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Title: Characterisation and identification of binding partners of non-LEE-encoded protein H of enteropathogenic E. coli
Author: Robinson, Keith Scott
ISNI:       0000 0004 2728 1180
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2012
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Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC respectively) are human, diarrhoeal pathogens that intimately adhere to host intestinal epithelia, characterised by effacement of brush border microvilli. Both pathogens encode a filamentous type 3 secretion system that is used to deliver virulence factors, termed effectors, into the host cell. These effectors disrupt a wide range of host signalling pathways including the immune response, cytoskeleton dynamics, phagocytosis, and apoptosis. Importantly, infected cells exhibit early stage, but not late stage, features of apoptosis indicating an inhibition of apoptotic cascades. Identified by secretomic and genomic analysis NleH effectors were shown to impact gut colonisation in vivo, with nleH mutants being competitively disadvantaged. Furthermore, nleH genes were shown to be conserved among all A/E pathogens and found on two prophage islands; encoding NleH1 and NleH2. In this study I demonstrate that cultured cell lines infected with EPEC nleH mutants have elevated levels of apoptotic characteristics, including cell detachment, nuclear condensation, ER disruption, and caspase-3 cleavage in comparison to cells infected with wild type EPEC. Furthermore, ectopic expression of NleH1 alone inhibited nuclear condensation and caspase-3 cleavage induced by TNFα, Fas ligand, staurosporine, Clostridium difficile toxin B, tunicamycin, brefeldin A (BFA), it also inhibited the cell rounding and detachment induced by other EPEC effectors. Furthermore, NleH effectors bear a class I, C-terminal PDZ binding motif that is largely essential for NleH anti-apoptotic activity. We showed, following a yeast two hybrid assay, using a HeLa human cDNA library, that NleH interacts with ER anti-apoptotic protein Bax inhibitor 1 (BI-1). Knockdown of BI-1 inhibited NleH cytoprotectivity, following infection, seemingly due to an inability of NleH to reduce cytosolic Ca2+ levels in a BI-1 dependent manner. Furthermore, we show that bi-1-/- cells ectopically expressing NleH1 could no longer protect against ER stress induced by tunicamycin or BFA. The NleH:BI-1 interaction was independent of the PDZ binding motif, as was tunicamycin and BFA protection, yet EPEC infection with NleH PDZ binding motif mutants had similar levels of cell detachment to the NleH mutant. Using a PDZ array, overlay, pull-down and co-immunoprecpipitation we identified two interacting partners; both belonging to the membrane associated guanylate kinase (MAGUK) family; synapse associated protein 97 and membrane associated guanylate kinase with inverted domains 3. We show that NleH1, in response to Fas ligand, blocks mitochondrial outer membrane permeabilisation by stopping pro-apoptotic Bax from undergoing conformational change. This is dependent upon both MAGUK interaction and the Akt pathway. Furthermore, cells ectopically expressing NleH1 PDZ binding motif mutants could no longer inhibit Fas Ligand, staurosporine, and TcdB induced apoptosis. Together our results demonstrate that NleH effectors are global inhibitors of apoptosis. The employment of anti-apoptotic effectors may offer a competitive advantage by preventing host shedding of infected cells for prolonged infection.
Supervisor: Frankel, Gad Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available