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（由申请方提供）：阴道毛滴虫是一种性传播的专性细胞外寄生虫，定植于人类泌尿生殖道。尽管对寄生虫的生存至关重要，但人们对其在宿主体内建立感染和茁壮成长的机制知之甚少。T.迷走神经必须附着在宿主泌尿生殖道的上皮层上才能存活。尽管T.迷走神经表面蛋白作为病原体-宿主相互作用的关键界面，参与这些过程的表面蛋白的身份仍然未知。该提案的总体目标是表征两种表面蛋白，这两种表面蛋白是四跨膜蛋白（TSP）家族的成员，含TSP 1的外泌体是一种丰富的表面蛋白，以确定它们在这种寄生虫与人类宿主的相互作用中所起的作用。哺乳动物TSP以膜复合物的形式存在，调节粘附、迁移、细胞内信号传导和运动[我们的初步数据表明T。迷走神经TSP参与寄生虫迁移]。在具体目标1中，我们将确定四跨膜蛋白6（TSP 6）在宿主-寄生虫相互作用期间的作用。我们的初步数据表明，TSP 6的目标，质膜和鞭毛的寄生虫和改变其定位后，暴露于宿主细胞。[此外，其16个氨基酸的C-末端胞内尾的丢失导致TSP 6的鞭毛定位的丢失和减少的寄生虫迁移。我们建议使用敲低方法进一步分析TSP 6功能，以测试其在粘附、迁移和对宿主细胞的细胞毒性中的作用。此外，由于TSP通过与其他细胞表面蛋白形成复合物而充当分子支架，因此我们将进行免疫共沉淀实验以鉴定TSP 6伴侣蛋白。这些研究将首次提供单细胞寄生虫四跨膜蛋白的分子分析，并加深我们对T。迷走神经宿主-病原体相互作用在具体目标2中，我们将表征含有TSP 1的外泌体。TSP 1存在于细胞表面和细胞内多泡体和寄生虫释放的外来体上。外泌体的纯化使我们能够显示寄生虫外泌体诱导宿主细胞释放细胞因子。我们将使用TSP 1过表达、TSP 1-KD和WT寄生虫确定外泌体的蛋白质组成及其对宿主：寄生虫通信的影响。除了提供有关TSP 1在宿主：病原体相互作用中的作用的信息外，这些研究是第一次检查T。迷走神经外泌体是介导T.寄生虫和它的宿主这项研究将主要在阿根廷Chascomus的Fundaci <$n Instituto de Investigaciones Biotecnol <$gicas（IIB-INTECH）与娜塔莉亚de Miguel博士合作进行。