time an uncharacterized enterotoxin. They showed that the capacity to produce this enterotoxin could be transmitted to E. coti K-12 by incubation with cell-free extracts of strain H19, t4
but they were unable to demonstrate that either a plasmid or a bacteriophage was involved. Nine years later, Scotland et al. (1980) demonstrated that both strain H19 and the above toxigenic K-12 derivative produced SLT. Subsequently, Scotland ef al. (1983) transferred the capacity to produce SLT from strain H19 to another K-12 stra¡n and isolated and purified a bacteriophage from culture filtrates of the toxigenic derivative. These phage particles had elongated hexagonal heads and non-contract¡le flexible tails, and lysogeny of K-12 with the purified phage conferred the ability to produce SLT. Subsequently, two different toxin-converting temperate phages were isolated from strain H19 and these were designated H19A and H198 (Smith et al., 1983). Smith et al. (1984) also isolated SlT-converting bacteriophages from two different Q157 H7 strains, and superinfection immunity studies suggested that these two phages were different both from each other and from those of strain H19. Thus, SLT is similar to certain other bacterial toxins such as E colí enterohaemolysins, diphtheria toxin, staphylococcal enterotoxin, botulinum toxin and streptococcal erythrogenic toxin, all of which are encoded (directly or indirectly) on the genome of bacteriophages that have established a lysogenic relationship with their respective bacterial hosts (O'Brien and Holmes, 19S7). Further evidence that a widely disseminated family of SLT- converting phages might exist in isolates of E. coliwas provided by O'Brien ef at. (1984). This group reported the isolation of two different SlT-converting phages from E. coti serotype O157:H7 strain 933, which was a high producer of SLT from a case of haemorrhagic colitis. These two phages were designated 933J and 933W. They also induced a phage, from E. colí O26:H11 strain H19, that was designated H19J. Comparative analyses of these phages as well as the previously isolated phages H19A and H19B (Smith et al., 1983) based on thermal stability, superinfection immunity, restriction analysis of phage DNA, electron microscopy and electrophoretic 15
analysis of phage proteins, revealed that phages H 1 9A and H 1 9J were probably independent isolates of the same phage. Phage H198 could be distinguished from the other two phages on the basis of superinfection immunity (O'Brien et al., 1984). They also concluded that phages H19J and 933J were very similar. Phage 933W behaved differently from phages H1gA/J and H19B in that it was more thermolabile and it could superinfect E coti C6OO lysogenized with either of the other phages. Phages H19J and 933J were morphologically indistinguishable by electron microscopy, each having elongated hexagonal heads and long non-contractile tails (O'Brien ef al., 1 984). However, Willshaw et al. ( 1 987) described a second morphological group of SLT-converting phages from several E. coli 0157:H7 strains, which had regular hexagonal heads and shorter contractile tails, O'Brien et at. (1989) subsequently reported that phage 933W belonged to this latter group, but they were no longer able to isolate phages with the same morphology as phage 933J from E coli strain 933, or from strain 933D (a derivative of strain 933 that had been cured of 933W). Both strains contained sequences that hybridized extensively with labelled phage 933J DNA, but Southern blot analysis identified differences in the labelling pattern of restriction fragments between phage 933J and the (possibly defective) prophage. They suggested that phage 933J may not have come from strain 933 at all and may have arisen as a result of contamination with phage H1gA/J (O'Brien et al., 1989).
1.7 Evidence for the Existence of Multiple Forms of SLT
O'Brien and LaVeck (1983) purified the cytotoxin produced by one of Konowalchuk's original verocytotoxic isolates (EPEC strain H30; serotype 026). The SLT from E. coti O157:H7 strain 933 was also purified and the properties of these two SLTs were compared with those of the SHT produced 16 by S. dysenteríae type 1 strain 6OR (O'Brien et al., 1983a;l O'Brien et al., X ì 1983b). This revealed that the respective SLTs produced by strains 933 and H3O were immunologically indistinguishable from SHT (as determined by Ouchterlony immunodiffusion) and were neutralized by anti-serum raised against SHT. Moreover, the three toxins had similar biological activities; they were cytotoxic for HeLa cells, enterotoxic for rabbit ileal loops and paralytic and lethal for mice (O'Brien ef al., 1983a; O'Brien and LaVeck, 1983). Whilst the purified SLT from strains 933 and H30 were immunologically indistinguishable from SHT (O'Brien ef al., 1983a), a later study (Scotland ef
êt1., 1985) found that anti-SHT could not neutralize all of the apparent SLT activity in crude culture filtrates of a number of 0157:H7 or O157:H- isolates, including strain 933, and indeed had no detectable impact on the cytotoxicity of one of the isolates, stra¡n E-32511 (O157:H-). The portion of cytotoxicity in the various filtrates which was refractory to anti-SHT could, however, be neutralized by an anti-serum raised against E32511. These results provided evidence that there might be more than one species of SLT, Strockbine et al. (1986) examined the SLTs produced by E. colí K-12 lysogenized with bacteriophages 933J and 933W to determine their biological and immunological relatedness. The SLTs encoded by both phages were indistinguishable from SHT in that they were cytotoxic to HeLa and Vero cells, they were enterotoxic in rabbit ileal loops and they were paralytic and lethal for mice. However, only the SLT from phage 933J could be neutralized with anti-SHT. Furthermore, crude ant¡-serum raised against the lysogen E. coli::933W could neutralize phage 933W SLT but not the SLT encoded by phage 933J, or SHT. lnterestingly, this group demonstrated that in E. coli strain 933, most of the cell-associated toxin could be neutralized by anti-SHT, but anti-933w toxin was required to neutralize the extra-cellular cytotoxin. Strockbine et al. (1986) went on to show that 11/60 strains isolated from cases of HC, HUS and diarrhoea, produced cell-associated toxins which could 17 also be neutralized by 933W anti-toxin. The SLT neutralized by anti-SHT was referred to as SLT-|, whilst the form neutralized by anti-933W was referred to as SLT-Il.
1.8 Cloning and Sequence Analysis of SLT Genes t.8. t sLT-t The structural genes of SLT-I of phage 933J were cloned from purified 933J DNA by Newland et al. (1985). Two other groups also reported the cloning of SLT-I genes from phage H198 (Willshaw et al., 1985; Huang et al., 1986). This enabled development of DNA probes for examination of the genetic relationship between SLT-I and SHT by Southern blot hybridization analysis, This showed that SLT-I probes hybridized under highly stringent conditions to sequences in S. dysenteriae type 1 (Newland et al., 1985), which is consistent with the antibody neutralization data reported above. lnterestingly, however, such probes only hybridized at lower stringency to phage 933W DNA and DNA from other E. coli strains producing the immunologically distinct SLT-ll (Willshaw et al., 1985; Strockbine et al., 1986). These studies provided independent confirmation of the existence of two distinct classes of SLT. The nucleotide and deduced amino acid sequences were determined for the SLT-I operon from phages 933J, H19B and H3O (Calderwood et al., 1987; De Grandis et al., 1987; Jackson et al., 1987b; Kozlov et al., 1988). Collectively, these studies showed that the SLT-l operon was a single transcriptional unit encoding first the A subunit followed by the B subunit, as shown in Figure 1.2. Both the 315 amino acid A subunit and the 89 amino acid B subunit had hydrophobic N-terminal signal sequences characteristic of secreted proteins. The respective M, values for the processed A and B predicted and Putative ribosome binding subunits were to be 32,217 491 . X Figure 1.2
Sequence of the SLT-I operon
The nucleotide and deduced amino acid sequences shown are those determined for the sLT-l operon from phage H19B by De Grandis et al. (1gg7). The coding regions for the putative A and B subunits are nucleotides
160-1 107 and nucleotides 1 1 17-1386, respectively. The amino acid translation is represented by single-letter code immediately above the first nucleotide of each codon, and the numbers in the left hand margin refer to the position in the processed polypeptide chain for each subunit. Possible signal peptidase cleavage sites are denoted "t". Putative ribosome binding sites for each subunit are denoted "rbs" and are underlined' Putative -10 and -35 promoter regions are double underlined, and a region of dyad symmetry spanning the -10 sequence believed to be involved in regulation by
Fur is shown in bold characters. -35 -10 1 GACCAGATATGTTAAGGTTGCAGCTCTCTTTGAATATGAT]AIEAITTTCATTACGTTATTGTTACGTTTATCCGGTGCGCCGTAAAACG a-22 TbSMKIIIFR 91 CCGTCCTTCAGGGCGTGGAGGATGTCAAGAATATAGTTATCGTATGGTGCTCAAGGAGfATTGTGTAATATGAAAATAATTATTTTTAGA T Y V D a- 15 V L T F F F V I F S V N V V ATK E F T L D F S T A K 18i GTGCTAACTTTTTTCITTGTTATCTTTTCAGTTAATGTGGTGGCGAAGGAATTTACCTTAGACTTCTC6ACTGCAAAGACGTATGTAGAT a16 SLNV I RSA I GTPLQTI SSGGTSLLl'4] DSGS 27r TCGCTGAATGTCATTCGCTCTGCAATAGGTACTCCATIACAGACTATTTCATCAGGAGGTACGTCTTTACTGATGATTGATAGTGGCICA a46 GDNLFAVDVRG I DP EEGRFNNLRL IVIRNN 361 GGGGATAATTTGTTTGCAGTTGATGTCAGAGGGATAGATCCAGAGGAAGGGCGGTTTAATAATCTACGGCTTATTGTTGAACGAAATAAT a76 LYVTGFVNRTNNVFYRFADFSHVTFPGTTA 451 TTATATGTGACAGGATTTGTTAACAGGACAAATAATGTTTTTTATCGCTTTGCTGATTTTTCACATGTTACCTTTCCAGGTACAACAGCG 14 R H qI a 106 VTL S G D S S Y TT L QR V AG I S RTG Q i N s41 CTTACATTGTCTGGTGACAGTAGCTATACCACGTTACAGCGTGTTGCAGGGATCAGTCGTACGGGGATGCAGATAAATCGCCATTCGTTG
a 136 TTSYL DLMS HSGTS LTQS VARAMLRFVTVT 631 ACTACTTCTTATCTGGATTTAATGTCGCATAGTGGAACCfCACTGACGCAGTCTGTGGCAAGAGCGATGTTACGGTTTGTTACTGTGACA M T A F a1 66 A E A L R F R,Q I q R G F R T T L D D L S G R S Y V I LL GCTGAAGcTTTAcGTTTTcGGòAAATAiAGAGGGGATTTcGTACAACACTGGATGATCTCAGTGGGCGTTCTTATGTAATGACTGCTGAA GR I S a1 96 D V D L T L N I,,G R L S S V L P D Y H GQ D S V R V 811 GATGTTGATCTTACATTGAACTGGGGAAGGTTGAGTAGCGTCCTGCCTGACTATCATGGACAAGACTCTGTTCGTGTAGGAAGAATTTCT a726 FGS 1 N A I LGSVAL I LNCHHHASRVARMASD 901 TTTGGAAGCATTAATGCAATTCTGGGAAGCGTGGCATTAATACTGAATTGTCATCATCATGCATCGCGAGTTGCCAGAATGGCATCTGAT I a256 EFPSl,4CPADGRVRG I THNK I L[,JDSSTLGA 991 GAGITTCCTTCTATGTGTCCGGCAGATGGAAGAGTCCGfGGGATTACGCACAATAAAATATTGTGGGATTCATCCACTCTGGGGGCAATT a286 L t'4 R R T I S S *TbS t.4 K K T L L I A A S L S F F S A S A 1 081 cTGATGcGcAGAAcTATTAGcÃGTTGAeeeeeTnnnATGAAAAAAACATTATTAATAGC-fGcATcccTTTcATTTTTTTcAGcAAGTGCG b-2 L ATT P D C V T G K V E Y T K Y N D D D T F T V K V G D K E ll7 I CTGGCGACGCCTGATTGTGTAACTGGAAAGGTGGAGTATACAAAATATAATGATGACGATACCTTTACAGTTAAAGTGGGTGATAAAGAA b29 L FTN Rl¡JN L Q S L L LS AQ I T GMTVT I KTNACH t?6t TTATTTAccAAcAGATGcAATcTTiReTcTcTTcTTcTCAGTGCccAAATTAcGGGGATGAcTcTAACcATTAAAACTAATGccTGTcAT b59 NGGGFSEVIFR* 1351 AATGGAGGGGGATTCAGCGAAGTTATTTTTCGTTGACTCAGAATAGCTCAGTGAAAATAGCAGGCGGAGATTCATAAATGTTAAAfACAT
I44I CTCAATTCAGTCAGTTGTTGCCGGTCTGATAATAGATGTGTTAGAAAATTTCTGCATGGTGAATCCCCCTGTGCGGAGGGGCGACTGGTG
1 53 1 AACGGTATGATCTCTTTGATGATCGTAAGCGAGAATACGCGGGTTTGGTGGCACCAGGCCGAACTCACGGGAGGCACCCGGCATCAIGCT
1 62 1 GTATACAGAGATTAGGCATATATCCAGGCTCCTCATCGCAGGAGCCTTTTTACATGCAAAAAAAAGCCGAGTGGGTTCGGGCAACAGCAT 18 sites (Shine-Dalgarno sequences) were located immediately upstream from both the A and the B subunit open reading frames and the position of the promoter site (-1O and -35 sequences) was determined by primer extension analysis. lnterestingly, a 21 base pair region of dyad symmetry spanning the -10 region, which was thought to be associated with iron regulation of SLT-I expression was also identified (Calderwood et al., 1987; De Grandis et al., 1987; Jackson et al., 1987b; Kozlov et al., 1988). lron regulation of SLT production will be discussed in Section 1.9.
t.8.2 SHT Cloning and sequence analysis of the structural genes for SHT (Kozlov et al", 1988; Strockbine et al., 1988) showed that the SHT operon was also a single transcriptional unit with an A and B subunit organization similar to that for SLT-|. Comparison of the nucleotide sequences of SHT and SLT-I indicated that there was greater than 99% homology. Strockbine et al. (1988) detected only 3 nucleotide differences, whilst Kozlov et al. (1988) detected 4 nucleotide differences between the two sequences. The single nucleotide difference between their two SHT sequences did not affect the deduced amino acid sequence, Both groups reported that the deduced amino acid sequence of SHT differed from SLT-I by a single amino acid; a threonine present at position 45 of the processed A subunit of SHT was a serine in the SLT-I amino acid sequence. lt was also shown that SHT and SLT-I A and B subunits were translated with identical signal peptides, and that they had identical ribosome binding sites. Also, SHT exhibited the same recognition sequences believed to be involved in iron regulation as was seen for SLT-I' Notwithstanding these similarities, there have been no reports of SHT- converting phages in nature and the SHT genes have been mapped to the chromosome of S. dysenteriae (Kozlov et al., 19BB; Strockbine et al., 1988). 19
Computer analysis of DNA sequence data bases identified a significant degree of homology between the deduced amino acid sequence of the A subunits of SHT and SLT-I and the A subunit of ricin, a toxin produced by the castor bean plant (Calderwood et al., 1987; Kozlov et al., 1988). Ricin is also a potent inhibitor of eukaryotic protein synthesis with a catalytic A subunit, which is capable of causing the inactivation of the 605 ribosomal subunit, and a B subunit responsible for binding to target cell receptors. The greatest homology was seen in one particular region of 73 amino acids (residues 138-210 for the SLT-I A subunit) in which there were identical residues at 32o/o of the positions and chemically similar residues at a further 21o/" of positions. Computer predictions of secondary structure in this region were almost identical for the two toxins and it was suggested that this conserved region was part of the catalytic site of both toxins (Calderwood et al., 1987; Kozlov ef al., 1988).
1.8.3 SLT-il The structural genes encoding SLT-ll were cloned from the SLT-ll- converting phage 933W (Newland et al., 1987). The genetic organization of the SLT-ll operon was the Same as that for SLT-I and SHT, i'e., separate A and B subunit genes forming a single transcriptional unit. Jackson et al' (1987a) determined the complete nucleotide sequence of SLT-ll and showed that overall it had only 58% nucleotide sequence homology and 560/o deduced amino acid sequence homology with SLT-1. The degree of homology between SLT-l and SLT-Il was similar for both the A and B subunits.
However, 29o/o of the amino acid changes between SLT-l and SLT-Il resulted in substitution of a chemically similar residue and the overall hydropathy plots for both the A and B subunits were similar for the two toxins. Like SLT-1, the A and B subunits of SLT-ll had hydrophobic N-terminal leader sequences' The mature SLT-ll A subunit was 3 residues longer than that of SLT-|, while 20 the mature SLT-Il B subunit was one residue longer than that of SLT-1. There was only limited sequence homology between SLT-l and SLT-ll in the promoter/regulatory sequences of the two toxins (Jackson et al., 1987a).
1.8.4 SLT-ll Variants SLT produced by SLTEC isolates from piglets with oedema disease has been shown to be a variant form of SLT-ll and was referred to as SLT-llv (Marques et al., 1987). SLT-llv differs from SLT-ll in that it is cytotoxic for Vero cells but less so for HeLa cells. However, SLT-llv can be neutralized by SLT-Il antiserum. There have been no SLT-Ilv-converting phages described so far and the SLT-llv genes have been mapped to the chromosome of the respective porcine SLTEC isolates. The cloning and nucleotide sequencing of SLT-llv genes isolated from chromosomal DNA has been reported by Gyles ef at. (1988) and Weinstein et al. (1988b). These groups reported 91o/o overall nucleotide sequence homology between SLT-Il and SLT-llv 194"/o and 79o/" homology for the A and B subunits, respectively). They also determined that the overall nucleotide sequence homology between SLT-llv and SLT-l was 550/0-600/u Weinstein ef at. (1988b) also established that, unlike SLT-I and SHT, SLT-ll and SLT-llv genes were not subject to iron regulation. The sequencing of a number of other variant forms of SLT-ll derived from human SLTEC isolates have also been described (Oku et al', 1989; Gannon et al', 1990; llo et al,, 1990; Schmitt et al., 1991; Meyer et al', 1992l.' These variants are discussed in Chapter 4 of this thesis.
1.9 Regulation of SLT Genes
The requirement for low concentrations of iron in culture media for the increased production of SHT and SLT-I by S. dysenteriae and SLTEC, respectively, was observed relatively early in their histories (Dubos and 2T
Geiger, 1946; O'Brien et al., 19821. The involvement of iron in the regulation of expression of toxin genes has been documented for other bacterial toxins (e.g.. Pseudomonas exotoxin A and diphtheria toxin) (Calderwood and Mekalanos, 1987). lndeed expression of other genes in E. colí, including genes for siderophores and inner and outer membrane proteins involved in siderophore transport and utilization, ate also repressed due to the presence of iron in culture media (Calderwood and Mekalanos, 1987). Regulation is thought to be mediated by the Fur pr,otein which acts as a repressor, with iron working in tandem as a co-repressor. The Fur protein and iron form a complex which binds to operator sites near the promoters of the iron- regulated genes (Calderwood and Mekalanos, 1987). Weinstein et al. (19BBa) examined the role of temperature and iron concentration in the regulation of SLT-I in E. coli C6OO::933J lysogens and in strains encoding SLT-I genes on a high-copy-number plasmid. The presence of iron suppressed production of SLT-I in E. coli C6O0::933J, but not in E' coli carrying the plasmid-encoded SLT-I genes. They also constructed fusions of the lac operon with the proximal region of the SLT-I A subunit gene and examined the effect of iron on the level of ß-galactosidase activity. However, there was no observed suppression of SLT-l promoter activity in the presence of iron and the authors concluded that in model systems involving high-copy-number plasmids, regulation of SLT-l production by iron could not be demonstrated. This finding would be consistent with the involvement of a repressor molecule of low abundance. lt was also observed that reduced growth temperature did not have any effect on production of SLT-I in either E. coliC6OO::933J or SlT-l-producing clones. Calderwood and Mekalanos (1987) also studied the role of iron regulation in the synthesis of SLT-1. They constructed a gene fusion between the promoter and proximal region of the SLT-I A subunit with the gene for bacterial alkaline phosphatase. They observed a 13-16-fold increase 22 in alkaline phosphatase production in conditions of low iron concentration. When a null mutat¡on was introduced into the før locus, high levels of alkaline phosphatase were detected regardless of the iron concentration. The authors suggested that the SLT-I operon was under negative regulation by lhe fur gene product. This group employed deletion analysis upstream from the fusion in order to determine the location of the SLT-I promoter sequence.. From these studies they found that the region of DNA between the -35 and -10 boxes is necessary for iron regulation of SLT-I expression. This DNA segment included a 21-bp region of dyad symmetry, which had homology with similar sequences in the promoter regions of other Fur-regulated genes. The authors also proposed that the dyad symmetry may be the operator binding site for the Fur protein/iron repressor/co-repressor complex. Calderwood and Mekalanos (1987) postulated that regulatory proteins such as Fur are important for fine-tuning responses of pathogens to their environment, permitting optimal timing of the deployment of virulence factors. The extremely low availability of free iron within intestinal mucus may thus act as a signal to the SLTEC that the host has been penetrated and that amplification of production of SLT-I was now indicated. Work reported by Huang et al. (1986) and Newland et al. (1985) suggested that there might be an independent promoter for transcription of the B subunit of SLT-|. This was based on the observation that recombinant plasmids encoding the B subunit gene were capable of directing production of B subunits even though they lacked the promoter upstream from the A subunit gene. De Grandis et al. (1987) also identified a separate SLT-I B subunit transcript using S1 nuclease protection analysis. Further evidence for this was obtained from primer extension studies involving examination of 5' termini of mRNA transcripts of the SHT operon (Kozlov et al., 1988)' Northern blot analysis has shown that there are two species of mRNA transcribed from the SHT operon. The first is a 1.7-kb transcript which codes 23
for the A and the B subunits of SHT. The second is an approximately O.7-kb transcript encoding only the B subunit, which seems to be transcribed not only more effectively, but independently from the larger transcript. This preferential transcription of the B subunit would explain how the toxin subunit stoichiometry of 1 A subunit molecule : 5 B subunit molecules can be achieved.
The existence of an independent promoter for the B subunit of SHT has also been described by Habib and Jackson (1992). They found two types of SHT transcript; a bicistronic mRNA encoding the A and B subunit transcribed from a promoter upstream of the A subunit gene, and a monocistronic mRNA of the B subunit transcribed from a promoter upstream of the B subunit. The B subunit promoter was not repressed by the presence of iron. ln contrast to the findings of Kozlov et al. (1988), however, transcriptional fusion studies revealed that the B subunit promoter was 6- times less active than the bicistronic promoter. Nucleotide sequence analysis has shown that the structural genes for
SLT-Il and SLT-Ilv are genetically organized with an A subunit preceding a B subunit as is the case for SHT and SLT-!. The promoters for SLT-|1 and SLT- llv have also been shown to be identical and have been mapped to 1 18 bases upstream from the SLT-ll A subunit gene, There are substantial differences between the promoter regions of SLT-¡I and SLT-llv and those of SHT and SLT-|, in that the former did not have Fur recognition sequences, suggesting that they are not subject to iron-regulation (Sung et al., 199O). Sung et al.
(199O) compared the expression of both SLT-I and SLT-ll in fur* or fuf E. coli lysogenized with phages H198 or 933W, in conditions of low or high iron concentration, Their results showed that SLT-I production was iron regulated in the fur* E. coti lysogenised with H19J but not in the fuf lysogen. lron regulation of SLT-ll production was not observed in the fur* or fuf E. coli 24
lysogenised with 933W, confirming that lhe fur gene product is not involved in the regulation of SLT-Il. Further work conducted by Sung et al. (199O) involving Northern blot analysis, revealed only one type of SLT-ll mRNA transcript. Primer extension studies also failed to detect an independent B subunit gene promoter as described above for SHT.
1.1O Structure-Function Analysis of SLTs
Although there is only 560/" homology between SLT-I and SLT-ll in terms of their primary amino acid sequences (Jackson et al., 1987a) the two toxins are clearly very similar in terms of tertiary structure. The A subunits of SLT-I and SLT-Il have identical catalytic actívity (modification of a specific base in 28S rRNA) and the B subunits both bind to the same glycolipid receptor (globotriaosyl ceramide). Moreover, lto et al. (1988) demonstrated that fully active hybrid SLT holotoxins could be reassembled from purified SLT-I A subunits and SLT-ll B subunits (and vice versa), suggesting strong structural conservation of the A:B interface. However, the homology between SLT-I and SLT-ll is not uniform throughout the full length of the primary sequence, and the most conserved zones presumably represent structurally and functionally important domains. These regions have been the targets of structure/function analyses based on site-directed mutagenesis.
1.1O.1 Structure and Function of the A (Catalytic) Subunit
DNA and amino acid sequence data have revealed some striking regions of sequence homology between the A subunit of SLTs and the plant toxin ricin (Hovde et al., 1988; Kozlov et al., 1988). Ricin has several structural similarities with SLT; it cons¡sts of a catalytic A chain and a B chain 25 involved in receptor binding. Ricin is a member of a family of structurally related RNA-binding proteins; these include cytotoxins such as abrin and modeccin and proteins which inhibit ribosomes including trichosanthin and pokeweed antiviral protein (Deresiewicz et al., 1992). lt has been shown that the mode of action of SHT, all the SLTs and ricin is identical; they are all single site RNA N-glycosidases (Endo et al., 1987:- Endo and Tsurugi, 1987; Saxena et â1., 1989). X-ray crystallographic analysis has identified a prominent cleft within the A chain of ricin which might represent its catalytic site. Furthermore, two regions of substantial amino acid sequence homology and one region of moderate homology have been identified between the A subunits of ricin, SLT-|, SLT-ll, and SLT-llv and these have been mapped to the putative active site cleft of ricin (Montfort et al., 1987; Yamasaki et al., I 1991). ì Í Hovde et al. (1988) observed that glutamic acid residues were found in T the region of the putative active site of ricin as well as the analogous regions of SLT-l and SLT-ll. Moreover, carboxylate side chains have been identified in the catalytic sites of several glycosyl hydrolases and transferases, and glutamic acid residues have been mapped to the active sites of the protein synthesis inhibitors diphtheria toxin and Pseødomonas aeruginosa exotoxin A (Hovde et al., 1988; Jackson, 1990; O'Brien et al., 19921. To investigate its role in catalytic activ¡ty, Hovde et al. (1988) used oligonucleotide-directed mutagenesis to change the glutamic acid codon at position 167 to aspartic acid in the cloned SLT-I gene. They reported a substantial reduction in specific activity of the mutant protein and postulated that this amino acid may be located in the active site of SLT-|. This group showed that the mutation did not affect tertiary structure of the toxin when compared to wild-type SLT- l. ln a later study, the same mutation was introduced at the analogous position (residue 166) in the SLT-ll A subunit gene (Jackson et al", 1990). This resulted in a significant reduction in both catalytic activity and ',1 26 t à cytotoxicty of the mutant SLT-ll. When the corresponding mutat¡on was I constructed in the ricin A-chain (at position 177]' significant reduction in toxin I ,l activity was also observed (Schlossman et al', 1989)' li Yamasaki et al. (1991) used oligonucleotide-directed mutagenesis to i generate SLT-I mutants with a variety of single amino acid substitutions in the i regions with greatest homology to ricin (amino acids 51-55, 166-172 and I I 2O2-2O71. Each mutant was tested for its ability to inhibit prote¡n synthesis (using a cell free rabbit reticulocyte lysate) and for cytotox¡city. They showed that two amino acid residues in the 166-172 region were particularly important for toxin activ¡ty: glutamic acid at position 167 (previously identified by Hovde et al. t19881) and arginine at position 170. Several other amino acid residues in the 166-172 region and in The 2O2-2O7 region were shown to affect cytotoxicity and protein synthesis inhibitory activity but to a J somewhat lesser extent. However, mutations in the 51-55 region had little ru impact (Yamasaki et al., 1991). May ef al. (1989) reported that removal of the first 13 amino acids of the N terminus of the ricin A chain abolishes its enzymatic activity. Perera et al. (1991a) have shown that the amino terminal of the A subunit of SLT-Il is also important, as deletion of amino acids 3-18 of the mature A subunit abolishes both toxicity and enzymic activity. A model system for the detection of spontaneous point mutants in ricin cloned in Saccharomyces cerevisiae was developed by Frankel et al. (1989). Plasmids encoding mutations which abolish toxin activity were identified by their inability to kill their host. Amino acid substitutions at positions 177, 211, 212 and 215 which resulted in loss of toxin activity were identified. The authors postulated that the two major regions of sequence conservation between ricin and SLTs might be required for binding to and depurinating 28S rRNA (Frankel et al., 1989; Jackson, 199O). Deresiewicz et al. (1992) cloned the SLT-I A subunit gene under the control of an inducible promoter into S. cerevisiae also used this system to select for mutations in the otherwise : 27 ,{ h^ lethal SLT-I A subunit gene. One such mutant was shown to have a subst¡tution at TyrTT,which was outside the regions previously identified as being close to the putative active site. However, an energy-minimized computer model of the SLT-I A subunit, based on the known crystal structure of the ricin A chain, identified a cleft on one face of the SLT-I A subunit. All the catalytically important residues identified above, including Tyr77, were clustered around this putative cleft (Deresiewicz et al., 1992). l.lO.2 Structure and Function of the B (Binding) Subunit SHT, SLT-I and SLT-ll have all been shown to bind to globotriaosyl ceramide (Gb3), a glycolipid containing a terminal Galø1-4Gal moiety present on microvillus membranes and on the cell membranes of Vero and HeLa cells (Jacewicz et al., 1986; Lindberg et al., 1987; Lingwood et al., 1987; Waddell et al., 1987). Lindberg et al. (1987) also showed that a receptor analogue prepared by conjugating Galø1-4Gal to bovine serum albumin competitively inhibited binding of SHT to HeLa cells. Further evidence that Gb3 is the functional receptor for these toxins was provided by Mobassaleh et al. (1988), who demonstrated that age-dependent increases in the secretory activity of ligated rabbit ileal loops in response to treatment with SHT correlated with the appearance of Gb3 in the microvillus membrane. Cohen et al. (1987) also reported that mutant Daudi cells, which lack the Gbg receptor, are refractory to SLT-I cytotoxicity. However, when this cell line was incubated with liposomes composed of phosphatidylethanolamine, phosphatidylserine and Gb3, some of the glycolipid became incorporated into the plasma membrane and the Daudi cells became susceptible to SLT-l (waddell et al., 1990). SHT, SLT-I and SLT-ll are all highly cytotoxic for Vero as well as HeLa cells. Porcine SLT-Ilv and some human SLT-Il variants are also highly cytotoxic for Vero cells, but they differ from the other toxins in that they 28 have significantly reduced cytotoxicity for HeLa cells (Marques et al., 1987; I Oku ef al., 1989). Weinstein et al. (1989) used subunit complementation and operon fusions to construct chimeric SLT molecules comprising various combinations of A and B subunits from SLT-|, SLT-ll and SLT-llv. Analysis of the comparative cytotoxicity of these proteins for Vero and HeLa cells established that differential cytotoxicity was a feature determined by the B subunit. Interestingly, the B subunit also determined the proportion of toxin that remained associated with E coli cells: SLT-ll is primarily cell-associated, whereas a major proportion of SLT-llv is released into the culture medium (Weinstein et al., 1989).
I .l O.2a Receptor Studies Two independent studies examined the interaction of these toxins with glycolipids separated by thin-layer chromatography (TLC) to determine whether the above differences might be explained on the basis of receptor specificity. De Grandis et al. (1989) reported that SLT-llv binds with greater affinity to the glycolipid globotetraosyl ceramide (Gba) than to Gbg. Samuel et al. (199O) showed that SLT-ll, but not SLT-llv was able to bind to the Gb3 receptor analogue Galø1-4Gal-BSA. They also showed that porcine SLT-llv bound to Gb3, Gb4 and another glycolipid referred to as Gb5 on thin-layer chromatograms of Vero cell lipid extracts, but only Gbg from HeLa cell extracts. SLT-ll bound mainly to Gb3 in extracts from both cell lines. Unlike Vero cells, neutral lipid extracts of HeLa cells did not appear to contain either Gb4 or Gb5 (Samuel et al., 199O). Thus, lack of the preferred receptor glycolipid might explain the reduced cytotoxicity of SLT-Ilv for HeLa cells. However, a degree of caution needs to be exercised when interpreting glycolipid binding specificity data obtained using these TLC overlay techniques. lt has recently been shown that polyisobutylmethacrylate, which was used by Samuel et al. (1990) to stabilize the silica gel prior to reaction of 29 the separated lipids with toxin, is capable of artifactually modifying the binding specificity of SLT-I to include Gb4 at the expense of Gb3, presumably by inducing conformational changes in the carbohydrate moieties of the glycolipids (Yiu and Lingwood, 1992).
l. 1 O.2b Functional Domains Structure/function analysis of the binding subunits of the SLT family has resulted in identification of regions involved in the localization or secretion of the different SLT classes, as well as regions crucial for receptor binding specificity and/or kinetics, which may have a major impact on tissue/organ specificity and indeed pathogenesis of SLT disease. Perera et al. (1991a) deleted the last four amino acids from the carboxyl terminus of the B subunit of SLT-ll, and this resulted in loss of holotoxin cytotoxicity. However, loss of only the last 2 residues d¡d not significantly affect toxicity of the holotoxin, Furthermore, a 21 amino acid extension to the carboxyl terminus of the B subunit also had little effect on cytotoxicity. ln another study by this group employing regionally directed chemical mutagenesis of the B subunit of SLT-ll, they identified three amino acids crucial for holotoxin cytotoxicity (Perera et al., 1991b). Mutation of either Arg32, Ala42 or GlySg in the mature SLT-ll B subunit resulted in complete loss of cytotox¡city. The residues at the analogous positions in the SHT B subunit (amino acids 33, 43 and 60) were identical, and when mutations were introduced at these positions a marked decrease in SHT cytotoxicity was observed (Perera et al., 1991b). To investigate differences in cell receptor specificity between SHT, SLT-|, SLT-ll and SLT-llv, Jackson et al. (199O) conducted a comparative study of SHT and SLT-llv B subunits to identify domains responsible for receptor binding and toxin localization in E. coli. They determined that mutations ¡ntroduced in a conserved region near the amino terminus of the 30
SHT B subunit abolished cytotoxicity without affecting holotoxin assembly or its immunoreactivity, as determined by neutralizing monoclonal antibodies. They also showed that changing a Glu to Gln five residues from the C- terminus of the SHT B subunit resulted in abrogation of receptor binding as well as toxin immunoreactivity. ln addition, these authors demonstrated that the carboxyl- terminus of the B subunit, particularly the Gln 5 residues from the end, influenced the cellular location of SLT-llv. lf this residue is mutated to Glu (the residue found in all the other members of the SLT-ll family), a significant proportion of the SLT-Ilv remained cell-associated, rather than being predominantly extracellular (Jackson ef al., 1990). lnterestingly, these same two amino acid variations in the B subunit of SLT-llv have recently been implicated as the basis for its different glycolipid receptor specificity and its different cytotoxicity for various cell lines compared with the other SLT-Ils (Tyrrell et al., 1992). Site-directed mutagenesis of the SLT-llv B subunit gene such that these two amino acids (Gln64 and Lys66 in the mature polypeptide) were changed to Glu and Gln, respectively (the analogous residues in SLT-Il), altered the predominanL in vitro binding specificity of the mutant toxin from Gb4 to Gb3 that is, to the same receptor binding phenotype as SLT-Il (Tyrrell et al., 1992l'. This resulted in changes in the relative cytotoxicity of the mutant SLT-Ilv for various cell lines, in accordance with their Gb3 and Gb4 content. Boyd et al. (1993) have since demonstrated that when this mutant SLT-llv was injected ¡ntravenously into pigs, the distribution of toxin to the various organs was different to that obtained with wild-type SLT-Ilv. This was accompanied by differences in the clinical characteristics of toxin-induced disease, but it did not effect the nature of the histological lesions (Boyd et al., 1993). However, the C- terminus is not the only part of the B subunit involved in the binding specificity of SLTs. Mutating Aspl g of SLT-I to Asn, the residue found at the analogous position (17) of SLT-llv, resulted in binding of the mutant toxin 31 to Gb4 as well as Gb3, although the reciprocal mutation did not affect the binding specificity of SLT-llv (Tyrrell et al., 1992). Recently, the crystal structure of the oligomeric B subunit of SLT-I has been published and this suggests that the site of carbohydrate receptor binding may be a cleft between adjacent monomers of the B pentamer (Stein et al., 1992t,. The two portions of the B subunit identified as impacting on specificity are found on opposite sides of this putative binding cleft (Stein et al', 1992).
1.11 The Role of SLTs in the Pathogenesis of Disease
It has been generally accepted that SHT and SLTs are cytotoxic for certain cell lines, that they are capable of inducing paralysis and death when administered intravenously to rabbits and mice, and they are enterotoxic in that they mediate fluid accumulation in ligated ileal loops (Tesh and O'Brien, 1991). However, these features by themselves are insufficient to directly link
SLTs in the pathogenesis of human and animal disease. There has been a steadily increasing body of circumstantial evidence which certainly points to an association of SLTEC and SLTs with disease. However, evidence that this correlation is due to a causal relationship must be carefully analysed before dogmatic statements supporting the link can be made.
I .1 1 .l Clinical and Pathological Features.
