Vesicle Trafficking and Bacteria Invasion

囊泡运输和细菌入侵

基本信息

批准号：
8423043
负责人：
Yehia Daaka
金额：
$ 34.08万
依托单位：
UNIVERSITY OF FLORIDA
依托单位国家：
美国
项目类别：
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/8423043
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 Targeting 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

项目摘要

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.

描述（由申请人提供）：尿路感染（UTI）是西方世界中最常见的严重致病感之一。 UTI会影响婴儿，儿童和老年人，但女性尤其面临更高的风险。大多数UTI是由大肠杆菌（E.）大肠杆菌引起的，该大肠杆菌表达了丝状粘附细胞器，称为I型纤维化。人们认为，纤维化可以通过介导大肠杆菌对膀胱上皮的依从性，然后侵袭细菌进入宿主细胞，从而引发慢性尿道。细胞内侵袭会导致静止感染，其中细菌无法访问宿主免疫细胞或可耐细胞的抗生素。新兴的证据暗示了细胞侵袭细菌（进入）进入宿主细胞中的细胞膜相关的内吞机械。这种通常参与膜结合受体内吞作用的细胞机制可能包括小窝和网格蛋白涂层的凹坑。值得注意的是，基于小窝蛋白和网格蛋白涂层的基于凹坑的内吞作用均被证明取决于无处不在的GTPase Dynamin2蛋白的酶促活性。 Dynamin2从质膜中执行芽囊泡的裂变的能力受到与伴侣蛋白的相互作用的影响。纯化蛋白质的最新发现表明，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，大肠杆菌侵入膀胱上皮细胞受Dynamin2和Enos的酶促活性控制。 Dynamin2在特定的半胱氨酸残基处进行S-亚硝基化，并且大肠杆菌侵袭需要这种修饰。该应用的中心假设是eNOS和Dynamin2通过调节内吞囊泡运输来协同调节大肠杆菌侵袭。相关的特定目的是：[1]确定大肠杆菌侵入膀胱上皮细胞如何促进dynamin2 s-硝基化，重点是ENOS介导的NO产生； [2]使用体外细菌浸润测定法评估Dynamin2 S-硝基化对大肠杆菌侵袭到膀胱上皮细胞中的影响； [3]使用动物模型来确定大肠杆菌侵入大肠杆菌侵袭到膀胱上皮的生理相关性。拟议的研究应更深入地了解涉及渗入尿中心的机制，并最终可能识别Dynamin2 S-硝基化作为限制频繁尿路感染的有效药物靶标。