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

宿主囊泡靶向弓形虫的液泡和脂质货物分选

• 批准号: 9178630
• 负责人: Isabelle Coppens
• 金额: $ 40.5万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-06-01 至 2020-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9178630
• 关键词: AIDS/HIV problem; Address; Animal Model; Biochemical; Biochemical Pathway; Biological; Biological Assay; Cell membrane; Cell physiology; Cells; Cellular biology; Cholesterol; Cytoplasm; Docking; Electron Microscopy; Encephalitis; Endosomes; Enzymes; Funding; Future; Genetic; Genetic Screening; Goals; Golgi Apparatus; Guanosine Triphosphate Phosphohydrolases; Immunocompromised Host; Immunologic Surveillance; In Vitro; Individual; Infection; Inflammatory; Intercept; Intervention; Light; Lipid Mobilization; Lipids; Low-Density Lipoproteins; Mammalian Cell; Mechanics; Mediating; Membrane; Membrane Fusion; Membrane Lipids; Microscopy; Microtubules; Mitochondria; Molecular; Morphology; Mus; Nutrient; Organelles; Parasites; Pathway interactions; Patients; Period Analysis; Phospholipases A; Plasma Cells; Play; Process; Proteins; Recruitment Activity; Recycling; Resources; Role; Route; Secretory Vesicles; Signal Transduction; Sorting - Cell Movement; Sphingolipids; Sterol O-Acyltransferase; Sterols; Structure; Surface; System; Techniques; Toxoplasma; Toxoplasma gondii; Toxoplasmosis; Transmembrane Transport; Transport Vesicles; Vacuole; Vesicle; Vesicle Transport Pathway; Yeasts; design; genetic manipulation; glycolipid exchange protein; human disease; inhibitor/antagonist; insight; lipid transport; microorganism; novel; obligate intracellular parasite; oxysterol binding protein; pathogen; public health relevance; rab GTP-Binding Proteins; response; therapeutic target; trafficking; uptake

 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.

 描述（由申请人提供）：膜运输途径在细胞生理学中发挥核心作用，包括对环境挑战（如微生物感染）的响应。一些病原体被这些途径摧毁，而另一些则破坏了它们的功能。刚地弓形虫是一种专性细胞内寄生虫，在哺乳动物细胞的细胞质中在一个自制的膜结合区室-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不与任何内吞或外吞细胞器融合。然而，我们发现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T.弓形虫分别从内吞细胞器和高尔基体囊泡中获取胆固醇和鞘脂，这就提出了一个令人困惑的问题：弓形虫是如何从细胞器和囊泡中获取胆固醇和鞘脂的？刚地虫可以在不融合的情况下获得这些细胞器的脂质含量。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。T.弓形虫靶向几种宿主途径，并拦截由Rab再循环和分泌囊泡介导的哺乳动物运输。所有这些Rab囊泡被重新路由到PV，然后完整地隔离在空泡腔中。被困在PV中的高尔基体Rab囊泡含有被寄生虫挽救的鞘脂。我们假设T.弓形虫通过充满营养物的囊泡进入PV的大胞吞作用获得所需的脂质。该应用程序的目标将是从机械细节上揭示这一过程的复杂性，并确定未来的干预目标。我们将进行遗传细胞生物学和生物化学方法来表征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膜并穿过PV膜的寄生虫和哺乳动物蛋白;并通过表征脂质转运蛋白的功能来分析寄生虫中清除的脂质的命运。.弓形虫病可导致免疫功能低下的人致命性脑炎，目前弓形虫病的治疗选择有限且耐受性差。Rab GTP酶和Rab调控的通路是人类疾病中的重要靶点，但作为治疗靶点的探索不足。以Rab GTPAses为标记物，了解T.弓形虫，我们希望确定从宿主细胞篡夺和/或表达的T.弓形虫暴露了寄生虫的新弱点。研究这种寄生虫用于控制Rab-mediated囊泡运输的机制可能会对这些GTP酶如何协调健康细胞中的膜运输产生有价值的见解。