As discussed previously, strong epidemiological associations between SLTEC and human diseases including HC and HUS have been documented (Smith and Scotland, 1988; Karmali, 1989). These syndromes often occur in
discrete outbreaks but may occur sporadically and are associated with a number of E. coli serolypes (particularly serotype O157:H7) (Karmali, 1989)' Furthermore, free toxin has been detected in faecal filtrates of individuals 32 presenting with diarrhoea, HC and HUS. SLT- and SHT-mediated tissue damage may be responsible for the development of bloody oedematous vascular lesions of the colon. lt has been suggested that these toxins directly kill colonic epithel¡al cells resulting in fluid secretion and hence diarrhoea (O'Brien and Holmes, 1987; Fontaine et al., 1988; Tesh et al., 1991). Although toxin damage to the gut appears to be an accepted phenomenon, system¡c damage due to absorption of toxin f rom the gut into the bloodstream has been difficult to establish. lndeed, neither SLTs nor SHT have ever been detected circulating in the blood of individuals with SLT- related disease or bacillary dysentery, respectively. However, rising titres of neutralizing anti-SHT and anti-SLT antibodies have been detected in such cases (Edelman et al., 1988; Karmali, 1989; Tesh and O'Brien, 1991), Unlike S. dysenteriae, SLTECs lack the capacity to invade cells of the mucosal surface of the colon and so SLT must presumably be absorbed from the gut lumen. lt has, however, been suggested that SLTEC may be able to adhere to colonic epithelial cells and thereby colonize the gut, permitting higher concentrations of toxin to come into contact w¡th the gut wall (Tesh and O'Brien, 1991). However, the role of adherence in SlT-associated diseases is still not fully understood. Epidemiological data have shown that during outbreaks of HC and bacillary dysentery, from 3o/" to 53o/o of individuals develop complications involving the kidneys or central nervous system (Tesh and O'Brien, 1991). Histopathological examination of lesions associated with the gut or those resulting from the secondary complications in individuals with either S. dysenteriae or SLTEC infections, have revealed damage to the vasculature of these tissues. The vascular endothelial cells of particular organs may well be specifically susceptible to damage by both SHT and SLTs. lt is known that vascular endothelial cells play a major role in the maintenance of a non- thrombogenic state, which is necessary for normal blood flow. Damage to 33 these cells results in progression to a pro-coagulant state and subsequent release of vasoactive substances and cytokines causing inflammation (Gerlach et al., 1990; Tesh and O'Brien 1991). Using the rat aorta model, Karch et al.
(1988) demonstrated that SLTs may inhibit the production of prostacyclin in endothelial cells. This has been proposed to result in an enhancement or induction of platelet aggregation observed in vivo in HUS patients (Rose ef al., 1985; O'Brien et al., 1992l'. A number of neurological sequelae have been identified in individuals with HC and bacillary dysentery including lethargy, severe headaches, convulsions and encephalopathy. However, it is thought that these neurological symptoms are caused by damage to capillaries supplying the central nervous system (Tesh and O'Brien, 1991).
l.l1.2 ln Vitro Effects on Endothelial Cells.
The characteristic kidney damage of HUS is evidence that SHT and SLTs may cause damage to particular target organs after dissemination via the bloodstream. lf indeed glomerular endothelial cells are damaged by these toxins, progression to a pro-coagulant state with subsequent inflammation is likely to occur, which could lead to the pathology observed in HUS (Tesh and O'Brien, 1991 ). Since SHT and SLT-I are essentially structurally and biologically identical, comparisons of the in vitro and in vivo effects of purified toxin are appropriate. However, as S. dysenteriae is known to be invasive, comparisons of the consequences of infection with this organism
and SLTECs, are less meaningful. ln vitro studies of the cytotoxicity of SHT and SLTs for human endothelial cells isolated from the saphenous vein (HSVECs) and umbilical vein (HUVECs) have been carried out (Obrig et al., 1988; Tesh et al., 1991). HSVECs and HUVECs were significantly less sensitive to the cytotoxic activity of SHT and SLTs than cell lines such as Vero and HeLa cells, which 34 are often used in in vitro cytotoxicity assays. lt has been suggested that the reduced sensitivity of the HSVECs and HUVECs is due to the reduced levels of toxin receptor Gb3 in their cell membranes. lndeed, it has been estimated thar HSVECs and HUVECs have 1O- to 3O-fold less Gb3 than human kidney cortex (Boyd and Lingwood, 1989; Tesh et al., 1991; Tesh and O'Brien, 1991). These studies suggest that increased concentration of Gb3 in target cell membranes increases the cytotoxic effect of SHT and SLTs, and thus the cells of the human kidney, possibly glomerular endothelial cells, may be particularly susceptible to these toxins because of their higher concentrations of Gb3.
1.1 1.3 Role of Endotoxin and Cytokínes ín Pathogenesis ln HUS, the kidney is the primary site of vascular endothelial cell damage. Swelling and detachment of renal endothelial cells results in intra- vascular coagulation and deposition of fibrin and platelets within the glomerulus. These events lead to interruption of normal blood flow to the kidney and the subsequent renal failure often observed in these patients (Louise and Obrig, 1991). A component of this damage is thought to be directly toxin-mediated, but there is increasing evidence that cytokines and
inf lammatory factors may contribute to endothelial cell damage and the disease process. Also, it has been suggested that colonic vascular damage sustained by individuals with HC may be a pre-requisite for the development of HUS by providing increased access of SHT and SLTs, as well as bacterial lipopolysaccharide/endotoxin (LPS) in the bloodstream (Louise and Obrig, 1991). Studies carried out by Barrett et al. (199O) using a strain of mice with defective macrophage responses to LPS, showed that the mean survival time following challenge with purified SLT-ll was significantly increased compared with that of similarly challenged mice with normal macrophage responses. 35
Frequently pat¡ents with HUS exhibit endotoxaemia and it has been documented that LPS is capable of causing physiological changes in endothelial cells (Louise and Obrig, 1992l,. Thus, it is important to determine the relative role of toxin and LPS in the endothelial cell damage characteristic to this disease. Louise and Obrig (1992) tested the in vitro cytotoxicity of
SHT and LPS in HUVECS. A synergistic cytotoxic effect (which was dose- dependent for both) was observed. Protein synthesis of HUVECs in the presence of LPS was transiently depressed. However, protein synthesis inhibition was synergistically enhanced when HUVECs were treated with SHT in the presence of LPS. Louise and Obrig (1991) investigated the in vitro effects of SHT, interleukin-1ß (lL-1ß) and tumour necrosis factor-ø (TNF-ø) on HUVECS. TNF- ø alone did not inhibit HUVEC protein synthesis, but did have a mild cytotoxic effect. However, TNF-ø significantly increased both cytotoxicity and inhibition of protein synthesis due to SHT. Moreover, pre-treatment of HUVECs with TNF-ø increased their sensitivity to SHT. ll-lß, on the other hand, had no deleterious effects on HUVECs on its own, and did not augment SHT toxicity (Louise and Obrig, 1991). lnterestingly, van de Kar et al. (1992) have shown that pre-incubation of HUVECs with TNF-ø resulted in a 10- to 1OO-fold increase in binding sites for 1251-lub.lled SLT-1. They also found that lL-|, tumour necrosis factor-ß (TNF-ß) and LPS also increased SLT-I binding. Analysis of glycolipid extracts of TNF-ø-treated HUVECS revealed that there was a marked increase in Gb3 content. This study suggests that treatment of HUVECs with TNF-ø may directly result in de novo Gb3 receptor synthesis. Thus, inflammatory mediators may play an important role in the pathogenesis of HUS. 36
1.1 1.4 Animal Models for SLTEC Disease
Pai et al. (1986) developed an experimental model to study the role of SLTEC in the pathogenesis of diarrhoeal disease. Two- to three-day-old rabbits were inoculated intragastrically with a strain of E. coli 0157:H7 which was a high producer of SLT, and E. colí 0157:H45 which was SLT negative. The two groups were examined for levels of intestinal SLT, bacterial colonization and clinical symptoms. Diarrhoea developed in the group infected with the SLT-producing O157;H7, but not in the other group. Those animals infected with the SLTEC were found to have colonic colonization, detectable levels of free colonic SLT, and histological damage to the colonic mucosal surface. Moreover, the clinical symptoms and mucosal abnormalities were also induced in infant rabbits which had been inoculated with intragastric doses of partially-purified SLT.
Studies using gnotobiotic piglets have been less conclusive in demonstrating a role for SLT in pathogenesis. Tzipori et al. (1987) examined the capacity of several (u^157:H7 strains and derivatives which had sp'ontaneously lost the ability to produce SLT, or had been cured of a 60-MDa EHEC-associated plasmid, to cause disease after oral administration. The presence or absence of either putative virulence determinant had no impact on the capacity of a given EHEC stra¡n to cause diarrhoea and characteristic mucosal lesions. Introduction of the 60-MDa plasmid or transduction with SLT-I- or SLT-Il-encoding bacteriophages did not cause E. colí K-12 to become virulent in this model, even though free SLT could be detected in the piglets'serum (Tzipori et al., 1987). ln a more recent study, Li et al. (1993) demonstrated that diarrhoea and changes in ion transport, resulting from oral administration of the above stra¡ns to infant rabbits, could not be fully attr¡buted to either SLT or the plasmid. The authors suggested that other factors, perhaps the chromosomally-encoded EHEC eae gene product, could 37 also contribute to pathogenesis. However, the E. coli K-12 derivative containing the plasmid and expressing SLT-I was capable of causing diarrhoea (Li er al., 1993).
Fontaine et al. (1988) constructed a derivative of S. dysenteriae type 1 in which the SHT gene had been insertionally inactivated. Macaque monkeys were infected intragastrically with either this strain or the wild-type parent to determine the role of SHT in the pathogenesis of bacillary dysentery. This study suggested that SHT increased the severity of disease by inducing colonic vascular damage (resulting in bloody stools) and intestinal ischaemia. However, both strains were capable of causing fulminant dysentery (Fontaine et al., 1988).
Studies employing a rabbit model for HC, have shown that intra- peritoneal infusion of purified SLT-ll, results in caecal lesions and diarrhoea analogous to the colonic diséase observed in humans. Acute focal tubular necrosis of the kidneys was also observed in these animals (Barrett et al., 1989). Further animal studies have found that streptomyc¡n-treated mice died 4 or 5 days following ingestion of E. coli K-12 containing SLT-ll genes cloned into a high-copy-number expression vector. Subsequent histopathological examination of the kidneys of these mice revealed bilateral cortical tubular necrosis (Wadolkowski ef a/., 1990). ln another experiment, streptomycin-treated mice died after being fed an SLTEC strain (which was capable of colonizing the mouse gut), which produced SLT-l and SLT-ll. Interestingly, passive transfer of neutralizing monoclonal antibodies directed against either the A or B subunit of SLT-ll imparted protection to these mice, whilst passive transfer of anti-SLT-l antibodies did not (Wadolkowski et al., 1990; Tesh and O'Brien, 1991). Most SLTEC from cases of HC have been shown to produce SLT-I and SLT-ll, or SLT-ll alone, lsolates producing SLT-I only are less common (Smith and Scotland, 1988). Similarly, the incidence of 38
ser¡ous renal or circulatory complications is higher in individuals infected with SLTEC producing both SLT-I and SLT-ll or just SLT-ll, than in individuals infected with sLTEC producing sLT-l only (scotland et al., 1988; ostroff ef al., 1989; Tesh and O'Brien, 1991). lnterestingly, commercial immunoglobulin preparations are known to contain both SHT- and SLT-I- neutralizing antibodies, but very rarely contain neutralizing antibodies to SLT- ll (Ashkenazi et al., 1988; Tesh and O'Brien, 1991). This might imply that the human antibody response to SLT-ll in humans is rather poor, but the possible role of such a differential immune response for SLT-I and SLT-ll in the development of HUS or other SLTEC-related diseases is not understood. Nevertheless, these findings suggest the possibility that SLT-I| is a more important determinant than SLT-I in SlT-related colonic disease, as well as the more serious classical sequelae (Tesh and O'Brien, 1991). Recent work (Richardson et al., 1992l' describes the establishment of experimental verocytotoxaemia in rabbits as an animal model for investigation of the pathogenesis of SLTEC-related disease, in particular HUS. Purified 125¡-labelled SLT-I was injected intravenously into either non-immune or SLT- t-immunized rabbits. lt was found that labelled toxin rapidly disappeared from the bloodstream and was specifically concentrated and localized in the tissues of the gastrointestinal system (GlS) and the central nervous system (CNS)' ln the non-immune rabbits the highest concentrat¡on of labelled toxin was found in the caecum, brain, small intestine, colon and spinal cord. Uptake of toxin in both the CNS and GIS was inhibited in the immunized rabbits, but organs involved with processing of immune complexes including the spleen, liver and lungs showed the highest uptake. The immunized rabbits also exhibited increased SlT-l-neutralizing antibody titres and all survived the challenge with toxin. The non-immunized rabbits developed a range of symptoms including non-bloody diarrhoea and paralysis, followed in most cases by death. Histopathological examination revealed oedema and haemorrhage in the 39
mucosa and submucosa of the caecum, as well as oedema, haemorrhage and neuronal necrosis in the brain and spinal cord (Richardson ef al., 1992). The above study provides further evidence for the targeting of specific organs in SLT-related disease states. lmmunof luorescence studies of damaged tissues of the spinal cord and caecum (where high concentrations of 1251-lub"ll"d toxin were demonstrated), confirmed that there was specific binding of toxin to vascular endothelial cells. As suggested in earlier work reported above, differences in distribution of Gb3 (the receptor for SLTs) would account for higher concentrations of toxin in specific tissues (Boyd and Lingwood, 1989). However, ¡nterpretation is complicated by the fact that there are major differences in the distribution of Gbg receptors between humans and rabbits. For example, Gb3 is a major component of glycolipids in human renal tissue but not of rabbit kidney (Boyd and Lingwood, 1989; Richardson et al., 1992]l. Thus, the rabbit is not an ideal model for human HUS. However, despite this, the work provides evidence for a link between the tissue damage sustained, the concentration of labelled toxin used and the receptor distribution, which is directly relevant to the study of pathogenesis of SLTEC-related diseases.
1.'l 1.5 Vaccínation Against SLTEC Dísease. Animal studies have established a role for SLT-llv in the pathogenesis of piglet oedema disease. The characteristic lesions and symptoms associated with this disease can be induced in healthy pigs by intravenous injection of purified SLT-llv (MacLeod et al., 1991). The possibility of protecting pigs from
oedema disease by immunization against SLT-Ilv has been investigated. Work carried out by Macleod and Gyles (1991) involved passive immunization (with a porcine-derived immunoglobulin preparation raised against purified SLT-Ilv) of 6 week old piglets, and the active immunization with a toxoided preparat¡on of purified SLT-llv of 6- and 2-week-old piglets. Both actively and 40 pass¡vely immunized animals, and a group of un-immunized, age-matched controls, were challenged intravenously with purified SLT-llv (6ng/kg body weight). All of the immunized group survived the challenge but animals which had the poorest post-immunization antibody response exhibited mild symptoms of oedema disease. All of the control animals died within 30 hours of challenge (Macleod and Gyles, 1991). Recently, Gordon et al. (1992) used oligonucleotide-directed mutagenesis to alter Glu167 of the A subunit of SLT-llv to Gln and demonstrated that .this resulted in a reduction in enzymatic activity by approximately 15OO-fold, and in the cytotoxic activity by tO6-totd (Gordon ef al., 1992). Pigs were immunized with a purified preparation of this mutant toxin and they developed high neutralizing antibody titres, lmportantly, the immunized animals did not develop pathological lesions usually associated with oedema disease. This group has reported that future work will involve the assessment of the suitability of this candidate vaccine for protection of neonatal piglets against oral challenge with an oedema disease strain (Gordon et al., 1992l.. The work described above outlines the advances that have been made in the development of a veterinary SLT-llv vaccine. SLTEC related disease in humans is serious and geographically widespread and human populations would derive benefit from the development of an efficacious vaccine. Boyd et al. (1991) outlined evidence that the B subunit of SHT/SLT-I might be a suitable immunogen; several B subunit-specific SlT-neutralizing monoclonal antibodies have been reported (Strockbine et al., 1985), and neutralizing polyclonal antibodies were isolated from hyperimmune sera using affinity chromatography with immobilized B subunit (Donohue-Rolfe et al., 1984). Moreover, synthetic peptides equivalent to the N-terminal and C-terminal regions of the B subunit of SHT have been shown to be immunogenic in mice (Harari et al., 1988, Harari and Arnon, 1990). Boyd et al. (1991) 4t demonstrated that polyclonal rabbit antibodies against a synthetic peptide corresponding to residues 28-40 of the B subunit neutralized the SLT-I cytotoxicity for Vero cells. lnterestingly, however, antibodies directed against either N-terminal or C-terminal peptides were less effective and it was suggested that the precise mode of presentation of the pept¡de antigen to the
immune system was a critical factor. They proposed that the (non-toxic) B subunit might be safer than an alternative vaccine based on a toxoid of the holotoxin and would not involve complicated conjugation of peptides to carrier molecules. Rabbits immunized with purified SLT-l B subunit were protected from challenge with a lethal dose of purified SLT-I (Boyd et al., 1991). An alternative strategy for developing vaccines against SHT and SLTEC disease involves the expression of protective SLT or SHT epitopes on the cell surface of live attenuated vaccine carrier organisms. Su et al. (1 992) described the construction of fusions of the complete B subunit gene, or N- terminal segments thereof, with the gene for the E coli ouTer membrane protein LamB. The SHT sequences were inserted at a point in the LamB gene which encoded a surface exposed loop of the protein. These constructs directed the expression of the hybrid LamB/SHT protein on the surface of E coti K-12 and wild type Salmonella typhimurium, but when the attenuated S. typhimurium aroA vaccine strains were used, the recombinant antigen was confined to the cytoplasm, presumably a consequence of membrane export defects in these strains. In spite of this, oral immunization of mice with these latter constructs resulted in production of significant levels of SHT B subunit- specific antibodies, both in serum and in intestinal washings. lntraperitoneal immunization with the same strains also resulted in a significant humoral immune response to the SHT B subunit. Ryd and Lindberg (1992) also employed the LamB expression system to express a 14 amino acid epitope (residues 13-26) of the B subunit of SHT on the surface of an attenuated 42
vaccine strain of Shígetta flexneri. lntravenous immunization of three month- old rabbits with the recombinant S. flexneri strain elicited the production of specific antibodies directed against the 14 amino acid peptide and against the whole B subunit molecule.
1.12 Diagnosis of SLTEC lnfection
1.12.1 Detection of Faecal SLT and SLTEC Diagnosis of SLTEC infection originally involved testing cell-free faecal extracts and/or culture filtrates of pure isolates for cytotoxicity using either Vero or HeLa cells in tissue culture. lt has been reported that in a significant o/o proportion of patients with HUS, SLTEC may account for less than 1 of their faecal flora (Smith and Scotland, 1988; Karmali, 1989) and this has necessitated the testing of large numbers of individual colonies. Such procedures are labour intensive, and not always successful, as the numbers of SLTEC in faeces are even lower ¡f the sample has been collected one to two weeks after the onset of symptoms. Karmali et al. (1985) improved the chances of detection of low numbers of SLTEC, by testing mixed faecal cultures which had been treated with polymyxin B to release cell-associated SLT. lt was determined that this method could detect 1 SLTEC colony in
1 00. It is possible that the cytotoxicity observed when testing culture filtrates of individual isolates or faecal filtrates may be due to the effects of other bacterial products or toxins. Therefore, definitive toxin identification requires demonstration of toxin neutralization by specific antisera raised against the various SLTs (Smith and Scotland, 1988). Although detection of SLT by tissue culture cytotoxicity is a valuable diagnostic method, it is time consuming and cumbersome, and not all microbiological diagnostic laboratories are appropriately set up for tissue culture work. 43
1.12.2 Sorbitol-MacConkey Agar for Detection of EHEC This method of detection of SLTEC was based on the strong association of SLTEC-related disease with the isolation of the otherwise rare E. coti serotypes O157:H7 and 0157:H-. While 95o/" of E. coli are able to ferment sorbitol these serotypes cannot (Krishnan et al., 1987). Sorbitol- MacConkey agar plates are inoculated with the faecal specimen and examined after 18-24 hours incubation for the presence of colourless, sorbitol-negative colonies. These colonies can then be tested by slide agglutination with 0157 and H7 antisera, and then tested for SLT production in tissue culture assays (Karmali, 1989). Obviously this method is only useful for detecting SLTEC belonging to serotype O157. Since SLTEC disease has been associated with a number of other EHEC and EPEC serotypes, methods which are not serotype-specific are more useful in diagnosis. Ritchie et al. (1992) conducted a comparative study of direct faecal SLT assay with Sorbitol-MacConkey agar culture for diagnosis of SLT infection. As expected, the direct faecal toxin assay was more sensitive than sorbitol-MacConkey agil selection, because of the occurrence of non-O157 serotypes responsible for SLTEC disease, Also, it has been reported recently that sorbitol positive E. coli O157:H- isolates have been associated with both outbreaks and sporadic cases of HUS (Gunzer ef al., 1992). Furthermore, in a survey conducted by this group, 17 out of 44 HUS patients were identified to have E. coli O157 strains that were sorbitol fermenters and were positive for ß-D-glucuronidase activity (this biochemical test is negative in E. coli O157:H71. The 17 sorbitol positive strains were cytotoxic for Vero cells and confirmed as SlT-positive by molecular methods that will be discussed in Section 1.12.6. 44
1.12.3 Rapíd Biochemical Test It has been estimated that 96% of E. coli strains produce the enzyme ß-D-glucuronidase. This enzyme can be readily detected fluorigenically using the substrate 4-methylumbelliferyl-ß-D-glucuronide (MUG), cleavage of which generates the fluorescent product 4-methylumbelliferone. lsolates of E. colí Serotype O157:H7, however, have been shown not to produce the enzyme and are referred to as being MUG negative (Krishnan et al., 1987; Ratnam ef al., 1988). Thompson ef a/. (199O) examined 188 serotype O157 strains using this method; 166 isolates were MUG-negative and SlT-positive, while the remaining 22 isolates were all MUG-positive and SLT-negat¡ve. Again, this method is not useful in cases of SLTEC-related disease where the causative organism is of a serotype other than O157. Moreover, Gunzer et at. (1992) recently reported the isolation of a number of SLT-ll-producing E. coli strains that were MUG-positive.
1.12.4 Serological Methods of Detectíon SLTEC-related disease can also be diagnosed serologically, on the basis of rising antibody titres against SLT itself or O157 lipopolysaccharide (LPS) (Karmali, 1989; Bitzan et al., 1991). This can be particularly useful for aetiological diagnosis of HUS late in the course of illness, when the numbers of SLTEC in faeces may be extremely low. Antibodies to SLT can be demonstrated using tissue culture cytotoxicity neutralization assays (Karmali, 1989), while Bitzan and Karch (1992) have described an indirect haemagglutination assay based on anti-O157 LPS. Barrett et al. (1991) described the development of three enzyme-linked immunosorbent assays (ELlSAs) for the detection of antibodies to SLT-|, SLT-Il and E. cotí 0157 LPS. For the SLT ELlSAs, SLT-I- and SlT-ll-specific monoclonal antibodies were bound to plates and used as catching antibodies to immobilize the respective toxin from crude extracts (Strockbine et al., 1985; Downes et al., 1988). For 45 the anti-O157 LPS ELISA, LPS purified from E. coli 0157:H7 strain 933 was used to coat plates (Barrett et al., 1991). Antibody levels were determined by incubating dilutions of serum in these antigen-coated plates, followed by colorimetric detection using anti-human immunoglobulin-enzYme conjugates (Barrett et al., 1991).
1.12.5 ELtSAs for the Direct Detectíon of SLTs ELISAs have also been developed for direct detection of SHT and SLT-I and sLT-ll (Donohue-Rolfe et al., 1986; Kongmuang ef al., 1987; Downes ef al., 1g8g). These assays involved use of immobilized monoclonal or affinity- purified antibodies to the toxins as catching antibodies, with bound toxin detected by a second-antibody-enzyme conjugate. Whilst these assays are highly specific and very useful for testing E. coli isolates for SLT production, they are not sufficiently sensitive to reliably detect low levels of SLT in faecal extracts (Downes et al., 1989). Ashkenazi and Cleary (1989, 199O) have developed a slightly different ELISA for detection of SHT and SLT-I from pure and mixed cultures using the natural receptor Gb3 as a capturing ligand. Monoclonal antibodies specific for SHT or SLT-I were then used to label the immobilized toxin and these were in turn detected using an anti-mur¡ne immunoglobulin-enzyme conjugate. An identical capture mechanism could be
used for SLT-ll, which also binds to Gb3.
'1. I 2.6 Molecular Bíologícal Diagnosis The availability of cloned SLT-l and SLT-ll genes enabled the development of DNA probes for the detection of SLTEC (Willshaw et al., 35S 1987; Newland and Neill, 1988). Probes labelled with 32P or could be used for testing large numbers of faecal E coli isolates, or for the direct screening of colonies on primary isolation plates, for the presence of SLT genes, by colony hybridization (Scotland ef al., 1988; Karmali, 1989; Thomas 46 ¡ t et al., 1991). These procedures were both highly sensitive and specific, and when stringent washing conditions were used, strains producing SLT-|, SLT-
ll, or both could be differentiated. However, radioactively-labelled probes do
have disadvantages for clinical laboratories, such as the time lag before a result is available (long autoradiographic exposures may be required to maximize sensitivity), limited probe life due to the short half-life of the labels (particula ¡V 32n and the problems associated with handling and disposing of radioisotopes. However, these can be largely overcome by the use of non- radioactive labels such as digoxigenin (DlG) and biotin and SLT probes employing these have been used for detection of SLTECs without loss of sensitivity or specificity (Thomas ef al., 1991). The availability of nucleotide sequence data for SLT genes has permitted the design of synthetic oligonucleotide probes for detection of * SLTEC (Brown et al., 1989; Karch and Meyer, 1989a). Some oligonucleotide tr probes were based on sequences which are highly conserved amongst the various SLTs, and so permitted detection of all classes of the toxin genes. Other probes were directed against less conserved regions, which, under the
appropriate hybridization/washing conditions, distinguished between SLT-¡, SLT-ll and SLT-llv genes (Brown et al., 1989). Access to sequence data for the various SLT genes has also permitted design of a variety of oligonucleotide primer sets for amplification of SLT genes using the polymerase chain reaction (PCR) (Karch and Meyer, 1989b;
Pollard et al., 1990; Brian et al., 1992; Gannon et al., 1992l'. Use of PCR technology permits detection of SLT genes from samples which are microbiologically complex (such as faeces or foodstuffs), including samples containing non-viable organisms (Jackson, 1992). PCR assays are extremely
sensitive and if secondary Southern blot or dot-blot hybridization with labelled oligonucleotides is used to detect PCR products, as little as I SlT-containing bacterial genome per assay can be detected (Brian et al., 19921. This I 47 I ,L'|. Þ^- sensit¡vity is very important, because the numbers of SLTEC in the faeces of patients with serious SlT-related diseases such as HUS are often very low
(Smith and Scotland 1988; Karmali, 1989). Some of the SLT PCR assays
described so far combine different primer pairs for SLT-I and SLT-Il, or SLT-Il and SLT-Ilv in the same reaction, that direct the amplification of fragments which differ in size for each toxin type (Johnson et al., 199O; Pollard et al.,
1990; Brian et al., 1992; Gannon et al., 1992). Other SLT PCR assays use single pairs of primers based on consensus sequences, which are capable of amplifying all SLT types, with subsequent identification of SLT type requiring Southern or dot-blot hybridization with labelled oligonucleotides directed
against type-specific sequences within the amplified fragment (Karch and Meyer, 1989b). Apart from the added sensitivity, secondary hybridization steps act as independent confirmation of identity of the amplified product.
Apart from its use in direct detection of SLT sequences, PCR can also be
used for preparation of DlG-labelled DNA probes by performing the PCR reaction in the presence of DIG-labelled nucleotides (Jackson, 19921.
1.13 Aims of the Work in this Thesis
Whilst a number of epidemiological surveys of the prevalence of SLTECs in human gastrointestinal disease have been carried out overseas (Smith and Scotland, 1988; Karmali, 1989), there is little or no such information for the Australian population, An unpublished survey conducted at the Adelaide Children's Hospital during 1988-90, involving screening of sorbitol-negative E. coli isolates from 5O0 cases of acute diarrhoea in children, failed to detect any serotype O157:H7 organisms. Bettelheim et al. (199O) examined the gut contents of babies who died in Adelaide and Melbourne of Sudden lnfant Death Syndrome (SIDS) as well as the faeces of healthy babies, for the presence of E. coli strains producing cytotoxins. 48
Strains which were cytotoxic for Vero cells were isolated from 13 of 46 SIDS I babies but were not detected in the healthy control group. This study did
not, however, include confirmation of the identity of the putative cytotoxin by antibody neutralization, or any molecular biological analysis of the isolates.
As discussed earlier in this chapter, sequence variations have been detected in SLT-ll genes from a number of human and animal SLTEC isolates. Some of these variations alter the in vitro and in vivo properties of the toxins themselves, including their antigenic structure. For this reason, information concerning sequence variation within SLT genes is important for the future development of vaccines for prevention of SLTEC disease in humans or animals. All of the SLT genes sequenced to date have been derived from SLTEC isolates from the northern hemisphere and these may not necessarily be representative of those from other parts of the world. The aim of the
I present study was to obtain more detailed information on the prevalence of SLTECs in Adelaide children, and to conduct a comprehensive molecular biological analysis of the SLT genes of such strains. 49
CHAPTER TWO I MATERIALS AND METHODS
2.1 Bacterial Strains Shigella dysenteriae type 1 was a clinical isolate provided by Dr. J. Lanser, Division of Clinical Microbiology, lnstitute of Medical and Veterinary Science, Adelaide.
E. coli K-12 strain JM109 lgyrA96, recA-1, relA-1, endA-1, thÌ- I , hsdRl T, supE44, lambda-, A,Uac-proABl, IF', traD36, proAB, lacl9, lacZAM /51) has been described by Yanisch-Perron et al. (1985).
E. colí K-12 strain C60O (F-, thi-1, thr-1 , leu76, lacYl , tonA21, supE44, mcrA, lambda-) has been described by Appleyard (1954).
E. coli strain H30 (serotype 026) was isolated from an infant with diarrhoea, described by Konowalchuk (1977), and was obtained from Dr. K. Bettelheim, Fairfield Hospital, Victoria.
E. coli strain 031 (serotype OX3:H21l' was isolated from the small bowel contents collected post-mortem from a 7 month-old male infant who died suddenly and unexpectedly, and in the absence of any obvious cause of death at autopsy, was classified as a case of SIDS.
E. coli strain PH (serotype O111:K-:H-) was isolated from the faeces of a 12 month-old male infant admitted to the Adelaide Children's Hospital with
HUS. 50
E. coti strain 87028 was a SLT-Il-producing reference strain obtained from Dr. K. Bettelheim, Fairfield Hostpital, Victoria'
2.2 Bacteriophages SlT-l-encoding bacteriophages H198, H19J, H19A and 933J and the
SLT-¡ l-encoding bacteriophage 933W, were provided as E coli C6OO lysogens by Dr. A. O'Brien, Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA. E. coli C6OO lysogenized with bacteriophage lambda was obtained from the Dept. of Microbiology and lmmunology, University of Adelaide.
2.3 Cell Lines Vero cells (African green monkey kidney cells) (ATCC CCL 81) and HeLa 229 cells (Human cervical carcinoma cells) (ATCC CCL 2'1l' were obtained from the Division of Medical Virology, lnstitute of Medical and Veterinary Science, Adelaide.
2.4 Bacterial Growth Media All bacterial growth media were prepared using water which had been deionized by passage through a Hi-pure deionization system (Permutit, Australia).
2.4.1 Luria-Bertani Medium (LB) Bacteria were routinely grown in LB medium, which contained 1% Bacto-tryptone (Difco Laboratories, Detroit, Michigan, USA.), O.5o/o Bacto- yeast extract (Difco), 1% NaCl , pH 7.5, with or without 1.5% Bacto-agar (Difco). LB broths and LB agar were sterilized by autoclaving ar. 121oC for 15 minutes. Where appropriate, ampicillin (Commonwealth Serum Laboratories, Parkville, Vic.) was added to growth media at a concentration of 50 lglml' 51
2.4.2 LB Ptates Supplemented with Ampicillin, IPTG and X-gal A solution containing 1OO ¡tl of sterile water, 20 ¡tl of 25 mg/ml IPTG (Boehringer Mannheim) and 30 ¡tl of 20 mg/ml X-gal (Boehringer Mannheim) in dimethyl formamide (DMF) was spread over the surface of LB agar plates
supplemented with 50 Uglml of ampicillin. These plates were stored for later use for up to a week at 4oC.
2.4.3 Soft LB Agar Soft LB agar for use in isolation of bacteriophages was prepared as for LB agar, except that the concentration of Bacto-agar was O.7o/". Three ml oC aliquots were dispensed into tubes and autoclaved at 121 for 1O minutes'
2.4.4 MacConkey Agar No.l MacConkey agar No. 1 was prepared by mixing 50g of Bacto MacConkey agar (Difco Laboratories) with one litre of water and was
autoclaved at 1 21oC for 15 minutes.
2.4.5 MacConkey.Agar No. 2 MacConkey agar No.2 consisted of 51.5 g of Oxoid MacConkey agar No. 2 (Unipath Ltd., Basingstoke, England) in one litre of water and was autoclaved at 121oC for 15 minutes.
2.5 Cell Culture Media Cell culture growth medium (GM) for HeLa 229 and Vero cells consisted of Dulbecco's Modified Eagles Medium (DMEM) buffered with 20 mM HEPES (Cytosystems, castle Hill, N.s.w., Aust.) supplemented with 10% (v/v) foetal calf serum (FCS) (Cytosystems), 1o/o (vlvl of a 200 mM solution ofL-glutamine(Cytosystems)and1o/o(v/v)ofasolutioncontaining5000 52 lU/ml penicillin and 5 mg/ml streptomycin (Cytosystems). Cell monolayers were maintained in maintenance medium (MM) which was the same as GM, except that the concentration of FCS was 2o/" lvlvl. Cell culture media were prepared using pyrogen-free deionized water (surgical irrigation grade, Baxter Healthcare Pty. Ltd., N.S.W., Aust.), and were sterilized by filtration through a O.45 pm membrane (Millipore Corporation, Milford, Mass., USA.).
2.6 Monoclonal Antibodies Culture supernatants of hybridoma lines 13C4 (specific for SHT and
SLT-I) and 11E10 (specific for the A subunit of SLT-ll) were used as a source of SlT-neutralizing monoclonal antibodies. The hybridomas were obtained from the American Type Culture Collection, Rockville, Maryland, USA., (catalogue Nos. ATCC CRL 1794 and ATCC CRL 1907, respectively) and supernatants were provided by Dr. K. Bettelheim.
2.7 Cloning Vectors The cloning vector pUC19 has been described by Yanisch-Perron et al. (1g85) and pBLUESCRIPT SKTM was obtained from Stratagene, La Jolla, Ca.,
USA
2.8 Routine Chemicals and Reagents Tris(hydroxymethyl)aminomethane (Tris) was purchased from
Boehringer Mannheim (Aust.), North Ryde, N.S.W., Austral¡a.
Sodium dodecyl sulphate (SDS) and N-lauroylsarcosine (sarkosyl) were purchased from Sigma Chemical Co., St. Louis, Mo., USA' 53
Unless otherwise stated, all other chemicals and solvents were at least AR grade and were obtained from Ajax Chemicals, Auburn, N.S.W.
2.9 Solutions and Buffers All buffers and solutions were prepared using water which had been deionized by passage through a Hi-pure deionization system (Permutit). Where required, these were sterilized by autoclaving at 121oC for 15 minutes, or by filtration through O.22 pm ü O.45 ¡rm membranes (Millipore). Tris-EDTA buffer (TE) consisted of 1O mM Tris-HCl, 1 mM EDTA, pH 8.0. Standard Saline Citrate (SSC) consisted of 15 mM tri-sodium citrate, 150 mM NaCl, pH 7.O. This was prepared as a 20x stock solution and diluted as required. lOOx Denhardt's solution consisted of 2o/o lwlvl Bovine serum albumin
(Commonwealth Serum Laboratoriesl, 2o/o (w/v) Ficoll (Pharmacia-LKB), 2o/o (w/v) polyvinyl-pyrolidone.
2.1O Restriction Endonucleases and other Enzymes All restriction endonucleases were obtained from Boehringer Mannheim. DNA polymerase I (Klenow fragment), terminal transferase, S1 nuclease, RNase-free DNase l, pronase and proteinase K were also obtained from Boehringer Mannheim. Pancreatic RNase A and DNase I were obtained from Sigma. Taq polymerase (DNA polymerase from Thermus aquaticus) was supplied by Bresatec, Thebarton, S.4., or Boehringer Mannheim. 54
2.1 1 Hybridization Membrane The nylon hybridiztion membrane used for Southern and dot blot hybridization analyses was Hybond N+, purchased from Amersham, U.K.
2.12 lsolation of Bacterial Strains Faeces or gut contents were directly plated onto MacConkey No. 1 or MacConkey No. 2 plates and incubated overnight at 37oC. Typical E. coli colonies were picked off onto LB plates or grown in LB broth in 96-well (U- bottomed) microtitre trays, and preserved as described in Section 2.13. For mixed cultures, gut contents or faeces were inoculated directly into 1O ml LB broths and incubated overnight at 37oC with vigorous shaking. One ml aliquots of each mixed culture were processed for subsequent PCR analysis or preserved in glycerol, as described in Sections 2.42 and 2.13, respectively.
