Role of tetraspanin proteins in Trichomonas vaginalis pathogenesis

描述（由申请人提供）：阴道毛滴虫是一种通过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，但人们对在宿主体内建立感染和繁殖的机制知之甚少。作为一种细胞外生物，阴道毛滴虫必须粘附在宿主泌尿生殖道的上皮衬里才能生存。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非常重要，但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四跨膜蛋白 (TSP) 家族成员的两种表面蛋白和含有 TSP1 的外泌体作为丰富的表面蛋白，以确定它们在这种寄生虫与其人类宿主相互作用中所发挥的作用。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阴道毛滴虫 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 (TSP6) 在宿主-寄生虫相互作用中的作用。我们的初步数据表明，TSP6 靶向寄生虫的质膜和鞭毛，并在暴露于宿主细胞后改变其定位。 [此外，其 16 个氨基酸 C 端胞内尾部的缺失导致 TSP6 鞭毛定位的缺失并减少寄生虫的迁移。我们建议使用敲低方法进一步分析 TSP6 功能，以测试其在对宿主细胞的粘附、迁移和细胞毒性中的作用。]此外，由于 TSP 通过与其他细胞表面蛋白形成复合物来充当分子支架，因此我们将进行免疫共沉淀实验来鉴定 TSP6 伴侣蛋白。这些研究将首次对单细胞寄生虫中的四跨膜蛋白进行分子分析，并增强我们对阴道毛滴虫宿主与病原体相互作用的理解。在具体目标 2 中，我们将表征含有外泌体的 TSP1。 TSP1 存在于细胞表面、细胞内多泡体和寄生虫释放的外泌体上。外泌体的纯化使我们能够证明寄生虫外泌体诱导宿主细胞释放细胞因子。我们将使用 TSP1 过表达、TSP1-KD 和 WT 寄生虫来确定外泌体的蛋白质组成及其对宿主：寄生虫通讯的影响。]除了提供有关 TSP1 在宿主：病原体相互作用中的作用的信息之外，这些研究首次检查了阴道毛滴虫外泌体，这是一种可能介导阴道毛滴虫及其宿主之间通讯的关键细胞器。这项研究将主要在阿根廷查斯科穆斯的生物技术研究基金会 (IIB-INTECH) 与 Natalia de Miguel 博士合作进行。

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.