Proteomic interrogation of the parasite vacuole using autoSTOMP

使用 autoSTOMP 对寄生虫液泡进行蛋白质组学研究

• 批准号: 10307153
• 负责人: Sarah E. Ewald
• 金额: $ 20.19万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-11-23 至 2023-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10307153
• 关键词: Affinity Chromatography; Animal Model; Apoptosis; Binding Proteins; Biological Response Modifiers; Cell Death; Cell Survival; Cells; Cessation of life; Chorioretinitis; Chronic; Communities; Data Set; Detection; Encephalitis; Future; Growth; Guanosine Triphosphate Phosphohydrolases; Homeostasis; Human; Image; Immune; Immune signaling; Immunity; Immunization; Individual; Infection; Infection Control; Infectious Agent; Inflammasome; Inflammation; Interferon Type II; Interferons; Life; Mediating; Membrane; Membrane Biology; Membrane Proteins; Microscope; Molecular; Mus; Nutrient; Optics; Parasite Control; Parasites; Pathway interactions; Phagolysosome; Pharmacologic Substance; Population; Proteins; Proteome; Proteomics; Regulation; Research Personnel; Rest; Rodent; Signal Transduction; Signaling Molecule; Stream; Stress; System; TLR2 gene; Technology; Testing; Therapeutic; Tissue Model; Toll-Like Receptor Pathway; Toll-like receptors; Toxoplasma; Toxoplasma gondii; Toxoplasmosis; Vacuole; Viral; Virulence; abortion; biochemical tools; cell type; guanylate; immune clearance; innate immune mechanisms; innate immune sensing; liquid chromatography mass spectrometry; new technology; novel; pathogen; recruit; sensor; tool

Project Summary Toxoplasma gondii (Tg) is an incurable, obligate intracellular protozoan parasite that naturally infects human and rodent hosts for life. Toxoplasma’s extraordinary ability to persist inside almost any nucleated cell depends on an intact parasite vacuole membrane (PVM). Secreted parasite effectors localized to the PVM interface subvert cell-intrinsic immune detection and import nutrients for growth. However, biochemical tools have not been available to identify the complete sets of host and parasite components recruited to the PVM. To recognize and clear Tg the host relies on two complementary cell autonomous immunity pathways. The interferon-y regulated GTPases (IRG) system detects the PVM as foreign and Toll-like receptors (TLRs) prime inflammasome cytosolic sensor recognition of Tg. Sub-cellular localization of the IRG system is a major point of regulation, however, the precise mechanism of PVM recognition, Tg clearance and control of host cell fate downstream of the IRG and TLR pathways are unclear. We hypothesize that immune recognition at the PVM controls parasite fate and host cell survival. To identify the critical regulators of host and parasite survival at the PVM we have developed a novel proteomics technology called automated spatially targeted optical micro proteomics or autoSTOMP. AutoSTOMP uses the confocal microscope to image the PVM and attach photo- activatable affinity purification tags to all PVM proteins so that they can be precipitated and identified by liquid chromatography and mass spectrometry (LC-MS). This tool allows us to easily compare PVM localized proteins across the three canonical types of Tg that differ in virulence by several logs. Here autoSTOMP will be used to understand how immune stimulation regulates protein recruitment to the PVM and controls the fate of Tg and host cells.

项目摘要 刚地弓形虫（Tg）是一种无法治愈的，专性细胞内原生动物寄生虫， 人类和啮齿动物的宿主弓形虫在几乎任何有核细胞内都能存活的非凡能力 依赖于完整的寄生虫空泡膜（PVM）。位于PVM的分泌型寄生虫效应物 接口颠覆了细胞内在免疫检测和输入生长营养。然而，生化工具 还没有确定完整的主机和寄生虫组成部分招募到PVM。到 宿主识别和清除Tg依赖于两条互补的细胞自主免疫途径。的 干扰素-y调节的GTPases（IRG）系统检测PVM作为外来和Toll样受体（TLR）的启动子 炎性小体胞质传感器识别Tg。IRG系统的亚细胞定位是研究IRG系统的一个要点。 然而，PVM识别、Tg清除和宿主细胞命运控制的精确机制 IRG和TLR通路的下游尚不清楚。我们假设PVM的免疫识别 控制寄生虫的命运和宿主细胞的存活。为了确定宿主和寄生虫生存的关键调节因子， 我们已经开发了一种新的蛋白质组学技术，称为自动空间靶向光学显微镜 蛋白质组学或autoSTOMP。AutoSTOMP使用共聚焦显微镜对PVM进行成像，并附上照片- 所有PVM蛋白质的可活化亲和纯化标签，使得它们可以通过液相色谱法沉淀和鉴定。 通过色谱和质谱法（LC-MS）分析。这个工具使我们能够很容易地比较PVM定位的蛋白质 在毒力相差几个对数的三种典型类型的Tg之间。在这里，autoSTOMP将用于 了解免疫刺激如何调节蛋白质募集到PVM，并控制Tg的命运， 宿主细胞