2.13 Preservation of Bacterial Strains One or 2 ml of an overnight LB culture (with or without ampicillin as appropriate) was transferred to sterile cryotubes (Nalgene, Nalge Co., Rochester, NY, USA) and glycerol was added to a final concentration of 15o/o. Duplicate vials were prepared for each bacterial isolate; one was stored at 7O"C for long term storage and the other was stored at -15oC for routine
use. Mixed cultures from faeces or gut contents were also preserved and stored as described above. Alternatively E. coli strains, including gene banks, were preserved in 96-well microtitre plates in LB supplemented with 15o/o (v/v) glycerol and stored at -7OoC. 55
2.14 Serotypin g of E. coli lsolates Serotyping of E coli isolates was carried out by Drs. F. and l. Orskov or Dr. F. Scheutz, International Escherichia and Klebsiella Centre, Statens Seruminstitut, Copenhagen, Denmark.
2.15 Cell Culture Vero and HeLa 229 cells were grown in 20 ml of GM in 75 sq. cm. tissue culture flasks (Corning, Corning Glass Works, New York, USA) and incubated at 37 oC in an air incubator. Cells were passaged once a week, or when they had formed a confluent monolayer. Old GM was poured off and cell layers were washed twice with 1O ml of calcium-magnesium-free phosphate buffered saline (PBS) (8 g NaCl, O.2 g KCl, 1.15 g Na2HPO4 and O.2 g KH2PO4 per litre of water, pH 7.4l'. Cells were then trypsinized by addition of 1.0 ml of O.25% trypsin-EDTA solution. This contained 10 ml of 2.5o/o trypsin (Cytosystems) with 1.O ml of 2o/" (w/v) EDTA and 89 ml of diluent (8 g NaCl, O.4 g KCl, 0.06 g Na2HPO4, O.O6 g KH2PO4 and 0'5 g NaHCO3 per litre of water). Flasks were gently rocked such that the cell monolayers were coated with trypsin. Excess trysin was removed and flasks were incubated at room temperature for 2 minutes, or until the cells detached from the plastic. Cells were then resuspended in GM and dispensed into new flasks. Vero and HeLa cells were routinely diluted 1:10 at each passage. When required, confluent cell monolayers could be maintained in MM at 37"C for up to 4 days. When cells were required for toxin assays, trypsinized cells were resuspended in GM at a density of 106 cells/ml. Two hundred ¡tl of cell suspension was dispensed into each well of 96-well flat-bottomed microtitre trays (Falcon Microtest lll, Becton Dickinson, New Jersey, USA.) and incubated overnight at 37 oC for use the following day. 56
2.16 Storage of Cells and Recovery from Liquid Nitrogen Freshly trypsinized Vero and HeLa cells (in exponential phase of growth) were resuspended in DMEM containing 20% (vlv) FCS and 1O% (v/v) dimethyl sulphoxide and 1-2 ml aliquots (containing approximately 106 cells) were dispensed into cryogenic ampoules (Nalgene). Cells were then held at -7O"C tor 24 hours and subsequently transferred into liquid nitrogen. To recover cells from liquid nitrogen, ampoules were thawed rapidly in warm water, and cells were dispensed into centrifuge tubes and made up to 10 ml in GM. Cells were pelleted by centrifugation at 1,500 x g for 10 minutes. Cell pellets were resuspended in 2O ml GM, dispensed into a tissue culture flask and incubated at 37oC in air.
2.17 Cytotoxicity Assays To prepare extracts of E. coli and S. dysenteriae for cytotoxicity assays, ten ml overnight LB cultures were centrifuged at 3,0O0 x g for 10 m¡nutes, The supernatants were transferred to new centrifuge tubes and the pellets were resuspended in 1O ml of PBS. The bacterial pellets in PBS were then lysed by passage through an Aminco Model FA-073 French pressure cell (SLM Aminco, Urbana, ll., USA.) operated at 16,000 psi. The supernatants and the French press lysates were then filtered using a syringe-mounted O'2
¡rm filter unit (Millipore) and stored on ice for use in toxin assays on the same day. Where required, 1 ml aliquots of supernatants and French press lysates were also stored at -7OoC for later use. SLT cytotox¡city was assayed using Vero cells or HeLa 229 cells (cultured in GM), which had formed confluent monlayers in 96-well flat- bottomed microtitre trays. The GM from these trays was removed by aspiration and the cell monolayers were washed with of PBS. The PBS was removed by aspiration and the monolayers were treated with 50pl of
I either culture supernatants or French press lysates which had been serially 57 diluted in MM. The microtitre trays were incubated at 37 oC for 3O minutes and then the volume in the wells was made up to 2OOltl with MM. Cytotoxicity was assessed after 3 days of incubation at 37oC. The CD59 titre was defined as the maximum dilution of sample producing a cytopathic effect on at least 5Oo/" of the cells, A permanant record of SLT cytotoxicity assays could be made by fixation and staining of the cells in the microtitre trays. Detached cells were removed by vigorous shaking for 1-3 minutes in a Dynatech microplate shaker (Dynatech Laboratories, Alexandria, Virginia, USA.) and removal of culture media by drainage on absorbent towelling. The remaining cells were fixed by the addition of 1 drop/well of a 2o/o solution of formalin in PBS. This fixative was removed after one minute and cells were stained with 0.13% crystal violet in PBS, supplemented with 57o ethanol and 2o/o tormalin for 20 minutes (1-2 drops/well). Excess stain was removed by gently washing the microtitre trays with de-mineralized water. The trays were then air dried and stored.
2.18 SLT Neutralization Assays Serial ten-fold dilutions (final volume/well was 9O pl) of French pressure cell extracts or supernatants of SHT or SLTs were prepared in U-bottomed microtitre trays. Ten pl of undiluted SHT/SLT-l- or SLT-ll-neutralizing hybridoma supernatants (13C4 or 11E1O, respectively) were added to each well, and the microtitre tray was incubated at 37 oC for 30 minutes. The cytotoxicity of the pre-incubated toxin/monoclonal antibody mixtures for Vero cell monolayers was then determined as described in Section 2.17.
2.19 UV lnduction and lsolation of Bacteriophages The procedure for induction of bacteriophages from E coli cultures was adapted from that described by O'Brien et al. (1984). Ten ml overnight LB broth cultures were centrifuged for 10 minutes at 3,000 x g. The 58
supernatants were poured off and the bacterial pellets were resuspended in 2 ml of 1O mM CaCl2. The bacterial suspensions were then transferred to plastic Petri dishes and irradiated with UV on a UVP model TM15 transilluminator (UV Products lnc., San Gabriel, Ca., USA.)' One ml aliquots were removed after 1 and 2 minutes irradiation and added to 10 ml of LB. The irradiated bacteria were then incubated at 37"C for 3 hours with vigorous shaking. After this, 3-4 drops of chloroform were added to each culture and they were stored overnight at 4oC. The cultures were then centrifuged for 1O minutes at 3,O00 x g. The supernatants were retained and filtered using a syringe mounted O.45 ¡tm filter (Millipore). Ten-fold serial dilutions (1O-1 - 10-6) were prepared in SM buffer (1OO mM NaCl, 1 mM MgSO4, 50 mM Tris-HCl (pH 7.5), O.O1o/" gelatin) and 1OO Al of each of these were combined in 1O ml test tubes with 1OO ttl of a mid-exponential phasecultureofE.coliC6oo(A600=o.2)andincubatedat3ToCfor30 minutes. These were combined with 3 ml of molten soft LB agar, which had been cooled to approximately 45oC, and then poured evenly over the surface of LB agar plates. After the soft agar had set, the plates were incubated overnight at 37oC. Phage plaques on the C6O0 lawn were excised from the agar using a ster¡le scalpel blade. These were transferred to eppendorf tubes containing 5OO Ul of SM buffer and stored overnight at 40C. Eluted bacteriophages were plaque-purified by two cycles of dilution in SM followed by re-plating on E coli C6OO and re-isolation of single plaques. High titre stocks were prepared by plating 300 øl of purified eluate on E colí C6OO, and harvesting the phage. This involved the addition of 5 ml of SM buffer to the plates and incubation at room temperature with gentle agitation tor 2-3 hours. The eluted phage were removed and transferred to 1O ml centrifuge tubes' Filty ltl of chloroform was added to each tube and they were then vortexed briefly and centrifuged at 3,OOO x g for 10 minutes. The supernatants were transferred to new sterile 1O ml centrifuge tubes and stored at 4oC. Phage 59 stock titres were determined by plating serial dilutions on C600 as described above, or if higher titre stocks were required, further plating and harvesting of phage from plates showing confluent lysis was carried out. E. coli C6OO could not always be used for isolating bacteriophages from UV-induced E. coli isolates due to the production of colicins by the clinical isolates. In such circumstances, further aliquots of the induction extracts were plated on colicin-resistant C600 derivatives isolated from the original plating experiment. Plaque purification could then be performed using E. coliC600 as host.
2.2O Preparation of E colí C6OO Lysogens To prepa re E. coli C600 lysogens, purified phage (approximately 107 pfu) were plated on C60O and the resultant growth was streaked for single colonies on a fresh LB plate. Ten ml LB broth cultures were inoculated with 6 representative single colonies and incubated overnight at 37 oC with vigorous shaking. The bacterial cultures were induced with UV irradiation and tested for the presence of phage by plating onto E. coli C6OO, as described in Section 2.19. Those colonies which tested positive for phage were retained.
2.21 Bacteriophage lmmunity Studies
ln order to test for bacteriophage superinfection capacity, QO31 or øPH (approximately 7OO pfu in lOO ¡tl of SM), were combined with 1OO ttl of mid- exponential phase cultures of E. coli C60O and various C6OO lysogens, Tubes containing the bacteriophage and bacterial cells, were incubated at 37oC for 30 minutes and were then combined with 3 ml of molten soft LB agar, which was poured evenly over LB agar plates. Plates were incubated at 37oC overnight and scored for plaques. One hundred Ul of each lysogen alone was also plated as above, to control for spontaneous lysis. 60
2.22 Extraction of Bacteriophage DNA
E. coli C6OO was infected with an appropriately diluted phage stock such that confluent lysis of the bacterial lawn resulted after overnight incubation. Five ml of SM buffer, supplemented with 10 ttl of 10 mg/ml DNase I was added to each of the plates, which were then gently rocked for 1-2 hours at room temperature. The SM containing the eluted phage was then transferred to 10 ml centrifuge tubes. Each plate was rinsed with a further 2 ml of SM which was combined with the first eluate. Tubes were centrifuged at 3,000 x g for 1O minutes. The supernatant was removed and filter-sterilized using 0.45 gm syringe-mounted filter units (Millipore). Ten lrl of 10 mg/ml RNAse A and 10 ttl of 10 mg/ml DNAse I was added to the phage-containing filtrate and incubated for 3O minutes at 37oC. An equal volume of SM containing 2OY" polyethylene glycol 6000 (BDH Ltd', Poole, England) and 2 M NaCl was added and the tubes were kept on ice overnight. Precipitated phage were recovered by centrifugation at 15,O0O x g lor 20 minutes at 4oC. The supernatant was removed by aspiration and centrifuge tubes were drained on absorbent paper to remove residual fluid. The pellets were resuspended in 7OO pl of SM and transferred to sterile screw-capped eppendorf tubes. Seven ¡tl of 10% SDS and 7 ¡tl of 0.5 M EDTA (pH 8.0) were added to each tube and these were then incubated at 68oC for 15 minutes. The phage DNA was extracted three times with TE-saturated phenol, or until the aqueous phase was clear, and then once with chloroform. Phage DNA was then precipitated with an equal volume of propan-2-ol and tubes were held at -15oC overnight, or at -70oC for 30 minutes. Tubes were then thawed and microfuged for 20 minutes. The supernatant was removed and pellets were washed in 0.5 ml of 7Oo/" ethanol and microfuged for 1O-15 minutes. The supernatant was discarded and the phage DNA pellets were dried in vacuo. DNA was dissolved in 1OO ttl of TE and stored at 4oC. 6t
2.23 Extraction of Ghromosomal DNA Overnight LB cultures (10 ml) of E. coli were pelleted by centrifugation
(3,OOO x g, 1O minutes), washed once with 1O ml of 5O mM Tris-HCl, 5 mM EDTA, 50 mM ruaCl (pH 8.O), and resuspended in 2 ml of 257o sucrose, 50 mM Tris-HCl (pH 8.0). One ml of 1O mg/ml lysozyme,0.25M EDTA (pH 8.0) was added and after 20 minutes on ice, 0.75 ml of TE buffer and O.25 ml of 50 mM Tris-HCl, 5 mM EDTA, 5% N-lauroylsarcosine (pH 8.O) was added. Ten mg of solid pronase was then added and the mixture was heated at 60oC for t hour. The lysate was then extracted 3-4 times with TE-saturated phenol, twice with chloroform, and finally dialysed overnight against TE buffer. The chromosomal DNA was recovered and stored at 4oC.
2.24 Plasmid DNA Extraction Plasmid DNA was extracted using the Triton X-1OO lysis technique described by Kahn et al. (1979). Briefly, pelleted bacterial cells from 1O ml overnight LB cultures were frozen at -70oC for at least 30 minutes and then resuspended in 0.4 ml of 25o/o (w/v) sucrose in 50 mM Tris-HCl, pH 8.O. These cells were transferred to eppendorf tubes and 50 ttl of lysozyme solution (1O mg/ml in O.25 M EDTA, pH 8.O) was added. Tubes were then vortexed and chilled on ice for 1O minutes, after which 5OO øl of TET buffer (50 mM Tris-HCl,66mM EDTA, pH 8.O, O.4o/o Triton X-1OO [v/vl) was added. Tubes were mixed by inversion and chromosomal DNA was pelleted by microfuging for 20 minutes. The supernatant was extracted tw¡ce with TE- saturated phenol, followed by a single chloroform extraction. Plasmid DNA was precipitated by the addition of an equal volume of cold propan-2-ol and storage of tubes at -7OoC for at least 30 minutes. Tubes were thawed and microfuged for 20 minutes and plasmid DNA pellets were washed in 0.5 ml of coldTOo/o ethanol and microfuged for a further 1O minutes. DNA pellets were 62 then dried at 65"C for 1O minutes, resuspended in 30 pl of TE and stored at 4"C.
2.25 Restriction Endonuclease Digestion of DNA DNA was digested with one or more restriction endonucleases as required, using the recommended buffers supplied by the manufacturer (Boehringer Mannheim). Reactions were routinely carried out us¡ng 2 units of restriction enzyme in a final volume of 20 pl at 37 oC for 2 hours, or overnight where convenient. Reactions were stopped by heating tubes at 65oC for 1O
m¡nutes or as recommended by the supplier.
2.26 Agarose Gel ElectroPhoresis Electrophoretic analysis of DNA was carried out at room temperature in a horizontal Pharmacia GNA2OO electrophoresis apparatus (Pharmacia-LKB, Uppsala, Sweden) at 5-8 Vicm using a Tris-borate-EDTA buffer system (TBE) (89 mM Tris-base,89 mM boric acid,2 mM EDTA) (Maniatis, 1982). The TBE was supplemented with O.5 ttglml ethidium bromide (Sigma). Gels were prepared using DNA grade agarose (Progen lndustries, Datra, Old.) at concentrations of 0.87o, 1.Oo/o, 1.2, 1.5o/" and 2o/" (w/v) depending on the expected fragment sizes. Samples were loaded onto the gel with 5 ¡rl of loading buffer (TE supplemented with O.05% xylene cyanol, O.05% bromophenol blue and 50% glycerol) per 20 pl sample. Ethidium bromide- stained DNA was visualized using a UVP model TM15 transilluminator. Gels were photographed with a Polaroid MP-4 camera using Polaroid type 667 film. DNA molecular size markers were included in each gel and were purchased from Bresatec. These included: bacteriophage lambda DNA restricted with Hindlll (fragment sizes of 23'1, 9.4, 6.6, 4.37, 2'3, 2'O, 0.564, and 0.125 kb), bacteriophage SPP-1 DNA restricted with EcoRl 63
(fragment sizes of 8.51, 7.35,6.11, 4.84,3.59, 2.81, 1.95, 1.86, 1'51, 1.39, 1.16,0.9b, O.72,0.48, and 0.36.kb) and pUC19 DNA restricted with Hpall (fragment sizes of 501 ,489,4O4,331,242, 190, 147,111,11O,67, 34 and 26 bp). DNA molecular size markers pre-labelled with Digoxigenin (DlG) (Boehringer Mannheim) were used when gels were to be used for Southern hybridization analyses. These included DIG-labelled lambda DNA restricted with Hindlll (fragment sizes l¡sted above) and D¡G-DNA molecular weight marker Vl. The latter was a mixture of pBR328 DNA digested separately with BgA and Hinfl, yielding fragments ot 2176, 1766, 1230, 1O33, 653, 517,
453, 394, 298, 234, 22O, and 1 54 bP. .DNA fragment sizes were calculated from plots of logto MW vs mobility for the above markers.
2.27 Band lsolation of DNA Fragments Restriction fragments or PCR products were excised from agarose gels using a scalpel blade, after visualization with a model UVL-56 long wave (366 nm) UV light source (UV Products). The method for isolation of DNA from the agarose gel was adapted from that of Heery et al. (1990). The gel slice was placed in an eppendorf tube which had been punctured at its base and plugged with sterile siliconized glass wool. This tube was placed inside another eppendorf tube and the DNA was collected in the bottom tube after microfuging for 10 minutes at 6,500 rpm. The eluate was transferred to another tube and 2OO ttl of TE was added to the upper tube which was microfuged again, as above. The eluates were combined and the DNA was extracted once with 30O ltl of phenol, followed by one chloroform extraction. A one-tenth volume of 0.3 M sodium acetate and an equal volume of cold propan-2-ol were added to precipitate the DNA, and the tubes were stored at -7OoC for 30 minutes. The tubes were then thawed and microfuged for 20 64
minutes and the supernatants discarded. Pellets were washed with 5O0 ¡tl of cold 7Oo/o ethanol and the tubes were microfuged again for 15 minutes. The supernatants were removed and the DNA pellets were dried by heating at 65oC for 1O minutes. lf required, protruding termini of restriction fragments or PCR products were removed by treatment with 51 nuclease and/or end- filled with DNA polymerase I (Klenow fragment) as described in 2.44.
2.28 Ligation of DNA Fragments DNA fragments and appropriately restricted vector DNA were ligated using 1 unit of T4 DNA ligase (Boehringer Mannheim) in 20 mM Tris-HCl, 10 mM MgC12, 10 mM dithioerythritol, O.6 mM ATP, pH 7.6 in a final volume of 20 ttl. However, when "blunt-end" ligations were performed, 5o/o polyethylene glycol was included in the ligation reaction and the concentration of ligase was doubled. Ligations were routinely incubated at room temperature for two hours and then the tubes were transferred to 4oC overnight.
2.29 Preparation of Competent E. colí JM109 Cells Ten ml LB broths were inoculated from a fresh overnight plate of E. coli JM109. Cultures were incubated at 37oC with vigorous shaking in a Ratek orbital shaking incubator (Ratek lnstruments, Mitcham, Vic.) until the A699 reached O.2. Bacterial cells were then centrifuged at 3,OOO x g lor 10 minutes and pellets were resuspended in 3.3 ml of cold O.1 M MgCl2 and centrifuged again. Bacterial pellets were then resuspended in 750 pl of cold O.1 M CaCl2 and stored on ice for at least 90 minutes before use. Competent cells could be stored at 4oC for up to two days. 65
2.3O Transformation ot E. coli JM 109 Cells Transformation of E. coli JM109 with plasmid DNA was carried out using CaCl2-treated cells, according to the method of Brown et al. (1979)' Briefly, this involved the addition of 1OO pl of competent JM1O9 cells to the ligation mix and storage on ice for 30 m¡nutes. The cells were then subjected to heat shock at 42oC for two minutes, followed by a further 30 minutes on ice. Five hund red pl of LB broth was added to the transformation mix and tubes were incubated aT37oC for 60 minutes. One hundred pl aliquots of the transformation mix were spread on LB/ampicillin or LB/ampicillin/IPTG/X-gal plates and these were incubated overnight at 37oC'
2.31 Synthesis of Oligodeoxynucleotides Oligodeoxynucleotides (oligos) were synthesized using an Applied Biosystems Model 391 DNA Synthesizer (Applied Biosystems, Foster City, Ca., USA.).
2.32 Ammonia Gleavage and Deprotection of Oligos A 1 ml syringe was attached to the bottom end of the synthesis column and 3OO-4OO ¡tl of concentrated ammonium hydroxide Qgo/ol was added to the column using another 1 ml syringe attached at the other end' The column was left at room temperature for 30 minutes and the ammonium hydroxide solution containing eluted oligo DNA was removed to a screw top eppendorf tube. This procedure was repeated twice and the final volume of the pooled oligo solution was approximately 1 ml' Tubes were then incubated overnight at 55oC. One hundred pl of the extract was removed and 1 ml of butanol was added to precipitate the oligo (the remainder of the extract was stored at -15oC for later processing). The butanol/oligo DNA solution was microfuged for 1 minute, the supernatant was discarded and the pellet wâS rê-dissolved in 1OO ¡tl of ster¡le water. A further 1 ml of butanol 66 was then added and this solution was microfuged for 1 minute. Following removal of the supernatant the pellet was dried under vacuum and the pellet was resuspended in 1OO øl of sterile water (final concentration of the oligo was approiimately 1 ttqltrll.
2.33 Labelling of Oligos with Digoxigenin The sequences of synthetic oligos used as probes for SLT genes are shown in Table 2.1. Oligos were labelled with Digoxigenin (DlG) using a D|G-oligo tailing kit purchased from Boehringer Mannheim. The reactions were set up on ice in eppendorf tubes, and contained 4 pl of 5x ta¡ling buffer (t M potassium cacodylate, 125 mM Tris-HCl, 1.25 mg/ml bovine serum albumin, pH 6.6), 6 ¡tl oÍ 25 mM CoCl2, 1 øl (approximately 1 Ugl of oligo, 2 ¡tl ot 1 mM DIG-1 1- dUTP, 2 ¡tl of 2.5 mM dATP, made up to 19 ¡tl with sterile water. One¡rl (10 units) of terminal transferase was added to the reaction mix, which was then incubated at 37oC for 5 minutes, microfuged and made up to 1OO pl with sterile water. The probe mixture was then added to 20 ml of oligo- hybridization solution and stored at -15oC until required.
2.34 Random Primer Labelling of DNA Fragments with Digoxigenin DNA fragments were labelled with D¡G using a modification of the random primer method described by Feinberg and Vogelstein (1983), using a DIG DNA labelling kit purchased for Boehringer Mannheim, Briefly, linearized DNA was denatured by heating for 1O minutes at 95oC, followed by rapid chilling in a 5Oo/" ice-ethanol bath held at -15oC. The sample was microfuged briefly and the tube was placed on ice. Two ttl of 10x hexanucleotide solution was added to the denatured DNA, followed by 2 ¡tl ot dNTP labelling mixture (1 mM dATP, 1mM dCTP, 1 mM dGTP, 0.65 mM dTTp,0.35 mM DIG-11-dUTP, pH 6.5). The volume of the reaction was X ',:
,t
È
)
! Table 2.1 :l I genes 't Sequences of s¡mthetic oligonucleotides used as probes for SLT I
!t I I
I
I i 0ligo Sequence (5'-3') Specificitya Reference
(1989) A-I ATACTGAATTGTCATCATCA sLT-I,II,IIv Brown et al. Meyer (1989b) 428-r GATAGTGGCTCAGGGGATAA SLT- I Karch &
428-Ll AACCACACCCACGGCAGTTA SLT- I I
AGAAC GCC CACTGAGATCATC SLT- I This study SLTl ID ;, il [, SLTzID ATACACAGGAGCAGTTTCAGA SLT-II,IIv Ë il 0x3 AAGAGTGGGCCCTGCGA SLT-OX3 il 0x3/2 AAAACTGTGACTTTCTG sLT-ox3/2
Section 2.38' a Hybridjzation was carried out at 45'C, as described in
t 67 I !t¡ à- made up to 1g pl with water and 1 ¡tl of Klenow enzyme (2 unitslt'tl) was ¡ for at tube was microfuged briefly and then incubated ¡ added. The reaction t { least t hour (up to a maximum of 16 hours) at 37oC. The reaction was stopped by the addition of 2 ¡tl of O.2 M EDTA (pH 8.O) and the probe was t -15oc' then added to 20 ml of DNA-hybridization solution and stored at i
I
1 2.35 Preparation of Digoxigenin-labelled sLT Probes by PCR SLT-l and SLT-ll-specific DNA probes were labelled w¡th DIG by PCR- amplification of bacteriophage H19B or 933W DNA, respectively, in the presence of DIG-11-dUTP (Boehringer Mannheim). The use of a single pair of oligo primers and amplification conditions, which direct the production of 227- and 224-bp PCR products from an analogous region of the SLT-I and genes, respectively, have been described by Karch and I SLT-ll A subunit ii il Meyer (1g8gb). The PCR reaction was carried out as described in Section 2.43, except that 8 ¡i of the dNTP labelling mixture described in Section 2.34 above, was substituted for the unlabelled PCR dNTP mix. After amplification, pCR products were electrophoresed on 2o/o gels and band-isolated. "satose The D|G-labelled DNA was extracted from the gel as described in Section 2.27. The recovered DNA was then added to a DNA-hybridization solution and stored at -15oC for later use.
2.36 preparation of Filters for Dot Blot Hybridization Analysis of Bacterial Lysates E. coliclinical isolates or clones for dot blot hybridization analysis had
been stored in 96-well (U-bottomed) microtitre plates (Disposable Products) at -7OoC. An MIC2OOO lnoculator (Dynatech Laboratories) was used to inoculate duplicate microtitre plates containing 2OO ttl of LB per well, with or oC, without ampicillin as appropriate. After overnight incubation at 37 the plates were centrifuged (2,OOO x g, 1O minutes) and pellets were 68
¡^. resuspended in 10 pl of TE buffer. Ten ¡rl ol 1Oo/" SDS was then added to r pl The plates were I' each well, followed by 50 of O.5 M NaOH, 1.5 M NaCl. I I then centrifuged again and 5 ¡rl aliquots of the supernatants were spotted ti onto nylon filters (Hybond N*), which were fixed with alkali, as described in Section 2.37. Microtitre plates containing the bacterial lysates were stored at -15 0C.
2.37 Preparation of Filters for Southern Hybridization Analysis DNA fragments separated by agarose gel electrophoresis were transferred to nylon filters (Hybond N * ) essentially as described by Southern (1975). Prior to transfer, gels were gently agitated in o.25 M HCI for 15 minutes, followed by a 30 minute incubation in O.4 M NaOH, 0.6 M NaCl and then a 30 minute incubation in 1.5 M NaCl, O.5 M Tris-HCl, pH 7.5. Transfer was achieved overnight by capillary action, as described by Maniatis et al. (1982), using 1Ox SSC as the transfer buffer. Following overnight transfer, the position of each well was marked on the membrane with pencil. DNA was then fixed by placing the membrane (DNA side up) on two pieces of Whatman filter paper No. 1, which had been soaked in O.4M NaOH, for 20-30 minutes. Membranes were gently agitated for one minute in 100 ml of 2x SSC. Membranes were either pre-hybridized and hybridized with the desired probe immediately after fixation, or dried, wrapped in plastic film and stored at 4oC for later use.
2.38 Hybridization of Membranes with Oligo Probes Following alkali fixation, Southern transfer or dot blot membranes were incubated with 20 ml of oligo-pre-hybridization solution (5x SSC, 1% SDS, O.5o/" polyvinyl-pyrolidone [Sigmal, O.2o/" skim milk powder) at 45oC for at least one hour with gentle agitation. This mix was removed and replaced with 2O-4O ml of oligo-hybridization solution (5x SSC, 1% SDS, O.5o/" bovine 69 serum albumin, 2mM Na4pyrophosphate, O.5% polyvinyl-pyrolidone) I containing the appropriate D|G-labelled oligo probe and incubated at 45oC (or other appropriate temperature) overnight with gentle agitation. The probe mix was removed and membranes were washed twice with 1OO ml of 5x SSC for 30 minutes at the hybridization temperature. This was followed by a single wash with 100 ml of 2x SSC for 30 minutes at room temperature. The membranes were then developed as described in Section 2.4O.
2.39 Hybridization of Membranes with DNA Probes Following alkali fixation, Southern transfer or dot blot membranes were incubated with 20 ml of DNA-prehybridization solution (6x SSC, 5x Denhardt's solution,ly" SDS, 5Oo/" formamide, 1OO ltglml salmon testes DNA IBoehringer Mannheim]) at 42oC for at least one hour with gentle agitation. This solution was then removed and 2O-4O ml of DNA-hybridization fluid (6x SSC, 1x Denhardt's solution, 1% SDS, 5Oo/" formamide, 1OO pglml salmon testes DNA, 1OO ¡tglml heparin lSigmal) containing the DIG-labelled DNA probe (which had been pre-heated at 80-9OoC for 1O minutes) was added. Membranes were then hybridized overnight with gentle agitation at 42oC. The prob'e was removed, and the membranes were washed twice with 1OO ml of 2x SSC for 15 minutes at 65oC. This was followed by a further two washes in O.2x SSC, O.1o/o SDS for 15 minutes at 65"C. The membranes were then developed as described in Section 2.4O.
2.4O Development of D|G-Labelled Membranes Buffers used for development of D|G-labelled filters were as follows:- DIG Buffer 1:0.1 M Tris-HCl,0.15 M NaCl, pH 7.5.
DIG Buffer 2:1o/o (w/v) "blocking reagent" (Boehringer Mannheim) in DIG
Buffer 1. 70
DIG Buffer 3: 0.1 M Tris-HCl, O.1 M NaCl, 50 mM MgCl2, pH 9.5
Colour/Substrate solution: This solution was prepared freshly on the day of use and contained 45 ttl of 75 mg/ml nitroblue tetrazolium salt (NBT) (Boehringer Mannheim) in 7Oo/o (v/v) DMF and 35 ltl of 5O mg/ml S-bromo-4- chloro-3-indolyl phosphate (X-phosphate) (Boehringer Mannheim) in DMF, in
1O ml of DIG buffer 3.
Washed, hybridized membranes were incubated with 30 ml of DIG buffer 2 for 30 minutes at room temperature with gentle agitation. This was followed by incubation with 30 ml of DIG buffer 1 containing 6 pl of polyclonal sheep anti-digoxigenin Fab-fragments conjugated to alkaline phosphatase (Boehringer Mannheim) for 30 minutes at room temperature. Membranes were then washed twice in 1O0 ml of DIG buffer 1 for 15 minutes at room temperature. This was then removed and 1O ml of Colour/Substrate solution was added to the surface of the membranes. They were then kept at room temperature in the dark, without agitation, until the colour precipitate developed on the filters (1O minutes to several hours, depending on the intensity of labelling). The colour development was stopped by rinsing membranes in TE for 5 minutes. Membranes were dried, photographed if necessary and then wrapped in plastic film and stored.
2.41 Stripping of Filters for Re-hybridization Where re-hybridization of previously developed filters was required, membranes were incubated twice, for 15 minutes each, in approximately 20 ml of DMF to remove the coloured precipitate. To remove the probe, DMF- treated membranes were washed in deionized water and then incubated for 30 minutes at 37oC in 50 ml of stripping solution (0.2M NaOH, O.1% SDS 7t
[w/v]). Membranes were then rinsed gently in 2x SSC for one minute and
either pre-hybridized and hybridized with the desired probe, or dried and stored at 4oC for later use.
2.42 Rapid DNA Extract¡on for PCR Analysis
Ten ml LB broth cultures were inoculated with pure E. coli isolates, or with approximately 5OO mg of faeces or gut contents, and incubated for 4 hours or overnight in an orbital shaking incubator at 37oC. One ml aliquots of these cultures were microfuged for 5 minutes and the pellets were resuspended in 95 ¡tl of 25o/o sucrose, 50 mM Tris-HCl (pH 8.O). Five ¡tl of 1O mg/ml lysozyme in 0.25 M EDTA was added to each and the tubes were incubated for 30 minutes at 37oC. The lysed bacteria were then transferred to tubes containing3 pl of 20 mg/ml proteinase K and incubated at 65oC for 60 minutes, followed by incubation at 95oC for 20 minutes. DNA was precipitated by the addition of 0.5 ml of 7Oo/o ethanol, followed by storage at -7OoC for 30 minutes. Samples were thawed and microfuged for 15 minutes,
Supernatants were then removed and the pellets were dried by heating at 65oC for 1O-15 minutes. The dried DNA pellets were resuspended in 300p| of TE. Five pl of the DNA sample was routinely tested by PCR. Aliquots of each DNA sample were stored at 4oC and -15oC.
2.43 PCR-Amplification
Nucleotides dATP, dCTP, dGTP, and dTTP were of molecular biology grade and were purchased as 1OO mM solutions from Boehringer Mannheim.
PCR-amplification of SlT-related DNA sequences was carried out using a variety of primer sets as set out in Table 2.2. Table 2.2 0ligonucleotide primers used for PCR-amplification of SLT genes
Prirner Sequence (5'-3'¡a Gene regionb Reference
Ia GGAGAGTCCGTGGGATTACG SLT- I Pollard et al. (1990)
Ib AGCGATGCAGCTÀTTAATAA nt 860-989
il IIa TTAACCACACCCACGGCAGT SLT- I I
IIb GCTCTGGATGCATCTCTGGT nt 190-536
IIIa TTTACGATAGACTTCTCGAC SLT-I & II Karch & Meyer (1989b)
IIIb CACATATAAATTATTTC G CTC nt 73-298
IVa ATACAGAG I GA] G I GA] ATTTCGT SLT-I & II This study
IVb TGATGATG I AG ] CAATTCAGTAT nt 586-797
Va GATGGCGGTCCATTATC SLT- I I This study
Vb AACTGACTGAATTGTGA nt -192-1311
a Bracketed nucleotides denote positions at which two alternative
nucleotìdes were incorporated during oìigo synthesis to accommodate
known sequence variations between varjous SLT types.
b Nucleotides are numbered from the first base of the init'iation codon of the A subunit coding sequence. 72
Samples (up to 20 ltll were amplified in 50 pl reactions containing 200 pM dNTPs, approximately 1 UM of each primer, and 1 U Taq polymerase (Boehringer Mannheim), in 1O mM Tris-HCl (pH 8.3),50 mM KCl,2 mM MgCl2, O.1% gelatin, O.1o/" Tween 20, O.1% NP-40, Samples were overlaid with 50 ¡tl of mineral oil (Sigma) and were subjected to 35 PCR cycles, each consisting of 1 minute denaturation at 94oC, 3 minutes annealing at 47oC and 4 minutes elongation at 72oC, using a Hybaid lntelligent Heating Block (model IHB2O24, Hybaid Ltd., Teddington, Middlesex, England). PCR reactions were electrophoresed on agarose gels and stained with ethidium
bromide.
2.44 Construction of Nested Deletion Derivatives
Unidirectional nested deletions of cloned DNA in pUC19 or pBLUESCRIPT SKTM, were constructed by the method of Henikoff (1984) using an Erase-a-Base kit (Promega Corporation, Madison, Wi. USA.). Plasmid DNA was first linearized within the polylinker region on one side of the DNA insert by digestion with restriction enzymes to generate 3'and 5' protruding termini, which are resistant or susceptible to Exonuclease lll digestion, respectively. For pUC19-derived clones, SacllBamHl and SphllSaA were used for generating sequence data using forward and reverse primer sequencing, respectively. For pBLUESCRIPT SKTM clones, KpnllClal and SacllBamïl were used for forward and reverse reactions, respectively. Approximately 5 ¡rg of restricted plasmid DNA in a reaction volume of 40 ¡tl was heated at 65 oC for 1O minutes to inactivate restriction enzymes. Meanwhile S1 nuclease mix containing 172 Ul of sterile water, 27 ¡tl of 7.4x S1 nuclease buffer (0.3 M potass¡um acetate [pH 4.6J, 2.5 M NaCl, 10 mM
ZnSQ4, 5Oo/o glycerol), and 60 units of 51 nuclease was dispensed (7.5 ¡¡ll tube) into 25 eppendorf tubes and held on ice. Thirty ¡tl of the restriction mix t3 was added to 6 ¡tl of 1Ox exonuclease lll buffer (660 mM Tris-HCl [pH 8.OJ, 6.6 mM MgCl2) and 24 ¡tl of sterile water and was equilibrated at 3OoC in a Hybaid lntelligent Heating Block for 3 minutes. A 2.5 ¡rl aliquot of this mix was added to the O minute 51 nuclease time point tube and stored on ice.
