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Title: Virulence factors of verotoxin-producing Escherichia coli O157:H7
Author: Urabi, Iftikhar
ISNI:       0000 0001 3541 2468
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 1993
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Escherichia coli O157:H7 is one of several E.coli serotypes that produce Verocytotoxins (VTs); they are collectively called "Verocytotoxin-producing E.coli" (VTEC). VTEC are medically important bacteria which have been implicated in cases of haemorrhagic colitis and haemolytic uremic syndrome. Two distinct VTs are known, VT1 and VT2, and variants of VT2 have been described. They are potent exotoxins which kill mammalian cells by inhibiting protein synthesis. The virulence properties manifested by these organisms include the elaboration of VT1 or VT2 (or both), and the adherence to intestinal epithelial cells via an attaching-effacing mechanism. Many strains carry a 60 MDa plasmid which is thought to be involved in adhesion. Initial data demonstrated that the VTEC O157:H7 isolates under study possess two virulence factors, production of VTs and adherence to epithelial cells. However, effort has focused on investigating bacterial adherence, largely because attachment of VTEC is thought to be an important pathogenic mechanism since it allows colonisation, which facilitates toxin delivery, and adherence may be sufficient to cause diarrhoea in experimental animals in the absence of VTs. Moreover, a better understanding of the adhesion mechanism should help in finding ways by which adherence can be prevented. Since the bacterial-mucosal interactions are complicated in vivo by events and conditions that are not reproduced in current in vitro tests, a series of experiments were designed to investigate bacterial adherence to epithelial cells under conditions which are as close as possible to the in vivo situation. Significantly different data were obtained when quantitative adherence assays were performed under different physiological conditions, (different growth media, growth phase, pH values, low iron and oxygen limitation). Both iron-restricted, and oxygen- limited media induced a reduction in the final cell density, however, anaerobiosis significantly increased the adherence capacity of VTEC O157:H7 to HeLa cells while low iron caused a reduction in the number of adherent bacteria. Actively growing cells in the exponential phase were more adherent to HeLa cells than cells in the stationary phase. Since adhesion results from mutual recognition of surface structures from both the bacterial cell (adhesin) and the host cell (receptor), the bacterial cell envelope, and the HeLa cell outer membranes were investigated. Results of the preliminary characterisation of VTEC 0157:H7 surface components which have been implicated as adherence factors indicated that these strains are not fimbriated, however, they have been shown to be capable of binding to epithelial cells. Further studies were therefore, focused upon the identification of nonfimbrial adhesin(s). The use of competitive inhibitors, such as bacterial outer membrane extracts (OMPs), isolated lipopolysaccharides (LPS) and rabbit antisera to the H-7 flagella, OMPs, and LPS suggested that the role of H-7 flagella is insignificant, the LPS may in part be involved, but the OMPs seemed to have the major role in mediating attachment of O157:H7 to HeLa cells. The expression of OMPs under variable cultural conditions was examined, and significant differences were detected by the SDS-PAGE analysis of these extracts. The expression and repression of certain proteins was apparent under anaerobiosis, iron-restriction, different pH values and different bacterial growth phases. HeLa cell outer membranes were studied to identify the receptors on the host cell. Purified outer membranes were analysed by SDS-PAGE and used as inhibitors of bacterial adherence. Two proteins were identified by immunoblotting as a potential receptors.
Supervisor: Not available Sponsor: Science and Engineering Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QR Microbiology