The Role of Toxoplasma Rhoptries in Host Cell Infection

描述(由申请人提供)：弓形虫是尖端复合体门中的一种专性细胞内寄生虫，可导致免疫受损(艾滋病/移植/淋巴瘤)患者的严重中枢神经系统疾病，并导致全球先天性感染新生儿的出生缺陷。这种寄生虫还作为研究细胞内顶体复合体寄生虫的模式系统，这些寄生虫引起各种具有全球医学和兽医意义的疾病，包括疟疾的病原体恶性疟原虫。这些寄生虫主动入侵并驻留在寄主细胞质中一个独特的寄生液泡(PV)内。PV的入侵和建立部分是通过棒状体的作用来调节的，棒状体是Apicomplexans特有的分泌细胞器。棒状病毒在这一过程中的确切作用尚不清楚，很大程度上是因为到目前为止只发现了少数几种棒状病毒蛋白。为了解决棒状病毒的功能，我们纯化了棒状病毒，并使用蛋白质组学和单抗方法分析了它们的含量。我们已经确定了棒状病毒的主要蛋白质成分，其中之一是一种蛋白磷酸酶2C样蛋白(TgPP2L)，它在感染过程中定位于宿主细胞的细胞核，表明棒状病毒在宿主-寄生虫相互作用中发挥了新的作用。我们将集中在我们的蛋白质组分析中发现的TgPP2L蛋白和两个新的杆状蛋白，以确定它们在侵袭中的作用。具体地说，我们将1)根据宿主核定位来鉴定TgPP2L，并直接评估其磷酸酶活性；2)通过分析TgPP2L敲除寄生虫在体内外以及TgPP2L在人类细胞中的表达来确定TgPP2L的功能；以及3)通过确定新的杆状蛋白ROP14和RON3在分泌后的最终目的地、确定相互作用伙伴和干扰其基因来确定其功能。这里提出的研究为Apicomplexan寄生虫建立细胞内生存环境提供了重要的见解，并提供了对宿主病原体相互作用的独特方面的理解。

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.