3OO-5OO units of Exonuclease lll were added to the mix and 2.5 ¡rl time samples were removed from the reaction and added to 51 tubes on ice, at various intervals (usually every 30 seconds). When all time points had been taken, the tubes were incubated at room temperature for 30 minutes. At this point, 1 øl of 51 stop buffer (O.3M Tris base, 50 mM EDTA) was added to each tube and they were heated at TOoC for 10 m¡nutes. Two ¡tl o'n each time point was electrophoresed on a 1 o/o agarose gel to determine how much DNA had been deleted and hence the suitability of each time point for ligation and transformation. one ¡tl of Klenow enzyme mix (30 ltl of 20 mM Tris-HCl pH 8.O, 1OO mM MgCl2, containing 5 units of Klenow fragment of DNA polymerase l) was added to each tube, and these were incubated at 37oC for 3 m¡nutes. One pl of dNTP mix (O.125 mM each of dATP, dCTP, dGTP and dTTP) was added to each tube, followed by further incubation for 5 minutes at 37oC. All or selected time points were then ligated (Section 2.281 and transformed into E. coli JM109 (Section 2.3O). Plasmid DNA was extracted from the resultant clones (Section 2.241 and subjected to restriction analysis and agarose gel electrophoresis to determine the size of the residual inserts in the nested deletion derivatives.
2.45 DNA Sequencing Double stranded DNA sequencing was carried out using dye-labelled primers on an Applied Biosystems model 3734 automated DNA sequencer. 74
2.46 Analysis of Sequence Data DNA and deduced amino acid sequence data were analysed using DNASIS version 6.0 and PROSIS version 6.0 software pachages (Hitachi Software Engineering Co., Yokohama, Japan). Comparisons were made with known sequences contained on CD-ROM discs containing GenBank R73,0 (August, 1992l., EMBL R32.0 (September, 1992l., PIR R33.O (June, 1992) and Swiss-PROT R23.0 (August, 1992) databases (Hitachi). The program BLASTX (Altschul et al., 1990) was also used to translate DNA sequences and conduct similarity searches of the prote¡n databases available at the National Center for Biotechnology Information, Bethesda, MD., USA.
2.47 Preparation of RNA Water and all solutions used in the extraction and analysis of RNA were treated with diethylpyrocarbonate (DEPC) to inhibit RNase activity, as described by Maniatis et al. (1982). For preparation of RNA, 10 ml of LB was inoculated with 2 ml of overnight LB broth cultures and incubated at 37 oC with vigorous shaking until mid-exponential phase (AOOO = O,5) was reached. Cultures were chilled on ice for 10 minutes and then centrifuged at 30OO x g for 10 minutes. The supernatants were decanted and 2 ¡tl of 4O units/gl RNase inhibitor (Boehringer Mannheim) was added to the bacterial pellets. The pellets were then resuspended in O.5 ml of lysis solution (2O mM sodium acetate, 1 mM EDTA, O.5o/" SDS, pH 5.5) and transferred to screw-capped eppendorf tubes. The lysates were then extracted with 0.5 ml of phenol equilibrated against RNA buffer (20 mM sodium acetate, 20 mM KCl, 10 mM MgCl2, pH 5.5) and heated for 5 minutes at 65oC. The samples were briefly vortexed, and were then microfuged for 5 minutes. The samples were re- extracted 4-times as above and the RNA was then precipitated with 3 volumes of lOOo/o ethanol (t ml) and a one tenth volume of 3 M sodium 75
acetate. The RNA samples were then stored overnight at -2OoC. These samples were then microfuged for 15 minutes at 4oC and the RNA pellets were dried in vacuo. To eliminate any contaminating DNA from the samples, the RNA pellets *"r" then resuspended in 50 ¡tl of DNase buffer (20 mM Tris- HCl, 1O mM MgCl2, pH 8.O) to which 1 ¡tl of RNase-free DNase (Boehringer Mannheim) and 1 ¡tl of RNase inhibitor were added. The samples were then incubated al 37 oC for 20 minutes, extracted once with an equal volume of phenol, and heated for 5 minutes at 65oC. The aqueous phase was then microfuged, the RNA precipitated with 3 volumes of 10O% ethanol (150 øl) and a one tenth volume of 3 M sodium acetate and held at -20oC overnight. The precipitated RNA was microfuged at 4oC for 15 minutes and the pellets were dried in vacuo and resupended in 30 ¡tl of water and 1 pl of RNase inhibitor. RNA samples were stored at -2OoC.
2.48 Gel Electrophoresis of RNA RNA samples were subjected to agarose gel electrophoresis in a buffer system consisting of 20 mM 3-(l/-morpholino)propanesulphonic acid (MOPS), 40 mM sodium acetate, and 5 mM EDTA (pH 7.0). Agarose gels (1.2%l were prepared by melting O.9 g agarose in 46.6 ml of DEPC-treated water. When this had cooled to approximately 6OoC, 15 ml of 5x concentrated MOPS electrophoresis buffer and 13.4 ml of formaldehyde (4Oo/o solution) were added and the gel was poured at once. lmmediately before analysis,
RNA samples were denatured by combining 4.5 Ul of each sample with 2 ¡tl of 5x electrophoresis buffer, 3.5 ltl of formaldehyde (4Oo/"1 and 1O Ul of formamide. The samples were heated at 65"C for 15 minutes, chilled on ice and then loaded onto the gel with 2pl of loading buffer (5Oo/" glycerol, 1 mM
EDTA [pH 8.Oì, O.25% bromophenol blue, O.257o xylene cyanol, in DEPC- treated water). Gels were run at 5V per cm for approximately 3 hours. 76
2.49 Northern Hybridization Analysis of RNA lmmediately after electrophoresis, formaldehyde was removed from the gels by two 15 minute washes, with gentle agitation, in DEPC-treated water, The RNA was then transferred (without further treatment of the gels) to nylon membranes, as described in Section 2.37. The membranes were fixed by placing them on 2 pieces of Whatman No. 1 paper, which had been soaked in O.O5 M NaOH for 5 minutes. Membranes were then rinsed in 2x SSC with gentle agitation for less than 1 minute, pre-hybridized and then hybridized with DIG-labelled oligo probes (Section 2.38).
2.5O Reverse Transcription PCR Reverse transcriptase-dependent PCR analysis of RNA samples was carried out using reagents contained in a GeneAmp RNA PCR kit (Perkin Elmer Cetus, Norwalk, Ct., USA.). Reverse transcripiion reactions were carried out in a final volume of 20 gl, comprising 3 ¡tl of RNA sample,2 ¡tl of 10x PCR buffer ff (0.5 M KCl, O.1 M Tris-HCl, pH 8.3), 4¡tlot 25 mM MgCl2, 2uleach of 1O mM dATP, dCTP, dGTP and dTTP, 1 ttl (2O units) of RNase lnhibitor, 0.5 pl (approximately O.5 ¡rg) of each oligo primer and 1 pl (50 units) of Moloney Murine Leukaemia Virus reverse transcriptase. Samples were overlayed with 5O ltl of mineral oil and incubated at 42oC for 15 minutes. Reverse transcriptase was then inactivated by heating at 99oC for 5 minutes. Each tube was then supplemented with a further 4 pl of 25 mM MgCl2, 8 pl of 1Ox PCR buffer ll, 67.5 ¡tl of deionized water and 0,5 ttl (2.5 units) of Taq polymerase (Boehringer Mannheím). Tubes were then subjected to 35 PCR cycles (Section 2.43l'.
2.51 Protein Assay Total protein concentration was assayed as described by Bradford (1976), using bovine serum albumin as standard. 77
CHAPTER THREE
ISOLATION AND PRELIMINARY ANALYSIS OF SLT.PRODUCIN G ESCHERICHIA COLI
3.1 lntroduction
As mentioned in Section 1.13, little information is available on the incidence of SLTECs in Adelaide children or on the nature of the SLTECs themselves. As part of an on-going study, at the Adelaide Children's Hospital, in which the possible role of toxigenic E coli in SIDS has been examined, a large number of E. coli isolates have been collected from the gut contents of infants dying of SIDS or other causes, as well as from the faeces of healthy babies. The majority of these strains have been tested for the production of Vero cell cytotoxins (Bettelheim et al., 1990; K. Bettelheim and
P.N. Goldwater, unpublished observations). The work which is described in this chapter was to provide a more detailed molecular examination of the above strains. In addition, isolates from children with illnesses associated with SLTEC (e.9., bloody diarrhoea and HUS) were also examined for the presence of SLT genes.
3.2 Results
3.2.1 Source of Bacterial Strains Tested A total of 1,475 E. coli isolates from either the gut contents or faeces of 2OO individuals were available for testing. These individuals were predominantly less than 12 months old and the number of strains isolated for each patient category is shown in Table 3.1, Tabìe 3.1 Source of f. colf isolates
Group Site l{o. individuals No. isolatesa
Heaì thy bab'ies faeces 57 97
D'i arrhoea faeces 4 104
HUS cases faeces 5 164
SIDS cases gut contents 107 949 non-SIDS gut contents 27 161
a The number of E. coli isolates collected from each sampìe varied
from I to approximateìy 100 for one of the HUS cases. 78
3.2.2 Screening for SLT Genes As an initial survey, a total of 160 E. coli isolates (from 83 individuals) were selected from the collection and grown overnight on LB agar plates and directly blotted onto nylon filters. These were fixed, pre-hybridized and then hybridized overnight with D|G-labelled oligo A-l (this probe hybridizes with both SLT-I and SLT-ll sequences lBrown et al., 1989]). Filters were washed and developed using anti-DlG-alkaline phosphatase conjugate and an X- phosphate/NBT substrate system. This procedure resulted in a high level of background labelling of all E. coli strains, presumably due to non-specific adsorption of the probe to cellular debris on the filter, or to endogenous E coli alkaline phosphatase activity. Nevertheless, one of the 160 strains tested produced a hybridization signal which was significantly above background. This strain, designated 031, had been isolated from the small bowel oÍ a 7- month old male infant who died of SIDS. To confirm the above result, diluted culture supernatant from an overnight culture of strain 031 was tested for cytotoxicity on Vero cell monolayers. Culture supernatants of S.dysenteriae type 1 and E. coli C600 were used as positive and negative controls, respectively. Both the 031 and S. dysenteriae samples were strongly cytotoxic, but no cytotoxic effect was observed for C600 (Figure 3.1). Further confirmation of the presence of SLT-related sequences was obtained by Southern hybridization analysis of genomic DNA extracted from strain 031, as well as from S. dysenteriae type 1 and E. coli C6OO. EcoRl- digested DNA was electrophoresed and transferred onto a nylon filter and probed with DIG-labelled oligo A-1. The probe hybridized with DNA fragments of approximately 17 kb and 4.7 kb in the O31 and S. dysenteriae digests, respectively. The probe did not react with E. colí C6OO DNA (Figure 3.2t.. Figure 3.1 Effect of culture supernatants on Vero cell monolayers
Confluent monolayers of Vero cells were treated with Maintenance Medium (A) or with culture supernatants of E. coli strain C6OO (B), Shigella dysenteriaetype 1 (C), or E. coli strain 031 (D), diluted 1:1OO in Maintenance Medium. Monolayers were photographed after incubation at 37 oC for 3 days
(Magnification 4OOx). ..v3 a:
+ ì t^ *:f û
: ')
4 .::. ,i
It a 4 ) e' fì .a ,È ''l.it 3 F' '4, .a ì ' l¡ þ (¡ a 1
a a a - Figure 3.2 Southern blot hybridization of genomic DNA with an SlT-specific oligo probe
Genomic DNA from E coli C6OO (Lane 1), E. coli O31 (Lane 2l,, and S' dystenteriae type 1 (Lane 3) was digested with EcoRl, electrophoresed on a
O.Bo/o agarose gel, and transferred onto a nylon filter. The filter was hybridized with DIG-labelled oligo probe A-l and washed at high stringency, (23.1, as described in Section 2.38. Lane M: D|G-labelled DNA size markers 9.4, 6.6, 4.37,2.9,2.O and 0.56 kb, respectively, from top to bottom). 123M
ala
.¡- -
rl
- 79
lndependent confirmation of the presence of SLT genes in strain 031 was obtained by PCR analysis. The PCR protocol utilized a single set of oligo primers (llla and lllb) and the amplification conditions used direct the production of 227- and 224-bp PCR products from an analogous region of the SLT-I and SLT-ll A subunit genes, respectively (Karch and Meyer, 1989b). Crude proteinase-K-treated culture extracts of strain O31 as well as several SlT-producing reference strains, (S. dysenteriae type 1 and E coli strains H30 and 87028) were subjected to PCR analysis and were electrophoresed on 2o/o agarose gels (Figure 3.3). All directed the amplification of DNA fragments of the expected size, but no amplified products were observed when extracts of E. colí C600 were tested. Aliquots of the 031-derived PCR reaction mix were then spotted onto nylon filters and probed with DIG- I labelled oligos 428-l and 428-ll, which are directed, respectively, against SLT- t l- and SlT-ll-specific sequences within the amplified region (Karch and Meyer, 1989b). Only the latter probe hybridized with the PCR product, suggesting that 031 contained SLT-Il-related sequences (result not shown). Whilst PCR is highly specific, it was considered too expensive and labour intensive for testing very large numbers of isolates for the presence of
SLT genes and so it was decided to persist with dot blot hybridization as a primary screening method. However, in order to reduce non-specific adsorption of probe to cell debris, SDS/NaOH-treated cell lysates were prepared in microtitre trays and 3-5 ¡rl aliquots were spotted onto filters after centrifugation of the trays to remove debris. Moreover, a higher specific activity probe was prepared by PCR-amplification of E. coli DNA containing SLT-l or SLT-ll genes in the presence of DIG-11-dUTP. This revised protocol resulted in much higher signal to background ratios (a representative filter is shown in Figure 3.4) and was used to screen the remaining 1,315 isolates for the presence of SLT genes. This resulted in the detection of a further 3 SLT- positive strains; strains 106, 234, and PH, which were isolated from the gut .1 I I .rl È
I
I ù
ü '{
'l
I I
I
I I
Figure 3.3 Detection of SLT genes bY PCR
Genomic DNA was subjected to PCR-amplification employing SLT-I- and SLT- ll-specific primers (llla and lllb) as described in Section 2.43. Twenty gl
L aliquots of each reaction were electrophoresed on a 2o/" agarose gel and & stained with ethidium bromide. Ë Tracks: 1: Negative control (E. colistrain C6OO).
2: S. dysenteriae tyPe 1 .
3: E. colistra¡n O31 . 4: E. colistrain H3O. 5: E. colistra¡n 87028. M: DNA size markers (500, 4O4, 331, 242, 190, 147, and 110 bp, respectively, from top to bottom). I
"l' I
I I t
'l
I I t 2 3 4 5M
- - - - - '224bP Figure 3.4 Dot blot hybridization analysis of E. coli lysates
Aliquots of 96 E. coli cell lysates were spotted onto a nylon filter and hybridized to a PCR-DlG-labelled SLT-I- and SLT-Il-specific DNA probe, prepared as described in Section 2.35, and washed at high stringency. Position A1 is a tysate from strain 031 (posit¡ve control) and position 81 is a lysate from E. coli C6OO (negative control). I l0 2 I 2 3 45 67 A 7a'-' \r' # (i B c
D
E
F
G
H 80
contents of a SIDS case, the faeces of a healthy baby and the faeces of an infant w¡th HUS, respectively. The presence of SLT genes in these strains was confirmed by PCR and culture supernatants of each strain were cytotoxic for Vero cell monolayers (results not shown).
3.2.3 Serotyping of SLT-positive Strains
Serotyping of the four SlT-positive strains was carried out by Drs. l. and F. Orskov and Dr. F. Scheutz, International Escherichia and Klebsiella Centre, Statens Seruminstitut, Copenhagen. Strain 031 belonged to serotype
OX3:H21; OX3 is a relatively new O serotype, which is yet to be assigned a number and is more commonly associated with domestic animals than with humans (F. and l. Orskov, pers. comm.). Strains 234 and 106 belonged to serotypes 036:K"C788":H- (K"C788" is the designation of a K antigen which has not been officially recognized tF. and L Orskov, pers. comm.l) and
O2:K1:H7, respectively. Strain PH belonged to serotype O111:K-:H-, a serotype previously associated with HC and HUS (Karmali, 1989).
3.2.4 lnduction of Temperate Bacteríophages from SLT-positive Strains At the time that this preliminary work was carried out, the SLT genes from the majority of SLTEC isolated from humans were thought to be encoded on toxin-coverting bacteriophages (O'Brien et al., 1992). For this reason, the four SLTECs were subjected to UV irradiation in order to induce toxin-converting phages (Section 2.191. Serial dilutions of filtrates of irradiated cells were plated on E. coli C6OO. Low numbers of plaques were present on the C6OO lawns infected with each filtrate (equivalent to < pfu per ml of f¡ltrate). Representative single plaques were picked off, diluted in SM buffer and replated on C6OO. Single plaques were again picked off and used for preparation of high titre phage stocks (Section 2.191. These phages 81
were designated øO31, Q234, Q1O6, and ØPH. C60O lysogens were also prepared for each of these phages.
3.2.5 Restriction .Analysis of Bacteriophage DNA As a first step in the characterization of these phages, phage DNA was purified and subjected to restriction analysis. Restriction patterns were compared with those of DNA purified from SlT-converting phages H198, 933J, and 933W (Figure 3.5). Restriction patterns of Q234 and Ø106 using Bam\l and Ctal were indistinguishable from respective digests of H198, lt was therefore concluded that these phages are either identical or very closely related. However, there was little similarity between the restriction patterns
of @031 and pPH and any of the SlT-encoding reference phages.
3.2.6 Superinfection Analysis
To further characterize QO31 and @PH, their capacity to superinfect E
coti C6OO lysogenized with either of the above phages or various reference SlT-converting phages was examined (Table 3.2). @031 had maximal plating efficiency on all lysogens tested except for C6OO::@O31. On the other hand,
@PH was unable to superinfect any of the lysogens tested (Efficiency of plating 12x 1O-3).
3.2.7 Cytotoxícity of C6OO Lysogens ln view of the lack of similarity of @031 and pPH to known SLT- converting phages, their capacity, as well as that of Q234 and @106, to convert E. colîC6OO to SLT production was examined . E. coli C6OO or C600 lysogenized with each of the above phages were grown overnight in LB. Filtrates of culture supernatant were then tested (either neat or diluted 1:100) for cytotoxicity on Vero cell monolayers. Both neat and diluted filtrates of C6OO::Q234 and C60O::ø106 were highly cytotoxic, resulting in complete Figure 3.5 Restriction analysis of phage DNA
DNA from ø933W (1), @933J (2l,, @H198 (3t', Q1O6 (4), ø234 (5), øPH (6),
and @O31 (7), was digested with either Bam\l or Clal, electrophoresed on a
O.8o/o agarose gel and stained with ethidium bromide. Track M: DNA size markers (23.1,9.4,6.6,4.37,2.3,2.O and 0.,56 kb, respectively, from top to bottom). amHl Clal Table 3.2
Superinfection ana'lysis of p03l and gPH
Infect ing bacteriophage
Host f. col f 9031 9PH
c600 + +
C600::9031
C600::gPH +
C600: : I ambda +
C600: :933t'l +
C600: :933J +
C600::HI9A +
C600::H198 +
Superinfection analysis was carnied out as described ìn Section 2.21.
+ No s'ignificant reduction in number of pìaques with respect to C600
(Efficìency of plat'ing =1). No p'laques observed (Eff ìc'iency of plating < 2 X l0-3). 82 destruction of the cell monolayer after 3 days incubation, but no cytotoxic effects were observed for C6OO, C600::QO31 and C6OO::@PH. However, the possibility remained that the two lysogens were capable of producing SLT, but not of secreting it into the culture medium. To examine this, filtrates of French pressure cell lysates of overnight cultures of C600,
C6OO::@O31 and C6OO::@PH were tested for cytotoxicity, but again no effect of the lysates on Vero cell monolayers was observed, even when used undiluted.
3.2.8 Southern Hybrídízation Analysis of Bacteriophages
Although the lysogens for QO31 and @PH did not produce SLT under the experimental conditions employed, it was important to determine whether these phages contained unexpressed SlT-related genes. BamHl digests of purified DNA from these phages as well as DNA from 933W, 933J and Q234 were electrophoresed, transferred onto nylon filters and probed with DIG- labelled oligo A-l (Figure 3.6). The probe hybridized strongly with DNA fragments of > 25, 20 and 20 kb, respectively, in the digests of 933W, 933J and Q234, but reacted weakly with a 2.7-kb fragment in the ø031 digest, and not at all with the QPH digest. Ethidium bromide staining of the electrophoresed phage DNAs prior to Southern transfer indicated that the total amount of DNA in the ø031 track was similar to that for the other phages (result not shown) and hence DNA loading could not account for the weaker reaction with the SlT-specific probe. This implied that the reactive sequences in pO31 might be substantially different from those in either of the SLT-I or SLT-Il-encoding reference phages. Additional Southern hybridization analysis was carried out to identify the most suitable restriction enzyme for use in cloning of the probe-reactive sequences. Clal was chosen because it generated a 7-kb reactive fragment, which had a greater probability of Figure 3.6 Southern hybridization analysis of phage DNA w¡th ol¡go A-l
(5)' DNA from øO31 (1), @PH (2), Q234 (3), @lambda (wild type) (4)' ø933J and @933W (6), was digested with Bam{l and electrophoresed on a O.8o/o agarose gel. The phage DNA was transferred to a nylon filter and hybridized with D¡G-labelled oligo A-l and washed at high stringency. The mobility of DNA size markers (Hindlll-digested lambda DNA) is indicated' | 2 3 4 5 6 kb
*_ ÊÞ
¡j rü - 23.\ 9 4 - 6 6
I
I ¡ - 4.4 I
- 2.3 - 2.4 83 incorporat¡ng a complete copy of the putative SLT operon than the smaller BamHl fragment (result not shown).
3.2.9 Cloníng and Sequence Analysis of Q03l DNA ln order to clone the øO31 DNA which reacted with the probe, phage DNA was digested with C/al, electrophoresed and the 7-kb fragment was excised from the gel and purified. The isolated fragment was then ligated with Accl-digested pUC19 DNA and transformed into E colí JM1O9. Plasmid DNA was extracted from recombinant clones carrying OO31 DNA inserts (white colonies on IPTG/X-gal plates) and these were subjected to restriction analysis using Hindlll and BamHl. This resulted in restriction fragments of approximately 1 and 6 kb, in addition to the 2.7-kb vector (Figure 3.7A). Only the 1-kb fragment was detected when these digests were subjected to Southern hybridization analysis using DIG-labelled oligo A-l (Figure 3.78). Single restriction digests using Hindlll and Bam\l indicated that there was an internal BamHl site in the insert DNA (result not shown). Digestion of plasmid DNA with BamHl enabled isolation of the reactive 1-kb fragment, which was subcloned into pUC19 and the recombinant plasmid was designated pJCP5OO. The nucleotide sequence of the insert of pJCPSOO was determined using double stranded plasmid DNA as template and both forward and reverse sequencing primers (Figure 3.8). Comparison of this sequence with those in the GenBank and EMBL databases identified a region (nucleotides 1 to 192) with strong (93.3%) homology to a portion (nucleotides 22,346To 22,538) of the genome of bacteriophage lambda. This region encodes "orf-194", the function of which is unknown (Hoess et al., 19781 Sanger et al., 1982t.. The homologous portion of the insert of pJCPS0O appears to be the distal portion of an open reading frame (ORF) which terminates at nucleotide 2O2. There was no significant overall sequence homology between the insert of pJCP5OO Figure 3.7 Restriction and Southern hybridization analysis of recombinant plasmids pUC19 derivatives containing the 7 kb fragment from QO31 were digested with Hindlll and Bam\l (Tracks 1, 2, 3 and 4), electrophoresed on a 1% agarose gel and stained with ethidium bromide (A). Track M: DNA size markers (23.1, 9.4, 6.6, 4.37, 2'3, 2.O, and 0.56 kb, respectively, from top to bottom). DNA was transferred to a nylon filter and hybridized with DIG- labelled oligo A-l and washed at high stringency (B). The mobility of the 1 kb fragment is indicated for each panel. B
432t
A
M t 2 3 4
ú ¡ ì. ,lJ-¡! U r¡ ¡rrl -
I-æf -1.0kb - t¡ '¡'¡ Ö Figure 3.8 Nucleotide sequence of the 1042-bp insert of pJCPSOO
The nucleotide sequence of the insert was determined by sequencing double stranded template using both forward and reverse sequencing primers, as described in Section 2.45. The sequence is shown 5'-3' commencing at the Bam1l site. The region with homology to the genome of bacteriophage lambda is underlined and the region with part¡al homology to oligo A-1 is double underlined. The distal portion of a possible open reading frame (denoted "...orf*") is shown in bold type. I BamHi 6l GCCGCTTCAGGATGCTGCAGATCTGGAAATTGCAACGGAGGAAGAAATCTCGTTGCTGGA
t2l AGCATGGAAAAAGTATCGG GTATTGCTGAAC C GTGTTGATACGTCAACTGCACAGGATAT
181 TGAATGGCCAGCACTGCCGTAG G GTAAAAC ATATAAATTC TATAATTAGATG TATCTTTC
24t CATTTAC GGCAAGGAAGGGGGCiTGGAAGAC GTAAAGCATCTCACAC CGAGATTATTTTT
301 TATATGTCAGGTGTCTGAAGGTTTTGCTTTGGCTC TTAAAATGGTTTGC CGCGAGGTTTT
361 GAATTCC CGGGCAATGGCACTTATACTTACACCTGACTTAATTC GTTCGAATAC CACCTG
42t TTTCTGTTCTTCATTTTACCACAGGTGGGCGGCCCAAAACGTTTCCCTGCGGCCGGGGGC
481 TCTTACTATC CCGGAAATGAGGGC G GTTCAAGGTAAAAGGCTC GTCAAATTCAGC G GACT
541 GCTGAAATTACTTGGATTCATCATTTTC C CTGTTGG C CAGGTCAGGTCAATGC CACC CAA
601 TGCTAAGCMTGCACTCTGATAC CTGTTCGGTCAGTTGTC CACTGTTTTCCTGATATC CA
661 TTGcATTAcAAcCAAGGcGATccAGTTTTGTcACAATCAAT@Gc
727 GAGCAAGCAAC CGGTTAAAAC CAGGACGCTCACTG GTTGCTG CTGAG C C GC TAATGTGTT
781 CTTC GATTATTTGCTGAGGTTTGATTTTAAAAC CTG CACTTTC GATTTCCC G GCGTTGAT
841 TTTCGGTGGTCTGATC CAGC GTTGATATC CGACAGTAAGCAAAAATTC GAGACATAGTGA
901 GACTCTATACGAAATTGGTGTTCATATCATAATGCATCTCAGAAAATAATTATGATTATT
961 TTTGTGCATATTTGTATGTACACGTTCGAAAATAAAC GAATG CGTATGCAAC CCCGTAAT
1021 TTTGGTGAGAC CCAAAATC GAT 84 and the published sequences for the genes encoding SLT-I or SLT-Il. There was, however, partial homology 176.5o/"1 between the sequence of oligo A-l and a region within the @O31 fragment (nucleotides 702-7181, which may have been responsible for the apparently artifactual labelling by the oligo probe. The region of the insert of pJCP5OO from nucleotides 20O to 1042 had approximately 63o/" DNA homology to the E coli transposon Tn2SOl (Michiels et al., 19871. BLASTX analysis identified three regions of significant amino acid homology (encoded on the minus strand) between the translated pJCPSOO sequence and the Tn2íOl resolvase. For these three regions, 68/86 (79o/ol, 26137 (7O./"1 and 15120 (75o/"1 amino acids are identical, although they are in different reading frames; this is most probably due to the fact that the pJCPSOO DNA sequence was derived from data for one strand only.
3.2.1O Further Attempts to lsolate SLT-converting Phages from E. coli Strains 03l and PH
Although eO31 and @PH did not appear to encode SLT-related genes, this did not exclude the possibility that the parental E. coli strains were also lysogenized by other phages which were indeed capable of toxin-conversion. Only two representative phages had been plaque-purified after primary induction of each strain and analysed: restriction analysis of the two phages from each strain indicated that they were indistinguishable from each other (result not shown). To determine whether the primary induction filtrates contained a small proportion of an SlT-converting species, in addition to
ø031 and @PH, these filtrates were diluted such that approximately 200-400 plaques per plate resulted after transfection into E. coli C6O0. Two plates from each filtrate were directly blotted with nylon filters, fixed and hybridized with SLT-l- and SLT-ll-specific DNA probes labelled with DIG using PCR 85
(Section 2.35). No plaques from either strain hybridized with the probe (result not shown).
ln a final attempt to detect other phages from strains O31 and PH, undiluted primary induction filtrates from each strain were plated on E. coli C6O0 lysogenized with QO31 or QPH, respectively. This would enable specific detection of low numbers of a putative bacteriophage, which was sufficiently different to permit superinfection of the C6O0 lysogen, regardless of high background levels of the other phage species. However, no plaques were obtained for either filtrate when tested in this manner.
3.3 Discussion
I 3.3.1 lsolation of SLTEC from Adelaide Chíldren á ¡ The aim of the SlT-screening phase of this study was to test as many T strains of E. coli as were available, in an attempt to isolate SLTECs from
Adelaide children for subsequent molecular analysis, rather than to conduct a comprehensive epidemiological analysis of the prevalence of SLTEC in specific patient groups. Thus, a high proportion of the strains tested were isolated by others in an earlier study (Bettelheim et al., 199O). Moreover, the number of
E. coli isolates from a given patient varied; the previous study selected a maximum of 10 isolates from each primary culture, whereas up to 100 isolates were tested from some patient samples obtained during the course of the present study. Clearly, given the low numbers of SLTEC sometimes found in faeces of patients with HUS or HC (Smith and Scotland, 1988; Karmali, 1989) there is a strong possibility that SLTECs may not have been isolated from infected patients from whom only a limited number of strains were tested. Thus, the isolation rate of 4 SLTECs from a total of 2OO individuals is likely to be an underestimate. i 86 ¡ à
The production of SLT and/or the presence of SLT genes in the 4 I Vero cell cytotoxicity, as well as by hybridization strains was confirmed by i t w¡th oligo and DNA probes, and by PCR. lnterestingly, many of the strains reported to produce Vero cell cytotoxins by Bettelheim et al. (199O), were ) not judged to be SLT positive using any of the above criteria, when re-tested i in the present study. This implies that the Vero cell bioassay employed by I I these workers lacked specificity, andlor that the SLT genes were extremely unstable and were lost during storage or subsequent subculture. lnterestingly, Karch et al. (1992) have recently reported that SLT genes are frequently lost from clinical isolates of E. coli during subculture. One third of all SLTEC isolates tested in their study lost their SLT genes after a single subculture, with further losses on subsequent subculture. One possible
,l;' reason for this instability could be spontaneous curing of SLT-encoding þ bacteriophages (Karch et al., 1992). Spontaneous curing of an SLT-Il- encoding bacteriophage has also been proposed as an explanation for the apparent production of only SLT-llc bV E. coli strain E-32511 by Hü et al. (1991), whereas Schmitt et al. (1991) reported the presence of both SLT-ll and SLT-llc genes in this strain. The same phenomenon was obseived in the present study for two of the SLTECs isolated. Strain s 234 and 106 were lysogenized with bacteriophages which were indistinguishable from the SlT-l-encoding phage HlgB. However, when subcultures of the stored strains were subsequently tested, both cytotoxicity and SLT probe-reactive sequences could no longer be detected (result not shown). Moreover, SlT-converting bacteriophages could no longer be induced by UV treatment. SLT production by strains 031 and PH, however, was Stable even after repeated subculture. Clearly, the apparent instability of SLT genes in clinical isolates has important ramifications as far as diagnosis of SLTEC infection is concerned. lndeed, it may account for the low numbers of SLTEC found in the faeces of 87 t 'Þ"
patients with SLTEC-related diseases such as HUS, and the fact that in many I a number of such cases, the presence of SLT able to be neutralized by t¡ t specific monoclonal antibody could be demonstrated in faeces, but SLTECs were not isolated (Karmali, 1989). ln these circumstances, diagnostic t methods aimed at direct detection of SLTECs in faecal or other samples, i rather than subsequent analysis of pure isolates, are likely to provide a more
I accurate indication of the presence or otherwise of SLTECS. This question will be addressed further in Chapter 6 of this thesis.
3.3.2 Analysis of Bacteríophages Strains 106 and 234 were shown in the present study to be lysogenized with bacteriophages capable of converting C600 to SLT production. Restriction analysis indicated that these bacteriophages were
t indistinguishable from phage H198, which encodes SLT-|. ln view of this
similarity, ø1 06 and O234 were not further investigated. lnterestingly, although bacteriophages were induced from each of strains 031 and PH they did not convert E. coli C6OO to SLT production following transfection. No SLT-related sequences were detected in øPH using oligo A-l as a probe, but weakly reactive sequences were detected in a portion of the genome of
øO31, However, when the reactive @O31 DNA fragment was cloned and subjected to sequence analysis, no overall homology to either SLT-l or SLT-Il could be found. This fragment did have significant homology, however, with the portion of the genome of the bacteriophage lambda, which encodes "orf- 194" (Hoess et al., 1978; Sanger et al., 1982l'.
A further attempt was made to isolate SLT-encoding phages from UV- induced cultures of 031 and PH, by plaque hybridization analysis using SLT probes. Primary induction filtrates were also plated on C600 lysogenized with the bacteriophage already isolated from the respective host stra¡n. 88
However, these attempts were unsuccessful. Thus, it seems likely I both of I that the SLT genes in these strains are encoded on the E colí chromosome. Whilst classical SLT-I and SLT-ll genes have been shown to be phage- encoded (Smith et al., 1983; O'Brien et al., 1984; Willshaw et al., 19871, variant SLT-ll genes from both porcine and human derived SLTECs are chromosomally encoded (Gyles et al., 1988; Weinstein et al., 1988b), as is the SHT gene in S. dysenteríae type l(Strockbine et al., 1988)' ln the
present study, the presence of QO31 and @PH prophages in the chromosome of the respective E coli strains complicates assessment of whether or not these contain other, possibly defective, prophages which might encode SLTs. Southern hybridization analysis using random primer-labelled total øPH DNA revealed extensive homology with multiple restriction fragments in digests of DNA from other bacteriophages, including OO31, lambda, 933W, 933J and H198 (result not presented). Thus, although toxin-converting bacteriophages were not detected in strains 031 and PH, the possibility that the SlT-related genes in either of these strains are encoded on defective phage particles cannot be eliminated. 89
CHAPTER FOUR
CLONING AND NUCLEOTIDE SEOUENCE OF VARIANT SHIGA.LIKE TOXIN II GENES FROM ESCHERICHIA COLI OX3=H21 STRAIN 031.
4.1 lntroduction
As mentioned in Chapter 3, SLTEC strain O31 was isolated from the gut contents of a case of SIDS, and belonged to serotype OX3:H21. OX3 strains are more commonly isolated from domestic animals than humans and many produce SLT (F. and l. Orskov, pers. comm.). Hybridization of O31 DNA with oligo probes and hybridization analysis of PCR products obtained using SlT-specific primers, suggested that this strain contained SLT-Il-related genes. Like the variant SLT-ll genes from animal (and some human) sources, these genes did not appear to be phage-encoded. Thus, the possibility existed that 031 might also produce a variant SLT-ll. This chapter describes the molecular characterization of the SlT-related genes from this strain.
4.2 Results
4.2.1 Further PCR Analysis
As an initial means of characterizing the SLT type produced by O31, genomic DNA was subjected to further PCR analysis using a different protocol to that used in Chapter 3. This utilizes two pairs of oligo primers (la & lb and lla & llb) which direct the amplification of 130- and 346-bp DNA fragments from SLT-I and SLT-ll genes, respectively (Pollard et al., 1990). DNA from 031 as well .as DNA from reference bacteriophages H198 and 933W, was 90
amplified and electrophoresed on 2o/o agarose gels (Figure 4.11. Amplification of 031 DNA yielded a single PCR product of approximately 346 bp. Amplification of the DNA from H198 generated an approximately 130-bp fragment, but both 13o-bp and 346-bp PCR products were obtained using the 933W DNA template. The presence of the two PCR products in the 933W reaction was unexpected, and may be a function of variations in the
heating/cooling rates and/or temperature sensors of the PCR cycler used in this study compared with that used by Pollard et al. (199O). Nevertheless, DNA from O31 directed the production of only the 346-bp PCR product, unequivocally indicating that it contains sequences closely related to SLT-Il.
4.2.2 Southern Hybridization Analysis.
Genomic DNA from 031 was digested with either Pstl, BamHl or BgAl and subjected to Southern hybridization analysis using DIG-labelled bacteriophage H198 or 933W PCR products as SLT-l- or SLT-ll-specific probes, respectively. As expected from the PCR results above, no DNA bands in the O31 digests were labelled w¡th the SLT-I probe (result not
shown). However, with the SLT-ll probe, two fragments were labelled in each of the Pstl, BamHl and Bglll digests of 031 DNA (Figure 4.2Ì'. Two
fragments were also labelled with the SLT-ll probe in digests of DNA from E. coli C6OO lysogenised with bacteriophage 933W, but the sizes of the fragments were different from those obtained with the respective 031 DNA digest (Figure 4.2l.. No fragments were labelled by the SLT-l probe in digests of E. coli C6OO::933W DNA and furthermore, no bands were labelled by either probe in digests oÍ E. coli C600 DNA (result not shown). These results confirm that the SlT-related gene (or genes) in strain O31 is more closely related to SLT-Il than SLT-|, and that there are sequence differences in the E coli chromosome, close to or within the SLT genes themselves, resulting in restriction polymorphisms. Figure 4.1
Detection of SLT genes by PCR
DNA samples were subjected to PCR-amplification using SLT-l-specific (la and lb) and SLT-Il-specific (lla and llb) primers, as described in Section 2.43. Aliquots of each react¡on were electrophoresed on 2o/o agarose gels and stained with ethidium bromide. Tracks: M: DNA size markers (50O, 4O4, 331, 242, 190, 147, and 110 bp, respectively, from toP to bottom). -: Negative control (E. coliC6O0 DNA). 1; E. colistrain O31 DNA.
