Intimate interactions between enteropathogenic and enterohemorrhagic Escherichia coli and intestinal epithelium in vitro
Enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) are diarrhoeagenic human pathogens that colonize intestinal epithelial cells via an attaching and effacing (A/E) lesion. Intimate adherence is mediated through binding of the intimin adhesin to the bacterial translocated intimin receptor, Tir. EPEC adheres to all regions of the human intestine in the in vitro organ culture (IVOC) system. In contrast, although EHEC is associated with colonic pathology in human infections, a prototypical strain of EHEC has shown a restricted tropism towards follicle-associated epithelium (FAE) of the terminal ileum without evidence of colonic adhesion. This thesis used the human IVOC system to further examine the intestinal interaction of EHEC 0157:H7 and related EPEC serotypes. To address the apparent paradox of non-adherence of EHEC to colonic tissue in the IVOC experimental system, the role of environmental, host and bacterial factors in modulating EHEC colonisation and tissue tropism were studied. No environmental factor (modulation of IVOC system, bicarbonate, serum, and mannose) was found to induce colonic adhesion. The investigation of the hypothesis that prior host-bacterial interactions might enhance subsequent EHEC colonic adhesion found that exposure to FAE promoted subsequent colonic adhesion, but in a non-intimate manner, demonstrating a novel form of interaction with human intestine. Great diversity was found in EPEC and EHEC in relation to the presence and sequence of tir, tccP and espJ fccP-negative strains expressing TireHEc were identified indicating that novel Nck-like molecules may be awaiting discovery. Tir was essential for EPEC and EHEC adhesion in IVOC but its phosphorylation (EPEC) and its interaction with TccP (EHEC) were not necessary for colonisation and A/E lesion formation on IVOC, questioning the role of pedestal formation in enterocyte infection. This is in direct contrast to cell culture findings and demonstrates the importance of IVOC in establishing 0157:H7 human pathogenesis.