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（由申请方提供）：刚地弓形虫是一种专性细胞内寄生虫，在侵入宿主细胞膜后内陷，形成寄生虫空泡，寄生虫在空泡内复制并存活。与相关的疟疾寄生虫恶性疟原虫一样，我们最近发现T。刚地虫摄取宿主胞质蛋白，并将它们靶向在称为空泡室或VAC的溶酶体样细胞器中降解。这一发现挑战了T.刚地虫缺乏内吞作用，因为它被隔离在非融合液泡中。初步研究表明，摄食缺陷型寄生虫在体内表现出毒力减弱和慢性感染缺陷.此外，这些寄生虫更容易被干扰素γ依赖性机制杀死，这意味着在免疫逃避中的作用。细胞内寄生虫的遗传和活细胞成像研究表明，从液泡内网络，寄生虫液泡中的专门结构的吸收囊泡途径。免疫荧光分析表明，摄入的物质穿过寄生虫的晚期内体到达VAC。我们的中心假设是，宿主蛋白质含有囊泡，产生的液泡内网络，被贩运到VAC通过网格蛋白介导的内吞作用。我设计了两个具体的目标来检验这个假设。在目标1中，我将使用活细胞成像直接可视化从寄生虫空泡膜到VAC的囊泡运输，以确定空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尽显微镜来定位野生型寄生虫或表达显性阴性形式的疑似内吞作用因子的寄生虫的寄生虫内体系统内的摄入的宿主蛋白，以阐明T.刚地