Regulation of uropathogenic E. coli invasion by dynamin

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DESCRIPTION (provided by applicant): Regulation of uropathogenic E.coli invasion by dynamin: Uropathogenic E. coli utilizes adherent oraganelles termed type I fimbriae to attach to and invade the uroepithelium, leading to persistent and recurrent infections of the urinary tract. Recent studies suggest that invasion (or internalization) of E. coli into uroepithelial cells is mediated by the membrane-associated endocytotic machinery that is typically linked to entry of membrane-anchored receptors through the formation of endocytotic vesicles. The large GTPase dynamin is involved in the fission of budding endocytotic vescicles from the plasma membrane and, as such, acts as the gatekeeper of entry of extracellular particles into cells. Ability of dynamin to execute the fission of budding vesicles from the plasma membrane is controlled, at least in part, by its self-assembly and binding to partner proteins. Emerging results show that dynamin directly interacts with nitric oxide (NO) synthase (NOS) and regulates NOS activity. Recent findings in our laboratories show that dynamin self-assembly and function in active receptor-mediated intemalization is regulated by NO-mediated S-nitrosylation of a key cysteine residue within the dynamin pleckstrin homology domain. This finding is important in light of results showing that bacterial infections lead to the activation of NOS, which produces NO to S-nitrosylate target proteins. The central hypothesis of this proposal is that dynamin regulates bacterial invasion of the uroepithelium by mediating fission of E. coli-containing endocytic vesicles. We hypothesize that NO-mediated S-nitrosylation of dynamin regulates its function in modulating bacterial invasion. The associated specific aims are: [1] To demonstrate that dynamin is a key regulator of invasion of E. coli into bladder epithelial cells; and [2] To demonstrate that E. coli infection of bladder epithelial cells activates NOS, leading to its interaction with and S-nitrosylation of dynamin, and to identify the cysteine residue(s) in dynamin that become S-nitrosylated in ordet to assess their role in bacterial invasion. The proposed studies should provide greater insight into the mechanisms involved in invasion of the uroepithelium and may ultimately identify NOS as effective drug targets that limit urinary tract infections.