2: @933W DNA.
3: øH1 98 DNA. M - 12 3 rt
-Ò Figure 4.2 Southern hybridization analysis of E coli C6OO::933W and E coli strain 031
Genomic DNA from E. colí C6OO::933W (A) and E. coli 031 (B) was digested with Psfl, Bamïl or BgAl, electrophoresed on a O.8o/" agarose gel, and transferred onto a nylon filter. The filter was hybridized with a PCR DIG- labelled SlT-ll-specific probe at high stringency, as described in Section 2.39. The mobility of various DNA size markers is indicated. Bgl TI BamHI Psf I
.-^-\ 4\r\-^ A B ABAB -,\â
a kb
tr - 23-1 ttD -lþ - 9.4 - 6.6 -{.1 -
- 2.3 - 2.0
(I
0. 56 91
4.2.3 DNA Sequence of the O31 PCR Product To examine the possibility of sequence variations within the SLT coding sequences of 031 and bacteriophage 933W, the 346-bp PCR product derived from O31 DNA was purified from agarose gels, treated with S1 nuclease and cloned into the Smal site of pUC19. The DNA sequence of the inserted fragment was then determined and compared with the published sequence of the respective region of the SLT-ll gene from bacteriophage 933W (Jackson et al., 1987a) (Figure 4.3). There was one base difference in this region and it was consequently decided that cloning and sequencing of the complete SLT gene from 031 was warranted.
4.2.4 Cloning of the SLT-related Gene Genomic 031 DNA was digested with Psfl, ligated with Psfl-digested pUC19 and transformed into E. coli JM109. Psfl was chosen because the Southern hybridization data indicated that the genes of interest were located on fragments of 4.6 kb and 1.45 kb suitable for direct cloning into plasmid vectors, whereas the reactive fragments ¡n the other enzyme digests were too large. Lysates of the resultant clones were screened for the presence of SlT-related genes by dot-blot hybrid¡zation with the SLT-¡I probe. One clone, out of a total of approximately 750, reacted strongly with the probe (see Figure 4.41 and this contained a recombinant plasmid with both 1.S-kb and 4.6-kb Psfl inserts. Further Southern hybridization analysis indicated that only the 4.6-kb fragment was reactive with the probe (result not shown), and this fragment was isolated and subcloned (in both orientations) into pUC19, These recombinant plasmids were designated pJCPSOI and pJCP502. Both the culture supernatants and French pressure cell lysates of E. coli JM109 harbouring either of these plasmids were cytotoxic for Vero cells. The effect of a 1:1OO dilution of E. colí ¡lVllO9IpJCPSO1l lysate is compared with that
Figure 4.3 Comparison of the DNA sequence of the PCR product from 031 with that of
SLT-¡I from bacteriophage 933W
The nucleotide sequence of the 346 bp PCR product derived from amplification of O31 DNA was determined as described in Section 2.45. The Sequence is shown 5'-3' and is numbered from 1. The sequence of the corresponding portion of the SLT-Il gene from bacteriophage 933W, commencing at nucleotide 426 (Jackson ef al., 1987a), is shown beneath the 031 sequence, with identical nucleotides represented by dots. The regions homologous, or complementary to the two oligo primers are underlined. 031 I TTAAC CACACCCACGGCAGTTATTTTGCTGTG GATATACGAG G GC TTGATGTCTATCAGG sl tII 426
031 6l CGCGTTTTGAC CATCTTCGTCTGATTATTGAG CAAAATAATTTATATGTG GCTGGGTTC G sl tII 486 c
127 031 TTAATAC GGCAACAAATACTTTCTAC CGTTTTTCAGATTTTACACATATATCAGTGC C C G sl tII 546
031 l8l GTGTGACAACGGTTTCCATGACAACGGACAGCAGTTATACCACTCTGCAACGTGTCGCAG sl tII 606
031 247 CGCTGGAACGTTCCGGAATGCAAATCAGTCGTCACTCACTG GTTTCATCATATCTGGCGT sltII 666
031 301 TAATGGAGTTCAGTGGTAATACAATGACCAGAGATGCATCCAGAG C sl tII 726 Figure 4.4 Dot-blot hybridization of clone lysates
Aliquots of lysates of clones from the E. coli strain 031 gene bank were spotted onto a nylon filter, and hybridized with a PCR-DIG-labelled SLT-Il- specific probe and washed at high stringency. The results for 192 of the 75O clones tested are presented and the location of the single.strongly positive clone is arrowed. *t € ' çrj ;tÊ ii.:ä'r: Þ
¿. i? Figure 4.5 Cytotox¡city of E. coli ¡M109tpJCPsO1I
Confluent Vero cell monolayers were treated with or without French pressure cell lysates of the indicated bacterial cultures, diluted 1OO-fold in Maintenance Medium. Monolayers were photographed after incubation at 37 oC for 3 days (Magnificat¡on 4O0x). A: Untreated monolaYer. B: E. coti ¡M109tpUC19l. C: E. colistrain 031. D: E. coli ¡M109tpJCPsO1l. Ai" li¡' ;'{ ',
_ù ì
',-
,1
;'f -r - I
Figure 4.6 Map of the insert of pJCPSOl and scheme for sequencing
The solid portion of the map denotes vector DNA. Restriction sites are indicated as follows: A, Accl; E, EcoRl; H, Hindlll; K, Kpnl; P, Psfl. Nested deletions were generated as described in Section 2.44. The portion of insert DNA retained in various nested deletion derivatives, and the Vero cell cytotoxicity (tox * or tox-) of E. coli JM1O9 harbouring these plasmids is indicated. The box labelled "SLT" indicates the location of the putative SLT coding sequence, deduced from the above data, The arrows beneath the map indicate the portions of the plus (-))and minus ((-) strand which were sequenced in the various nested deletion derivatives of pJCPSOI or pJCP5O2. EKP A A A K E K PH \r I I I I I l,/
S1 T
tox+ tox- tox - tox + tox+ tox- € _+_--_| _+ -+# -+ + + + <_ <- <-- +- F- - 1.0kb Figure 4.7 Nucleotide sequence of the the insert of pJCP5O1
A, Nucleotid es 1-2O7O including the SLT coding region, are shown in section while nucleotides 2O71-4644 are shown in section B (overleaf). The amino acid translation (represented by single letter code above the first nucleotide of each codon) is shown above the coding regions for the putative A and B subunits (nucleotides 570-1529 and 1542-1805, respect¡vely)' Putative signal peptidase cleavage sites are indicated by arrows. Putative ribososme binding sites (rbs) for each subunit and putative -1o and -35 promoter regions are underlined. The location of two possible transcription terminator sequences (nucleotides 1BG6-19o9 and 2074-2093) are double underlined' The position of an open reading frame downstream from the sLT gene
{nucleotides 3059-3718} is indicated in botd type' A
i CTGCAGCAGATAATCAGTGCGAGCAGTCGTCCGGGTGACCfGGTTGCAGATTTCTTCATGGGGTCAGGTTCGACAGTCAAAGCAGCGATG
9 1 GCGCTGGGGCGTCGTGTAACTGGCGTTGAGCTGGAGACTGAACGTTTTGAGCAGACGGTCAGGGAAGTTCAGGATTTAGTCAGTCAGAAC
1 B 1 GGATGATATTGCAGGAT TAGTTACGTACCGTTATTATCCTGCGCCCGGCCCTTTAGCTCAGTGGTGAGAGCGAGCGACTCATAATCGCCA
27 I GGTCGCTGGTTCAAATCCAGCAAGGGCCACCATATCACATACCGCCATTAGCTCATCGGGATAGAGCGCCAGCCTTCGAAGCTGGCTGCG 35 -10 3 6 I CGGGGTTCGAGTCCTCGATGGCGGTCCATTATCGGTATTCAGCGTTGTTAGCTCAGCCGGACAGAGCAATTGCCfTCTAAGCAATCGGTC
451 ACTGGTTCGAATCCAGTACAACGCGCCATACTTATTTTTTCTGGCTCGCTTTTGCGGGCCTTTTTTATATCTGCGCCGGGTCTGGTGCTG rbs MKCILFKI4,VLCLLLGFSSVSY 541 ATTACTTCAGCCAAAAGGAACACCTGTATATGAAGTGTATATTATTTAAATGGGTACTGTGCCTGTTACTGGGTTTTTCTTCGGTATCCT STR E F M I D F S T A A S Y V S S L N S I R T E I S T P L E 631 ATTCcCGGGAGTTTATGATAGACTTTTCGACCiAAiAAAGTTATGTCTCTTCGfTAAATAGTATACGGACAGAGATATCGACCCCTCTTG H I SQGTTSV SV I N HTP PGSYFAVD I RGLDV 72r AAcATATATcTiAGGGGAccAcATCGGTGTcTGTTATTAACCACACCCCACCGGGCAGTTATTTTGCTGTGGATATACGAGGGCTTGAÏG YQARFDHLRL I I EQNNLYVAGFVNTATNTF 811 TCTATiAGGcGCGTTTTGACCATCTTCGTCTGATTATTGAGCAAAATAATTTATATGTGGCTGGGTTCGTTAATACGGCAACAAATACTT Y R F S D FÌ H I S V P G V T T V S I4T T D S S Y TT L Q R 901 TCTACCGTTTTTCAGATTTTACACATATATCAGTGCCCGGTGTGACAACGGTTTCCATGACAACGGACAGCAGTTAfACCACTCTGCAAC VAAL ERSGMQ I SRHSLVS SYLALME FSGNT 991 GTGTcGcAGcGcTGGAAcGTTccGGAATGiAAATCAGTCGTCACTCACTGGTTTCATCATATCTGGCGTTAATGGAGTTCAGTGGTAATA l4TRDASRAVLRFVTVTATALRFRQIQREFR t0B1 CAATGACCAGAGATGCATCCAGAGCAGTTCTGCGTTTTGTCACTGTCACAGCAGAAGCCTTACGCTTCAGGCAGATACAGAGAGAATTTC Q A L S E T A P V YTMT P E E V D L T L N I.,GR I S N V L ),171 GTiAGGcACTGTCTGAAACTGCTccTGTGTATACGATGACACCGGAAGAAGTGGACCTCACACTGAACTGGGGGAGAATCAGCAATGTGC P E F R G E G G V R V G R I S F N N i S A 1 L' G T V A V I L I26I TTCCGGAGTTTCGGGGAGAGGGGGGTGTCAGAGTGGGGCGAATATCCTTTAATAATATATCAGCGATACTGGGCACAGTGGCGGTTATAC N C H H Q G A R S V R A V N E E I Q P E C Q I T G D I I-.Y I 13 51 TGAATTGCCATCATCAGGGGGCGCGTTCCGTTCGCGCCGTGAATGAAGAGATACAACCAGAAfGTCAGATAACTGGCGACAGGCCAGTTA R i N NTLl/llE S NTAAAFLNRRAHS LNTSGE* t44L TAAGGATAAACAATACTTTATGGGAAAGTAATACCGCAGCTGCTTTTCTGAATCGCAGGGCCCACTCTTTAAATACATCCGGAGAATAAC rbS t4 K K I F V A A L F A F V S V N A l'l ATA D C P K G K I 1531 AGGAGTTAAATATGAAGAAGATATfTGTAGCGGCTTTATTTGCTTTTGTTTCTGTTAATGCAATGGCAGCTGATTGTCCAAAAGGTAAAA E F S KY N E N DT FT V KV AG KE YlrlT N RI^/ N L Q P L 1621 TTGAGTTCTCTAAGTATAATGAGAATGATACATTCACAGTAAAAGTGGCCGGGAAAGAGTACTGGACTAACCGCTGGAATCTGCAACCGC LQSAQLTGl4TVT I KSNTCASGSGFAEVQFN r7]j TAC-TGCAAAGCGCACAGTTAACAGGAATGACGGTAACAATCAAATCAAATACCTGTGCGTCAGGTTCAGGATTTGCTGAAGTGCAGTTTA
1BO1 ATTAATATCAGAAGCATTGCTGGTTTCGTGGTGTGCAGCAATGTAGTTACAGTGTAATCAATGTCACAATTCAGTCAGTTG
189 1 TGCCCGACTGAGAATTTGTTAAAAAAAAATCCTGCATGGTGAATCCCCCTGAGCGGCGGGGCATATCAGCGTCACAGGTGTTTCTGTTTT
1SB1 ACCTCTATCCTTTCTGTGCGGGTTCAGGTGCTGATACTGAACTCACCGGGAGGCACCCGGCACCATGCATGAACGGTACATAGCGCATAC ',:
,t à
I B I t
I 207 r ATCAG!çee]e]ee.EGAeçGGc]TTcTTGTGGGcAAAAAAAAAAAAAGCCcGCGCCGGGAGACGCGGGCGGcAAGGAATAAAcAACAAAA I
2161 CGTGAGGTAATATTTCAGCTGGCGAATAATTCCCGCCAGTAATCAATCTTGCGCAACTTCGTGGCCTTTITCGTATTGCGGGCTGTAGTC I 225t TTCCTCCTGTCATTGTCCTGTAACTTCCGGACTTCAGCCCGCTCCTTATCCGACTAACAACATTATCCCGACCGGGAGGATTCATGACAT I I 2341 TfAAACATTACGATGTGGTCAGGGCGGCATCGCGTCAGACCTTGCTGATGCACTTGCGCAAAAAATTCGTGAAGGATGGCAACCATACGG
?431 TGGGCCGTTTTCTTCGTATACGGATGATGGTGCAGCACTTATTCAGGCGATTGTCGCAGAAGGTGATGTGAGCACACCTGTTGTGGTGAA
2521 GCCGACAGGTGGAGAAGGTGCAGTAATCAGCGCCACCAGCGACCCGGAGTATTACTTTGTTGTGGCTCTTGCAGGGCAGTCAAACGGCAT
261 I GTCGTATGGTGAAGGTCTTCCGCTGCCGGAGACATATGACCGTCCGGACCCGCGTATTAAGGAAGCTTGCGCGCCGAAGTACGGTGAACA
21 07 CCAGGACGAGAGCAGAATGTAAAGTATAACGACATCATTCCGGCGGACCATTGTATGCATGATGTGCAGGACATGAGCCGCCTTAACCAT
2791 CCGAAAGCGGACCTGTCAAAGGGGCAGTACGGTACCGTGGGGCAGGGGCTGCATATCGCCAAAAAACTGCTGCCGTTTATACCGGCGAAT
2BB 1 GCGGGfATTCTGCTGGTTCõGTGCTGTCGTGGTGGTTCAGCGTTCACCACCGGAGCTGATGGCACATACAGTGACGCTGGCGGTGCCTCG
297 r GAGAAfTCAACCCGCTGGGGTGTGGACAAGCCGCTGTATAAGGACCTTATCGGTCGAACAAAAGCAGCACTGAAGAAGAACCCGAAAAAT
3061 GTGCTGTTTGCCCGTGGTGTGGATGCAGGGGGAATITGATTTTGGCGGTACGCCGGTAAATCACGCCGCACAGTTTGGCGCGCTGGTTGA
3i51 TAAATTCCGTGCAGÀCCTGGCGGATATGGCAGGCCAGTGCGTCGGTGGCTCTGCTGGTGGTGTTCCCTGGATATGCGGGGACACGACGTA
324r TTTcTGcAAGcAGAAcAAcGAATccAcGTAccAcÄcGcrGTAcGGcAccrATAAiw{cAAAAcGGAAAAGI4ÄTATccATTTcGTAccGTÏ
333 I CATGACGGATGAGAACGGGGTGAATGTGCCGACGAACAAACCGGAAGAAGACCCGGACATTCCGGGTATCGGATATTÀCGGTTCGÁÀATG
3421 GCGTGACAGCTCAGCCACCTGGACGTCACAGGACAGGGCCACCCATÍTCAGCACCTGGGCACGCCGGGGGÄTTÁTTTCCGACCGTCTGGC
3511 AACGGCGATTCTGATTCATCCGGGÀCGAACCACAGTAAAAGTCGATGCTCCÀTCTTCTGAAACTGGCGCACCGACGCCATCACCGTCAGA
3 601 AAATGA/IGCGGTAAGCACAACAACACTGCTGTCTIACCGTGCCAGTGAGTCTGAÄGGGAGGCTGÁCCGAACAGGGCTGGAGTGCTGGCGG
369i AGGTAAAGCGGAAATTGTTGCCGATTGAAGGTGCAACAGGTGGTACCGCGATGAAGCTGAGCAAGCAGACAGGAGTTGGGGTCATGGTAT
3781 CTGGAGCATGATGCCGGTACTGGCGCTGAACTGCTGAAAAATGGCGGTTTAATCAGCTGCCGCTTTAAGGCATCCGGTGAACTGGTAGCA
387 1 AATCAGTATGTCATTGCACTTTACTGGCCGGTTTCCTCTCTGCCGCAGGGTGTCACCCTGACAGGGGATGCAGGGAATAATCTGCTGGCA
3961 GCGTTTTACATCCAGACAGATGCAAAAGACCTGAATGTGATGTACCACAATGCGAAGGTGGCGACAAATAACCTGAAACTGGGAAGCTTT
405 1 GGCGCATTTGATAACGAATGGCATACGCTGGCfTTCCGCTTTGCCGGGAATAACCCTGGGTCCCCGGTTTAAGGTCGGGATGGCACCCCG
4t4t TCAACTGACCAGTCACGGGCAGTGCATTTCGGCGGATAATTGCATGTTACAGATATTCCCAAAAGTGCGCTTACCCGGTGCTGATTGACA
423I AGCATTCGGTGGAAGTGAACAACCGGATGCCGCGGCATGATAAAAAAGCCGCCAGTGCCTGAGACAACCGGCGGTGTGAGATTCATGGAG
43?I AATCAAGGAAAGATACCATTACTTTCGTCACTGGCATTTTTAAACGAAAACTGTTfACTTAGTCAACCATAACGGTAAGAAACTAT6ACG
44),1 TTTATTCATCAGCTGATGCTGTACTTCTGTACGGTGGTCTGTGTGCTGTATCTTCTTTCGGGTGGGTACAGGGCAGTGCGCGATfGCTGG
4501 CGCAGGCAGATTGATAAAAGGGCCGCAGAGAAAATCAGCGCCAGTCAGTCAGCCGGAAGCAAACCCGAAGAGCCCATTACTCCTTAATAA
459 1 CCCCTTTCAACGAGAAAATCCCATGTCAGAAATTACATCCCTGGTTCACTGCAG .f, t'
't{
., I Table 4.1 ¡ I
genes DNA sequence homology between sLT-0x3 and reìated
DNA sequence homoì ogY
0peron Asubunit. Bsubunit
vtx2ha 95.9% 88.6%
SLT- I Iv 92.0% 79.7%
SLT- I I 95.6% 85.9%
for DNA sequence homology between sLT-0x3 and the published sequences vtx2ha (Ito et â1., 1990), sLT-II (Jackson et dl., 1987a) and SLT-IIv (t.leinste'in et â7., 1988b) was calculated separately for the A and B subunit coding regions. 93 patient with HUS (lto ef al., 1990) (95.9% and 88.6%, homology for the A and B subunit genes, respectively). Analysis of the region downstream from the SLT-OX3 coding sequence indicated the presence of a possible rho-dependent transcription terminator sequence (nucleotides 1866-1909) with a Gibbs Free Energy of -17 '1 kcal/mole, the structure of which is shown in Figure 4.8. A second possible transcription term¡nator sequence, with a Gibbs Free Energy of -19' 1 kcal/mole, was found further downstream from the SLT gene (nucleotides 2074-209$¡ (Figure 4.8). Another oRF, sufficient to encode a 23.6 kDa polypeptide, was located from nucleotides 3059 to 3718, but BLASTX analysis (Altschul et al., 1990) of the sequence from nucleotides 1850 to 4644 failed to detect a significant degree of deduced amino acid sequence homology between this region and any other known protein sequences' However, a significant degree of DNA homology Q5.5o/"1 was detected between a 45O-bp region (nucleotides 1850-2300) immediately downstream from the SLT-OX3 B subunit coding sequence and the analogous region of the SHT operon of S. dysenteríae type 1 (Kozlov et al., 1988). The SLT-OX3 toxin-coding region was less similar and shared only 59.7o/o DNA homology with the SHT coding sequence. To determine whether the observed DNA sequence variations re,sulted in amino acid sequence variation, the deduced amino acid sequence of the SLT-OX3 gene was compared with that reported for SLT-ll and other related
genes (Figure 4.9). The %" amino acid sequence homology amongst SLT-OX3
and the other SLT-ll-related toxins is also summarized in Table 4.2.
4.2.6 Cloning of a Second SLT-ll-related Gene from Straín O31 The Southern hybridization analysis shown in Figure 4.2 is strongly suggestive of the presence of a second SLT-Il-related gene in strain 031 (4.6- kb and 1.45-kb Pstl fragments were labelled by the SLT-Il-specific probe). A Figure 4.8 Structure of possible transcription terminator sequences
The upper figure represents the region from nucleotides 1863 to 1912, with base pairs denoted ":". The Gibbs Free Energy of this structure is -17.1 kcal/mole.
The lower figure represents the region from nucleotides 2O71 to 2O96. The
I Gibbs Free Energy of this structure is -19.1 kcal/mole. 1863 -G *T.^ a-ç 4-a J-T. t"-A-C-A.A-T-T-C T-C-A-G-T-T-G G b G. T T-T-G-T-T A-A-G-A-G-T-C-A-G-C-C-C. C' -A-A'' Ì' 'G-T' t9t2
2071 -¡'T' c-R-e-c- -T- c-c-c -T-c C t -G-G -A-G c ,T-T-C-G-G -6, -¿-T 2096 Figure 4.9
Deduced amino acid sequence of SLT-OX3 and comparison with related SLT- ll sequences
The deduced amino acid sequences of the A and B subunits of SLT-OX3 are shown, numbered from the first residue in the mature polypeptide. Arrows denote the putative signal peptidase cleavage sites. Published sequences for SLT-ll (Jackson et al., 1987a1, SLT-llv (Weinstein et al., 1988b) and vtx2ha (l1o et al., 1990) are also shown. Amino acids are represented by single letter code; dots denote amino acids identical to that for SLT-OX3; 'r-r! denotes absent residues. A SUBUNIT
-1¿t SLT - OX3 _2? MKC I L FKl^lV LCL L L GF SS VSYSR E F M IDFSTQQSYVSSLN VTX2 h a T SLT-IIv LI a T SLT- I I T SLT_ OX3 19 S I RTE I STPLEH i SQGTTSVSV I NHTPPGSYFAVDI RGLD VTX2 h a T SLT-IIv SLT_ I I ::: 1: :::::: -ú:::1ì:Î::::: S LT- OX3 59 VYQARFDHLRLI I EQNN LYVAG FVNTATNTFYR F SD FTH I VTX2h a SLT-IIv E .RTA SLT_ I I SLT- OX3 99 S VPGVTTVSl'1TTDSSYTTLQRVAAL ERSGMQ I SRHS LVSS VTX2h a SLT- I Iv SLT_ I I SLT - OX3 I39 YLALMEFSGNTMTRDASRAVLRFVTVTAEALRFRQ I VTX2h a QREF SLT-IIv SLT_ I I
SLT.OX3 179 RQ A S T A T M T E V D I l,J b S V P FRGEGGV VTX2h a D YD SLT_IIv .L D YA SLT- I I D YD SLT-OX3 219 RVGR I SFNN I SA i LGTVAV I LNCHHQGARSVRAVNEE I QP VTX2 h a s SLT-IIv S SLT- I I S SLT-OX3 259 EC Q I TGDRPV I R I NNTLt,JESNTAAAF L NRRAHS L NT SGE VTX2h a K KSQ. T SLT-IIv K KSQ T SLT- I I K KSQF TK
B SUBUNIT -lrl SLT. OX3 M -I9 K K I F V A A L F A F V S V N A M A A D C P K G K I I F S K Y N E N D T F T V VTX2 ha M.M.V L A SLT-IIv MIV L A DN SLT- I I M M.V LA A D
SLT-OX3 22 K VAG K EYl.,TNRl.lN L Q P L L q SA Q L T G MT V T I KS N TC AS G S G VTX2ha s S E SLT-IIv S R S SLT. I I D S E
SLT-OX3 62 FAEVQFN-- VTX2 h a ND SLT-IIv QK SLT- I I ND Table 4.2
Amino acid sequence homology amongst SLT-OX3 and related toxins
A subunit
Toxin % homology wìth: vtxZha SLT-II SLT-IIv
SLT-OX3 95.9 95.3 91 .8
vtx2ha 98.7 94.4
SLT- I I 93.7
B subunit
Toxin % homology w'ith: vtx2ha SLT-II SLT-IIv
SLT-OX3 88.8 85.4 85. I
vtx2h a 96.6 85.4
SLT- I I 85.4
Deduced amìno acid sequence homoìogy amongst aìl possible paìrs of SLT- 0X3, vtx2ha (Ito et a7., 1990), SLT-II (Jackson et al., 1987a) and SLT-IIv (t{einste'in et â1., I988b) was calculated separateìy for the A and B
subun ì ts . 94
further 2OOO clones from the 031 genomic library were screened in an attempt to isolate a clone carrying the smaller fragment, but all four SLT-Il- reactive clones that were isolated, contained 4.6-kb rather than 1'45-kb Pstl inserts. An additional 1OO0 clones from separate gene banks, constructed using either pUC19 or pBLUESCRIPT SKTM vectors and size-fractionated 031 DNA (1.2 to 1.7 kb), also did not include any SLT-reactive recombinants with the 1.45-kb Psfl insert. Thus, it appeared that this particular fragment was refractory to cloning by conventional techniques. Moreover, given the size of the fragment and the fact that at least 1.2 kb of DNA is required to encode both the A and B subunits of SLT, there was a strong possibility that the 1.45-kb PsÍl fragment d¡d not include the entire SLT operon. This possibility was all the more likely given the fact.that the gene for SLT-ll contains an internal Psfl site in the A subunit coding region (Jackson et al., 1987a). To overcome the above difficulties, oligo primers which would enable direct amplification of complete SlT-ll-related operons from genomic DNA by PCR were designed. Two 17-mer oligonucleotide primers (5'- GATGGCGGTCCATTATC-3' and 5'-AACTGACTGAATTGTGA-3',) were synthesized. The former is homologous to a region 177-194 bp upstream from the ATG initiation codon of the A subunit gene of SLT-ll, while the latter is complementary to a region approximately 45-62 bp downstream from the SLT-ll B subunit termination codon. Thus, these primers direct the amplification of an approximately 1.s-kb DNA fragment which incorporates the entire SLT-Il operon, including the putative -1O and -35 promoter regions. Computer analysis showed that the two primer target sequences appeared to be conserved amongst the majority of the SLT-ll variants whose sequences are contained in the GenBank or EMBL databases. When O31 genomic DNA was subjected to PCR-amplification, a heavy band approximately 1.5 kb in length was seen on agarose gels. When this amplicon was digested to completion with Pstl, DNA fragments of 95 approx¡mate¡y 1.5 kb, 1.1 kb and O.4 kb were seen (Figure 4.10t-. This implied that the PCR reaction mix contained two different amplicons, one with an internal Psfl site and one without, which would be consistent with the Southern hybridization data. To examine this, further aliquots of undigested PCR reaction mix were electrophoresed and the 1.s-kb PCR product was excised from the gel and cloned into E. coli JM1O9 using pBLUESCRIPT SKTM. Recombinant plasmids with 1.s-kb inserts, which either lacked or contained an internal Psrl site, were isolated and representatives of each type were designated pJCPS2O and pJCPS21, respect¡vely, French Pressure cell extracts from E. coli JM109 harbouring either of the above plasmids were cytotoxic for Vero cell monolayers (CDSO titres were approximately 640 and 128O, respectively) (result not shown). Since the insert of pJCPS20 did not contain an internal Psfl site, and the Southern hybridization data (Figure 4.21 indicated the presence of no more than two SlT-related genes, ¡t was concluded that pJCPS2O contained the already sequenced SLT-OX3 gene. Therefore this plasmid was not analysed further,
4.2.7 Sequencíng of the Second SLT Gene from Strain O31 To determine the complete DNA sequence of the insert of pJCPS21, nested deletion derivatives from either end of the insert were constructed, The scheme for sequencing both strands of the SlT-ll-related gene is shown in Figure 4.11 and the complete nucleotide sequence is shown in Figure 4.12. This SLT gene is designated SLT-OX3|2. Comparison of these sequences with those for other SlT-ll-related genes in the GenBank or EMBL databases revealed a total of 1 1 nucleotide differences between the A and B subunit coding regions of SLT-OX3|2 and the amino acid coding region of the most closely related SLT sequence, which was a SLT-ll variant from E coli strain 32511 referred to as SLT-Ilc (Schmitt et al., 1991); (GenBank accession Figure 4.1O PCR-amplification of complete SLT-ll operons
Chromosomal DNA from E. colístrain 031 and E coli C6OO was subjected to PCR-amplification using PCR primers Va and Vb. Aliquots of each reaction
(with or without digestion with Psfl) were electrophoresed on a 1 .2%" agarose gel and stained with ethidium bromide. Tracks: M: DNA size markers (8.51, 7.35,6.11,4.84,3.59, 2.81, 1.95, 1.86, 1.51, 1.39, 1.16,0.98, O.72, O.48, and 0.36 kb, respectively, from top to bottom). 1: E. colí 031 DNA (undigested). 2: E. coli 031 DNA digested with Ps¡l after amplification. 3: Negative control (8. colí C60O DNA).
Figure 4.1 1
Map and scheme for sequencing the insert of pJCP521
The broken line denotes vector DNA, while the $olid line denotes insert DNA. Restriction sites are ¡ndicated as follows: P, Pstl,B, BamHl, (S), former vector Smal site into which the blunt-ended PCR product was cloned. The boxes indicate the location of the SLT A and B subunit coding regions, as determined from the sequence data. The arrows below the map indicate the portions of the plus (->) and minus ((-) strands sequenced in the various nested deletion derivatives of pJCPS21. pJCP521
P (S) P (S) B \ V I slr-n ll slr-s I
1.0 kb Figure 4.12 Nucleotide sequence of SLT-OX3l2 and comparison with related sequences
The nucleotide sequence of the insert of pJCP521 (encoding SLT-OX3/2) is compared with that of SLT-llc (Schmitt et al., 1991) and SLT-OX3. The coding regions for the putative A and B subunits (labelled sltA and sltB, respectively) are represented in bold type. Putative ribososme binding sites (Shine-Dalgarno sequences) for each subunit are labelled "rbs" and are underlined, and putative -10 and -35 promoter regions are double underlined' Nucleotides in the latter two sequences which are identical to SLT-OX3/2 are shown as dots; the symbol ^ beneath a nucleotide indicates that it has been inserted at that position; - indicates absence of a nucleot¡de. The maximally divergent portions of SLT-OX3l2 and SLT-OX3 (nucleotides 11OO to 1116), targeted to distinguish respective transcripts (see Section 4.2.81 are underlined. The 17 nucleotides at the 5'and 3'termini corresponding to the
PCR primer sequences have been deleted. -35 -10 SLT-OX3/2 GGTATTCAGCG-r TGTTAGCTCAGCQGGÂCAGAGCAATTGCCTTCTAAGCAATCGG-TCACTGGTICGAAICCAGTACAACGCGCCATACTT SLT-l Ic SLT -OX3 rbs sìtAr TC IGGT GCTGAT TAC T TCAGCCAAAAGGAACACCTGIAIAIGA sLl-0x3/2 9 I ATT T TT CCTGGCI C6C T TTT GCGGGCCT TT I I TAT ATC TGCGCCGGG SLT-l lc sL-t -0x3
sLT-ox3/2 I 8 I AGTGTATATTATTTAAATGGGTACT6T6CCTGTTACTGGGTTTTCCTTCGGTATCCTATICCCGGGAGTTTATGATAGACTTTTCGACCC SIT I Ic SLl-OX3
SLT-OX3/2 2/r AÁCAAA6TTATGTCTCITCGTTAAATAGTATACGGACAGAGÂTATCGACCCCTCTTGAACATATATCTCAGGGGACCACATCCGTGTCTG SLT-l Ic SLT -OX3
SLT-OX3/2 36 I TTATTAACCACACCCCACCGGGCAGTTATTTTGCTGTGGATATACGAGGGCTTGATGTCTATCAGGCGCGTTITGACCATCTTCGTCTGA SLT-l Ic
S LT -OX3
slt-0x3/2 451 TTATTGAGCAAÁATAATTTATATGTGGCTGGGTTCGTTAÃTACGGCAÁCAAATACTTTCTACCGTTTTICAGATTTTACACAIATATCAG SLT-l Ic SLT-OX3
SLT-OX3/2 541 TGCCCGGTGTGACAACGGTTTCCATGACAACGGACAGCAGTTATACCACTCTGCAACGTGTCGCAGCGCTGGAACGTTCCGGMTGCAAA SLT-llc
S L T_OX3
SLT-OX3/ 2 63 I TCAGTCGTCACTCACTGGTTTCATCATATCTGGCGTTAATGGAGTTCAGTGGTAATACAATGACCAGAGATGCATCCAGAGCAGTTCTGC SLT-llc SLT-OX3
StT_OX3/2 72I GTTTTGTCACTGTCACAGCAGAAGCCTTACGCTTCAGGCAGATACAGAGAGAATTTCGTCAGGCACTGICTGMACTGCTCCTGTGIATA SLT- I lc
SL T -OX3
sLr 0x3/2 8u SLT- I Ic
S LT_OX3
sLT-0x3/2 9 O I TGGGGAGAATAICCTTTAATAATATAICGGCGATACTGGGCACTGTGGCCGTTATACIGAATTGTCATCATCAGGGGGCGCCTTCTGTTC SLT-I Ic
SL T _OX3
slr-ox3/2 99 I GCGCCGTGÂATGAÁGAGAGTCAÂCCAGAATGICAGATMCTGGCGACAGGCCCGTTATAAAÁATAAACÁATACATTATGGGAAAGTAATA StT-l lc SLT OX3 rbs sl t8' sLt-0x3/2 TOsI CAGCTGCAGCGTTTCIGAAçAGÁ44GIEÂEAG'L[f]TATATACMCffiGTAAATMA!ç48]TAAGTATGAÁGAÁGATGTTTATGGCGGT SLI- I Ic SLT OX3 C-,A- T T TC.CGGC.CC. A TC AG. C A GA C
sLf-0x3/2 I 1 7 I TTTATTTGCATTAGTTTCTGTTAATGCMTGGCCGCGGATICCGCTMAGGTAAAATTGAGTTIICCMGIATAATGAGAATGATACATT SLT-l Ic SLT-OX3
sLT-0x3/2 1261 CACAGTAAAAGTGGCCGGAAAÃGAGTACTGGACCAGTCGCTGGAATCTGCAACCGTTACTGCAAAGTGCTCAGTTGACAGGMTGACTGT SLT- I Ic SLT_OX3 T.AC
sLT-0x3/2 I3sI CACAATTAAATCCAGTACCTGTGAATCAGGCTCCGGATTTGCTGMGTGCAGTTTAATMTGÁCTGAGGCATAACCTGATTCGTGGTATG SLT- I Ic SLT-OX3 A. .C...44..
sLT-0x3/2 1441 TGGGTAACAAGTGIAA-ICTGTG SLT-llc
5 LT -UX3 CA CA IbT,. _ OAG A 96 number M59432). A further six nucleotide differences were found in the non-coding regions (Figure 4.12l,.
The deduced amino acid sequences of the A and B subunits of SLT- OX3l2 is compared with those for SLT-llc, SLT-I| and SLT-OX3 in Figure
4.13. This indicates that the 1 1 nucleotide differences between the coding regions of SLT-OX3|2 and SLT-llc result in only two amino acid changes in the A subunit (Pro instead of Ser at residue -6, and Met instead of Thr at residue 4). There were no amino acid changes in the B subunit. The fact that SLT-OX3|2 retains cytotoxic activity for Vero cells implies that these amino acid substitutions do not have a significant impact on the structure or function of the toxin.
