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病原性感染之一。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(E.)表达丝状黏附细胞器的大肠杆菌，称为I型菌毛。菌毛被认为是通过介导大肠杆菌与膀胱上皮的黏附，随后细菌入侵(或内吞)到宿主细胞而引发慢性尿路感染。细胞内的入侵导致静止性感染，宿主免疫细胞或细胞不渗透的抗生素无法接触到细菌。新出现的证据表明，与细胞膜相关的内吞机制与细菌入侵(进入)宿主细胞有关。这种细胞机制通常涉及膜结合受体的内吞作用，可能包括小窝和笼状蛋白包裹的小窝。值得注意的是，小窝和笼状蛋白包被的基于小窝的内吞作用都被证明依赖于普遍存在的GTP酶Dynamin2蛋白的活性。Dynamin2从质膜上执行萌发小泡分裂的能力受到与伙伴蛋白相互作用的影响。最近对纯化蛋白的发现表明，Dynamin2与内皮型一氧化氮合酶(ENOS)形成复合体，并调节eNOS的活性。我们的初步结果表明，E.Coli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经历S亚硝化，这一修饰是大肠杆菌入侵所必需的。这一应用的中心假设是eNOS和Dynamin2通过调节胞内囊泡的运输来协同调节大肠杆菌的侵袭。相关的具体目标是：[1]确定大肠杆菌侵袭膀胱上皮细胞如何促进动力蛋白2 S亚硝化，重点是eNOS介导的NO的产生；[2]通过体外细菌侵袭试验评估动力蛋白2 S亚硝化对大肠杆菌侵袭膀胱上皮细胞的影响；以及[3]通过动物模型确定动力蛋白2 S亚硝基化对大肠杆菌侵袭膀胱上皮的生理相关性。这项拟议的研究应该能够更深入地了解尿路上皮细胞侵袭的机制，并可能最终确定Dynamin2 S-亚硝酸酯为一种有效的药物靶点，以限制频繁的尿路感染。 公共卫生相关性：尿路感染是最常见和最严重的病原性感染之一。我们发现一氧化氮合酶/一氧化氮和动力蛋白2是大肠杆菌侵袭膀胱细胞的调节因子。靶向eNOS和Dynamin2可能为干预目前无法治愈的复发性尿路感染提供了机会之窗。

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.