Calcium signaling in the parasitophorous vacuole of Toxoplasma gondii

 描述（由申请方提供）：弓形虫是一种专性细胞内寄生虫，已知可慢性感染三分之一的人群，是免疫功能低下个体和先天性感染者发生毁灭性疾病的原因。弓形虫被美国疾病控制中心认为是一种被忽视的寄生虫病，迫切需要新的有效治疗方法。发现新的药物靶点的关键是表征寄生虫所必需和独特的事件和蛋白质。弓形虫生存所需的事件，如入侵和外出，是由钙信号传导过程，包括寄生虫特有的蛋白质。有趣的是，弓形虫的排泄依赖于寄生虫和宿主细胞中的钙信号。寄生虫内的钙流和信号传导如何与宿主内发生的钙流和信号传导相关并转化为宿主内发生的钙流和信号传导，反之亦然，目前尚不清楚。而在其宿主细胞内，弓形虫在一个专门的寄生虫空泡（PV）内分裂，该空泡保护其免受细胞清除机制的影响，并为其提供所需的营养。我们假设，鉴于其位置在寄生虫和宿主之间的接口，PV也发挥了关键作用，整合控制出口和其他钙依赖性事件的信号事件。为了验证这一假设，我们将基因候选方法与PV信号蛋白的新筛选相结合。使用生物信息学的方法，我们已经确定了两个分泌蛋白，包含EF手结构域，这是发现在许多钙信号蛋白，包括钙调蛋白和寄生虫特异性钙依赖性蛋白激酶。有趣的是，这两种蛋白质都定位于PV。我们的第一个目标是确定这两个假定的钙结合蛋白在寄生虫的溶解周期的功能。我们的第二个目标是 鉴定和表征与PV相关的信号蛋白。PV蛋白的完整补体远未被了解，因为在将其内容物与寄生虫和宿主细胞的内容物分离方面存在重大挑战。我们的新方法来确定PV的蛋白质组成将是使用基于邻近的BioID蛋白质-蛋白质相互作用陷阱来鉴定相互作用或接近已知液泡蛋白的蛋白质。一旦我们确定了推定的新型PV蛋白，我们将重点关注那些可能参与信号事件的六个特征。同时，我们的研究结果将揭示宿主和寄生虫信号通路是如何整合的，并利用PV和出口的重要作用，为这种重要而被忽视的寄生虫发现新的药物靶点。

 DESCRIPTION (provided by applicant): Toxoplasma gondii, is an obligate intracellular parasite known to chronically infect a third of the human population and the cause of devastating disease in immunocompromised individuals and in those infected congenitally. Toxoplasma is considered a neglected parasitic disease of the United States by the Center for Disease Control and there is a dire need for new and effective therapeutics. Critical to the discovery of new drug targets is the characterization of events and proteins that are essential and unique to the parasite. Events required for Toxoplasma survival such as invasion and egress are regulated by calcium signaling processes that include proteins unique to the parasite. Interestingly, Toxoplasma egress depends on calcium signaling in both the parasite and host cell. How calcium fluxes and signaling within the parasite relate and translate to those occurring in the host and vice versa is not understood. While inside its host cell Toxoplasma divides within a specialized parasitophorous vacuole (PV) that protects it from cellular clearance mechanisms and provides it with the required nutrition. We hypothesize that, given its position at the interface between parasite and host, the PV also plays a critical role in integrating the signaling events that control egress and other calcium dependent events. To test this hypothesis we are combining a gene candidate approach with a novel screen for PV signaling proteins. Using a bioinformatics approach we have identified two secreted proteins that contain EF hand domains, which are found in numerous calcium signaling proteins including calmodulin and the parasite specific calcium dependent protein kinases. Interestingly, both of these proteins localize to the PV. Our first aim consists of determining the function of these two putative calcium-binding proteins during the lytic cycle of the parasite. Our second aim will be to identify and characterize signaling proteins associated with the PV. The full complement of PV proteins is far from known given significant challenges in separating its content from that of the parasite and host cell. Our novel approach to determine the protein makeup of the PV will be to identify proteins that interact or are near known vacuolar proteins using the proximity based BioID protein-protein interaction trap. Once we have identified putative novel PV proteins we will characterize six of them focusing on those likely to be involved in signaling events. In conjunction, our results will shed light on how host and parasite signaling pathways are integrated and exploit the essential roles of the PV and egress to discover new drug targets for this important and neglected parasite.