4.2.8 Expression of SLT-OX? and SLT-OX3/2 Genes in E. coli Straín O3l The results presented above indicate that both SLT-OX3 and SLT- OX3l2 are constitutively expressed from recombinant plasmids in E. coli
JM109. lt was not known, however, whether both genes were expressed in E. coli strain 031. Monoclonal antibodies capable of distinguishing between the products of these two SLT-ll-related genes were not available. The nucleotide sequences of the two genes were therefore compared to identify regions of minimal homology, which might serve as targets for specific oligo probes in Northern hybridization analysis. From Figure 4.12 it can be seen that the greatest sequence divergence occurs between nucleotides 1 1O0 and
1 1 16 (part of the A subunit gene), with only 9117 nucleotides in common. Two 17-mer oligos (designated OX3 and OX3l2l complementary to this portíon of the SLT-OX3 and SLT-OX3|2 genes, respectively, were therefore synthesized, labelled with DIG and used as operon-specific probes. The specificity of the two oligo probes was confirmed by probing Southern blots of restricted pJCPS2O and pJCPS21 DNA; after washing filters at high Figure 4.13 Deduced amino acid sequence of SLT-OX3/2 compared with related sequences
The deduced amino acid sequences of the A and B subunits of SLT-OX312 are shown, numbered from the first residue of the mature polypeptides. The arrows denote the putative signal peptidase cleavage sites. The deduced amino acid sequences of SLT-Ilc (Schmitt et al., 1991), SLT-Il (Jackson et al., 1987a1, and SLT-OX3 are also shown. Amino acids are represented by single letter code; dots denote residues identical to SLT-OX3/2; - denotes absent residues. A SUBUNIT
-1.t 1 sLT-0x3/2 -22 MKC I L FKI,JV L C L L LG F P S V SY SR E FM I D F S SLT-IIc s T SLT- I I S T SLT- OX3 S sLT- 0x3/2 9 TQQSYVSSLNSIRTIISTPLEHISQGTTSV SLT- I Ic SLT- I I SLT_OX3 sLT - 0x3/2 39 SV I NHTPPGSYFAVD I RGLDVYQARFDHLR SLT- I Ic SLT- I I . -H SLT- OX3 sLT- 0x3/2 69 Li IEQNNLYVAGFVNTATNTFYRFSDFTHI SLT-IIc SLT- I I SLT- OX3 sLT-0x3/2 99 S V P G V TT V S MTT D S S Y T T L Q R VAA L I R S G M SLT-IIc SLT- I I SLT. OX3 sLT-0x3/2 r29 Q I SRHSLVSSYLALME FSGNT¡4TRDASRAV SLT-IIc SLT- I I SLT- OX3 sLT-0x3/2 ls9 LR FVTVTATALR FRQ I QRE FRQALS ETAPV SLT- I Ic SLT- I I SLT-OX3 sLT-0x3/2 189 YTMTPGDVDLTLNl,JGR I SNV L P EYRGEDGV SLT-IIc SLT- I I SLT-OX3 .F G sLT-0x3/2 2r9 RVGRI SFNN I SAI LGTVAV I LNCHHQGARS SLT-IIc SLT_ I I SLT- OX3 sLT-0x3/2 24e VRAVNEESQPECQI TGDRPV I K I NNTLl^JES SLT-IIc SLT_ I I SLT_OX3 T R sLT-0x3/2 27e NTAAAFLNRKSQFLYTTGK SLT-IIc SLT- I I SLT- OX3 .RAHS N S I
B SUBUNIT
-1+1 sLT-0x3/2 -'e SLT- I Ic TiiT:TiY::i:Y:Yi1Ti1?:1i:il : :: SLT- I I .A SLT-OX3 .I V A F P sLT-0x3/2 12 KY N E N D T FT V K VAG K E Yl^lT S R l.J N L Q P L L Q S SLT-IIc SLT- I I .DD SLT- OX3 .N sLT-0x3/2 42 AQLTGMTVT I KSSTCESGSG FAEVQFNND SLT-IIc SLT- I I SLT-OX3 97
stringency (2x SSC at 50"C), OX3 hybridized exclusively with the former and OX3l2 hybridized exclusively with the latter (result not shown). Total cellular RNA was extracted from an overnight culture of strain 031. Aliquots of RNA were denatured with formaldehyde, electrophoresed and transferred onto nylon filters (Sections 2.48 and 2.49t.. Filters were then probed with each of the above oligo probes (end-labelled with DIG-11-dUTP using terminal transferase). Replicate blots of 03t celluår RNA were also A probed with a DIG-labelled positive strand oligo (A-l) capable of recognizing either SLT gene. This was necessary to determine whether the preparation was contaminated with DNA (which unlike SLT mRNA, would contain minus strand SLT sequences). No hybridization signal was observed with any of the probes. One possible explanat¡on for the above result was that the specific activity of the DIG-labelled oligo probes was too low to permit detection of possibly low abundance SLT mRNA species. To overcome this, an SLT operon-specífic RNA-dependent PCR assay was developed. Strain 031 RNA was subjected to PCR-amplification following reverse tianscription in the presence of either one of two pairs of oligo primers (OX3 & lVa and OX3/2 & lVa) (see Tables 2.1 and 2.2t'. Aliquots of RNA were also amplified in the absence of reverse transcriptase to control for trace DNA contamination of the preparation. Aliquots of pJCPSOl (encoding SLT-OX3) and pJCPS21 (encoding SLT-OX3/2) were also subjected to PCR-amplification to confirm the specificity of the primer pairs (see Figure 4.14l.. The former primer pair directed the amplification of a 355-bp fragment (the expected size) from the pJCP5Ol template. The pJCP521 template did not appear to direct the amplification of a 355-bp fragment, but several larger and smaller PCR products were obtained, possibly a result of non-specific priming. The latter primer pair directed the amplification of a 35S-bp fragment from pJCP521, but not from pJCPSOI . Both primer pairs directed the amplification of 355-bp Figure 4.14 Detection of the presence of SLT-OX3- and SlT-Ox3/2-specific RNA by reverse transcription PGR
,/-;, Total cellular RNA from strain 031 was extracted and subjected/ pCn amplification, with or without reverse tanscriptase (+RT or -RT), employing oligo PCR primers OX3 + lVa or OX3l2 + lVa. Aliquots of each react¡on were electrophoresed on a 2o/o agarose gel and stained with ethidium bromide. The expected mobility of a 355 bp DNA species is indicated, Tracks: 1: Negative control, no DNA template (primer pair OX3 + lVa). 2: Negative control, no DNA template (primer pair OX3/2 + lVa). 3: O31 RNA, -RT, (primer pair OX3 + lVa). 4: 031 RNA, -RT, (primer pair OX3/2 + lVa). 5: 031 RNA, +RT, (primer pair OX3 + lVa). 6: O31 RNA, +RT, (primer pair OX3/2 + lVa). 7: pJCP5O1, +RT, (primer pair OX3 + lVa). 8: pJCP5O1, +RT, (primer pair OX3/2 + lVa). 9: pJCP521, +RT, (primer pair OX3 + lVa). 10: pJCP521, +RT, (primer pair OX3/2 + lva). M: DNA size markers (501, 4O4, 331, 242, 19O, 147 and 110 bp, respectively, from top to bottom). M 1 2 3 4 5 6 7 8 910
O !¡- t -¡ -355 bp 98
fragments from the 031 RNA template in the presence of reverse transcriptase, but no PCR product was seen when reverse transcriptase was omitted (Figure 4.14l.. These results indicate that the two primer pairs were specific for their respective SLT operons and that the 031 RNA preparation contained both SLT-OX3 and SLT-OX3|2 mRNA species.
4.3 Discussion
4.3.1 Presence of Two SLT-related Genes rn E. coli Strain O3l Southern hybridization analysis presented in this Chapter indicated that E. coli strain 031 contains two genes which are related to SLT-ll. The presence of two SLT genes has been demonstrated for a number of SLTECs from both human and animal sources, either on the basis of antibody neutralization studies (Scotland ef al., 1985; Strockbine et al., 1986) or DNA hybridization/sequence analysis (Jackson et al. 1987a; lto et al., 1990; Schmitt et al., 1991). Indeed, Schmitt et al. (1991) reported that 5 of 19 SLT-Il-producing SLTECs which they tested contained two such genes. The Southern hybridization data presented here also suggests the possibility of a second SLT-Il-related gene in E. coli C60O::933W, as at least two bands were labelled with the SLT-ll probe in BamHl and Pstl digests of E coli C6OO::933W DNA (Figure 4.2l.. Further Southern hybridization analysis of EcoRl and Clal digests of C6OO::933W DNA indicated that with each
enzyme, two bands were labelled (5 and 1 1 kb for EcoRl and 7.3 and 15 kb for Ctall, while no bands were detected in digests of E. coli C60O DNA (results not shown). These results cannot be explained by the presence of cleavage sites for the above enzymes within the region of the SLT-I¡ gene against which the probe was directed. lt is possible that the C600::933W culture (which was obtained from Dr. A. O'Brien) had become infected with 99 an addit¡onal SlT-ll-encoding bacteriophage (although no such phages have been handled in our laboratory) or had undergone a gene duplication event (perhaps mediated by lS elements) at some stage during handling. However, it is also possible that the existence of two SLT-ll-reactive restriction fragments in C6OO::933W could be a result of "flip-flop" of a reversible DNA element within the bacteriophage, as has been shown for bacteriophages Mu and P1 (Glasgow and Hughes, 1989).
4.3.2 SLT-ll Sequence Variatíon There are a number of reports in the literature of variations in the nucleotide and derived amino acid sequence of SLT-ll genes. Oku et al.
(1989) purified an SLT from E. coli B2F1, an 091:H21 strain isolated from a patient with HUS. This toxin was found to be immunologically relâted to SLT- l¡, but behaved similarly to the piglet-derived SLT-llv in that ¡t was less cytotoxic for HeLa cells. Therefore this SLT was predicted to be a human variant form of SLT-Il. Two separate SLT sequences were cloned from the cellular DNA extracted from stra¡n B2F1 and their nucleotide sequences were determined (lto et al., 1990). The two SLT-Il-related sequences (designated vtx2ha and vtx2hb) were almost identical (99% nucleotide sequence homology with each other). The A and B subunit genes of vtx2ha and SLT-ll shared 98.6% and 95.5% nucleotide sequence homology, respectively. Vtx2ha exhibited 94.5o/o and 82.5Y" nucleotide sequence homology, respectively, with the A and B subunit genes of the piglet-derived SLT-Ilv. lt was also noted that the nucleotide sequences in the vicinity of the putative promoter and ribosome binding sites for the two human SLT-llv sequences, a piglet SLT-llv and SLT-ll were all identical. Schmitt et al. (1991) sequenced two SLT-Il-related genes from E. coli 0157:H- strain E3251 1. The first of these was identical to SLT-ll with the exception of one nucleotide. However, this nucleotide difference did not 100 result in a change in the predicted amino acid sequence. lnterestingly, the deduced amino acid sequence of the A subunit of the second SLT gene was identical to SLT-Il, but that of the B subunit was identical to the B subunit of vtx2ha, described by lto et al. (1990). This second variant SLT-Il sequence from strain F32511 was designated SLT-Ilc. Differences in the neutraliz¡ng capacity of various monoclonal antibodies and a polyclonal anti-serum directed against SLT-ll suggested that there were antigenic distinctions between SLT-Il and SLT-Ilc (Schmitt et al., 1991). A further human SLT-ll variant reported by Meyer et al. (1992) was isolated from E. coli C157:H7 strain 7279. This toxin was not neutralized by anti-SLT-l or anti-SLT-ll antibodies and was designated SLT-llvhc. The deduced amino acid sequence of the A subunit of SlT-llvhc differed from the A subunits of vtx2ha and SLT-llc by two and three amino acids, respectively. The predicted amino acid sequence of the B subunit of SlT-llvhc was identical to that for SLT-Ilc and vtx2ha. Gannon et al. (1990) also reported the cloning and nucleotide sequence of another human variant SLT-ll (designated SLT-llva) from one of Konowalchuk's original strains lE. colí H.1 .8. serotype |U-128:812l. (Konowalchuk et al., 19771. Although this strain was isolated from a case of infantile diarrhoea, SLT-llva was similar to porcine SLT-llv in that it had reduced cytotoxicity for HeLa cells. The nucleotide sequence homology between SLT-llv and SLT-llva was 98Y" for the A subunit genes, but remarkably, only 70.60/" for the B subunit genes.
4.3.3 Sequence Analysis of SLT-OX? from E. coli Strain O31 In the present study, the gene encoding one of the SlT-ll-related toxins from E. colí 031, denoted SLT-OX3, was cloned into pUC19 as a 4.6-kb Pstl fragment and subjected to sequence analysis. Comparison of the DNA sequence of SLT-OX3 with published data for related genes (as well as those tít
101
contained in GenBank and EMBL databases) indicated that SLT-OX3 ¡ t closely related to vtx2ha from E colí O91:H21 strain B2F1 (lto er al., 1990), At both the DNA and deduced amino acid sequence level, there was approximately 96% and 89o/o homology for the A and B subunit coding
regions, respect¡vely. There was an identical degree of homology with SLT- llvhc (Meyer et al., 1992l., which differed from vtx2ha by only 2 amino acid residues in the A subunit. The difference between SLT-OX3 and vtx2ha was more substantial (1 3 and 1O amino acids in the A and B subunits, respect¡vely). Moreover, 11 of the A subunit sequence variations and 6 of the B subunit variations are so far unique to SLT-OX3. However, the majority of the substitutions are conservative in nature and there is very little difference in the hydrophobicity plots for these two variants (data not presented).
There is also a high degree of DNA homology (950/"1 between SLT-OX3 and vtx2ha for the region of the respective genes 2OO bp upstream from the A subunit coding sequence, with absolute homology in the regions identified as putative promoter (-1O and -35) sequences and the ribosome binding site (lto ef al., 1990). However, there was less homology l7o%l for the region 2OO bp downstream from the B subunit coding region. This region of the SLT-OX3 gene included a possible rho-dependent transcription terminator sequence (bases 1866 to 1909), with a Gibbs Free Energy of -17.1 kcal/mole. lnterestingly, De Grandis et al. (1987) and Gannon ef a/. (1990) have both reported the presence of putative rho-independent transcription terminators in the SLT-I operon of phage H198 and a variant SLT-ll operon (SLT-llva) of E. coli strain H.1.8, respectively. A second possible transcription terminator sequence, with a Gibbs Free Energy of -19.1 kcal/mole, was found further downstream from the SLT-OX3 gene. This sequence (nucleotides 2O74-2093) is identical to the putative rho-independent terminator of the SHT gene of S. dysenteriae type 1 (Kozlov et al., 1988). .i r02 I { ù
The amino acid sequences of both the A and the B subunit of SLT-OX3 I have typical hydrophobic leader sequences, with a signal peptidase cleavage ¡ 'l site on the C-terminal side of 22nd and 1gth residues of the primary t translation products, respectively, Both signal sequences agree with the rules i of von Heijne (von Heijne, 1984 and 1985). i
I
I i 4.3.4 Sequence Analysis of the Second SLT-ll-related Gene ISLT-OXS/2) from Strain O3l Attempts to isolate clones containing the second SLT-Il-related gene from the plasmid gene banks of strain 031 DNA were not successful, perhaps indicating that any such clones were unstable. Furthermore, the 1.45-kb Psrl fragment containing the probe-reactive sequences may not have encoded the
ú' complete toxin operon. For these reasons, PCR was used to amplify the år, dl |'!i complete SlT-ll-related operon. Primers were designed after examination of 1,', existing DNA sequence data for other SLT-ll-related genes for conserved regions 5' to the promoter site and 3' to the B subunit coding region. Meyer et al. (1992) have also used PCR to sequence SLT-ll-related genes, but their primer pair amplified only the A and B subunit coding regions and did not permit analysis of the promoter region 5' to the SLT gene. In this study the chosen primers directed the amplification of two distinct 1.s-kb DNA fragments from strain 031 genomic DNA. Amplicons derived from SLT-OX3 lacked an internal Psfl restriction site, unlike those derived from the second SLT gene (denoted SLT-OX3/2). The deduced amino acid sequence of SLT-OX312 was very similar to that for SLT-Ilc (Schmitt et al., 1991), differing by only two residues in the A subunit. ln SLT-OX3|2 the residue at position -6 is Pro, whereas for all other members of the SLT-ll family (including SLT-OX3), the residue at this position in the signal pept¡de is Ser. Thus, the SLT-OX312 A subunit signal peptide conforms more closely to the rules of von Heijne, by having a helix-breaking 103 J ,i Þ- res¡due at position -6 (von Heijne, 1984 and 1985). The second amino acid I difference with respect to SLT-llc (Met instead of Thr at residue 4) is also r found in SLT-OX3. The fact that a lysate o'f E. colí JM1O9IpJCPS21l (which t encodes SLT-OX3/2) is highly cytotoxic for Vero cells suggests that this t substitution does not have a dramatic impact on cytotoxicity. The B subunits I' of SLT-OX3|2 and SLT-Ilc were identical. : Some caution must be exercised when interpreting sequence data derived from cloned PCR fragments. Taq polymerase has been reported to have an error frequency of 2 x tO-5 (Stratagene Catalogue Í1gg2l, p. 126, Stratagene, La Jolla, California). The impact of this problem can be minimized by direct sequencing of purified PCR product using appropriate synthetic oligonucleotides as sequencing primers (and dye-labelled dideoxynucleotides when using automated DNA sequencing protocols). Assuming the original DNA template contained many copies of the SLT sequence, incorrect nucleotides inserted at any given point due to polymerase infidelity, would be present in only a very small proportion of amplicons and thus, would not produce a detectable signal in subsequent sequencing reactions. However, this approach was not possible in the present case, as the 1.s-kb PCR product derived from amplification of 031 DNA contained two distinct amplicon species, which would have resulted in ambiguities at any positíon in the nucleotide sequence where SLT-OX3 and SLT-OX3|2 varied. Attempts were made to amplify the 1.s-kb SLT operons using Pfu polymerase, which is reported to have a 12-fold higher fidelity than Taq polymerase (Stratagene Catalogue Í19921, p. 1261. However, no full length
PCR products were obtained using this enzyme (results not presented). Thus, although the probability that certain of the nucleotide variations detected in the SLT-OX3|2 sequence are Taq polymerase-induced artifacts is low, this cannot be eliminated altogether. Recently VENT polymerase, which has proof-reading as well as thermostable polymerase activity has become 104 commercially available and use of this enzyme is likely to produce higher I fidelity data.
As stated in Section 4.3.1, the presence of two SLT-ll-related genes in a single SLTEC strain is quite common (Schmitt et al., 1991). lto et al. (1990) have suggested that in SLTEC strains such as B2F1, which encode two very similar SlT-ll-related genes (99.2% homology at the DNA level), may have arisen as a consequence of insertion sequence-mediated gene duplication. However, this is unlikely for strain 031, as SLT-OX3 and SLT- OX3l2 share only 94% overall DNA sequence homology within the toxin coding region.
4.3.5 Functional Significance of Amíno Acid Sequence Variation. The precise functional domains of SlT-related toxins have not yet been determined. lt is known, however, that within the A subunit of SLT-1, Glu167 and Arg 17O ate both esSential for enzymic activity and toxicity (Yamasaki et al., 1991), Perera et al. (1991a) have shown that the amino terminal of the A subunit is also important, as deletion of amino acids 3-18 of the mature SLT-ll A subunit abolishes both toxicity and enzymic activity. lt has also been demonstrated (Pererc et al., 1991a, 1991b) that deletion of the last 4 amino acids from the B subunit of SLT-ll (but not tn" last 2 residues) abolishes toxicity of the holotoxin, as does mutation of the Arg32, Ala42 and Gly5g in the SLT-ll B subunit. As would be expected, none of the sequence variations found in SLT-OX3 occur in any of the above regions, with the exception of an Ala4 -' Phe substitution in the B subunit. lnterestingly, several of the sequence variations in SLT-OX3 are also found in SLT-Ilv from porcine E. coli isolates (Weinstein et al., 19BBb; Gyles et al., 1988). This may be of epidemiological significance since OX3:H21 is a serotype more commonly found in domestic animals than humans (F. and l. Orskov, pers. comm.). ln particular, the B subunits of both toxins are two 105 amino acids shorter than SLT-ll (Jackson ef al., 1987a) and a number of SLT- ll variants (vtx2ha, vtx2hb, SLT-llc and SLT-Ilvhc) (lto ef al., 1990; Schmitt ef al., 1991; Meyer et al., 1992Ì, derived from human SLTECs. However, Gannon et al. (1990) have reported that the B subunit of a SLT-ll variant (SLT-llva) from a human-derived E coli strain H.¡.8, also lacks the two C- terminal residues found in SLT-Il. The carboxyl terminal of the B subunit gene of SLT-llv, particularly the Gln 5 residues from the end, is known to influence the cellular location of the toxin (Jackson et al., 199O). lf this residue is mutated to Glu (the residue found in all the other members of the SLT-ll family), the major proportion of the SLT-llv remains cell-associated, rather than being predominantly extracellular. The fact that in this study, a substantial proportion of the SLT- OX3 produced by ¡M1O9tpJCP501l remained cell-associated in spite of the lack of the last two amino acids in the B subunit, implies that, in SLT-llv, it is variations in the 3rd and 5th residues from the C-terminus (rather than the lack of the last two residues), which are primarily responsible for the difference in toxin release. Interestingly, these same two amino acid variations in the B subunit of SLT-llv have recently been implicated as the basis for its different glycolipid receptor specificity and its different cytotoxicity for various cell lines compared with the other SLT-Ils (Tyrrell et â1., 1992). Site-directed mutagenesis of the SLT-llv B subunit gene such that these two amino acids (Gln64 and Lys66) were changed to Glu and Gln, respect¡vely (the analogous residues in SLT-ll), altered the predominant in vitro binding specificity of the mutant toxin from Gb4 to Gb3 (Tyrrell er al., 1992) i.e., to the same receptor binding phenotype as SLT-ll. This resulted in changes in the relative cytotoxicity of the mutant SLT-Ilv for various cell lines, in accordance with their Gb3 and Gb4 content. Boyd et al. (1993) have since demonstrated that when this mutant SLT-Ilv was injected intravenously into pigs, the distribution 106 of toxin to the various organs was different to that obtained with wild-type SLT-!lv. This was accompanied by differences in the clinical characteristics of toxin-induced disease, but it did not effect the nature of the histological lesions (Boyd et al., 1993). However, the C terminus is not the only part of the B subunit involved in the binding specificity of SLTs, as mutating the Aspl g of SLT-I to Asn (the residue found at the analogous position (17l' of SLT-llv) resulted in binding of the mutant toxin to Gb4 as well as Gb3, although the reciprocal mutation did not affect the binding specificity of SLT-Ilv (Tyrrell et al., 19921. Recently, the crystal structure of the oligomeric B subunit of SLT-l has been published and this suggests that the site of carbohydrate receptor binding may be a cleft between adjacent monomers of the B pentamer (Stein et al., 1992ì-. The two portions of the B subunit identified as impacting on specificity are found on opposite sides of this putative binding cleft (Stein et al., 1992). ln SLT-OX3, the third and fifth amino acids from the C terminus (positions 64 and 66) are the same as those of SLT-ll and vtx2ha, which is consistent with the cytotoxicity of SLT-OX3 for both Vero and HeLa cells. For the other site implicated in receptor binding (position 17), SLT-Il, SLT-OX3 and vtx2ha all have Asp, while SLT-llv has Asn. However, the preceding residue is Asp for SLT-Il and SLT-Ilv, but Asn for SLT-OX3 and vtx2ha. Such variability in the immediate vicinity of the receptor binding cleft could well result in subtle changes in receptor affinity and target tissue specificity for the latter two toxins. It is also interesting that the amino acids near the C-terminus identified as being directly involved in carbohydrate receptor binding, have also been shown to be involved in the release of toxin from the E. coli cells, as discussed above. ln the light of the crystal structure data (Stein et al., 1992l', it therefore appears that the binding cleft could also be involved in interaction with components of the E coli cell surface. t07
4.3.6 Expression of SLT-OX3 and SLT-OX3/2 The fact that an SLTEC contains more than one SLT gene does not necessarily imply that both toxins are produced in vivo, or that both play an equal role in pathogenes¡s. The two SLT genes in strain 031 are both expressed when separately cloned in E. coli JM109. Monoclonal antibodies capable of distinguishing between SLT-OX3 and SLT-OX3/2 were not available for testing extracts of strain 031 either by toxin neutralization or by Western blot analysis. Differences in the level of in vivo expression of the two genes in strain O31 were not expected because putative -35 and -10 promoter sequences and ribosome binding sites for the A and B subunits were identical. However, there were sufficient sequence variations between a portion of the A subunit coding regions to develop operon-specific oligos. Attempts to detect SLT-OX3- and SLT-OX3/2-specific RNA species by Northern hybridization analysis using D|G-labelled oligos were unsuccessful, possibly due to low specific activity of the probes. Notwithstanding this, the two oligos were successfully employed to demonstrate the presence of transcripts of both genes in 031 RNA preparations by reverse transcription-
PCR analysis. PCR signals obtained using either primer were not due to trace contamination of the RNA preparation with genomic DNA, as no signals were obtained when the RNA template was PCR-amplified without prior reverse transcription. Thus, both genes appear to be expressed constitutively in strain O31. However, this may be a basal level of expression and it is possible that toxin production might be amplified in vivo.
4.3.7 Clinical Sígnífícance It is not possible at this stage to draw any conclusions about the possible clinical significance of the isolation of an SLTEC from the gut contents of a case of SIDS. Unfortunately, the original sample of gut contents 108 had not been preserved, and so it was not possible to test th¡s for free toxin. However, a sample of serum collected postmortem from this SIDS case had been stored at -8OoC, but it did not contain detectable levels of free SLT. This of course, was not surprising, as free SLT has not been detected in sera from HUS cases (Edelman et al., 1988; Karmali, 1989; Tesh and O'Brien, 1991). As mentioned in Chapter 1, SLTECs have been reported to be associated with asymptomatic carriage in animals and humans and in the present study an SLTEC (strain 234) was isolated from a healthy baby (see Chapter 3). Furthermore, toxin has been detected in the faeces of other individuals who are apparently well (Edelman ef al., 1988; Brian et al., 1992l'.
The clinical presentation and pathogenesis of SLTEC-related diseases such as HUS is complicated. The causative SLTEC is confined to the gut, with toxin presumably being absorbed into the bloodstream, but the onset of disease may in some cases occur in the absence of preceding gastrointestinal symptoms (Karmali, 1989). Moreover, SLTECs have been reported to represent less than 1o/o of faecal flora in a significant proportion of HUS cases (Smith and Scotland, 1988), and SLT genes have also been reported to be highly unstable in some strains (Karch et al., 1992l,. Both of these factors have compounded the problems involved in isolating SLTECs even in active cases of HUS or HC. Thus, failure to isolate SLTECs from cases of HUS or HC does not exclude an aetiological role for these organisms. Conversely, isolation of SLTECs from patients with other conditions (e.9.
SIDS) does not necessarily imply a causal relationship. 109
CHAPTER FIVE
CLONING AND NUCLEOTIDE SEOUENCE OF VARIANT SHIGA-LIKE TOXIN GENES FROM ESCHERICHIA COU O111:H- STRAIN PH.
5.1 lntroduction
The work described in Chapter 3 indicated that E. colí 0111:H- strain PH, isolated from a 12 month old boy with HUS, produced at least one type of SLT, as judged by cytotoxicity for Vero cell monolayers and hybridization with DNA and oligo probes. Attpmpts to isolate toxin-converting bacteriophages from this strain were unsuccessful. This chapter describes
the cloning and sequence analysis of two SLT-related genes from strain PH.
5.2 Results
5.2.1 Cytotoxicity of SLT Produced by Straín PH and Neutralization with Monoclonal Antibodies Culture supernatant and French pressure cell extracts of overnight cultures of strain PH were assayed for: cytotoxicity using Vero cells; both
extracts were strongly cytotoxic (CD5O titres after 3 days incubation of cell
cultures were 1:640 and 1 :5120, respectively). This cytotoxicity was almost completely neutralized by incubation with culture supernatant from hybridoma 13C4, which produces a SlT-l-specific monoclonal antibody (Strockbine ef â1., 1985) (see Section 2.181, but there was no detectable neutralization
using supernatant from hybridoma 11E10, which produces an SLT-I|- and 110
SlT-llv-neutralizing monoclonal antibody (Strockbine ef al., 1985) (result not shown). This suggested that PH produces SLT-I.
5.2.2 Southern Hybridizatíon Analysis
To further examine the SLT genes in strain PH, genomic DNA was extracted. Southern hybridization analysis, using a probe mix capable of reacting with both SLT-I and SLT-Il sequences, was then carried out on
EcoRl, Hindlll, Sphl, and Bglll digests of this DNA and DNA from S. dysenteriae type 1, E. coli strain H3O (which is known to produce SLT-I) and E. coli C6OO lysogenised with either of the SLT-l-encoding bacteriophages H198 and 933J (Figure 5.1). The S. dysenteriae, E. coli H3O, E. coli C6OO::933J and E. coli C6OO::H198 DNA all contained single SlT-related DNA fragments in all four restriction digests. However, the PH DNA contained two SlT-hybridizing DNA fragments in the EcoRl digest, and three bands in each of the other three restriction digests, E. coli C6OO DNA did not contain any SlT-hybridizing DNA fragments. These results suggest that strain PH contains at least two SLT genes. When replicate Southern blots of EcoRl digests of PH DNA were hybridized separately with SLT-I- (oligo 4281) or SLT-ll- (oligo 428-lll specific probes, the higher fragment (approximately 8.5 kb) hybridized exclusively with the SLT-I probe and the lower fragment (approximately 4.5 kb) hybridized exclusively with the SLT-¡I probe (Figure
5.2). The apparent size of the SlT-l-hybridizing fragment in the EcoRl digest of PH DNA was fractionally larger than that in the digests of C60O::H198 and C6OO::933J (approximately 8,1 kb). Moreover, the sizes of the hybridizing fragments in Hindlll digests of the two C600 lysogens were markedly different from that of strain PH. Southern hybridization patterns for the various restriction digests of S. dysenteriae and E. coli H30 DNA were also dissimilar to those of PH. Thus, there are clear differences in the DNA Figure 5.1 Southern hybridization analYsis
Chromosomal DNA was digested w¡th Hindlll (l-l), EcoRl (E), Spltl (S) or Bglll (B), and electrophoresed on 0.8% agarose gels. DNA was transferred to nylon filters, hybridized with a PCR-DlG-labelled SLT-I- and SLT-Il-specific probe mix and washed at high stringency. Tracks: 1: E. colistrain PH.
2: S. dysenteriae tyPe 1 . 3: E. coli H3O. 4: E. coliC60O::H198. 5: E. colíC6OO::933J. 6: E. coliC6OO.
M: Df G-labelled DNA size markers (23.1, 9.4, 6.6, 4.4, 2.3, 2.O, and O.56 kb, respectively, from top to bottom). H E Ml23/.56 123/.56M
- l= It- O - || r-r ,rt -- - --Q-t - -
S B
M1 2 3 /.56 123/.56M
"{D (Þ .- ü -r !** *; e 0ú I -r- II
{.d, Figure 5.2 Southern hybridization analysis of PH DNA using SLT-l and SLT-Il-specific oligo probes
Genomic DNA was digested with EcoRl and electrophoresed on a O.8% agarose gel. DNA was transferred onto nylon filters, hybridized with either DIG-labelled oligos 428-l (panel A) or 428-ll (panel B) and washed at high stringency. Tracks:
1 : E. coli C60O DNA. 2: E. colistrain PH DNA. M: D|G-labelled DNA size markers (23.1,9.4,6-6,4.37,2.3,2.O, and O.56 kb, repectively, from top to bottom). A B
12M 12M
(I¡
-
-
t- t- 111 structure in the vicinity of the SLT-I operon in PH compared with those of other SLT-I- or SHT-encoding strains.
5.2.3 Cloning of the SLT-l-related Gene from E. coli Straín PH ln order to clone the SLT-related genes from E. coli strain PH, genomic DNA was digested with EcoRl, ligated with EcoRl-digested pUC19 and transformed into E. colí JM109. EcoRl was chosen because the Southern blot data (Figure 5.1) indicated that both the SLT genes were located on fragments of 4.5 kb and 8.5 kb, suitable for direct cloning into plasmid vectors. The other enzymes were judged less suitable, because the hybridizing fragments were too large for direct cloning and the presence of three reactive fragments suggested the possibility of restriction sites within the SLT coding sequence, such that single fragments may not have contained complete copies of one of the putative SLT genes. Lysates of the resultant clones were prepared and spotted onto nylon filters, which were fixed and hybridized to the SLT-l/SLT-ll probe. Three clones, from a total of approximately 11OO, reacted strongly with the probe.
One of these contained a recombinant plasmid (designated pJCPSlO) with an
8.s-kb EcoRl insert. The other two clones contained plasmids with only a single EcoRl site and therefore presumably were the result of DNA deletions, Southern hybridization analysis (Figure 5.3) indicated that the SLT-related sequences in pJCPSlO were contained on a 3.75-kb Hindlll fragment and a 3.O-kb SphllEcoRl fragment, as shown in Figure 5.4. These fragments were isolated and subcloned into appropriately-digested pBLUESCRIPT SKTM or pUC19, respectively, and recombinant plasmids were designated pJCP511 and pJCP512, respectively. Two further subclones of pJCPS12 (designated pJCPS13 and pJCPS14l are also shown in Figure 5.4. French pressure cell lysates of E. coli JM109 harbouring these plasmids were tested for cytotoxicity on Vero cell monolayers. Extracts from cells harbouring Figure 5.3 Southern hybridization analysis of pJCPSlO to localize the SLT gene w¡th¡n the 8.5 kb insert
pJCPSl O DNA was digested with various restriction enzymes, electrophoresed on a 1o/o agarose gel and stained with ethidium bromide (panel A). DNA was transferred onto a nylon filter, hybridized with a PCR- DIG-labelled SlT-l-specific probe and washed at high stringency (panel B). Mobilities of DNA size markers are indicated. Tracks: 1: Hindll. 2: Híndlll. 3: Clal. 4: Pstl. 5: Sacl. 6: Smal. 7: Sphl. A B
12345677654321kb
--.Ò--f. hrw -23.1 f¡- at -9.4-6.6 3- -4.4 å -2.3 I - 2.O Figure 5.4 Map of the insert of pJCPSlO and subclones, and scheme for sequencing
Restriction sites are indicated as follows: E, EcoRl; H, Híndlll; S, Spå|. The box in the pJCPSlO insert labelled "SLT-A" and rrBtr denotes the region encoding the SLT gene (A and B subunits). The boxes labelled "lS" and
"LAM" indicate the location of regions with homology to an lS element and a portion of the bacteriophage lambda genome, respectively. The arrows beneath the map indicate the portions of the plus (->) and minus ((-) strand which were sequenced, using nested deletion derivatives of pJCP511, pJCP513 and pJCPS14 as templates. tkb
E H S H E PJCP5r0 I I I I I I rs tl LAM I I srT-A I B I H S H PJCP5il s H E PJCP5t2 s H PJCP5r3 H E PJCP514 rt2
pJCPSlo and pJCPS12 were highly cytoroxic (cDso titres were both > 1:1OO,0OO). No effect on Vero cells was observed for similar fractions from
JM109 cells harbouring pJCPS1 1, pJCPS13, pJCPb14, pUC19 or pBLUESCRIPT SKTM (result not shown).
5.2.4 Nucleotide and Amino Acid Sequences of the SLT-t-retated Operon ín Strain PH
The above studies localized the SLT coding sequences to within the 3.0-kb insert of pJCP512 and the complete DNA sequence of both strands of this region was determined, using nested deletion derivatives of both pJCP513 and pJCPS14. ln addition. nested deletion derivatives of pJCP511 were used to obtain single stranded sequence data for a further 2.57 kb ot DNA immediately upstream (see Figure 5.4). The nucleotide sequences for these two regions are shown in Figure 5.54 & B.
Analysis of the data in Figure 5.58 indicated that ORFs encoding the A and B subunits of a SlT-l-related toxin were located from nucleotides 3187 to 4136 and 4146 to 4415, respectively. Putative ribosome binding sites were located immediately upstream from each ORF. Putative -10 and -35 promoter sites, as well as a region of dyad symmetry spanning the -1o sequence believed to be involved in regulation of expression by Fur (Calderwood et al., 19871, were also located approximately 11o-14o bp upstream of the A subunit coding sequence.
The nucleotide sequences for SLT-l encoded by three different bacteriophages (H198,933J and H3O) have previously been shown to be identical (Calderwood et al., 1987; De Grandis et al., 1987; Jackson et al., 1987b; Kozlov et al., 1988). However, the SLT-I A subunit gene from E. coli PH differed from these published sequences by five nucleotides, which resulted in two amino acid substitutions (see Figure 5.58). lnterestíngly, the PH SLT-I gene was more similar to SHT, with a total of three nucleotide .t f
,{ Þ
I
I' ¡ 'l t
'l
t I
I I
Figure 5.5 A Nucleotide sequence of the DNA contained in the inserts of pJCPS11 and pJCP51 2
Double stranded DNA sequencing of nested deletion derivatives of pJCPS11, I $i pJCPS13 and pJCPS14 was carried out. Panel A shows the region from the Hindlll site at the 5' end of the insert of pJCP511 to the point of symmetry of the Spl,1 s¡te. The sequence is numbered (see left hand margin) from the first nucleotide of the HindllJ site (1) to the third nucleotide of the Sphl site (nucleotid e 2570). The region of DNA encoding a putative lS element is underlined, whilst the region with a high degree of homology to a portion of bacteriophage lambda is double-underlined. An ORF within this latter regiori' (nucleotides 1446-2027], is shown in bold type. f I ,f, t^.
