Trafficking of host proteins into Toxoplasma via a novel ingestion pathway

通过新的摄入途径将宿主蛋白运输到弓形虫中

• 批准号: 8908567
• 负责人: Olivia Lauren McGovern
• 金额: $ 3.4万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8908567
• 关键词: Acute; Attenuated; Caspase; Cathepsin L; Cell membrane; Chronic; Chronic Phase; Clathrin; Disease; Dominant-Negative Mutation; Endocytosis; Genetic; Goals; Guanosine Triphosphate Phosphohydrolases; Hemoglobin; Immune; Immunofluorescence Immunologic; Individual; Infant; Infection; Ingestion; Interferon Type II; Left; Life; Lysosomes; Malaria; Mediating; Membrane; Microscopy; Organelles; Parasites; Pathway interactions; Pharmaceutical Preparations; Plasmodium falciparum; Proteins; Role; Route; Staging; Structure; System; Testing; Therapeutic Intervention; Toxoplasma; Toxoplasma gondii; Toxoplasmosis; Vacuole; Vesicle; Virulence; Work; cellular imaging; design; in vivo; killings; late endosome; latent infection; novel; obligate intracellular parasite; protein degradation; public health relevance; relating to nervous system; research study; trafficking; uptake

 DESCRIPTION (provided by applicant): Toxoplasma gondii is an obligate intracellular parasite that invaginates the host plasma membrane upon invasion to form a parasitophorous vacuole inside which the parasite replicates and survives. Like the related malaria parasite, Plasmodium falciparum, we recently discovered that T. gondii ingests host cytosolic proteins and targets them for degradation in a lysosome-like organelle called the vacuolar compartment or VAC. This discovery challenges the longstanding notion that T. gondii lacks endocytosis, because of its isolation in a non-fusogenic vacuole. Preliminary studies show that ingestion- deficient parasites display attenuated virulence and defective chronic infection in vivo. Further, these parasites are more vulnerable to killing by interferon gamma-dependent mechanisms, implying a role in immune evasion. Genetic and live cell imaging studies of intracellular parasites suggest a vesicular pathway for uptake from the intravacuolar network, a specialized structure in the parasitophorous vacuole. Immunofluorescence analysis indicates that ingested material traverses the late endosome of the parasite en route to the VAC. Our central hypothesis is that host protein-containing vesicles, generated from the intravacuolar network, are trafficked to the VAC via clathrin- mediated endocytosis. I have designed two specific aims to test this hypothesis. In Aim 1, I will use live-cell imaging to directly visualize vesicular trafficking fromthe parasitophorous vacuole membrane to the VAC to determine if the intravacuolar network is a conduit for ingestion. In Aim 2, I will use state of the art Stimulated Emission Depletion Microscopy to localize ingested host proteins within the parasite endosomal system of wild type parasites or parasites expressing dominant negative forms of suspected endocytic players to elucidate the pathway of trafficking within T. gondii.

 描述(申请人提供)：弓形虫是一种专性细胞内寄生虫，入侵时侵入宿主质膜形成寄生空泡，寄生虫在空泡内复制并存活。像相关的疟疾寄生虫恶性疟原虫一样，我们最近发现弓形虫摄取宿主胞浆蛋白，并将它们定位于一种名为空泡室或VAC的溶酶体样细胞器中进行降解。这一发现挑战了长期以来的观点，即弓形虫缺乏内吞作用，因为它被隔离在一个非融合产生的液泡中。初步研究表明，摄食缺陷寄生虫在体内表现出毒力减弱和有缺陷的慢性感染。此外，这些寄生虫更容易被干扰素依赖的伽马机制杀死，这意味着在免疫逃避中发挥了作用。对细胞内寄生虫的遗传和活细胞成像研究表明，从空泡内网络摄取寄生虫的囊泡途径，液泡内网络是寄生虫空泡中的一种特殊结构。免疫荧光分析表明，摄取的物质在进入VAC的途中穿过寄生虫的晚期内体。我们的中心假设是，从液泡内网络产生的含有宿主蛋白的囊泡通过网状蛋白介导的内吞作用被输送到VAC。我设计了两个具体目标来检验这一假设。在目标1中，我将使用活细胞成像来直接显示囊泡从寄生虫空泡膜到VAC的运输，以确定液泡内网络是否是摄取的管道。在目标2中，我将使用最先进的刺激发射耗竭显微镜来定位摄取的宿主蛋白在野生型寄生虫或表达显性阴性形式可疑内吞蛋白的寄生虫的内体系统中，以阐明弓形虫内转运的途径。