Vesicle Trafficking and Bacteria Invasion

囊泡运输和细菌入侵

• 批准号: 8042587
• 负责人: Yehia Daaka
• 金额: $ 36.26万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-03-15 至 2015-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8042587
• 关键词: Adherence; Adhesions; Affect; Animal Model; Antibiotic Resistance; Antibiotic Therapy; Antibiotics; Bacteria; Bacterial Infections; Biological Assay; Bladder; Caveolae; Cell membrane; Cells; Child; Chronic; Clathrin; Complex; Cyclic AMP-Dependent Protein Kinases; Cysteine; Data; Disease; Drug Delivery Systems; Dynamin; Elderly; Endocytic Vesicle; Endocytosis; Enterococcus; Epithelial Cells; Epithelium; Escherichia; Escherichia coli; Female; Genus staphylococcus; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Hydrolysis; Immune; In Vitro; Incidence; Infant; Infection; Invaded; Klebsiella; Knowledge; Mediating; Membrane; Membrane Microdomains; Modeling; Modification; Nature; Neck; Nitric Oxide; Nitric Oxide Synthase; Organelles; Phosphorylation; Physiological; Production; Proteins; Proteus; Pseudomonas; Recurrence; Role; Staging; Time; Urinary tract infection; Uropathogenic E. coli; Vesicle; Virus Receptors; Western World; base; coated pit; fimbria; high risk; human NOS3 protein; insight; men; pathogen; public health relevance; receptor binding; self assembly; trafficking

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.

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性感染之一。UTI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埃希氏菌（E.）大肠杆菌表达的丝状粘附细胞器称为I型菌毛。菌毛被认为是通过介导E.将大肠杆菌感染至膀胱上皮，随后细菌侵入（或内吞）宿主细胞。细胞内侵入导致静止感染，其中细菌无法接近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在细菌入侵（进入）宿主细胞。这种细胞机制通常参与膜结合受体的内吞作用，可能包括小窝和网格蛋白包被的小凹。值得注意的是，已显示小窝和网格蛋白包被的基于坑的内吞作用都依赖于普遍存在的GT3发动蛋白2蛋白的酶活性。dynamin 2从质膜分裂出芽囊泡的能力受到与伴侣蛋白相互作用的影响。最近的发现与纯化的蛋白质表明，dynamin 2与内皮型一氧化氮（NO）合酶（eNOS）形成复合物，并调节eNOS的活性。初步结果表明，E.大肠杆菌进入膀胱上皮细胞是由动力蛋白2和eNOS的酶活性控制的。动力蛋白2在特定的半胱氨酸残基上发生S-亚硝基化，这种修饰是E.大肠杆菌侵袭。本申请的中心假设是eNOS和dynamin 2协同调节E.大肠杆菌入侵通过调节内吞囊泡运输。相关的具体目标是：[1]确定E。大肠杆菌侵入膀胱上皮细胞促进dynamin 2 S-亚硝基化，重点是eNOS介导的NO产生;大肠杆菌侵入膀胱上皮细胞使用体外细菌侵入测定;和[3]为了确定动力蛋白2 S-亚硝基化在大肠杆菌上的生理相关性。大肠杆菌侵入膀胱上皮的动物模型。拟议的研究应提供更深入的了解，在泌尿上皮细胞的入侵所涉及的机制，并可能最终确定dynamin 2 S-亚硝基化作为一个有效的药物靶点，以限制频繁的尿路感染。 公共卫生相关性：尿路感染（UTI）是最常见和最严重的病原性感染之一。我们确定NOS/NO和dynamin 2是E.大肠杆菌侵入膀胱细胞。eNOS和dynamin 2的靶向作用可能为干预目前无法治疗的复发性尿路感染提供了机会。