Kinase regulation of trafficking at the Toxoplasma intravacuolar network

项目摘要/摘要 弓形虫几乎可以感染几乎所有温血动物的任何细胞类型。 可以说是地球上最成功的寄生虫，已经感染了大约三分之一的 全球的人类。虽然最初的感染通常不会出现并发症，但寄生虫能够持续存在。 为其宿主的生命，并可再次出现在免疫功能受损和免疫抑制而导致致命 疾病。弓形虫和其他顶端复合体寄生虫一样，必须入侵宿主细胞才能生存和复制。 一旦进入宿主细胞，寄生虫就会在一个名为 寄生性液泡。空泡的破坏会导致寄生虫的死亡，寄生虫会分泌一种 蛋白质进入液泡的电池，以促进其生物发生和调节营养物质的运输 效应器蛋白。寄生性液泡中的一个主要结构是液泡内网络 膜小管(IVN)，被认为是主要的运输工具。IVN生物发生是 由破坏IVN的寄生虫蛋白和突变体的寡聚复合体直接作用而形成 在感染的动物模型中显示毒力降低。我们已经鉴定出一种寄生虫特异的蛋白激酶 它调节与寄生虫相关的蛋白质子集的膜结合 空泡和IVN膜，该激酶的缺失会导致IVN管形成异常的空泡。 虽然我们已经确定了激酶底物和磷酸化的位置，但 被这种磷酸化调控的基因是未知的。拟议研究的目标是确定 磷酸化调控分子间和分子内的精确分子机制 推动IVN生物发生的相互作用。首先，我们将确定蛋白质的成分是如何 推动IVN生物发生的复合体在寄生虫分泌过程中发生变化 系统并插入IVN膜中。我们将使用分子遗传学、细胞学和生物化学方法 以确定磷酸化调节这些蛋白质-蛋白质的分子机制 促进IVN发展的互动。此外，我们将使用创新的生物物理方法来 生成这些关键寄生虫蛋白质复合体的第一个结构模型，以确定生物物理 它们诱导IVN形成的机制。

PROJECT SUMMARY/ABSTRACT Toxoplasma gondii has the remarkable ability to infect virtually any cell type of almost all warm-blooded animals and is arguably the most successful parasite on earth, having infected an estimated one-third of humans globally. While initial infection typically resolves without complication, the parasite is able to persist for the life of its host, and can re-emerge in the immunocompromised and immunosuppressed to cause fatal disease. Toxoplasma, like other apicomplexan parasites, must invade a host cell to survive and replicate. Once inside a host cell, the parasite survives and replicates within a specialized organelle called the parasitophorous vacuole. Disruption of the vacuole results in parasite death, and the parasite secretes a battery of proteins into the vacuole to facilitate its biogenesis and regulate trafficking of nutrients and effector proteins. A principle structure within the parasitophorous vacuole is the intravacuolar network of membranous tubules (the IVN), which is thought to act as a major trafficking apparatus. IVN biogenesis is formed by the direct action of oligomeric complexes of parasite proteins and mutants that disrupt the IVN show reduced virulence in animal models of infection. We have identified a parasite-specific protein kinase that regulates the membrane association of a subset of the proteins that associate with the parasitophorous vacuolar and IVN membranes, and deletion of this kinase results in vacuoles with aberrant IVN tubulation. While we have identified the kinase substrates and the sites of phosphorylation, the interactions that are regulated by this phosphorylation are unknown. The goal of the proposed studies is to determine the precise molecular mechanisms by which phosphorylation regulates the inter- and intra-molecular interactions that drive IVN biogenesis. First, we will determine how the components of the protein complexes that drive IVN biogenesis change as the complexes progress through the parasite secretory system and insert into the IVN membrane. We will use molecular genetic, cellular, and biochemical methods to determine the molecular mechanisms by which phosphorylation regulates these protein-protein interactions to facilitate IVN development. Furthermore, we will use innovative biophysical methods to generate the first structural models of these critical parasite protein complexes to determine the biophysical mechanism by which they induce IVN formation.