Enterohaemorrhagic Escherichia coli colonisation of the human colonic epithelium and the innate immune response to infection

Enterohaemorrhagic Escherichia coli (EHEC) are of great public health importance, causing diarrhoea, haemorrhagic colitis and haemolytic uraemic syndrome. A wealth of evidence from cell line and animal studies has linked colonic pathogenesis with EHEC induction of mucosal attaching/effacing (A/E) lesions, which are characterised by intimate bacterial attachment to intestinal epithelial cells (IECs), microvillous effacement and actin polymerisation. However, the clinical relevance of A/E lesions has been called into question by the fact that adherent bacteria have not been located on colonic biopsies taken from EHEC-infected patients. In addition, in vitro organ culture (IVOC) of human intestinal biopsies with EHEC has not yielded signs of bacterial colonisation or A/E lesion formation. In this study, we have re-evaluated the colonic IVOC model and demonstrated for the first time that EHEC colonise the colonic epithelium ex vivo and induce A/E lesion formation. High levels of oxygen, as used in previous IVOC studies, were found to inhibit EHEC-IEC interactions. Adherence was dependent on expression of the bacterial adhesin intimin and the type III secretion system (T3SS). In contrast, EHEC adherence to the widely-used T84 colonic carcinoma cell line was associated with microvillous effacement but not actin polymerisation, and was not dependent on intimin or the T3SS, highlighting the importance of using physiologically relevant intestinal models when conducting EHEC research. EHEC pathogenesis has also been linked to induction of an inflammatory response in the colonic mucosa, characterised by recruitment of neutrophils to the epithelium. This process has been associated with expression of the neutrophil chemokine interleukin-8 (IL-8) by cultured IECs following exposure to EHEC flagellin. However, other aspects of the innate immune system, such as expression of antimicrobial peptides (AMPs), have been neglected. We have demonstrated that EHEC induces expression of the AMP human β-defensin-2 (hBD2) and IL-8 in human colonic IECs both in vitro and ex vivo. Induction was dependent on exposure of IECs to flagellin, and was mediated by mitogen activated protein kinase (MAPK) signalling. Furthermore, we present evidence that IL-8 is secreted apically in polarised T84 cells following apical stimulation, suggesting that this cytokine may be secreted into the lumen during infection. Put together, this PhD project has shed new light on EHEC-IEC interactions and has re-established the IVOC model as an important tool for future EHEC research