Vesicle Trafficking and Bacteria Invasion

描述（申请人提供）：尿路感染（UTI）是西方世界最常见的严重致病性感染之一。尿路感染影响婴儿、儿童和老年人，但女性的风险尤其高。大多数尿路感染是由大肠杆菌引起的，大肠杆菌表达被称为I型菌毛的丝状粘附细胞器。被认为是通过介导大肠杆菌对膀胱上皮的粘附，然后侵入（或内吞作用）进入宿主细胞而引发慢性尿路感染。细胞内侵入导致静止感染，在这种感染中，细菌无法被宿主免疫细胞或细胞不渗透的抗生素接近。新出现的证据暗示了细菌侵入（进入）宿主细胞时细胞膜相关的内吞机制。这种细胞机制通常参与膜结合受体的内吞作用，可能包括小泡和网格蛋白包覆的凹坑。值得注意的是，小窝和网格蛋白包被的凹坑内吞作用都依赖于普遍存在的GTPase dynamin2蛋白的酶活性。dynamin2在质膜上进行出芽囊泡裂变的能力受到与伴侣蛋白相互作用的影响。最近对纯化蛋白的研究表明，dynamin2与内皮型一氧化氮合酶（eNOS）形成复合物并调节eNOS活性。我们的初步结果表明，大肠杆菌对膀胱上皮细胞的侵袭是由dynamin2和eNOS的酶活性控制的。dynamin2在特定的半胱氨酸残基上进行s -亚硝基化，这种修饰是大肠杆菌入侵所必需的。本应用的中心假设是eNOS和dynamin2通过调节内吞囊泡运输来协同调节大肠杆菌的入侵。相关的具体目的是：b[1]确定大肠杆菌入侵膀胱上皮细胞如何促进动力学2 s -亚硝基化，重点是enos介导的NO产生；[2]采用体外细菌侵袭法研究dynamin2s -亚硝基化对大肠杆菌侵袭膀胱上皮细胞的影响；通过动物模型研究dynamin2 s -亚硝基化与大肠杆菌侵入膀胱上皮的生理关系。这些研究将进一步深入了解尿路上皮侵袭的机制，并可能最终确定dynamin2 s -亚硝基化作为限制尿路感染的有效药物靶点。

DESCRIPTION (provided by applicant): Urinary tract infections (UTI) are among the most common serious pathogenic infections in the Western world. UTIs affect infants, children, and the elderly, but females are particularly at higher risk. Majority of UTIs are caused by Escherichia (E.) coli that expresses filamentous adhesion organelles termed type I fimbriae. The fimbriae are thought to initiate chronic UTIs by mediating adherence of E. coli to the bladder epithelium followed by invasion (or endocytosis) of the bacterium into the host cell. The intracellular invasion leads to a quiescent infection in which the bacteria are inaccessible to host-immune cells or cell-impermeable antibiotics. Emerging evidence implicates the cell membrane-associated endocytic machinery in the invasion (entry) of bacteria into the host cell. This cellular machinery, typically involved in endocytosis of membrane- bound receptors, may include caveolae and clathrin-coated pits. Significantly, both caveolae- and clathrin-coated pit-based endocytosis have been shown to be dependent upon enzymatic activity of the ubiquitous GTPase dynamin2 protein. Ability of dynamin2 to execute the fission of budding vesicles from the plasma membrane is influenced by interactions with partner proteins. Recent discoveries with purified proteins show that dynamin2 forms a complex with endothelial nitric oxide (NO) synthase (eNOS) and regulates eNOS activity. Our preliminary results demonstrate that the invasion of E. coli into bladder epithelial cells is controlled by the enzymatic activities of both dynamin2 and eNOS. The dynamin2 undergoes S-nitrosylation at specific cysteine residues and this modification is required for the E. coli invasion. The central hypothesis of this application is that eNOS and dynamin2 cooperatively regulate E. coli invasion by regulating endocytic vesicle trafficking. The associated Specific Aims are: [1] To determine how E. coli invasion into bladder epithelial cells promotes the dynamin2 S-nitrosylation with emphasis on eNOS-mediated NO production; [2] To assess effect of dynamin2 S- nitrosylation on E. coli invasion into bladder epithelial cells using in vitro bacteria invasion assays; and [3] To determine physiologic relevance of dynamin2 S-nitrosylation on E. coli invasion into bladder epithelium using animal models. The proposed studies should provide greater insight into the mechanisms involved in invasion of the uroepithelium and may ultimately identify dynamin2 S-nitrosylation as an effective drug target to limit the frequent urinary tract infections. PUBLIC HEALTH RELEVANCE: Urinary tract infections (UTIs) are among the most common and serious pathogenic infections. We identified NOS/NO and dynamin2 as regulators of E. coli invasion into bladder cells. The targeting of eNOS and dynamin2 may provide a window of opportunity to interfere with currently untreatable recurrent UTIs.