¡ t ti
'l A t I
I
1 I I
91
181 2/r
361
451 54t
631 72I
811
901
991
1081
IITI TCCTGTTTACCTCTTTCTCAGGGAGTTTAGTCTCCAGGATTTCCGGGGCGGTTCAGTCTAATCCACTCCGCGCCGCCATGATTGTCTTTC r261 TCATGATGCAGGACGCCAATAATGCTTAGCCCATCCCAATCCCTTCAATACCAGAAAGAAAGCGTCGAGCGGGCTTTAACGTGCGCTAAC
1351
1441
153 1 T62I t7 1L
1 801
189 1
1981
?07L 2I6I AAAGATACTGATGCGT
225r GATATCCGGCAGGTTCTTGAGTGCTGGGGGGCATGGGCGGCAAATAACCATGAGGATGTGACCCTGGTCACCCATTGCCGCCGGATTTAA
234L GGGACTGATCCCCGAAAAAGTAAAAICACGCCCGCAGTGCTGTGACGATGATGCGATGGTGATATGCGGTTGTATAGCCCGCCTTTACCG
243I GAACAATCGCGATCTGCATGACTTGCTGGTTGATTATTACGfGCTGGGGGGGACGTTCATGGCGCTGGCACGGAAACATGGGTGCTCTGA
252L CACCTGTATAGGTAAACGCCTTCACAAAGCGGAGGGGATTGTTGAAGGCA Figure 5.5 B Nucleotide sequence of the DNA contained in the inserts of pJGPS11 and pJCPS12 (continuedl panel B shows the sequence of the DNA insert of pJCPS12, commencing at the fourth nucleotide of the Spál site (nucleotide 2571) to the last nucleotide of the EcoRl site (nucleotide 5567). The coding regions for the putative A and B subunits are nucleotides 3187-4136 and nucleotides 41 46-4415, respectively. The amino acid translation is represented by single-letter code immediately above the first nucleotide of each oodon, and the numbers in the left hand margin refer to the position in the processed polypeptide chain for each subunit. Possible signal peptidase cleavage sites are denoted 'ú". putative ribosome binding sites for each subunit are denoted "rbs" and are underlined. Putative -10 and -35 promoter regions are also underlined, and a region of dyad symmetry spanning the -1O sequence believed to be involved in regulation by Fur is shown in bold characters. The location of a hypothetical rho-independent transcriptional terminator sequence is double underlined. Within the coding sequence of the A and B subunits, nucleotides and amino acids which are different to that previously published for SLT-l are underlined, those which are different to SHT are double-underlined, and those which are different to both SLT-I and SHT are double-underlined and shown in bold type. The position of an ORF downstream from the SLT coding sequence (nucleotides 4926-5312) is also shown in bold type. B
?5t I TGCTGATGATGCTGGGAGTGAGGCTTGAGATGGATCGGTATGTTGAGCGTGAATTGCCGGGAGGGAGAACCTCTGTATTTTATCAGCG
2 659 AAAAAATAGTTTACGATCGTAAAAATCTGCATATCATGATAAGAGTGGTTACATTGCCACGCTGCTTAACCCGCCGATGCGCGGGTTTTT
27 49 TTGTACCCAGAATCCTGTGAGCTATACGGAAAGTACACAGAAAGGAAGGTGCGACCACAATTAATAACAAAATCTTAAAAATTGCACATG 2839 GCACTATTAGTTTTCTAAATATTGTGTATTTATTGTATTGCAGGATGACCCTGTAACGAAGTTTGCGTAACAGCATTTTGCCCTACGAGT 2929 TTGCCAGCCTCCCCCAGTGGCTGGCTTTTTTATGTCCGTAACATCCTGTGTATCAATAAATGTTGTTGTCTACGTACGTCAAGTAGTCGC
3 019 ATGAGATCTGACCAGATATGTT AAGGTTGCAGC TCTCTTTGAATATAATTATCATTTTCATTACGTf ATTGTTACGTTIATCCGGTGCGC -35 -lorbsMKII A-22 3109 CGTAAAACGCCGTCCTTCAGGGGGTGGAGGATGTCAAGAATATAGTTATCGTATGGTGCTCAAGGAGTATTGTGTAATATGAAAATAATT
A_ 1B I F R V L T F F F V I F S V N V V ATK E F T L D F S T A K T 3 199 ATTTTTAGAGTGCTAACTTTTTTCTTTGTTATCTTTTCAGTTAATGTGGTTGCGAAGGAATTTACCTTAGACTTCTCGACTGCAAAGACG
Ai3 YVDSLNV I RSA I GTPLQT I SSGGTSLLl"lI D 3 289 TATGTAGATTCGCTGAATGTCATTCGCTCTGCAATAGGTACTCCATTACAGACTATTTCATCAGGAGGTACGTCTTTACTGATGATTGAT
443 SGTGDNLFAVDVRG I DP EEGRFNNLRL I VE 3379 AGTGGCACAGGGGATAATTTGTTTGCAGTTGATGTCAGAGGGATAGATCCAGAGGAAGGGCGGTTTAATAATCTACGGCTTATÏGTTGAA A/3 RNN LYVTGFV NRTN N VFYRFADFSHVTFPG 3469 CGAAATAATTTATATGTGACAGGATTTGTTAACAGGACAAATAAT6TTTTTTATCGCTTTGCTGATTTTTCACATGTTACCTTTCCAGGT
A 103 TTAVTLSGDS S YTTLQR VAG I SRTG|4Q I NR 3 559 ACAACAGCGGTTACATTGTCTGGTGACAGTAGCTATACCACGTTACAGCGTGTTGCAGGGATCAGTCGTACGGGGATGCAGATAAATCGC
A 133 HSLTTSYL DLMS HSGTLLTQSVARAMLRFV 3 649 CATTCGTTGACTACTTCTTATCTGGATTTAATGTCGCATAGTGGAACCTTACTGACGCAGTCTGTGGCAAGAGCGATGTTACGGTTTGTT
A 163 TVTAEALRFRQ I QRGFRTT LDDLSGRS YVM 3739 ACTGTGACAGCTGAAGCTTTACGTTTTCGGCAAATACAGAGGGGATTTCGTACAACACTGGATGATCTCAGTGGGCGTTCTTATGTAATG 4193 TAEDV DLTL N\4lGRL S SV L P DYHGQDSVRVG 3829 ACTGCTGAAGATGTTGATCTTACATTGAACTGGGGAAGGTTGAGTAGIGTCCTGCCTGATTATCATGGACAAGACTCTGTTCGTGTAGGA 4223 R I SFGS I NAI LGSVAL I LNCHHHASRVARM 39 19 AGAATTTCTTTTGGAAGCATTAATGCAATTCTGGGAAGCGTGGCATTAATACTGAATTGTCATCATCATGCATCGCGAGTTGCCAGAATG 4253 ASDEFP SMCPADGRVRG I THNKI Ll¡JDSSTL 4009 GCATCTGATGAGTTTCCTTCTATGTGTCCGGCAGATGGAAGAGTCCGTGGGATTACGCACAATAAAATATTGTGGGATTCATCCACTCTG *TbS A2B3 G A I L M R R T I S S M K K T L L I A A S L S F F S 4099 GGGGCAATTCTGATGCGCAGAACTATTAGCAGTTGAGGGGGTAAAATGAAAAAAACATTATTAATAGCTGCATCGCTTTCATTTTTTTCA
B-5 A S A I AtT P D C V T G K V E Y T K Y N D D D T F T V K V G 4189 GCAAGTGCGCTGGCGACGCCTGATTGTGTAACTGGAAAGGTGGAGTATACAAAATATAATGATGACGATACCTTTACAGTTAAAGTGGGT
ó1Y) DKEL FTNRl,lNLQSL L LSAQ I TGl'4TVTI KTN 4?/9 GATAAAGAATTATTTACCAACAGATGGAATCTTCAGTCTCTTCTTCTCAGTGCGCAAATTACGGGGATGACTGTAACCATTAAAACTAAT 856 ACHNGGGFSEVIFR* 4369 GCCTGTCATAATGGAGGGGGATTCAGCGAAGTTATTTTTCGTTGACTTAGAATAGCTCAGTGAAAATAGCAGGCGGAGATTCATAAATGT 4459 TAAATACATCTCAATTCAGTCAGTTGTTGCCGGTCTGATAATAGATGTGTTAGAAAATTTCTGCATGGTGAATCCCCCTGTGCGGAGGGG 4549 CGACTGGTGAACGGTATGATCTCTTTGATGATCGTAAGCGAGAATACGCGGGTTTGGTGGCACCAGGCCGAACTCACCGGGAGGCACCCG 4639 GCACCATGCAATGGCACATAGCGCCACTCTCCAGCCCCTCTCCGGAGGGGCTTTCTTATGGACAAAAAAAGCCCGCGCAGGGAGACGCTG 4729 GCGGCAAGGAATAAACAACAAAACGTGAAGTAATATTTCAGCTGGCGAATAATATCCGACAGTAATCACTCTGCGGAATTAGGCGGCCTT 4819 TTTCCGTATTGCGGGCTGTTGTCTCTGTTCTGCCATTGTCCTGTAACTTCCGGATTTCAAGCCCGCTCATCATTTTACCCACAATATTAT 49 09 CCCGGCCGGGAGGATTCÁTGGCATTÍAAACACTACGATGTGGTCAGGGCGGCATCGCCGTCAGACCTTGCTGATGCACTTGCGCAAAAAÁ 4999 ATCCTGAAGGÄTGGCÁACCATATGGTGGGCCGTTTTCTTCGTATACGGÁTGATGGCGCAGCACTTATTCAGGCGATTGTCGCAGMGGTG 5089 ATGTGAGCACACCTGTTGTGGTGÀAGCCGACAGGTGGAGAAGGTGCAGTAATCAGCGCCACCAGCGACCCCG6GTATTACTTTGTTGTGG 51/9 TTCTGGCAGGGCAGTCAAACGGCATGTCGTÂTGGTGAAGGTCTTCCGCTGCCGGAGACATATGACCGTCCGGACCCGCGCATTAAAGCAA 5269 CTGGCCCGTCGCAGTACGGTCACACCGGGCGGTGTCCCCTGTAAAÍATAACGACATCATTCCGGCGGACCATTGTCTGCATGATGTGCAG 5359 GACATGAGCCGCCTfAACCATCCGAAAGCGGACCTGTCAAAGGGGCAGTACGGAACCGTGGGGCAGGGGCTGCATATCGCCAAAAAACTG 5449 CTGCCGTTTATACCGGCGAATGCGGGCATTCTGCTGGTTCCGTGCTGTCGTGGTGGTTCAGCGTTCACCACCGGAGCTGATGGCACATAC 5539 AGTGACGCGAGTGGTGCCTCGGAGAATTC 113 differences in the A subunit gene, resulting a single amino acid substitution (Kozlov et al., 1988; Strockbine et al., 1988). Three of the nucleotide substitutions and one of the amino acid changes in PH SLT-|, with respect to the classical SLT-|, were the same as that seen in SHT. However, three other nucleotide differences and one amino acid substitution are unique to PH SLT- t.
Examination of the nucleotide sequence of the region 5' to the SLT-l gene (Figure 5.54) revealed two interesting features. Firstly, the region of DNA from nucleotides 1 to 1228 represents a putative lS element, which has 93o/o homology to lS34l/ (present in the E. coli citrate utilization transposon Tn34l 'll (lshiguro and Sato, 1988) and 95% homology with 15629 of Shigella sonnei (Matsutani et al., 1987). The lS element is located 1.96 kb upstream of the SLT-I coding region. To determine whether the PH genome contained further copies of the lS element, a HindllllHindll fragment containing nucleotides 1-772 of the sequence shown in Figure 5.5A was isolated and labelled with DlG. Southern hybridization analysis of PH DNA digested with various restriction enzymes, using this lS-specific probe is shown in Figure 5.6. A large number of DNA species in each d¡gest was labelled. Examinat¡on of the ethidium bromide-stained gel prior to Southern transfer d¡d not suggest incomplete restriction had occurred (result not shown). Thus, the hybridization data indicate that there are multiple copies of this or a very similar lS element in the PH genome. Different labelling intensities of various restriction fragments might imply that some fragments contain more than one copy of the lS element, or that lS elements with partial homology to the probe are present. The second noteworthy feature is the region from nucleotides 1309 to 2234, which is greater than 93% homologous to bacteriophage lambda DNA
(nucleotides 42,290 to 43,216l'. This region of lambda includes an ORF encoding a 2O4 amino acid polypeptide Nin204, the function of which is Figure 5.6 Southern hybridization analysis of PH DNA using an lS element probe
(1l'' chromosomal DNA from E. coli strain PH was digested with BamHl Bglll l2l, Eco1l (3), Hindlll (4), or Sphl (5), and electrophoresed on a O.8o/" agarose gel. DNA was transferred to a nylon filter and hybridized with the a DIG- labelled 772 bp DNA fragment from the PH lS element and washed at high stringency. Lane M contains DIG-labelled DNA size markers (23.1, 9.4, 6.6, 4.g7,2.3 and 2.O kb, respect¡vely, from top to bottom)' M12345
- t- .-
'F (r¡ l-a Gd - -
l-D
aæ
t- lt4
unknown (Kroeger and Hobom, 1982). An oRF was also present in the homologous region of the PH sequence (nucleotides 1446-2027), sufficient to
encode a 193 amino acid polypeptide, the first 1 79 of which were 94.4o/o homologous to Nin2O4. Sequence divergence at the C-terminal end of the ORF appeared to be due to a single extra nucleotide in the PH sequence,
which resulted in a frame shift. Given that this portion of the PH DNA sequence was based on single-strand sequence data, it is possible that this difference is artifactual.
5.2.5 Cloníng and Sequencing of a Second SLT-related Gene from Strain PH
Several attempts to clone the SlT-l|-related gene from strain PH as a
4.s-kb EcoRl fragment were unsuccessful. SlT-ll-reactive clones isolated from the library contained only single EcoRl sites and presumably had
undergone deletions, To overcome this, PH genomic DNA was PCR-amplified using oligo primers Va and Vb (Table 2.21 which generated a 1.5-kb fragment (including the complete SlT-l!-related operon), which was purified and cloned into the Smal site of pBLUESCRIPT SKTM {designated pJCp122l. A French pressure cell lysate of E. coli JM1O9tpJCP522l was weakly cytotoxic for Vero cell monolayers (the CD56 titre after 3 days incubation was 1:40) (result not Shown).
Nested deletion derivatives of pJCP522 were constructed and subjected to DNA sequence analysis. The scheme for sequencing is shown in
Figure 5.7 and the complete nucleotide and deduced amino acid sequence is shown in Figure 5.8. The PH SLT-ll gene (designated SLT-ll/O111)was very similar to SLT-OX3, one of the variant SLT-Il genes from strain 031 described in Chapter 4. There were only two nucleotide differences between the coding regions of the two genes, resulting in one amino acid difference in the A subunit (Gly instead of Arg, at residue 176ll. Figure 5.7 Map and scheme for sequencing the insert of pJCP522
The solid line represents insert DNA and the broken line indicates vector DNA. Restriction sites are indicated as follows: A, Accli B, BamHl; P, Pstl; (S), former Smal site to which the blunt-ended PCR product was ligated. The boxes indicate the location of the SLT A and B subunit coding regions, as determined from the sequence data. The arrows beneath the map indicate the portions of the plus (->) and minus ((-) strands sequenced in various nested deletion derivatives of pJCPS22. pJCP522
P (S) A A (S) B
SLT-A LT. B
1.0kb Figure 5.8 Nucleotide sequence of the insert of pJCP522
The nucleotide sequence of the insert of pJCPS22 (encoding SLT-ll/O111) is shown. putative ribososme binding sites (Shine-Dalgarno sequences) for each subunit are labelled "rbs" and are underlined, and putat¡ve -1O and -35 promoter regions are double underlined. The deduced amino acid sequence for the A and B subunits are shown (in single letter code) above the first nucleotide of each codon. The numbers in the left hand margin indicate the position in the mature polypeptide for each subunit and the putative signal peptidase cleavage site is denoted "t". Nucleotides for the SLT-OX3 gene which differ from SLT-ll/O111 are shown. beneath the sequence, while the amino acid of SLT-OX3 which differs from SLT-ll/O111 is shown above the latter sequence. The 17 nucleotides at the 5' and 3' termini corresponding to the PCR primer sequences have been deleted. -35 -10 i CGTATTCAGCGTTGTTAGCTCAGCCGGACAGAGCAATTGCCTTCTAAGCA4TCGGTCACTGGTTCGAATCCAGTACAACGCGCCATACTT
rbs ¡4 K 9 1 ATTTTTTCTGGCTCGCTTTTGCGGGCCTTTTTTATATCTGCGCCGGGTCTGGTGCTGAT TACTTCAGCCAAAAGGAACACCTGTATATGA
C I L F K I¡J V L C L L L G F S S V S Y S¿R E F M I D F S T A i 8 1 AGTGTATATTATTTAAATGGGTACTGTGCCTGTTACTGGGTTTTTCTTCGGTATCCTAT TCCCGGGAGT TTATGATAGACTTTTCGACCC
QS YVSSLNS i RT E I STPL E H I SQGTTSVSV 27t AACAAAGTTATGTCTCTTCGT TAAATAGTATACGGACAGAGATATCGACCCCTCTTGAACATATATCTCAGGGGACCACATCGGTGTCTG
I NHTPPGSYFAV D I RGL DVYQARFDHLRL I 3 6 1 TTATTAACCACACCCCACCGGGCAGTTATTTTGCTGTGGATATACGAGGGCTTGATGTCTATCAGGCGCGTTTTGACCATCTTCGTCTGA
] EQNNLYVAGFVNTATNTFYRFSDFTHI SV 451 TTATTGAGCAAAATAATTTATATGTGGCTGGGTTCGTTAATACGGCAACAAATACTTTCTACCGTTTTTCAGATTTTACACATATATCAG
PGVTTVSMTTDS S YT]LQRVAALERSG¡4Q i 541 TGCCCGGTGTGACAACGGTTTCCATGACAACGGACAGCAGTTATACCACTCTGCAACGTGTCGCAGCGCTGGAACGTTCCGGAATGCAAA
SRHSLVSSYLALME FSGNTMTRDASRAVLR 63 1 TCAGTCGTCACTCACIGGTTTCATCATATCTGGCGTTAATGGAGTTCAGTGGTAATACAATGACCAGAGATGCATCCAGAGCAGTTCTGC
R FVTVTAEALRFRQIQGEFRQALSETAPVYT 7 2I GTTTTGTCACTGTCACAGCAGAAGCCTTACGCTTCAGGCAGATACAGGGAGAATTTCGTCAGGCACTGTCTGAAACTGCTCCTGIGTATA A
l'4T P E E V DL T L N ì/G R I S N V L P E FRGE GGVR V 81 1 CGATGACACCGGAAGAAGTGGACCTCACACTGAACTGGGGGAGAATCAGCAATGTGCTTCCGGAGTTTCGGGGAGAGGGGGGTGTCAGAG
GR I SFNN I SA I LGTVAV I LNCHHQGARSVR 9O 1 TGGGGCGAATATCCTTTAATAATATATCAGCGATACTGGGCACAGTGGCGGTTATACTGAATTGCCATCATCAGGGGGCGCGTTCCGTTC
AV N E E I Q P E C Q I T G D R P V I R I N N T LI,JE S NT 99 I GCGCCGTGAATGAAGAGATACAACCAGAATGTCAGATAACTGGCGACAGGCCAGTIATAAGGATAAACAATACTTTATGGGAAAGTAATA
AAAFLNRRAHSLNTSGE* TbS MKKIFVAA 1 OB 1 CCGCAGCTGCTTTTCTGAATCGCAGGGCCCACTCTTTAAATACATCCGGAGAATAACAGGAGTTAAATATGAAGAAGATATTTGTAGCGG
L F A F V S V N A M ATA D C A K G K I E F S K Y N E N D T F II7 I CTTTATTTGCTTTTGTTTCTGTTAATGCAATGGCAGCTGATTGTGCAAAAGGTAAAATTGAGTTCTCTAAGTATAATGAGAATGATACAT c
T V KV AGK E Ytl/ T N R I.I N L Q P L L Q S AQ L T GMT V i 2 6 1 TCACAGTAAAAGTGGCCGGGAAAGAGTACTGGACTAACCGCTGGAATCTGCAACCGCTACTGCAAAGCGCACAGTTAACAGGAATGACGG
TIKSNTCASGSGFAEVQFN* 135 1 TAACAATCAAATCAAATACCTGTGCGTCAGGTTCAGGATTTGCTGAAGTGCAGTTTAATTAATATCAGAAGCATTGCTGGTTTCGTGGTG C
I44T TGCAGCAATGTAGTTACAGIGTAATTAATG c 115
The observed cytotoxicity of lysates of E. coli JM1O9[pJCP522l was significantly lower than JMlO9tpJCPSOll which encoded SLT-OX3 as a 4.6- kb Pstl fragment in pUC19. A more direct estimate of the comparative cytotoxicity of the two closely related toxins was obtained by testing lysates of JMlO9tpJCP522l and ¡MlO9tpJCPs2Ol. This latter clone contained the
SLT-OX3 gene as a 1.s-kb PCR-derived fragment in pBLUESCRIPT SKTM, and
restriction analysis using .Accl indicated that the orientation of the SLT operons with respect to vector promoter sequences was the same (result not shown). Respective CD5gs for the two lysates were 1:4O and 1:320, confirming the clear difference in cytotoxicity. This difference was not due to variation in growth or effifiency of lysis, as total protein concentrations in the two filtered lysates were not significantly different (result not shown).
SLT Strain PH 5.2.6 Detectíon of ,Transcripts in Neutralization studies reported in Section 5.2.1 failed to detect the presence of cytotoxicity ¡n strain PH lysates, which could be neutralized with an anti-SlT-ll-specific monoclonal antibody, against the high background SLT- I toxicity. To determine whether the SLT-IllO111 gene was constitutively expressed by strain PH, RNA was extracted and subjected to reverse transcription and PCR-amplification (see Section 2.50) using oligo primers lVa and lVb (see Table 2.2l'. The use of these primers for PCR-amplification of SLT sequences is described in Chapter 6 (Section 6.2.1l.. Aliquots of pJCPS12 and pJCP522 plasmid DNA were also amplified, as positive controls for PH SLT-l and SLT-Ill0111 sequences, respect¡vely (Figure 5.9 panel A). A
215-bp PCR product was generated from the pJCP512, pJCP522 and PH RNA templates. No amplified product was seen for the PH RNA preparation in the absence of reverse transcriptase, indicating that it was not contaminated with SLT DNA sequences. Southern hybridization analysis of the PCR products, using SLT-l- and SLT-ll-specific oligos (Figure 5.9 panels B Figure 5.9 Detection of PH SLT-I and SLT-ll/0111 transcripts in E coli strain PH by reverse transcription PCR and Southern hybribization analysis
Total cellular RNA from strain PH was extracted and subjected to reverse transcription in the presence or absence of reverse transcriptase (+ RT or - RT), followed by PCR-amplification, employing oligo PCR primers lVa and lVb. Atiquors of pJCP512 and pJCP522 plasmid DNA (diluted 1:1000) were also amplified as positive controls for PH SLT-l and SLT-ll/O111 sequences, respectively. Aliquots of each react¡on were electrophoresed on a 2o/o agarose gel and stained with ethidium bromide (panel A). DNA from replicate gels was transferred onto nylon filters and probed with DIG-labelled oligos SLTItD (panet B) or SLT2ID (panel C). The expected mobility of a 215 bp fragment is indicated.
Tracks: 1: Negative control (no DNA template), +RT. 2: PH RNA, -RT. 3: PH RNA, +RT. 4: pJCP512 DNA, +RT. 5: pJCP522 DNA, + RT. M: DNA size markers (5O1, 4O4, 331, 242, 1gO, 147 and 110 bp, respectively, from top to bottom). A B c
M 1234 5 1 2 34 5 12345
E
-2ì5 bp 116 and C) indicated thar rhe pJCPS12- and pJCPb22-derived PCR products contained only SLT-I- and SlT-ll-specific sequences, respectively. The PCR product from PH RNA contained sequences derived from both genes, although the signal obtained with the SLT-Il-specific probe was weaker than that for the SlT-l-specific probe. Thus, both PH SLT-l and SLT-ll/O111 appear to be transcribed in E coli strain PH.
5.3 Discussion
5.3.1 Seguence Analysís of a varíant sLT-l operon in strain PH Previous studies have described the cloning and sequencing of the SLT- loperons from phages 933J, H198 and H3O. The nucleotide sequences for all three were identical (Calderwood et al, 1987; DeGrandis et al., 1987¡ Jackson et al,. 1987b; Kozlov et al., 1988). Comparisons between the nucleotide and deduced amino acid sequences of SLT-I and SHT have shown that there is greater than 99o/o homology between the two sequences. Strockbine et al. (1988) detected only three nucleotide differences whilst Kozlov et al. (1988) detected 4 nucleotide differences. Both groups reported that the deduced amino acid sequence of SHT differed by a single amino acid (Thr45 in the A subunit of SHT is Ser in SLT-|). lnterestingly, the single amino acid difference between SLT-I and SHT was not observed by Takao ef at. (1988), who concluded that the two primary amino acid sequences were identical, on the basis of both fast atom bombardment mass spectrometry and Edman degradation analysis of purified tox¡ns. The SLT-I used in that study had been purified from an E. coli O157:H7 strain 83-1386. The phage- encoded SLT-I gene from this strain had also been cloned (Kurazono et al., 1987) and Takao et al. (1988) determined its nucleotide sequence. This revealed that there was a single nucleot¡de difference between this and the tt7 other SLT-I gene sequences referred to above, such that Ser45 reverted to Thr, the same residue found in SHT' ln this Chapter the coding sequences of SLT-I from E. coli strain PH was shown to vary compared to those previously published for the SLT-I genes of phages 933J, H19B and H3O by 5 nucleotides, resulting in two amino acid substitutions in the A subunit. The PH gene was more similar to that for SHT, from which it differed by only 3 nucleotides, resulting in one amino acid substitution in the A subunit. Although the unique amino acid substitution (Ser149 --) Leu) is not a conservative change, there did not appear to be any impact on toxicity, indicating that this site is not essential for catalytic activity. The nucleotide sequence of the B subunit gene for SLT- I from PH was identical to that for SLT=l from bacteriophages H3O, 933J and
H198, and for SHT. The sequence immediately upstream of the PH SLT-I A subunit gene which contains the putative promoter site was identical to that found in the analogous region of other SLT-I and SHT operons. This region includes the sequence (spanning the -1O region) purportedly involved in regulation of SLT-I expression by Fur. Strain PH expressed high levels of SLT-I in LB broth that had not been iron-depleted. Attempts to enhance SLT-I production by growth in the presence of 2,2'-dipyridyl (an iron chelator) resulted in very poor bacterial growth and an actual reduction in toxin production (result not shown). Smith and Scotland (1988) previously reported that any improvement ¡n cytotoxin production by growth of SlT-l-producing isolates in iron-limited media was also offset by reduced bacterial growth. The role of iron in regulation of SLT-I expression has been discussed in Chapter 1 (Section 1.9) but was not further examined in this study. 118
5.3.2 Analysís of Sequences Downstream from the PH SLT-I Operon The region immediately downstream from the PH SLT-I operon also appears to be more similar to the analogous region of the S. dysenteriae SHT locus than to either of the E. coli SLT-I downstream regions. There is >95% DNA sequence homology for about 250 bp following the SLT-I B subunit coding regions of PH and H198 and H3O, but beyond this point, the sequence homology is only of the order of 600/". However, the analogous region downstream of the SHT B subunit coding sequence maintains 95o/o DNA homology with PH for at least 48O bp, including lOOo/o homology in the region (nucleotides 4672-4691 for the PH SLT-I gene) encoding a putat¡ve rho-independent transcription termination sequence, with a Gibbs Free Energy of -19.1 kcal/mole. An open reading frame sufficient to encode a 128 amino acid polypeptide, immediately preceded by a ribosome binding site, is located from nucleotides 4926 to 5312, but no significant homology at either nucleotide or amino acid level was found with other known sequences, lnterestingly, there is also very strong DNA sequence homology (91.3%) in the downstream sequences of the SLT-I operon of strain PH (from nucleotides 4618 to 5567 lthe end of rhe insert of pJCPS10]) and that of the sequences downstream of the SLT-OX3 gene of strain 031 described in Chapter 4. This region of close homology includes the putative transcription terminator sequence for the PH SLT-I gene. However, there is only 59.5% homology between the A and B subunit coding sequences of PH SLT-I and SLT-OX3. These findings are consistent with the location of the two SLT genes at analogous chromosomal map positions in the respective E. colistrains.
5.3.3 Analysís of Sequences Upstream of the PH SLT-I Operon As described in Chapter 3, following the identification of strain PH as an SLTEC, a phage was isolated from the strain, but Southern hybridization analysis of phage DNA showed that it did not encode any SlT-related 119 sequences. Further attempts described in Section 3.2.1O, also failed to isolate an SlT-converting phage. lt was concluded therefore, that the SLT-I operon of strain PH also differed from other previously reported SLT-I genes in that they did not appear to be phage-encoded. However, a region of strong homology (greater than 93%) to a portion of bacteriophage lambda (nucleotides 42,290 to 43,216) was detected approximately 1 kb upstream from the SLT-I operon. This region of lambda includes an open reading frame which encodes a 2O4 amino acid polypeptide (Nin2O4) of unknown function (Kroeger and Hobom, 1982; Sanger et al., 19821. The presence of lambdoid sequences in the immediate vicinity of the SLT-I operon is a possible indication that it may have been phage-encoded at a previous point in time. Alternatively, it cannot be ruled out that the SLT-I operon may still be associated with a defective phage. The SHT operon and all porcine and human variant SLT-ll genes reported so far, have not been encoded on toxin- converting phages, but have been mapped to the chromosome of S. dYsenteriaeandE.coli,respectively(Gylesetal.,1988;Kozlovetal',1988; Strockbine et al., 1988; Weinstein et al., 1988b). Transposons and lS elements are mobile genetic elements which are known to be associated with plasmids, bacterial chromosomal DNA and the genomes of bacteriophages (lshiguro and Sato, 1988). The presence of an lS element immediately upstream from this lambdoid sequence (approximatelY 2 kb upstream from the SLT-I operon) is also suggestive of the possible involvement of a transposon in the present chromosomal localization of the SLT-I gene in strain PH. A second copy of the lS element was not found in
the 1 1OO-bp region downstream from the SLT-I gene for which sequence data were available. An attempt was made to locate another copy of the lS element by Southern hybridization analysis. A DIG-labelled 772-bp fragment of the PH lS element was hybridized with PH genomic DNA cut with various restriction enzymes. This probe reacted with multiple PH DNA fragments t20 indicating that there are at least 12 copies of the lS element in the PH genome. This was not an altogether unexpected result, as there have been several reports of multiple copies of other related lS elements in other strains of E. coli, S. dysenteriae, S. sonnei, and S. flexneri (Matsutani et al., 1987). However, the presence of multiple copies of the lS element has made the localization of the nearest lS element sequence downstream from the PH SLT- I sequence difficult. Pulsed-field gel electrophoresis of larger DNA fragments obtained using rare-cutt¡ng restriction enzymes might provide further information on the chromosomal distribution of the lS element. DNA sequence analysis indicated that the PH lS element has 95o/" homology with 15629 of S. sonnei, at least 6 copies of which are present in the genome of S. sonnei (Matsutani et al., 1987). The PH lS element also has 93% homology at the DNA level to 15347/, which is a component of the plasmid-encoded citrate utilization transposon -fn341 1 of E. coli (lshiguro and Sato, lg88). The largest ORF within the ls34l / sequence is sufficient to encode a 28.3 kDa polypeptide and this is believed to be the transposase (lshiguro and Sato, 1988). The PH lS element DNA sequence did not appear to contain any ORFs approaching this size, but this is most probably consequence of frame shifts resulting from errors in this region of the PH DNA sequence, which was based on data from only one strand. Clearly, sequencing of the other strand is required before any meaningful analysis of homologies between the PH lS element and lS34l I at the amino acid level can be undertaken. lnterestingly, Kozlov et al. (1988) found an lS element (which was almost identical to ls6oo found in s. sonnei lMatsutani ef al., 19871) 185 bp upstream of the SHT gene of S. dysenteriae. However, this lS element has only about 48o/o DNA homology to that found in the present study. Kozlov ef al. 11988) suggested that the S. dysenteríae lS element m¡ght be a structural component of a transposon responsible for amplification of the SHT operon in .t 12l i ,t ,I- the chromosome. They cited unpublished observations that there were more I than 1O copies of the SHT operon in S. dysenteriae (Kozlov et al., 1988). I t ü tÌ However, there are no other reports in the literature of the existence of such ,.]
,] multiple Indeed, in the present study, Southern hybridization analysis copies. ï t (Figure 5.1) indicated the presence of only a single SLT-probe-reactive DNA I fragment in S. dysenter¡ae type 1 DNA digested with either of four restriction ! I I enzymes. Moreover, Fontaine et al. (1988) successfully constructed SHT- negative S. dysenteriae by allelic exchange with an insertionally-inactivated derivative of the cloned SHT gene; th¡s would have been extremely difficult had there been more than one copy of the SHT gene in the chromosome.
5.3.4 Sequence Analysis of SLT-ll/Ol l I from Strain PH Attempts to directly clone the 4.s-kb EcoRl fragment containing the SLT-Il-reactive sequence from strain PH were not successful. Although SLT-¡l probe-react¡ve clones were isolated, these appeared to have undergone deletion events during the cloning process, and so were deemed unsuitable for analysis. The PCR-amplification/cloning strategy developed in Chapter 4 enabled cloning of this gene as a 1.s-kb PCR product. Sequence analysis indicated that this second SLT-related operon of'strain PH (designated SLT-
ll/O1 1 1) had strong homology with the SLT-OX3 gene from strain 031, described in Chapter 4. There were only two nucleotide differences between the coding region of the two sequences, which resulted in only one amino acid difference in the A subunit of the SLT-ll/O111 gene; Gly instead of Arg at residue 176. A comparison of the cytotoxicity for Vero cells was made between SLT-ll/O111 and SLT-ll-OX3. Extracts of E. coli JM109tpJCP522l, which expresses PH SLT-Il/O111, were approximately eight-fold less toxic than similar extracts of E. coli JMl09tpJCP52Ol (an otherwise identical construct with respect to orientation to the vector promoter, expressing SLT- OX3). There was no apparent difference in plasmid DNA yields from these t22 i Þ* two strains suggest¡ng that plasmid copy number is also similar. This
suggests that the single amino acid difference between the two toxins could ¡ { have a partial impact on specific activity. I Residue 176 is located at the C-terminal end of an 18 amino acid l I I sequence, which is identical for SLT-1, SLT-ll and all previously reported I variants of SLT-ll. This sequence spans one of the three regions of the SLT A subunit which have been shown to have homology with ricin, as discussed in
Chapter 1. All three regions in the ricin A subunit have been mapped to a cleft which forms the putative active site as determined by x-ray crystallographic analysis (Monfort et al., 1987; Yamasaki ef al., 1991). Therefore, any amino acid change within or flanking any of these regions has the potential to modify the catalytic activity or substrate binding efficiency of these toxins. Yamasaki et al. (1991) used site-directed mutagenesis of a number of amino acids in the analogous regions of SLT-1, and several of these (particularly Glu167, Atg179 and Arg¡2l' significantly reduced cytotoxicity. However, the effect of mutation of ArgtZ6 was not investigated. The apparently lower cytotoxicity of recombinant E coli expressing SLT-ll/O111 with respect to similar constructs expressing SLT-OX3, is strongly suggest¡ve that the residue at position 176 of SLT-Il is important for catalytic function, but this needs to be confirmed directly by toxicity testing of the respective purified toxins. As pointed out in Chapter 4, the interpretation of sequence data generated from cloned PCR products should be tempered with the knowledge of the possibility of low frequency errors by Taq polymerase. Notwithstanding this, it is clearly important to identify any amino acid substitution which results in either a reduction or increase in toxicity of SLTs, as these may have the capacity to ultimately affect the severity of SLTEC disease in humans or animals. r23
5.3.5 Expression of SLT Genes in Straín PH I As discussed in Chapter 4, it is not unusual for an SLTEC to encode two SlT-related sequences. lt has been demonstrated that both the PH SLT-I and SLT-lllO111 genes are expressed when they are separately cloned into E coli JM109. However, it was important to determine whether they were both expressed in strain PH. Neutralization studies employing monoclonal antibodies directed against SLT-!, revealed that E coli strain PH was a high constitutive producer of SLT-|. However, the high background level of SLT-I in extracts of strain PH complicated demonstration of the presence of toxin that could be neutralized by SlT-ll-specific monoclonal antibodies. Analysis of total cellular RNA of strain PH by reverse transcriptase-dependent PCR- amplification unequivocally demonstrated the presence of both PH SLT-I and SLT-ll/O111 transcr¡pts. The relative strengths of SLT-I- and SlT-ll-specific signals obtained from Southern hybridization analysis of the reverse transcription-PCR products, suggested that the SLT-I transcript may have been more abundant than the SLT-Il-related transcript.
