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Title: Bovine epithelial cell responses to colonisation by Escherichia coli 0157:H7
Author: Paton, Neil
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2009
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E. coli 0157:H7 was the first identified pathogen of a group that have come to be referred to as Enterohaemorrhagic E. coli (EHEC) and was identified in 1983 as being associated with Haemolytic ureamic syndrome (HUS). This bacterium carries the potent cytotoxin Shigalike toxin (Stx), also known as verotoxin, which through the inhibition of protein synthesis causes cell necrosis in the endothelium of the renal vasculature and this leads to the triad of symptoms - renal failure, thrombocytopenia and microangiopathic haemolytic anaemia. EHEC is also associated with Haemorrhagic colitis in humans as endothelium of both the colonic vasculature and the renal system express Gb3 receptors. These receptors bind the toxin and internalisation of the toxin allows the inhibition of protein synthesis. EHEC is a food borne zoonosis and its reservoir host is the bovine and faecal contamination of the environment, the food chain and direct contact with cattle are the most recognised routes for human infection to occur. The bovine host is asymptomatically colonised in the field and detection and removal is problematic, any future intervention strategy to remove this pathogen from the national herd is likely to be expensive and labour intensive. EHEC has a number of virulence factors that are involved in colonisation and proinflammatory responses. These were examined in a bovine model system. The flagellum was the only virulence factor that produced a proinflammatory response when measured by quantitative and traditional RTPCR. Commensal bacteria were unable to produce a response although one motile strain was included in the panel and was presumed to express flagella which were shown to be pro-inflammatory when associated with EHEC. In the host animal there is limited evidence for a pathological effect and to elucidate the mechanisms that explain this lack of response to pathogenic effects microarray was utilised to tease out these mechanisms. The data produced identified a limited set of genes that were differentially regulated including CyclinC Angiopoetin-1 like protein, Jumonji domain containing protein 2B, Zinc finger protein 161 and Est-lplike protein, all of which were thought to be involved in cell cycle regulation. Quantitative RTPCR was unable to confirm the data from the array; further work is therefore required to determine whether colonisation does in fact alter the expression of these genes. EHEC was therefore hypothesized to alter the proliferation rate within the epithelium and an immunohistochemical approach was used to assess this. Proliferating cell nuclear antigen (PCNA) was used as a marker to identify replicating cells and counts of cells in the epithelium demonstrated a reduction in proliferating cells in colonised epithelium. Further analysis suggested that retinoblastoma protein was a central protein that was involved in pathways influenced by the proteins already outlined. IHC was used to study this protein and differences in the number of cells expressing this protein and localisation within the cells. Retinoblastoma appears to be retained in the cytoplasm in colonised cattle which limits its ability to induce proliferation through release of E2F. It is suggested that E.coli 0157:H7 can manipulate the epithelial cell proliferation rate in the bovine host and increases the time that the bacterium is retained in the host. It was hoped that microarray data and QtRT-PCR would identify proteins involved in this phenotype but the lack of support from the real time data for targets identified by the array makes it impossible to conclude that these proteins are defiantly involved. This increase in time allows for a greater chance of spread to other host animals within the herd. Further work to clarify the details of this pathway will allow interventions which limit colonisation the National herd to be designed.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available