The EspF protein is translocated into host cells by the type III secretion system of enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC). EspF sequences differ between EPEC and EHEC serotypes in terms of the number of SH3-binding polyproline rich repeats and specific residues in these regions as well as residues in the amino domain involved in cellular localization. In this study we have compared the capacity of different espF alleles to inhibit: (i) bacterial phagocytosis by macrophages; (ii) translocation through an M-cell co-culture system; (iii) uptake by and translocation through cultured bovine epithelial cells. The espFO157 allele was significantly less effective at inhibiting phagocytosis and also had reduced capacity to inhibit E. coli translocation through a human-derived in vitro M-cell co-culture system in comparison to espFO127 and espFO26. In contrast, espFO157 was the most effective allele at restricting bacterial uptake into and translocation through primary epithelial cells cultured from the bovine terminal rectum, the predominant colonisation site of EHEC O157 in cattle and a site containing M-like cells. As functional differences could not be simply assigned to variation in established interactions of EspF with Sorting Nexin 9 and N-WASP, yeast-2-hybrid screening was used to identify additional host proteins that may interact with EspF. The anaphase promoting complex inhibitor, Mad2L2, was identified from this screen. Mad2L2 was then demonstrated to interact with EspF variants from EHEC O157:H7, O26:H11 and EPEC O127:H6 by Lumier assays. While Mad2L2 has been shown to be targeted by the non homologous Shigella effector protein IpaB to limit epithelial cell turnover, we presume that EspF interactions with this protein may indicate a similar function to promote EPEC and EHEC colonization. The final section of work addressed whether bacterial interactions can actually induce M-cell differentiation on follicle-associated epithelium. The work focused on bovine rectal primary cell cultures interacting with Salmonella enterica serovar Typhimurium. The type III secreted protein, SopB, was required for Salmonella to: III (i) activate parts of epithelial to mesenchymal transition (EMT) pathway; (ii) transform a subset of epithelial cells to a cell type that phenotypically and functionally resembles specialized antigen sampling M cells; (iii) induce RANKL and downstream RelB dependent NFkB signaling. The work suggests that Salmonella may induce this cellular transformation to promote its invasion and colonization of intestinal mucosa.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:563667 |
| Date | January 2011 |
| Creators | Tahoun, Amin M. Abd El Hady |
| Contributors | Gally, David. : Mahajan, Arvind |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/5899 |
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