Calcium signaling in the parasitophorous vacuole of Toxoplasma gondii

 描述(申请人提供)：弓形虫是一种专性的细胞内寄生虫，已知会慢性感染三分之一的人类人口，并导致免疫功能受损的个人和先天感染的人患上毁灭性疾病。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，迫切需要新的有效的治疗方法。对于发现新的药物靶点来说，关键是对寄生虫必不可少的和独特的事件和蛋白质进行表征。弓形虫生存所需的事件，如入侵和排出，由钙信号过程调节，其中包括寄生虫特有的蛋白质。有趣的是，弓形虫的出口依赖于寄生虫和宿主细胞中的钙信号。寄生虫体内的钙离子通量和信号如何与宿主中发生的钙离子通量和信号相关联和翻译，反之亦然，目前还不清楚。而在其宿主细胞内，弓形虫在一个专门的寄生性液泡(PV)内分裂，该液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。我们推测，由于其在寄生虫和宿主之间的界面位置，PV也在整合控制出口的信号事件和其他钙依赖事件中发挥关键作用。为了验证这一假设，我们结合了一种候选基因方法和一种新的PV信号蛋白筛选方法。利用生物信息学方法，我们鉴定了两个含有EF手区的分泌蛋白，它们存在于许多钙信号蛋白中，包括钙调蛋白和寄生虫特异的钙依赖蛋白激酶。有趣的是，这两种蛋白都定位于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裂解周期中的功能。我们的第二个目标是 鉴定和鉴定与PV相关的信号蛋白。鉴于在将其内容与寄生虫和宿主细胞的内容分离方面存在重大挑战，PV蛋白的完整补充还远未知晓。我们确定PV的蛋白质组成的新方法将是使用基于邻近的BioID蛋白质-蛋白质相互作用陷阱来识别与已知的空泡蛋白相互作用或接近的蛋白质。一旦我们确定了推定的新的光伏蛋白，我们将描述其中六个蛋白，重点放在那些可能参与信号事件的蛋白上。同时，我们的结果将阐明宿主和寄生虫信号通路是如何整合的，并利用PV和Exress的重要作用来发现这种重要和被忽视的寄生虫的新药物靶点。

 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.