The Role of Toxoplasma Rhoptries in Host Cell Infection

描述（由申请方提供）：弓形虫是一种专性细胞内寄生虫，属于顶复门，可导致免疫功能低下（AIDS/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。这种寄生虫还用作研究细胞内顶复门寄生虫的模型系统，所述顶复门寄生虫引起具有全球医学和兽医重要性的多种疾病，包括恶性疟原虫（疟疾的病原体）。这些寄生虫主动侵入并驻留在宿主细胞质中独特的寄生虫空泡（PV）内。PV的侵入和建立部分由顶复门特有的分泌细胞器棒状体的作用介导。棒状体在这一过程中的确切作用尚不清楚，主要是因为迄今为止仅鉴定了少数棒状体蛋白。为了解决棒状体的功能，我们纯化了棒状体，并使用蛋白质组学和单克隆抗体的方法分析其内容物。我们已经确定了棒状体的主要蛋白质成分，其中之一是蛋白磷酸酶2C样（TgPP 2L）蛋白，其在感染过程中靶向宿主细胞的细胞核，表明棒状体在宿主-寄生虫相互作用中的新作用。我们将专注于TgPP 2L，蛋白质和两个新的棒状蛋白在我们的蛋白质组学分析中确定，以确定其在入侵中的作用。具体而言，我们将1）表征TgPP 2L的宿主核定位并直接评估其磷酸酶活性; 2）通过在体外和体内分析TgPP 2L敲除的寄生虫以及通过TgPP 2L在人细胞中的表达来确定TgPP 2L的功能;和3）通过确定新棒状体蛋白ROP 14和RON 3分泌后的最终目的地来确定它们的功能，识别相互作用的伙伴，并破坏它们的基因。这里提出的研究提供了重要的见解Apicomplexan寄生虫建立细胞内生存环境的手段，并提供了独特的方面的主机病原体相互作用的理解。

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.