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Title: Pathogenicity studies on verocytotoxin-producing Escherichia coli : bacterial adhesion, toxin expression and uptake
Author: Colby, Susan Mary
ISNI:       0000 0001 3559 9920
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 1993
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Verotoxin-producing Escherichia coli (VTEC) are a group of medically important bacteria which have been implicated in cases of haemorrhagic colitis and haemolytic uraemic syndrome. They are characterised by the ability to produce Verotoxins (VT), potent exotoxins which kill mammalian cells by inhibiting protein synthesis. Two distinct VT are known, Verotoxin 1 (VT1) and Verotoxin 2 (VT2), and variants of VT2 have been described. VTEC adhere to intestinal cells via an attaching-effacing mechanism and many strains carry a 60-MDa plasmid which was thought to be involved in adhesion. Attachment of VTEC is thought to be an important pathogenic mechanism since it allows colonisation to occur, and facilitates delivery of toxin. The exact mechanisms of VTEC infection, however, especially the variation in severity of disease have not been established. This study aimed to investigate the putative virulence attributes of VTEC, namely toxin production and bacterial attachment. In order to study the toxins, it was first necessary to develop a reliable method of assay, and for this, two assays were chosen; a Vero cell assay which measured biological activity, and a colony ELISA, which detected the presence of toxin antigens. Since VT are produced by lysogenic phages, the effect of a phage inducer, mitomycin C, on VT production, was investigated. Increased levels of VT appeared to be detected and SDS-PAGE analysis of extracellular proteins revealed that the amount of toxin, and other phage proteins increased in the presence of mitomycin C, whilst bacterial protein levels remained constant. This effect was not seen when a non-VTEC strain was tested. The specific activity of VT increased inside the cell, where bacterial protein synthesis had ceased, but decreased extracellularly due to cell lysis. Extracellular phage particles were demonstrated by plaque assay. Mutants of 0157:H7 strain ATCC 35150, were produced by chemical mutagenesis. They did not possess the 60-MDa plasmid, found in the parent strain, and had an altered biochemical profile. Immunological assays revealed that they were altered in the O side chain of the LPS and it was shown by immunoblotting, that only the major outer membrane proteins reacted with anti-0157:H7 although they were not rough mutants. Analysis of the LPS neutral sugars showed that the mutants contained rhamnose, instead of fucose which was present in the parent strain. Despite these changes, however, the hydrophobicity of the mutants, or their ability to attach to colonic mucus or HeLa cells was unaffected, indicating that the 60-MDa plasmid is not significant in attachment. The mutants appeared to produce toxin with little activity, which was also biologically unstable. Secretion of the toxin appeared to be reduced, possibly as a result of the LPS mutations. The internalisation and intracellular processing of VT1 and VT2 was studied, and it was shown that the toxins were internalised by receptor- mediated endocytosis, but could probably by-pass this pathway. There was no requirement for exposure to an acidic environment, although fusion of endosomes with lysosomes was essential. It was also necessary for cells to possess physiologically functioning calcium channels for intoxication to occur.
Supervisor: Not available Sponsor: University of Warwick ; Nuffield Foundation ; Society for General Microbiology
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QR Microbiology