The Role of Toxoplasma Rhoptries in Host Cell Infection

描述（由申请人提供）：刚地弓形虫是顶复合体门的一种专性细胞内寄生虫，可引起免疫功能低下（艾滋病/移植/淋巴瘤）个体的严重中枢神经系统疾病和全球先天性感染新生儿的出生缺陷。这种寄生虫还可以作为研究细胞内顶复合体寄生虫的模型系统，这些寄生虫引起各种具有全球医学和兽医重要性的疾病，包括疟疾的病原体恶性疟原虫。这些寄生虫积极侵入并居住在宿主细胞质中独特的寄生液泡（PV）内。PV的入侵和建立部分是由顶复合体特有的分泌细胞器的作用介导的。菱形体在这一过程中的确切作用尚不清楚，很大程度上是因为迄今为止只有少数菱形体蛋白被确定。为了研究其功能，我们对其进行了纯化，并利用蛋白质组学和单克隆抗体方法对其含量进行了分析。我们已经确定了红体的主要蛋白质成分，其中一种是蛋白磷酸酶2c样（TgPP2L）蛋白，在感染期间靶向宿主细胞核，这表明红体在宿主-寄生虫相互作用中的新作用。我们将重点研究TgPP2L、蛋白和两个在我们的蛋白质组学分析中鉴定出的新型状体蛋白，以确定它们在入侵中的作用。具体来说，我们将1)表征TgPP2L的宿主核定位，并直接评估其磷酸酶活性；2)通过体外和体内TgPP2L敲除寄生虫的分析以及TgPP2L在人细胞中的表达来确定TgPP2L的功能；3)通过确定新的腺状体蛋白ROP14和RON3在分泌后的最终目的地、识别相互作用伙伴和破坏它们的基因来确定它们的功能。本文提出的研究对顶复合体寄生物建立细胞内生存环境的方式提供了重要的见解，并提供了对宿主病原体相互作用独特方面的理解。

DESCRIPTION (provided by applicant): Toxoplasma gondii is an obligate intracellular parasite in the phylum Apicomplexa that causes severe central nervous system disorders of immunocompromised (AIDS/transplant/lymphoma) individuals and birth defects to congenitally infected neonates worldwide. This parasite also serves as a model system for the study of intracellular Apicomplexan parasites that cause a variety of diseases of global medical and veterinary importance including Plasmodium falciparum, the causative agent of malaria. These parasites actively invade into and reside within a unique parasitophorous vacuole (PV) in the cytoplasm of the host. Invasion and establishment of the PV are mediated in part by the action of the rhoptries, secretory organelles unique to Apicomplexans. The precise role of the rhoptries in this process in not known, largely because only a few rhoptry proteins have been identified to date. To address the function of the rhoptries, we have purified rhoptries and analyzed their contents using proteomic and monoclonal antibody approaches. We have identified the major proteins constituents of the rhoptries, one of which is a protein phosphatase 2C-like (TgPP2L) protein that is targeted to the nucleus of the host cell during infection, indicating a novel role for the rhoptries in host-parasite interaction. We will focus on the TgPP2L, protein and two novel rhoptry proteins identified in our proteomic analysis to determine their roles in invasion. Specifically, we will 1) characterize TgPP2L with respect to its host nuclear localization and directly assess its phosphatase activity; 2) determine the function of TgPP2L by analyzing TgPP2L knockout parasites in vitro and in vivo and by the expression of TgPP2L in human cells; and, 3) determine the functions of the novel rhoptry proteins ROP14 and RON3 by determining their ultimate destination following secretion, identifying interaction partners, and disruption of their genes. The studies proposed here offer important insight into the means by which Apicomplexan parasites establish an environment for intracellular survival and provide an understanding of unique aspects of host pathogen interactions.