Regulation of uropathogenic E. coli invasion by dynamin

描述（由申请人提供）：动力蛋白对尿路致病性大肠杆菌侵袭的调控：尿路致病性大肠杆菌利用称为I型菌毛的粘附细胞器附着并侵入尿路上皮，导致尿路持续和反复感染。最近的研究表明，大肠杆菌侵入（或内化）尿上皮细胞是由膜相关的内吞机制介导的，该机制通常与膜锚定受体通过内吞囊泡的形成进入有关。大的GTPase动力蛋白参与了从质膜出芽的内吞囊泡的分裂，因此，作为细胞外颗粒进入细胞的看门人。动力蛋白从质膜上分裂出芽囊泡的能力，至少在一定程度上是由它的自组装和与伴侣蛋白的结合控制的。新的研究结果表明，动力蛋白直接与一氧化氮合酶（NOS）相互作用并调节NOS活性。我们实验室最近的研究结果表明，动力蛋白自组装和活性受体介导的内化功能是由no介导的动力蛋白pleckstrin同源结构域中关键半胱氨酸残基的s -亚基化调节的。这一发现很重要，因为结果表明细菌感染会导致NOS的激活，从而产生NO到s -亚硝基酸靶蛋白。该建议的中心假设是动力蛋白通过介导含有大肠杆菌的内吞囊泡的裂变来调节细菌对尿上皮的侵袭。我们假设no介导的动力蛋白s -亚硝基化调节其调节细菌入侵的功能。相关的具体目的是：b[1]证明动力蛋白是大肠杆菌入侵膀胱上皮细胞的关键调节因子；证明大肠杆菌感染膀胱上皮细胞激活NOS，导致其与动力蛋白相互作用和s -亚硝基化，并鉴定动力蛋白中发生s -亚硝基化的半胱氨酸残基，以评估其在细菌侵袭中的作用。拟议的研究应提供更深入的了解与尿路上皮侵袭有关的机制，并可能最终确定NOS是限制尿路感染的有效药物靶点。

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.