E. colt 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
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
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.