5.3.6 Relatíve lmportance of SLT-I versus SLT-ll ín Pathogenesis SLT-I is usually associated with SLTECs that produce high levels of cytotoxin (often exceeding t06 CO5gs per ml of culture lysate), while SLT-Il production is usually 1OO-fold lower in terms of CD56 (Strockbine et al., 1986). Weinstein ef al. (1988b) have proposed that one explanation for the lower levels of cytotoxin production in SLT-ll strains is that the strength of the promoters for the SLT-ll operons may be weaker than that of the SLT-I promoter, which more closely resembles the consensus sequences for established E. coli promoters (Rosenberg and Court, 1979). However, cytotox¡city as measured by CD56 for cell monolayers correlates poorly with i.p. LD5g for mice. Strockbine et al. (1986) demonstrated that the number of HeLa cell CD5gs corresponding to one LDSO was 4O-7O-fold greater for SLT-I t24 than for SLT-ll. Thus, apparently higher levels of expression of SLT-I genes, as measured by cytotoxicity, may be largely negated by reduced lethality. Interpretation of the significance of the relative cytotoxicities and mouse lethalities of various SLTs is complicated by substantial differences between the figures obtained in several studies using purified toxins. For SLT-|, the estimates of the HeLa cell CD5g range from 1 to 1O pg, and the estimates for the mouse i.p. LDSO range from 5 to 1OO ng (O'Brien and LaVeck, 1983; Karch et al., 1988). Moreover, O'Brien and LaVeck (1983) reported a 2O-fold difference between the mouse i.p. LDSO for SLT-I and SHT, although their HeLa CD5g was identical. These toxins differ by just one amino acid (Kozlov et al., 1988). For SLT-lls the situation is more complicated because of the substantial sequence variation that has been reported. Published figures for Vero cell CD59 range from O.2 pg to 0.6 ng, with estimates for mouse i.p. LD5g ranging from 1 pg to 2OO ng (Karch et al., 1988; Dickie et al., 1989; Macleod and Gyles, 199O; Samuel et al., 1990). Remarkablv, the highest and lowest estimates for mouse LD5g were both obtained for SLT-llv purified from porcine E. coli isolates (Macleod and Gyles, 1990; Samuel et al., 199O). lt is likely that the various purification protocols employed in the above studies may have had an impact on toxin specific activity. lt is also possible that the different strains of mice used in the LD59 studies may differ in their susceptibility to a given SLT type. Epidemiological data has shown that there is a strong correlation between the development of serious.sequelae to SLTEC infection (including HUS, HC, and TTP) and SLTEC strains which produce high rather than moderate or low levels of SLT (Tesh and O'Brien, 1991). lt has also been reported that these complications are more strongly associated with SLTEC encoding SLT-Il only, than with those encoding SLT-I and SLT-Il, or SLT-l only (Scotland et al., 1987; Ostroff et al., 1989; Tarr et al.,'1989; Wadolkowski ef 31., 199O; Schmitt et al., 1991; Tesh and O'Brien, 1991). lt has been 125 postulated that SLTEC encoding multiple SLT-Il genes may also have increased cytotoxicity and hence enhanced virulence, but this has yet to be demonstrated (Schmitt et al., 1991). lnterestingly, human immunoglobulin preparations routinely contain significant levels of antibodies capable of neutralizing SLT-I/SHT but do not contain detectable levels of anti-SlT-ll (Ashkenazi et al., 1988). Whether this provides a degree of protect¡on against SLT-l-producing strains, thereby biasing the apparent SLT type distribution, is not known. The high sequence variability of SLT-lls is known to result in antigenic variation (Schmitt et al., 1991) and Meyer et al. (1992) have suggested that this may assist evasion of the immune system. lnterestingly, Christopher-Hennings et al. (1993) have recently reported that infection of gnotobiotic pigs with SLT-l-producing E coli reduced peripheral lymphocyte counts and proliferative responses to mitogens, implying that SLTs might have immunosuppressive properties. Further animal studies also support the notion that more serious disease is associated with SLT-ll-producing strains. Wadolkowski et al. (1990) observed that streptomycin-treated mice died from acute bilateral cortical tubular necrosis after being fed an E. coli O157:H7 strain which was a producer of SLT-I and SLT-ll and was capable of colonizing the mouse gut.
Pre-treatment of mice with monoclonal antibodies directed against the A or B subunit of SLT-ll, protected them from this strain. However, when infected mice were pre-treated with anti-SLT-|, no such protection was observed (Wadolkowski ef a/. 1990). Furthermore, the level of SLT-Il production was crucial for virulence. Mice died after being orally-infected with recombinant E. coli strains carrying the SLT-ll genes on a high copy number plasmid (and expressing high levels of SLT-ll), but were not killed when fed a low SLT-ll- producing recombinant strain (Wadolkowski ef al., 1990). Recombinant E coli strains expressing high levels of SHT were avirulent. Wadolkowski et a/. (1 99O) investigated the apparent ín vívo avirulence of SHT-producing 126 recomb¡nant strains in this model. One possibility was that a higher proportion of SHT than SLT-ll remained cell-associated, but there was no increase in virulence when the SHT genes were expressed in E. coli strains with a leaky periplasmic phenotype (Wadolkowski et al., 199O). Furthermore, expression of the SHT gene s in fur* or fur strains had no impact, implying that iron repression was not responsible for the reduced virulence of SHT-
(and by inference SLT-|-) producers compared with that of SlT-ll-producing E colí (Wadolkowski et al., 1990). ln spite of the above epidemiological and experimental findings, E. coli strains producing SLT-l alone have certainly been associated with serious disease (Dickie et al., 1989). Moreover, HUS is a well recognized sequel of S. dysenteríae type 1 infection, and this organism is not known to produce SLT-ll-related toxins. The recent report of instability of SLT genes in clinical SLTEC isolates during routine isolation and subculture complicates the interpretation of the epidemiological studies (Karch et al., 1992l'. These workers suggested that spontaneous curing of SLT-convert¡ng bacteriophages might be responsible for this instability. With the possible exception of strain
PH, all SLT-I genes reported to date have been phage-encoded (O'Brien et al., 1992t', whereas a number of SlT-ll-related genes have now been reported to be chromosomally-encoded in E coli land may as a consequence be more stable) (Gyles et al., 1988; Weinstein et al., 1988b; Gannon et al., 1990; lto et al., 199O; Schmitt et al., 1991; Meyer et al., 1992). Thus, a selective loss of SLT-I genes from HUS- or HC-associated isolates prior to analysis could have resulted in an underestimate of the prevalence of SLT-l- versus SLT-Il- producing strains. Caution should also be excercised when extrapolating results obtained in animal models of SLTEC disease to the mechanism of pathogenesis of disease in humans. Strain PH was isolated from the faeces of a 12 month old boy with
HUS. Although it has been shown to encode and express both SLT-I- and t27
SlT-ll-related toxins, the latter appears to have lower specific cytotox¡c¡ty than other closely related SLT-lls. Moreover, its gene may be transcribed less efficiently than the PH SLT-I gene, collectively accounting for the fact that virtually all of the cytotoxicity in extracts of strain PH was attributable to SLT- l. These findings suggest that the contribution of SLT-I to pathogenesis should not be underestimated. t28
CHAPTER SIX
DEVELOPMENT OF A PCR ASSAY FOR DIRECT DETECTION OF SLT-RELATED GENES IN PRIMARY CULTURES OF FAEGES AND GUT CONTENTS
6.1 lntroduction
When SLTECs were first recognized as pathogens of humans and animals, laboratory detection of SLTs was dependent on demonstration of the presence of a Vero cell cytotoxin in extracts of faeces or E coli isolates, which could be neutralized by antibodies to the respect¡ve SLT type (Smith and Scotland, 1988). W¡th the advent of cloning and nucleotide sequencing of SLT genes, strains could be identified as SLTECS and the SLTs could be 4t, typed by hybridilon to DNA or oligo probes (Scotland et al., 1988; Brown ef \ al., 1989; Karch and Meyer, 1989a; Thomas et al., 1991; ), or by PCR- amplification (Karch and Meyer, 1989b; Pollard et al., 1990; Brian et al., 1992; Gannon et al., 19921. These techniques have been employed for detection and character¡zat¡on of SLTECs in the work described in Chapters 3, 4 and 5 of this thesis. However, it has been shown that testing individual isolates may result in poor sensitivity of detection, unless very large numbers of isolates are examined. As mentioned previously, in cases of SLTEC-related disease such as HUS, fewer than 1 o/o of t. cot¡ isolates from a given faecal sample may be SlT-positive (Smith and Scotland, 1988; Karmali, 1989). The probability of isolating SLTECs from faecal cultures also has been reported to diminish 3-fold during the week following the onset of gastrointestinal symptoms (Tarr et al., 1990). Furthermore, Karch et al. (1992) have recently r29 reported a high frequency of loss of SLT genes from clinical isolates of E. coli during subculture. Development of a rapid screening technique based on direct PCR-amplification of crude DNA extracts of primary faecal cultures would be expected to overcome these problems. Thus, the work described in this Chapter involves the development of just such a PCR assay, which has resulted in increased sensitivity of detection of SLTEC and provides a potentially improved rapid diagnostic test for SLTEC-related disease.
6.2 Results
6.2.1 Development of a Novel SLT-I- and SLT-ll-specific PCR Assay Work described in Chapter 3 of this study describes the use of the single PCR primer pair (llla & lllb) designed by Karch and Meyer (1989b) for amplification of both SLT-I and SLT-ll genes. This primer pair has been employed in the laboratory to synthesize PCR-DlG-labelled SlT-specific DNA probes and these probes have been used extensively in the open laboratory during the course of this study. They were therefore deemed unsuitable for use in a direct faecal PCR assay, as the þotential for contamination with PCR product was considered too high. This problem was avoided by designing novel oligo PCR primers, which direct the ampl¡fication of a region of the SLT coding sequence that does not overlap with that which is amplified by the Karch and Meyer primer set. The primers used were 5'- ATACAGAG(GA)G(GA)ATTTCGT-3' and 5'-TGATGATG(AG)CAATTCAGTAT- 3' (designated lVa and lVb), respectively. The bracketed nucleotides denote positions at which two alternative nucleotides were incorporated during oligo synthesis to accommodate known sequence variations between various SLT types. This enabled the same primer set to be used for amplification of a 215-bp region corresponding to nucleotides 586 to 8OO of the A subunit coding sequence of SLT-|, or a 212-bp region corresponding to nucleotides 130
583 to 794 ot the A subunit coding region of SLT-ll (including all SLT-ll variants).
The SLT-I and SlT-ll-related PCR products generated using the PCR primers lVa and lVb, were distinguished by hybridization with an additional pair of DIG-labelled oligos. The first of these was a 21-mer 5'- AGAACGCCCACTGAGATCATC-3' which is specific for SLT-I (designated SLTI lD) and the second was a 22-mer 5'-ATACACAGGGAGCAGTTTCAGA-3' which is specific for SLT-ll and SLT-ll variants (designated SLT2ID). To confirm the specificity of the PCR reaction employing primers lVa and lVb, crude DNA extracts (see Section 2.42!- were prepared from pure cultures of E. colî C6OO, E. coli C6OO lysogenized with the SlT-l-encoding phage H198, E. coli C60O lysogenized with the SlT-ll-encoding phage 933W, E. colí strain PH (encoding both SLT-¡ and SlT-ll-related genes) and E. coli strain O31 (encoding two SlT-ll-related genes). Samples of each extract were subjected to PCR-amplification in the presence of PCR primers lVa and lVb and aliquots of each reaction were electrophoresed on 2o/o agarose gels and stained with ethidium bromide (Figure 6.1). A discrete PCR product of the expected size 12151212 bp) was seen for all templates except the negative control (no DNA template) and the E. coli C600 extract. DNA from replicates of this gel was then transferred onto nylon filters, hybridized overnight at 45oC with D|G-labelled SLTIlD or SLT2ID oligo probes and washed at high stringency. The SLT-I probe hybridized strongly with the PCR products from the C6OO::H198 and PH extracts, while the SLT-¡I probe reacted strongly with the extracts from C6OO::933W, PH and 031, thereby confirming the specificity of the two oligos (Figure 6.2l.. Weak PCR products of higher than the expected size which were seen for the C6OO template (Figure 6.1, lane 2l did not react with either SLT probe, suggesting these are due to non-specific priming. Such non-specific amplicons were not seen for either of the C60O::H198 or C6OO::933W templates, presumably because the Figure 6.1
PCR of DNA extracts from pure E coli cultures
DNA extracts from pure E coli cultures (prepared as described in Section 2.421 were subjected to PCR-amplification employing SLT-I- and SLT-ll- specific primers (lVa and lVb). Aliquots of each reaction were electrophoresed on a 2o/o agarose gel and stained with ethidium bromide. Tracks: 1: Negative control (no DNA template). 2: E. coli C6OO. 3: E. coli C600::933W. 4: E. coli C6O0::H198. 5: E. coli PH. 6: E. coliO31. M: DNA size markers of 2176, 1766, 1230, 1033,653, 517,453,394, 298,234, 22O and 154 bp (from top to bottom). 123'45 6M
.-=
f-. a
t
Iltt + 2ì5 bp Figure 6.2 Southern hybridization analysis of PGR products
DNA from replicates of the gel shown in Figure 6.1 was transferred onto nylon filters, hybridized with either DIG-labelled oligo SLTI lD ("SLT-1") or SLT2ID ("SLT-ll") probes and washed at high stringency. The expected mobility of a 215 bp fragment is indicated. Tracks: 1: Negative control (no DNA template). 2: E. coli C6OO. 3: E. coliC6OO::933W. 4z E. coli C6OO::H198. 5: E. colistrain PH. 6: E. colí031. SLT.I SLT - II 65 43 2l 65 43 2 I
215 bp.- 'r#. 131 primers have much higher affinity for the correct (SLT-specific) target sequence present in those samples. The non-specific amplicons observed for the C6OO template could be eliminated by increasing the annealing temperature of the PCR reaction to 52oC, but at this temperature sensitivity was compromised, as the yield of PCR product obtained using the known SlT-positive templates was reduced (result not shown).
6.2.2 Preparation of Samples for PCR Analysis
The purpose of this PCR assay was to detect the presence of SLTECs in faecal or other samples (e.9. foodstuffs). Previous studies have shown that faecal samples may contain substances which inhibit Taq polymerase (Olive, 1989; Allard et al., 1990; Xu et al., 199O). Faecal samples from patients with SLTEC disease often contain blood, and haemoglobin has also been identified as an inhibitor of Taq polymerase (Saulnier and Andremont, 1992). ln the present study primary faecal cultures rather than unprocessed faeces were examined, and sample preparation included ethanol precipitation (see Section 2.42l. to minimise the above problems. To confirm that the sample preparation procedure removed inhibitory substances associated with blood, extracts were prepared from LB cultures of
E. coli strain PH or an E. coli C6OO culture supplemented with a O.1 7o volume of PH culture, with or without the addition of human whole blood at a final concentration of 1o/o. Samples were subjected to PCR-amplification, electrophoresed and stained with ethidium bromide. The presence of blood in the sample had no impact on the intensity of PCR product obtained for either the PH or C6O0/PH cultures (result not shown).
6.2.3 Sensitívity of Direct Detection of SLT-related Genes in Faecal Cultures To determine the sensitívity of the PCR assay when using crude extracts of primary faecal cultures as a template, 1 ml aliquots of a known r32
SLT-negative faecal culture (containing >1Og organisms per ml) were seeded with 1OO pl aliquots from 1O-fold serial dilutions of a culture from the SLT- positive E. coli strain PH. Crude DNA extracts were prepared and the samples were PCR-amplified, electrophoresed and subjected to Southern hybridization analysis using the SlT-l-specific oligo probe (Figure 6.3). The lower limit of detection was approximately 1O cfu/ml of SlT-positive E. colí against a background of >109 organisms (after correcting for the volume of each crude DNA extract assayed, this is equivalent to approximately a single SlT-positive organism per reaction). Thus, this method could be expected to yield positive results in samples in which the frequency of SLT-positive organisms in the sample is only 1O-8.
6.2.4 Detection of SLT Genes in Clinical Samples Samples of gut contents (from the small and large bowel) were obtained from fresh autopsy specimens of 22 babies who died of Sudden Infant Death Syndrome. Similar specimens were obtained from a further 14 babies who died from other causes. Fresh faecal samples were obtained from 13 healthy babies and from 6 babies with acute diarrhoea. Primary cultures from each of these samples were tested using the PCR assay for the presence of SLT-l- and/or SLT-ll-encoding organisms. Results of the Southern hybridization analysis for 7 of the faecal cultures from healthy bab¡es are shown in Figure 6.4. A result was scored as positive for SLT-I or SLT-ll if a hybridization signal with a fragment of approximately 215 bp was obtained using the respective oligo probe. Specimen 1 was positive for SLT-ll, specimens 3 and 4 were positive for both SLT-I and SLT-ll, while the other specimens were negative for both genes. The PCR/Southern hybridization results for all the samples tested are summarized in Table 6.1. Remarkably, SlT-related PCR products were detected in approximately half of the samples from each group. SLT-Il-related PCR products were detected in 23155 l42o/"1 Figure 6.3
Sensitivity of PCR assay
Aliquots of a (SLT-negative) primary faecal culture were seeded with serial dilutions of a culture of E coli sfrain PH of known density. DNA extracts were prepared immediately, subjected to PCR-amplification and Southern hybridization analysis using the SlT-l-specific oligo (SLTI lD) probe. Lanes: 1, negative control (no DNA template)i 2-6, SlT-negative faecal cultures seeded with approximately 1OO, 1O1 , 1O2, 103 and 1O4 E. coli PH per ml, respectively; F, unseeded faecal culture; M, DNA size markers pre-labelled with DIG (2176, 1766, 1230, 1033, 653, 517,453,394,298,234,22O, and 154 bp, respectively, from top to bottom). M 123156F t ; ö t.
2rs bp I Figure 6.4 PCR and Southern blot analysis of faecal extracts using SLT-I- or SLT-ll- specific oligo probes
DNA was extracted from mixed faecal cultures as described in Section 2.42, subjected to PCR-amplification using SLT-I- and SLT-Il-specific primers (lVa and lVb) and electrophoresed on 2Yo agarose gels. DNA was transferred ontonylonfilters,hybridizedtosLT-l-(SLTllD)orSLT-Il-(SLT2|D)specific oligo probes and washed at high stringency. The expected mobility of a 215 bp fragment is indicated. Tracks: M: D|G-labelled DNA size markers (2176, 1766, 1230, 1033, 653, 517, 453, 394, 298, 234, 22O, and 154 bp, respectively, from top to bottom). +: Positive control (extract of E. colistrain PH), 1-72 Extracts from primary faecal cultures. SLT.I +1231567
tu, l l.r
stT-ll ¡til 12 3 4 5 67
l{r Table 6.1
Detection of SLT-related genes in patient samples
ilumber of Results
Group (n) Specimen Negative SLT-I SLT-II SLT-I+II
Heaìthy (13) faeces 6 I 4 2
D'iarrhoea (6) faeces 4 0 0 2 srDS (22) gut contents ll I 8 2 non-SIDS (14) gut contents 7 2 4 I
r,1 t33 I
Þ. samples, 7 of which also contained SlT-l-related amplicons. A further 4 I samples (7%l contained only SLT-I-related PCR products. The higher I prevalence of SLT-Il than SLT-I is consistent with other studies (Smith and t were 28155 (51o/"1 of Scotland, 1988). Negative results obtained for 'f samples. There was no significant difference in SlT-detection rate between I any of the four sample groups. Two of the extracts shown in Figure 6.4 I contained higher molecular weight PCR products that reacted weakly with the i D|G-labelled oligo probes. The identity of these species is not known, but they may be a consequence of the presence of SlT-related sequences in other species of bacteria.
6.2.5 Amplification of Complete SLT-ll-related Operons from Faecal DNA
f/l Extracts rii
Chapters 4 and 5 of this thesís describe the amplification of the variant {,i SLT-ll genes SLT-OX3/2 and SLT-ll/O111 lrom E. coli strains 031 and PH, respectively. An attempt was made to apply this PCR protocol to directly amplify a complete SlT-related operon from an extract of a faecal culture 'for which had previously tested positive SLT-Il by PCR-amplification using primers lVa and lvb. Aliquots of the extract were subjected to PCR- amplification employing PCR primers Va and Vb. A 20 pl aliquot from one of the PCR reactions was electrophoresed on a 1.2o/o agarose gel, transferred onto a nylon filter, hybridized with a PCR-DlG-labelled SLT-ll probe and washed at high stringency (Figure 6.5). A discrete SLT-ll-reactive PCR product of the expected size (1.5 kb) was seen in the faecal extract track and therefore the remaining PCR product from the pooled reactions was purified and attempts were made to clone it into the Smal site of pBLUESCRIPT SKTM. No SlT-positive clones were obtained from three separate attempts. One possible explanation for this could have been the low concentration of 1.s-kb PCR product and the need to use blunt-end ligation. In an attempt to Figure 6.5 Southern hybridization analysis of a PCR-amplified putative SLT-Il operon from faeces
SLT-Il positive DNA extracted from a mixed faecal culture, was subjected to PCR-amplification employing SlT-ll-specific primers (Va and Vb). An aliquot of the reaction was electrophoresed on a 1.2o/o agarose gel and the DNA was transferred onto a nylon filter, hybridized with a PCR-DlG-labelled SLT-ll- specific probe and washed at high stringency. The expected mobility of a 1.5 kb fragment is indicated. Tracks: 1: Negative control (no DNA template). 2: Mixed faecal culture DNA sample. M: DIG-labelled DNA size markers {'2176, 1766, 1230, 1033, 653, 517, 453, 394, 298, 234, 22O, and 154 bp, respectively, from top to bottom). Mt 2
Ia - 1.5kb - - -
- ? t34 improve the efficiency of cloning, new Va and Vb primers were synthesized with the addition of the sequence 5'-GCCGGAATTC-3' at the 5' end of each oligo. These extensions incorporate EcoRl sites and permit the "forced" cloning of PCR products. Using the above primers, a 1.5-kb fragment was amplified from an extract prepared from 1 ml of a primary culture of an SLTEC-negative faecal sample (containing approximately 109 organisms) which had been seeded with approximately lO4 f. coli stain PH. The fragment was purified, digested with EcoRl and was successfully cloned into similarly digested pBLUESCR¡PT SKTM. Thus, the methodology permits direct amplification and cloning of SlT-ll-related genes from faecal cultures containing SLTECs at a frequency of 10-5.
6.3 Discussion
6.3.1 Direct Detectíon of SLT Genes in Faecal Samples Access to sequence data for the various SLT genes has permitted design of a variety of oligo primer sets for PCR-amplification of SLT genes (Karch and Meyer, 1989b; Pollard et al., 1990; Brian et al., 1992; Gannon ef al., 1992). PCR assays are extremely sensitive and if secondary Southern or dot-blot hybridization with labelled oligos is used to detect PCR products, as little as one SLT-containing bacterial genome per assay can be detected (Brian et al., 1992l'. Some of the SLT PCR assays described above combine different primer pairs for SLT-I and SLT-ll or SLT-ll and SLT-llv in the same reaction, which direct the amplification of different size fragments for each toxin type (distinguishable by their different electrophoretic mobilities) (Johnson et al., 199O; Pollard et al., 199O; Brian et al., 1992; Gannon et al., 1992l'. Other SLT PCR assays use single pairs of primers based on consensus sequences, which are capable of amplifying all SLT genes, with 135 subsequent typing requiring Southern or dot-blot hybridization with labelled oligos directed against type-specific sequences within the amplified fragment (Karch and Meyer, 1989b). Apart from the added sensitivity, secondary hybridization steps provide independent confirmation of the identity of the amplified product. The majority of the studies involving PCR-amplification of SLT genes described above has involved testing of individual isolates, rather than primary (i.e. mixed) faecal cultures. When such protocols are applied in the clinical laboratory, there is a risk that positive cases of SLT-related disease may be missed, due to either low abundance of the SlT-producing organism in the sample or if the SLT genes themselves are unstable, as demonstrated by Karch et al. (1992). These workers recommended direct testing of faecal samples to ensure maximum recovery. A second reason for direct examination of faecal samples rather than individual E. colí isolates is that there is now both immunological and molecular evidence that other enteric bacteria are capable of production of apparently SLT-related toxins. Some stains of Vibrio cholerae, V. parahaemolytícus, Salmonella typhimuríum, and Camplylobacter jejuni have been shown to produce a cell-associated HeLa cell cytotoxin which could be neutralized with polyclonal anti-SHT, andlor with a monoclonal antibody to SLT-l B subunit (O'Brien and Holmes, 1987). Recently, Schmidt et al. {1993}, using SlT-specific DNA probes, identified two SlT-positive strains of Citrobacter freundii from stools of patients with diarrhoea and a further five from samples of beef. The SlT-related gene from one of the human isolates was PCR-amplified, cloned and sequenced and was shown to be very closely related to SlT-llvhc (Meye( et al., 1992), differing by only 4 nucleotides (2 amino acids) in the A subunit (the nucleotide sequences for the B subunits were identical). Furthermore, antisera raised against SLT-llvhc completely neutralized the Vero cell cytotoxicity of the C. freundii strain (Schmidt et al., 136
19931. Thus, confining SlT-testing of stools and foodstuffs to E. coli isolates may exclude clinically significant SlT-encoding bacteria belonging to other species. Faecal samples could contain substances which inhibit the PCR reaction itself, necessitating extensive purification of the template. lndeed, Brian et al. (1992) found that when DNA extracted directly from SlT-positive faecal samples was analysed, the extracts had to be diluted 1OO-fold before positive PCR results were obtained. ln primary cultures, such inhibitory substances are diluted and are theoretically less likely to cause difficulties. Moreover, the total number of target sequences is amplified in vivo during the culture step, thereby potentially increasing sensitivity. Thus, the protocol described in this Chapter represents a compromise between SLT gene stability and factors relating to sensitivity of detection. The results of this study indicate that when extremely sensitive methods are used, SLT-related genes can be detected in a high proportion of babies (many of whom had no detectable gastrointestinal abnormality). ln view of the moderate to low intensity of the hybridization signals obtained from the PCR products from the faecal or gut cultures, it is probable that many of the specimens contained very low numbers of SLT-positive organisms. Attempts were made to isolate SlT-producing E coli from some of the PCR-positive primary faecal cultures from the healthy control group. Up to 1OO colonies from each of four directly-plated faecal samples were tested by dot-blot hybridization using a SlT-specific DNA probe, but no positive isolates were detected (result not presented). However, testing this number of colonies would only be expected to yield positive results if the SlT-positive organism accounted for more than 1o/o of total gut flora. The PCR methodology described here had an apparent sensitivity limit 1O6-totd lower than this. t37
The presence of SlT-related sequences in approximately half of the faecal samples from apparently healthy babies, as well in a similar proportion of the gut contents of dead infants, complicates the clinical interpretation of the significance of PCR results in isolation. Clearly, very sensitive, direct analytical techniques need to .be used to overcome the problems of low pathogen numbers in genuine cases, as well as the known instability of SLT a genes in some strains. However, it now appears that such techniques will also detect carriers. Testing direct faecal extracts for the presence of Vero cell cytotoxins might be expected to provide confirmation of the PCR result. However, the presence of free toxin in the faeces of asymptomat¡c individuals has been reported (Edelman et al., 1988). Indeed, Brian et al. (1992) have reported that the faeces of an asymptomatic child in their study was positive for SLT-I by both PCR and cytotoxin assay. lt ¡s possible that individuals differ in their susceptibility to SLT-producing organisms or the toxins themselves, or that disease-causing SlT-positive strains also produce other virulence factors (e.9. adhesins, enterohaemolysins, etc.). Thus, definitive diagnosis of SLT-related disease remains problematic and may necessitate consideration of both laboratory and clinical findings. The high SLT detection rate described in this Chapter contrasts with the f¡ndings of Chapter 3, where only 411475 E. colí strains (isolated from 2OO individuals) were SLT-positive. One of these (strain 234) was isolated from the faeces of an apparently healthy infant. This strain was lysogenized by a bacteriophage that was indistinguishable from H198. lt is probable that both low numbers of SLTECs in a given sample (as a proportion of total gut flora), as well as ¡nstability of SLT genes in some strains, collectively contributed to the low detection rate in the in¡tial survey. lt is unlikely that the high SLT detection rate in Chapter 6 is a consequence of PCR contam¡nation-induced artifacts. The oligo primers (lVa and lVb) were designed such that the portion of the SLT genes amplified did not overlap 138
with regions previously amplified (using primers la & lb, lla & llb or llla & lllb) which had been handled in the laboratory. Moreover, Southern hybrization analysis never resulted in detection of even trace amounts of SlT-probe- reactive PCR products of the appropriate size in reagent blanks and negative controls (usually extracts of E. coli C6OOI, which were included in every experiment. Thus, it seems likely that the SlT-detection rates in this Chapter may be a more accurate estimate of the frequency of SlT-containing organsims in Adelaide infants than that implied by the results of Chapter 3.
6.3.2 Direct Cloning of PCR-amplified SLT Genes from Extracts of Faecal Cultures
Until recently, molecular data on SLT-related toxins has been derived from proteins purified from, or genes cloned from, isolated E coli strains.
However, such strains may represent a more stable subset of the SLTEC
family. Moreover, most SLTECs studied at the molecular level have been isolated from cases of human or animal disease. Apparently asymptomatic carriage of SLTECS has been reported (Edelman et al., 1988; Karmali, 1989; Brian et al., 1992) and this feature is consístent with the findings of this
Chapter. However, the SLTs produced by these strains have never been characterized at the DNA sequence level. Thus, it is not possible to state whether any particular sequence variant is more strongly associated with disease than with carriage. In this Chapter it was demonstrated that using oligo primers Va and Vb carrying EcoRl site 5' extensions, the complete SLT-Il-related operon from strain PH could be PCR-amplified and successfully cloned from faecal cultures seeded with strain PH at a frequency of 1O-5. This methodology provides the capacity to rapidly clone and sequence SlT-related genes from faecal cuftures, as well as permitting purification of toxin from recombinant. E. coli.
This should be possible even in circumstances where the proportion of SLT- t39
producing orgarfurÀ' in the original faecal sample is so low as to virtually ,.{ exclude the possibility of isolation of the strain itself. 140
CHAPTER SEVEN
FINAL DISCUSSION
The work described in this thesis provides further evidence that SLTs are a diverse family of toxins in terms of their primary nucleotide and amino acid sequences. Three further variant SLT-ll operons (SLT-OX3 and SLT-
OX3l2 from E. coli strain 031, and SLT-ll/O1 1 1 from E. coli strain PH) were described. ln addition, the SLT-I gene from strain PH is the first report of an SLT-I gene which differs in its amino acid sequence from those previously reported for both SLT-I and SHT. Moreover, in strain PH, the SLT-I gene appears to be chromosomally-encoded, in contrast to all previously characterized SLT-ls.
The phylogenetic relationships between the various members of the
SLT family are shown separately for the A and B subunits in Figures 7.1 and 7.2, respect¡vely. The most notable feature is the marked phylogenetic difference between the SlT-l-related toxins (SLT-|, SHT and PH SLT-I) and all the other (SlT-ll-related) amino acid sequences. Moreover, there is a much greater degree of genetic variability within the latter group compared with the former. Clonal groupings within the family are generally similar for the two subunits, suggesting that extensive recombination within operons has not occurred. The only obvious exceptions to this are SLT-Ilv and SLT-Ilva (toxins from porcine and human SLTEC isolates, respectively), the B subunits of which appear to form a distinct subgroup within the SLT-ll family. However, the A subunits of these two toxins are less related, with SLT-Ilva forming a distinct subgroup of its own.
Given the fact that SLT-I and SLT-¡I share only about 60% sequence homology at the amino acid level, but have extremely similar biological Figure 7.1 Phylogenetic relationships between SLT A subunits
Phylogenetic relationships between the A subunits of various SLTs were determined using the PHYLIP (Version 3.5c) software package (J. Felsenstein, University of Washington, USA.). SLTs are designated as follows:
SLTOX3: SLT.OX3 (This study).
SLTOX32: SLT-OX3/2 (This study).
PHSLTI: PH SLT-I (This study).
PHSLTII: sLT-lr/o1 1 1 (This study).
SHT: SHT (Kozlov et al., 1988).
SLTI: SLT-I (Jackson et al., 1987b).
SLTII: SLT-II (Jackson et al., 1987a).
SLTIIC: SLT-llc (schmirt et al., 1991).
SLT2VHC: SLT-llvhc (Meyer et al., 1992). SLTIIVA: SLT-llva (Gannon et al., 1990).
SLTIIV: SLT-llv (Weinstein et al., 1988b) A SUBUNIT
PHSLTII
SLTOX3
S LTIIC
SLTII
SLTOXS2
SLT2VHC
SLTIIV
SLTIIVA
SLTI
SHT
PHSLTI Figure 7.2 Phylogenetic relationships between SLT B subunits
Phylogenetic relationships between the B subunits of various SLTs were determined using the PHYLIP (Version 3.5c) software package. SLTs are designated as described in the legend for Figure 7.1. B SUBUNIT
SLTIIV
SLTIIVA
PHSLTII
SLTOXS
SLT2VHC
SLTIIC
SLTOX32
SLTII
SHT
PHSLTI
SLTI r4l
properties (Jackson et al., 1987a1, it is unlikely that there are more rigid structural constraints operating on SLT-l genes. However, the lesser variability in SLT-I sequences might be explained if SLT-I had arisen more recently in evolutionary terms than SLT-ll. The apparently higher proportion of phage-encoded SLT-I genes could also account for this, through clonal expansion as a result of higher rates of horizontal transfer than might be expected for chromosomally-encoded (predominantly SLT-l l-related ) genes. Clearly, amino acid sequence variations in SLTs have the potential to modify the properties of the toxins. Previous studies using site-directed mutagenesis have located regions in the A subunit which are important for enzymic activity and hence toxicity of various SLTs, as discussed in Chapters 4 and 5. Naturally occurring sequence variations in SLTs have generally not been found in the immediate vicinity of these sites, The one exception to this is SLT-ll/O111 from strain PH. As stated in Chapter 5, this toxin differs from all other members of the SLT family described to date in that it has Gly instead of Arg at position 176 in the A subunit. Extracts of recombinant E coli carrying this gene had approximately 8-fold lower cytotox¡c¡ty than identical constructs expressing SLT-OX3, which differs from SLT-Il/O111 only at this residue. Amino acid sequence variations in the B subunit could potentially impact upon target cell receptor affinity and specificity as well as release of
the toxin from the E. coli cell. Previous studies have located regions of the B subunit implicated in these properties, as discussed in Chapter 4. Differences in residues towards the C-terminal end of the B subunit between SLT-Ilv from porcine-derived SLTECs and classical SLT-ll appear to account for the different glycolipid receptor specificities (and relative cytotox¡cities for different cell lines) of these two toxins (Tyrrell et al., 1992Ì,. A further study (Boyd et al., 1993) indicated that these sequence variations resulted in different organ specificity for different toxin types when administered t42 intravenously to pigs. This was accompanied by differences in the clinical characteristics of toxin-induced disease, but it did not effect the nature of the histological lesions (Boyd et al., 1993). Some of the SLT-ll variants derived from human SLTECs have been reported to have reduced toxicity for HeLa cells (Gannon ef al., 1990; lto et al., 1990). In the present study, SLT-OX3 from strain O31 was also shown to have only low cytotoxicity for HeLa cells. The glycolipid receptor specificities of these toxins have not been directly determined, but it remains a possibility that they may display different tissue tropism in humans to classical SLT-ll, with concomitant alteration in the spectrum of disease. The work described in this thesis also indicates that organisms containing SlT-related genes may be present in low numbers in the faeces of a significant proportion of apparently healthy individuals, This has important consequences for the d¡agnosis of SLTEC diseases such as HUS or HC. At present it is not known whether there are consistent differences in the sequences of SLT operons from SLTECs from diseases in which SLTs are known to be aetiologically involved (e.9., HUS or HC) and those from other groups, including apparently healthy individuals. lt would also be of value to determine whether any such differences impact on the properties of the toxins themselves, including in vivo and in vitro toxicity. Such studies would be facilitated by the application of the techniques developed in this thesis, particularly the direct amplification and cloning of SLT genes from SLTECS present in low abundance in faecal or other samples. A final consideration is whether or not SLTECs isolated from diseased individuals or healthy carriers differ in production of other virulence-associated factors such as adhesins or enterohaemolysins. Such studies would be expected to provide further information on the structure/function of this apparently highly variable family of toxins. ldentification of specif ic sequence types with increased toxicity, and/or t43
preferential associat¡on of given types with d¡sease rather than asymptomat¡c carriage, will also permit development of specific PCR assays for detecting the presence of strains encoding clinically significant toxin types in faecal samples. t44
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APPENDIX I
PUBLICATIONS
Paton, 4.W., Paton, J.C., Heuzenroeder, M.W., Goldwater, P.N. and Manning, P.A. (1992). Cloning and nucleotide sequence of a variant
Shiga-like toxin ll gene from Escherichia coli OX3:H21 isolated from a case of Sudden lnfant Death Syndrome. Microb. Pathogen. 13: 225- 236.
Paton, 4.W., Paton, J.C., Goldwater, P.N., Heuzenroeder, M.W. and Manning, P.A. (1993). Sequence of a variant Shiga-like toxin type-l
operon of Escherichia coli O1 1 1 :H-. Gene, 129: 87-92.
Paton, 4.W., Paton, J.C. and Manning, P.A. (1993). Polymerase chain reaction amplification, cloning and sequencing of variant Escheríchia
coli shiga-like toxin type ll operons. Microb. Pathogen. 15: ln Press.
Paton,4.W., Paton, J.C., Goldwater, P.N. and Manning, P.A. (1993). Direct detection of Escherichia coli shiga-like toxin genes in primary fecal cultures using the polymerase chain reaction. J. Clin. Microbiol. Accepted for publication. t76
APPENDIX II
SEOUENCE DATABASE ACCESSION NUMBERS
The nucleot¡de sequences of SlT-related genes determined as part of the present study have been deposited in EMBL or GenBank databases, with the following accession numbers:
SLT-OX3 EMBL x65949
SLT-OX3/2 GenBank L1 1 079
PH SLT-I GenBank 104539
sLT-il/O111 GenBank L1 1 078