Dissecting the role of Toxoplasma CDPK3 in parasite propagation and virulence

 描述(申请人提供)：弓形虫是一种专性细胞内寄生虫，已知会慢性感染三分之一的人类人口。弓形虫是一种机会性感染源，可导致艾滋病患者发生脑炎和其他严重症状。弓形虫入侵、出口和运动所需的事件是由钙信号过程调节的，其中包括与人类宿主显著不同的蛋白质和因子。要利用这些事件作为潜在的药物靶点，需要更好地了解弓形虫的钙信号级联。一个特殊的钙依赖蛋白激酶(CDPKs)家族受到了特别的关注，因为它在人类细胞中是缺失的，并且参与了关键过程。通过正向遗传方法，我们发现TgCDPK3的突变导致钙离子载体诱导的外泄(IEress)延迟，重要的是，小鼠大脑中潜伏期的形成存在缺陷。利用一种新的蛋白质-蛋白质相互作用的筛选，我们鉴定了几种可能作为功能伙伴或底物的蛋白质 TgCDPK3，包括一种蛋白磷酸酶2C，我们将其命名为TgPP2C4和另一种钙依赖蛋白激酶TgCDPK2a。我们的假设是，TgCDPK3、TgCDPK2a和TgPP2C4形成了对寄生虫毒力至关重要的“共同调节”网络的一部分。我们的第一个目标是分析TgPP2C4和TgCDPK2a的定位和功能。此外，我们将确定这两种酶的底物和功能伙伴，这将使我们能够定义TgCDPK3、TgCDPK2a和TgPP2C4共同调节的事件和蛋白质。我们的第二个目标是确定TgCDPK3在毒力中的作用。这将通过分析缺乏TgCDPK3或其共同调节伙伴TgCDPK2和TgPP2C的寄生虫在体内的传播和包囊来完成。此外，我们将在组织培养的包埋过程中确定TgCDPK3的底物，这将为了解TgCDPK3在体内繁殖和发育过程中的功能提供机制方面的信息。同时，我们的工作将阐明钙信号在生命周期中的作用 并有可能揭示抗击急性和慢性弓形虫病的新靶点。

 DESCRIPTION (provided by applicant): Toxoplasma gondii is an obligate intracellular parasite known to chronically infect a third of the human population. T. gondii is an opportunistic infectious agent that can cause encephalitis and other severe manifestations in AIDS patients. Events required for T. gondii invasion, egress and motility are regulated by calcium signaling processes that include proteins and factors significantly divergent from those of the human host. To exploit these events as potential drug targets would require a better understanding of T. gondii's calcium signaling cascades. A particular family of calcium dependent protein kinases (CDPKs) has garnered special attention, as it is absent from human cells and it is involved in key processes. Through a forward genetic approach we discovered that mutations in TgCDPK3 lead to a delay in calcium ionophore-induced egress (iiEgress), and importantly a defect in the formation of latent stages in the brains of mice. Utilizing a novel screen for protein-protein interactions, we identified several proteins that might act as functional partners or substrates of TgCDPK3, including a protein phosphatase 2C, which we have named TgPP2C4 and another calcium dependent protein kinase TgCDPK2a. It is our hypothesis that TgCDPK3, TgCDPK2a, and TgPP2C4, form part of a "co-regulatory" network essential for parasite virulence. Our first goal will be to analyze the localization and function of both TgPP2C4 and TgCDPK2a. In addition, we will determine the substrates and functional partners of these two enzymes, which will allow us to define the events and proteins co-regulated by TgCDPK3, TgCDPK2a and TgPP2C4. Our second goal will be to determine the role of TgCDPK3 in virulence. This will be accomplished by analyzing the in vivo dissemination and encystation of parasites lacking TgCDPK3 or its co-regulatory partners TgCDPK2 and TgPP2C. Furthermore, we will identify the substrates of TgCDPK3 during the process of encystation in tissue culture, which will provide mechanistic insight into the function of TgCDPK3 during in vivo propagation and development. In conjunction, our work will shed light on the function of calcium signaling during the life cycle of T. gondii and has the potential to reveal novel targets to combat both acute and chronic toxoplasmosis.