The molecular basis to Escherichia coli O157:H7 colonisation of the terminal rectum in cattle

The overall objective of the study is to identify the bacterial and host factors that enable E. coli O157 to colonise the recto-anal junction in cattle. The work is divided into three complementary sections:
1. Optimisation of in vitro methods to demonstrate E. coli O157 adherence to the rectal mucosa adjacent to the recto-anal junction. This section will start by optimising conditions using in vitro organ culture and then progress to testing the tropism using sorted cell subsets, epithelial cell culture and purified mucin. The experiments are also designed to investigate receptor variation between the tropic mucosa at the recto-anal junction and rectal mucosa 20 cm proximal to the junction which does not demonstrate the tropism.
2. Identification of bacterial factors required for E. coli O157 adherence to bovine terminal rectal mucosa. This section will compare adherence of different VTEC strains and mutants in the most appropriate in vitro assay identified in section 1. Multiple VTEC serotypes will be compared with O157 for the tropism. Defined and random transposon mutants of E. coli O157 will be tested for loss of binding using the optimised in vitro assay.
3. Detection of antigens expressed by E. coli O157 during colonisation of the bovine host at the recto-anal junction. Immunofluorescence and RT-RCR will be used to demonstrate which determinants are expressed in vivo at this site.

This project was funded to take forward our research finding that enterohaemorrhagic E. coli O157:H7 has a specific tropism for the final few centimetres of the bovine gastrointestinal tract. In vitro experiments with organ cultures, primary cell culture and mucus fractions have not demonstrated any obvious receptor based differences that would account for this tropism although the expression context of the bacteria for these experiments is critically important. Type III secretion (T3S) expression is essential for rectal colonisation and H7 flagellin binds to bovine rectal epithelium by comparison with other H types tested. We propose that the expression of the EHEC O157:H7 T3S is limited until the bacteria gain access to the epithelium or are in the correct micro-environment with the appropriate levels of e.g. quorum sensing molecules, carbonate, calcium, iron and oxygen. We propose that the environment of the colon switches on T3S but that access to the epithelium occurs on FAE at the terminal rectum where the mucus barrier is limited. This leads to an interaction with M-cells for which T3S and injected EspF are important. Adherence is facilitated by H7 flagella. Once established, A/E lesion formation is important for persistence and injected effector molecules along with shiga-toxin down-regulate inflammatory responses induced by micro-colony formation (our data). Ongoing research will lead to a complete gene expression profile of E. coli O157:H7 at the terminal rectum and development of a multi-component vaccine that should limit colonisation and reduce shedding levels.