Regulation of uropathogenic E. coli invasion by dynamin

描述(申请人提供)：动力素对致尿性大肠杆菌入侵的调节：致尿性大肠埃希氏菌利用被称为I型菌毛的黏附的管状菌毛附着并侵入尿路上皮，导致持续性和复发性尿路感染。最近的研究表明，大肠杆菌对尿路上皮细胞的入侵(或内化)是由膜相关的内吞机制介导的，这种内吞机制通常与膜锚定受体通过形成内吞小泡进入细胞有关。大的GTPase Dynamin参与了质膜上萌发的内吞囊泡的分裂，因此，它扮演着细胞外颗粒进入细胞的守门人的角色。Dynamin从质膜上执行萌芽小泡分裂的能力至少部分受其自组装和与伙伴蛋白结合的控制。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的活性。我们实验室的最新发现表明，Dynamin在活性受体介导的内化中的自组装和功能受NO介导的Dynamin pleckstrin同源结构域中关键半胱氨酸残基的S亚硝化调控。鉴于细菌感染导致一氧化氮合酶的激活，从而产生对S亚硝酸盐靶蛋白的一氧化氮，这一发现是重要的。这一提议的中心假设是，动力蛋白通过介导含有大肠杆菌的内吞囊泡的分裂来调节细菌对尿路上皮的侵袭。我们推测，NO介导的Dynamin的S亚硝化反应调节其在调节细菌入侵中的功能。相关的具体目的是：[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；[2]证明大肠杆菌感染膀胱上皮细胞激活一氧化氮合酶，导致其与动力蛋白的相互作用和S亚硝化，并鉴定动力蛋白中的半胱氨酸残基(S)，以评估它们在细菌入侵中的作用。这项拟议的研究应该能更深入地了解尿路上皮细胞侵袭的机制，并最终确定一氧化氮合酶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.