Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 is a foodborne pathogen that causes significant morbidity and mortality in developing and industrialized nations. EHEC infection of host epithelial cells is capable of inhibiting the interferon gamma (IFNγ) pro-inflammatory pathway through the inhibition of Stat-1 phosphorylation, which is important for host defense against microbial pathogens. The aim of this thesis was to determine the bacterial factors involved in the inhibition of Stat-1 tyrosine phosphorylation. Human HEp-2 and Caco-2 epithelial cells were challenged directly with either EHEC or bacterial culture supernatants, stimulated with IFNγ, and then protein extracts were analyzed by immunoblotting. The data showed that IFNγ-mediated Stat-1 tyrosine phosphorylation was inhibited by EHEC secreted proteins. Using 2D-Difference Gel Electrophoresis, EHEC Shiga toxins were identified as candidate inhibitory factors. EHEC Shiga toxin mutants were then generated, complemented in trans, and mutant culture supernatant was supplemented with purified Stx to confirm their ability to subvert IFNγ-mediated cell activation. I conclude that E. coli-derived Shiga toxins represent a novel mechanism by which EHEC evades the host immune system.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/34056 |
| Date | 13 December 2012 |
| Creators | Ho, Nathan |
| Contributors | Sherman, Philip M. |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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