Role of tetraspanin proteins in Trichomonas vaginalis pathogenesis

描述（由申请人提供）：阴道毛滴虫是一种性传播的专性细胞外寄生虫，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繁殖的机制知之甚少。作为一种细胞外生物，阴道绦虫必须附着在宿主泌尿生殖道的上皮上才能存活。尽管阴道绦虫表面蛋白作为病原体-宿主相互作用的关键界面具有重要意义，但参与这些过程的表面蛋白的身份仍然未知。本提案的总体目标是表征四联蛋白（TSP）家族成员和含tsp1的外泌体作为丰富表面蛋白的两种表面蛋白，以确定它们在该寄生虫与人类宿主相互作用中的作用。哺乳动物的tsp作为膜复合物存在，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. vaginalis的tsp参与了寄生虫的迁移]。在特定目标1中，我们将确定四联蛋白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同时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端胞内尾的缺失导致TSP6鞭毛定位丧失，寄生虫迁移减少。]我们建议使用敲低方法进一步分析TSP6的功能，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TSP6通过与其他细胞表面蛋白形成复合物而起到分子支架的作用，我们将进行共免疫沉淀实验来鉴定TSP6的伴侣蛋白。这些研究将首次提供单细胞寄生虫中四联蛋白的分子分析，并增强我们对阴道绦虫宿主-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存在于细胞表面和由寄生虫释放的细胞内多泡体和外泌体上。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。我们将利用过表达tsp1、TSP1-KD和WT寄生虫来确定外泌体的蛋白质组成及其对宿主与寄生虫交流的影响。除了提供有关TSP1在宿主：病原体相互作用中的作用的信息外，这些研究还首次研究了阴道绦虫外泌体，这是一个可能介导阴道绦虫与其宿主之间通信的关键细胞器。这项研究将主要在阿根廷Chascomus的Fundaci¿¿n Instituto de Investigaciones biotechnologicas （ibi - 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.