Toxoplasma endocytosis of host cytoplasm

描述(由申请人提供)：先天免疫防御有助于限制原生动物病原体弓形虫的生长，但其效力被寄生虫的反防御所抵消。例如，受感染的巨噬细胞试图通过表达免疫相关GTP酶(IRGS)来摧毁寄生虫，这些酶加载到寄生虫的膜上并拆除，但寄生虫通过磷酸化IRG蛋白来降低它们的活性来阻止这一点。由于磷酸化是可逆的，因此是暂时的，我们假设弓形虫还通过将这些宿主效应蛋白内化到其内吞系统来永久消除这些宿主效应蛋白。我们最近出人意料的发现，寄生虫很容易内化在宿主细胞质中表达的报告蛋白，这支持了这一假说。此外，报告蛋白在缺乏蛋白酶的寄生虫中含量最高，这表明内化的宿主蛋白通常在寄生虫的内吞系统中被消化。此外，寄生液泡内缺乏管状膜生物发生的寄生虫无法有效地内化报告蛋白，这表明管状膜是吸收所必需的。这些有缺陷的寄生虫也很容易被激活的巨噬细胞杀死。其具体目的是：(1)确认寄生虫的内吞系统是内化蛋白的目的地；(2)证明弓形虫利用管膜的空泡内网络内化IRGS以破坏其内溶体系统。我们希望证明这种寄生虫通过内吞和降解来消除IRGS，作为一种免疫逃避策略。这项工作有望带来令人兴奋的新机会，以减少寄生虫在感染期间的细胞内存活。

DESCRIPTION (provided by applicant): Innate immune defenses help restrict growth of the protozoan pathogen Toxoplasma gondii, but their efficacy is offset by parasite counter defenses. For example, infected macrophages attempt to destroy the parasite by expressing immunity-related GTPases (IRGs) that load onto and dismantle the parasitophorous membrane, but the parasite thwarts this by phosphorylating Irg proteins to diminish their activity. Since phosphorylation is a reversible, and therefore temporary, we hypothesize that T. gondii additionally eliminates these host effector proteins permanently by internalizing them into its endocytic system. Our recent unexpected finding that the parasite readily internalizes a reporter protein expressed in the host cytosol supports this hypothesis. Further, the reporter protein is most abundantly seen in protease-deficient parasites, suggesting that internalized host proteins are normally digested in the parasite endocytic system. Also, parasites deficient in the biogenesis of tubular membranes within the parasitophorous vacuole are unable to efficiently internalize the reporter protein, indicating that the tubular membranes are required for uptake. These deficient parasites are also highly susceptible to killing by activated macrophages. The specific aims are to: (1) confirm the parasite endocytic system as the destination of internalized proteins; and (2) show that Toxoplasma uses the intravacuolar network of tubular membranes to internalize IRGs for destruction in its endolysosomal system. We expect to show that the parasite eliminates IRGs by endocytosis and degradation as an immune evasion strategy. The work is anticipated to open exciting new opportunities to diminish parasite intracellular survival during infection.