Vesicle Trafficking and Bacteria Invasion

描述（由申请人提供）：尿路感染（UTI）是西方世界最常见的严重病原体感染之一。尿路感染会影响婴儿、儿童和老年人，但女性的风险尤其更高。大多数尿路感染是由表达称为 I 型菌毛的丝状粘附细胞器的大肠杆菌 (E.) 引起的。菌毛被认为通过介导大肠杆菌粘附到膀胱上皮，然后细菌侵入（或内吞）进入宿主细胞来引发慢性尿路感染。细胞内入侵导致静止感染，其中细菌无法接触宿主免疫细胞或细胞不可渗透的抗生素。新出现的证据表明，细胞膜相关的内吞机制参与了细菌侵入（进入）宿主细胞的过程。这种细胞机制通常参与膜结合受体的内吞作用，可能包括小凹和网格蛋白包被的小凹。值得注意的是，小窝和网格蛋白包被的小凹内吞作用均已被证明依赖于普遍存在的 GTPase dynamin2 蛋白的酶活性。 dynamin2 从质膜分裂出芽囊泡的能力受到与伙伴蛋白相互作用的影响。最近对纯化蛋白质的发现表明，dynamin2 与内皮一氧化氮 (NO) 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处发生 S-亚硝基化，这种修饰是大肠杆菌入侵所必需的。本申请的中心假设是 eNOS 和 dynamin2 通过调节内吞性囊泡运输来协同调节大肠杆菌入侵。相关的具体目标是： [1] 确定大肠杆菌侵入膀胱上皮细胞如何促进 dynamin2 S-亚硝基化，重点是 eNOS 介导的 NO 产生； [2] 使用体外细菌侵袭试验评估dynamin2 S-亚硝基化对大肠杆菌侵袭膀胱上皮细胞的影响； [3] 使用动物模型确定 dynamin2 S-亚硝基化对大肠杆菌侵入膀胱上皮的生理相关性。拟议的研究应能更深入地了解尿上皮侵袭的机制，并可能最终确定 dynamin2 S-亚硝基化作为限制频繁尿路感染的有效药物靶点。 公共卫生相关性：尿路感染 (UTI) 是最常见和最严重的病原体感染之一。我们确定 NOS/NO 和 dynamin2 是大肠杆菌入侵膀胱细胞的调节因子。 eNOS 和 dynamin2 的靶向可能提供干扰目前无法治疗的复发性尿路感染的机会之窗。

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.