The role of the DOC2.1 protein in Toxoplasma gondii Ca2+- dependent exocytosis

描述(由申请人提供)：弓形虫寄生虫是免疫功能低下患者中危及生命的脑炎的病原体，此外，如果感染是先天性感染，还可能导致各种出生缺陷。与疾病相关的病理起源于快速的裂解细胞内复制周期。利用遗传学方法，我们最近发现了DOC2蛋白(TgDOC2.1)在钙离子介导的微线粒分泌中的作用。微线虫含有成功侵入和排出宿主细胞所需的黏附分子，这是完成裂解周期所必需的。这项提议的目标是揭示TgDOC2.1在微线体分泌中的作用和功能。这将为了解一个鲜为人知的机制提供令人兴奋的新见解，该机制不仅对弓形虫的发病至关重要，而且对所有其他顶复合体寄生虫都是如此，因为依赖钙的微线体分泌在整个门中是保守的。同时，这将为这一途径作为新的特定药物靶点(分泌不是 以目前批准的药物为靶标)。在其他系统中，含有DOC2结构域的蛋白招募膜融合机制(如SNARE和MUNC蛋白)以促进分泌囊泡与质膜的融合。然而，在TgDOC2.1中，没有与分泌机制相互作用的保守结构域。TgDOC2.1的比较基因组学在顶层复合体中发现了四个保守的序列块，突出了这些结构域的潜在关键作用(块2包含DOC2结构域)。为了分析TgDOC2.1的S功能，我们将首先产生试剂，或者是特异性的抗血清，或者是融合报告，以建立其在整个钙依赖性胞外作用中的时空亚细胞定位模式。这将指示TgDOC2.1在哪个膜上发挥其作用。此外，我们还将建立一个有条件的TgDOC2.1敲除寄生虫系，可用于与TgDOC2.1结构域缺失突变体的功能互补研究。这将确定哪些结构域在膜转位和/或微线分泌中发挥关键作用。在同一模型中，我们将测试钙结合域的点突变。首先将使用已建立的异源哺乳动物组织培养模型来定位与钙结合的天冬氨酸残基。同时，为了了解TgDOC2.1在分泌过程中与哪些蛋白质合作，我们将使用两种平行的方法识别与TgDOC2.1相互作用的蛋白质。第一种是遗传酵母双杂交系统，我们将以TgDOC2.1为诱饵来筛选速殖子的cDNA文库。在第二部分中，我们将探索一种名为BioID的新方法。在这种方法中，我们将一个大肠杆菌Bira突变蛋白融合到TgDOC2.1中，并在寄生虫中表达它。Bira突变体导致同一复合体中蛋白质的混杂生物素化，随后可以很容易地通过链霉亲和素浓缩和质谱仪进行鉴定。任何一种方法确定的假定的TgDOC2.1相互作用伙伴都将通过寄生虫的共同定位研究进行验证。总之，我们将定义保守的微线体分泌过程背后的机制。

DESCRIPTION (provided by applicant): The apicomplexan parasite Toxoplasma gondii is the causative agent of life-threatening encephalitis in immunocompromised patients and in addition can cause a variety of birth defects if the infection is contracted congenitally. The pathology associated with disease originates in fast rounds of lytic intracellular replication cycles. Using genetic approach we recently identified a role for a DOC2 protein (TgDOC2.1) in Ca2+- mediated microneme secretion. Micronemes contain adhesion molecules required for successful host cell invasion and egress, which is essential to complete the lytic cycle. The goal of this proposal is to unravel the role and function of TgDOC2.1 in microneme secretion. This will provide exciting new insights into a poorly understood mechanism critical to the pathogenesis of not only Toxoplasma, but to all other apicomplexan parasites since Ca2+-dependent microneme secretion is a conserved across the phylum. At the same time, this will provide insights into the potential of this pathway as a new specific drug target (secretion is not targeted by currently approved drugs). In other systems DOC2-domain containing proteins recruit the membrane fusion machinery (e.g. SNARE and MUNC proteins) to facilitate fusion of the secretory vesicle with the plasma membrane. However, no conserved domains interacting with the secretory machinery are conserved in TgDOC2.1. Comparative genomics of TgDOC2.1 identified four conserved sequence block across the Apicomplexa, highlighting a potentially crucial role for these domains (block 2 contains the DOC2 domain). To dissect TgDOC2.1's function we will first generate reagents, either a specific antiserum or fusion-reporter, to establish its spatio-temporal sub-cellular localization pattern throughout Ca2+-dependent excocytosis. This will indicate at which membrane TgDOC2.1 exerts its function. In addition we will establish a conditional TgDOC2.1 knock-down parasite line that can be used for functional complementation studies with TgDOC2.1 domain deletion mutants. This will identify which domains play a critical role in membrane translocation and/or microneme secretion. In the same model we will test point mutations in the Ca2+-binding domains. The Ca2+- binding Asp residues will first be mapped using a well-established heterologous mammalian tissue culture model. Simultaneously, to learn with which proteins TgDOC2.1 cooperates in the secretion process we will identify proteins interacting with TgDOC2.1 using two parallel approaches. The first is a genetic yeast two- hybrid system wherein we will use TgDOC2.1 as bait to screen a tachyzoite cDNA library. In the second we will explore a new method, named BioID. In this method we will fuse an E. coli BirA mutant protein to TgDOC2.1 and express it in the parasite. The BirA mutant results in promiscuous biotinilyation of proteins in the same complex, which subsequently can be easily identified by streptavidin enrichment and mass spectrometry. Putative TgDOC2.1 interaction partners identified by either method will be validated by co-localization studies in the parasite. Altogether, we will define the mechanism behind the conserved microneme secretion process.