Kinase regulation of trafficking at the Toxoplasma intravacuolar network

项目概要/摘要 刚地弓形虫具有显著的感染几乎所有温血动物的任何细胞类型的能力， 动物，可以说是地球上最成功的寄生虫，感染了估计三分之一的 全球人类。虽然最初的感染通常没有并发症解决，寄生虫能够持续存在 它的宿主的生命，并可以重新出现在免疫功能低下和免疫抑制，造成致命的 疾病弓形虫和其他顶复门寄生虫一样，必须侵入宿主细胞才能生存和复制。 一旦进入宿主细胞，寄生虫就在一个称为细胞器的特殊细胞器中生存和复制。 寄生泡。空泡的破坏导致寄生虫死亡，寄生虫分泌一种 将一系列蛋白质送入液泡，以促进其生物发生并调节营养物质的运输， 效应蛋白寄生液泡内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装置。IVN生物成因是 由寄生虫蛋白质和突变体的寡聚复合物的直接作用形成，所述突变体破坏IVN 在动物感染模型中表现出毒性降低。我们已经鉴定出一种寄生虫特异性蛋白激酶 调节与寄生虫相关的蛋白质亚类的膜结合 空泡和IVN膜，并且该激酶的缺失导致具有异常IVN微管的空泡。 虽然我们已经确定了激酶底物和磷酸化位点，但 由这种磷酸化调节的是未知的。拟议研究的目标是确定 磷酸化调节分子间和分子内的精确分子机制 驱动IVN生物发生的相互作用。首先，我们将确定蛋白质的成分 驱动IVN生物发生的复合物随着复合物通过寄生物分泌的进展而改变 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的分子机制 互动，以促进IVN的发展。此外，我们将使用创新的生物物理方法， 生成这些关键寄生虫蛋白质复合物的第一个结构模型，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.