Role of tetraspanin proteins in Trichomonas vaginalis pathogenesis

四跨膜蛋白在阴道毛滴虫发病机制中的作用

• 批准号: 8410373
• 负责人: Patricia Jean Johnson
• 金额: $ 6.36万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-03-08 至 2016-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8410373
• 关键词: Adherence; Adhesions; Amino Acids; Argentina; Award; Biological; Biological Process; C-terminal; Cell Communication; Cell Surface Proteins; Cell membrane; Cell surface; Cells; Cellular biology; Cervicitis; Co-Immunoprecipitations; Collaborations; Communication; Complex; Cytoplasmic Tail; Data; Development; Doctor of Philosophy; Dominant-Negative Mutation; Electron Microscopy; Epithelial; Epithelial Cells; Exposure to; Extracellular Matrix; Family; Flagella; Foundations; Generations; Genitourinary system; Goals; HIV Infections; Human; Immune response; In Vitro; Individual; Infection; Institution; Knowledge; Laboratories; Lead; Light; Malignant neoplasm of cervix uteri; Malignant neoplasm of prostate; Mammalian Cell; Mass Spectrum Analysis; Measures; Mediating; Membrane; Membrane Proteins; Methods; Modeling; Molecular; Multivesicular Body; Nature; Organelles; Organism; Parasites; Pathogenesis; Pelvic Inflammatory Disease; Pharmacotherapy; Play; Postdoctoral Fellow; Predisposition; Process; Property; Proteins; Proteome; Proteomics; Publishing; Regulation; Research; Research Methodology; Role; Sexually Transmitted Diseases; Signal Transduction; Source; Students; Surface; System; Tail; Techniques; Testing; Trichomonas Infections; Trichomonas vaginalis; Urethritis; Vagina; Vaginitis; Vesicle; Visit; Work; base; cell motility; combat; cytokine; cytotoxicity; extracellular; human PHEMX protein; intercellular communication; interest; knock-down; matrigel; member; migration; novel; overexpression; pathogen; programs; protein expression; public health relevance; research study; scaffold; success; vaccination strategy

DESCRIPTION (provided by applicant): Trichomonas vaginalis is a sexually-transmitted, obligate extracellular parasite that colonizes the human urogenital tract. Despite being of critica importance to the parasite's survival relatively little is known about the mechanisms employed to establish an infection and thrive within its host. As an extracellular organism, T. vaginalis must adhere to the epithelial lining of the host's urogenital tract to survive. Despite the importance o T. vaginalis surface proteins as a critical interface for pathogen-host interactions, the identity f the surface proteins involved in these processes remains unknown. The overall goals of this proposal are to characterize two surface proteins that are members of the tetraspanin (TSP) family and TSP1-containing exosomes as an abundant surface protein to determine the roles they play in the interaction of this parasite with its human host. Mammalian TSPs exist as membrane complexes that regulate adhesion, migration, intracellular signaling and motility [and our preliminary data indicate that T. vaginalis TSPs are involved in parasite migration]. In specific aim 1 we will determine role of tetraspanin 6 (TSP6) during host-parasite interaction. Our preliminary data show that TSP6 targets to both the plasma membrane and flagella of the parasite and changes its localization upon exposure to host cells. [Moreover, the loss of its 16 amino acid C-terminal intracellular tail results in loss of flagella localization of TSP6 and reducd parasite migration. We propose to further analyze TSP6 function using a knock down approach to test its role in adherence, migration and cytotoxicity to host cells.] In addition, as TSPs act s molecular scaffolds by forming complexes with other cell surface proteins, we will perform co- immunoprecipitation experiments to identify TSP6 partner proteins. These studies will provide the first molecular analyses of tetraspanin proteins in unicellular parasites and enhance our understanding of T. vaginalis host-pathogen interactions. In specific aim 2 we will characterize TSP1 containing exosomes. TSP1 is present on the surface of the cell and on intracellular multivesicular bodies and [exosomes that are released by the parasite. Purification of exosomes has allowed us to show that parasite exosomes induce cytokine release by host cells. We will determine the protein composition of exosomes and their effect on host:parasite communication using TSP1-overexpressing, TSP1-KD and WT parasites.] In addition to providing information about the role of TSP1 in host:pathogen interactions, these studies are the first to examine T. vaginalis exosomes, a key organelle which may mediate communication between T. vaginalis and its host. This research will be done primarily at Fundaci¿n Instituto de Investigaciones Biotecnol¿gicas (IIB-INTECH) in Chascomus, Argentina in collaboration with Dr. Natalia de Miguel.

描述(申请人提供)：阴道毛滴虫是一种性传播的专性细胞外寄生虫，在人类泌尿生殖道定居。尽管对寄生虫的生存至关重要，但人们对用来建立感染并在其宿主内茁壮成长的机制知之甚少。作为一种细胞外有机体，阴道毛滴虫必须附着在宿主的泌尿生殖道上皮层才能生存。尽管阴道毛滴虫表面蛋白作为病原体-宿主相互作用的关键界面很重要，但参与这些过程的表面蛋白的特性仍不清楚。这项建议的总体目标是将属于TSP家族成员的两种表面蛋白和含有TSP1的外切体鉴定为一种丰富的表面蛋白，以确定它们在这种寄生虫与其人类宿主相互作用中所起的作用。哺乳动物的TSP以膜复合体的形式存在，调节黏附、迁移、细胞内信号和运动[我们的初步数据表明阴道毛滴虫TSP参与了寄生虫的迁移]。在特定目标1中，我们将确定TSP6(TSP6)在宿主-寄生虫相互作用中的作用。我们的初步数据显示，TSP6针对寄生虫的质膜和鞭毛，并在接触宿主细胞时改变其定位。[此外，TSP6的16个氨基酸C末端细胞内尾巴的缺失导致TSP6鞭毛定位的丧失和寄生虫迁移的减少。我们建议使用敲击法进一步分析TSP6的功能，以测试其在宿主细胞的黏附、迁移和细胞毒性中的作用。]此外，由于天冬氨酸蛋白通过与其他细胞表面蛋白形成络合物作为S分子支架，我们将进行免疫共沉淀实验来鉴定TSP6蛋白。这些研究将首次对单细胞寄生虫中的Tetraspanin蛋白进行分子分析，并加深我们对阴道毛滴虫宿主与病原体相互作用的了解。在特定目标2中，我们将描述含有外切体的TSP1。TSP1存在于细胞表面、细胞内多泡小体和寄生虫释放的外体上。外切体的纯化使我们能够证明寄生虫外切体诱导宿主细胞释放细胞因子。我们将确定外切体的蛋白质组成及其对宿主的影响：利用TSP1过表达的寄生虫、TSP1-KD和WT寄生虫进行交流。]除了提供有关TSP1在宿主-病原体相互作用中的作用的信息外，这些研究还首次检测了阴道毛滴虫外周体，这是一个可能介导阴道毛滴虫与其宿主之间沟通的关键细胞器。这项研究将主要在阿根廷查斯科莫斯的生物科技研究所(IIB-INTECH)与Natalia de Miguel博士合作进行。