Targeting of host vesicles to the vacuole of Toxoplasma and lipid cargo sorting

 描述(由申请人提供)：膜转运途径在细胞生理学中发挥核心作用，包括对环境挑战的反应，如感染微生物。一些病原体被这些途径摧毁，而另一些则颠覆了它们的功能。弓形虫是一种专性的细胞内寄生虫，在哺乳动物细胞的细胞质中繁殖，在自制的膜结合室-寄生虫空泡(PV)内繁殖。PV膜具有独特的脂质和蛋白质组成，并且PV不与任何内胞或胞外细胞器融合。然而，我们证明了寄生虫的细胞内生存依赖于哺乳动物细胞器中存在的营养物质。例如，弓形虫分别从内吞细胞器和高尔基囊泡中提取胆固醇和鞘磷脂，这就提出了一个令人困惑的问题，即弓形虫如何在不融合的情况下获取这些细胞器的脂肪含量。为了解决这个问题，我们分析了受感染的哺乳动物细胞中的囊泡运输途径。弓形虫以多种宿主途径为靶标，通过RAB循环和分泌小泡来拦截哺乳动物的交通。所有这些RAB囊泡都被重新路由到PV，然后原封不动地隔离在空泡腔中。被困在PV中的高尔基Rab囊泡含有被寄生虫抢救出来的鞘脂。我们的假设是，弓形虫通过将充满营养的囊泡大内吞到PV中来获得所需的脂类。这个应用程序的目标将是以机械的细节解开这一过程的复杂性，并确定未来的干预目标。我们将采用遗传、细胞、生物学和生化方法来表征PV与哺乳动物Rab GTP酶及其效应物的相互作用；鉴定介导Rab囊泡运输到PV膜和穿过PV膜的寄生虫和哺乳动物蛋白质；并通过表征脂质转运体的功能来分析寄生虫中清除的脂类的命运。。弓形虫可在免疫功能低下的人中引起致命的脑炎，目前弓形虫病的治疗选择有限且耐受性差。Rab GTP酶和Rab调节的通路是人类疾病的重要靶点，但作为治疗靶点的研究还不够深入。利用Rab GTP酶作为标记来了解弓形虫摄取宿主囊泡的过程，我们希望识别从宿主细胞窃取和/或弓形虫表达的关键因子，以暴露这种寄生虫的新脆弱性。研究这种寄生虫用来控制RAB介导的囊泡运输的机制，可能会对这些GTP酶如何协调健康细胞的膜运输产生有价值的见解。

 DESCRIPTION (provided by applicant): Membrane trafficking pathways play central roles in cell physiology, including in responses to environmental challenges like infection with microorganisms. Some pathogens are destroyed by these pathways, while others subvert their function. Toxoplasma gondii is an obligate intracellular parasite that multiplies in the cytoplasm of mammalian cells within a self-made membrane-bound compartment - the parasitophorous vacuole (PV). The PV membrane has a unique lipid and protein composition, and the PV does not fuse with any endocytic or exocytic organelles. However, we showed that the parasite's intracellular survival relies on nutrients present in mammalian organelles. For example, T. gondii retrieves cholesterol and sphingolipids from endocytic organelles and Golgi vesicles, respectively, which raises the perplexing question of how T. gondii can access the lipid content of these organelles without fusion. To address this issue, we analyzed vesicular trafficking pathways in infected mammalian cells. T. gondii targets several host pathways and intercepts the mammalian traffic mediated by Rab recycling and secretory vesicles. All of these Rab vesicles are re-routed to the PV and then sequestered intact in the vacuolar lumen. Golgi Rab vesicles trapped in the PV contain sphingolipids that are salvaged by the parasite. Our hypothesis is that T. gondii acquires the needed lipids by macroendocytosis of nutrient-filled vesicles into the PV. The goals of this application will be to unravel the complexity of this process in mechanistic detail and identify future targets for intervention. We will conduct genetic cell biological and biochemical approaches to characterize the interactions of the PV with mammalian Rab GTPases and their effectors; to identify the parasite and mammalian proteins that mediate the transport of Rab vesicles to and across the PV membrane; and to analyze the fate of the scavenged lipids in the parasite by characterizing the function of lipid transporters. . gondii can cause fatal encephalitis in immunocompromised individuals, and current treatment options for toxoplasmosis are limited and poorly tolerated. Rab GTPases and Rab-regulated pathways are important targets in human disease, yet are underexplored as therapeutic targets. Using Rab GTPAses as markers to understand the process of host vesicle uptake by T. gondii, we expect to identify key factors usurped from the host cell and/or expressed by T. gondii to expose new vulnerabilities for the parasite. Studying the mechanisms used by this parasite to control Rab-mediated vesicle trafficking may yield valuable insights into how these GTPases coordinate membrane transport in healthy cells.