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dc.contributor.authorKumar, Manoj-
dc.descriptionPh. D. Thesis.en_US
dc.description.abstractDisease associated with enteropathogenic Escherichia coli (EPEC) depends on a Type 3 Secretion System (T3SS) that delivers ‘effector’ proteins into infected cells. The EPEC E2348/69 strain has approximately 6Mb of horizontally acquired DNA provided, mostly, by 21 prophage (PP) and integrative element (IEs). The T3SS, six effectors and two chaperones - latter aid the export of T3SS substrates - are encoded on an IE named LEE (Locus of Enterocyte Effacement). The remaining effectors are encoded on 7 other IE/PPs. EPEC inhibits the activity of a host kinase, Akt, that regulates important cellular processes, including cell survival, but the responsible effectors and inhibitory mechanism remain undefined. Previous work discounted roles for 17 of 21 known effectors but linked the inactivation process to Akt dephosphorylation in a manner requiring the LEE chaperone, CesT. Here, additional screening strategies undertaken to identify the responsible effector(s) uncovered key but redundant roles for two, previously unexamined, T3SS substrates: LifA (3223 residue, IE6-encoded) and LifA-like (2624 residue, IE2- encoded) proteins. These proteins have putative glycosyltransferase and protease activities with LifA thought to be delivered into host cells. Other studies support CesT dependence of the inhibitory process and, surprisingly, revealed LifA’s inhibitory activity requires IE2-encoded factor(s). Screening IE2-related fragments implicated factor(s) on a cloned 1.7kB region but further studies are needed to confirm the results and provide mechanistic insights. Studies on LifA’s inhibitory mechanism revealed its T3SS- and CesT-dependent accumulation with host membrane proteins with dephosphorylation of Akt not requiring motifs needed for its known glycosyltransferase and protease activities. By contrast, immunoprecipitation studies linked the inhibitory mechanism to O-glycosylation of Akt and revealed infection-induced O-GlcNAc of an Akt-sized band that gradually disappeared in a T3SS-dependent manner. Studies described in this thesis not only identify the EPEC effectors responsible for inhibiting Akt activity, but also provide important insights into the inhibitory mechanism.en_US
dc.publisherNewcastle Universityen_US
dc.titleDefining the effector(s) and mechanism(s) by which enteropathogenic E. coli (EPEC) inhibits Akt signalling upstream of PI3 kinaseen_US
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