The Anti-Autophagy Arsenal of Legionella pneumophila

嗜肺军团菌的抗自噬阿森纳

• 批准号: 10679185
• 负责人: Kevin Reyes Parducho
• 金额: $ 4.77万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10679185
• 关键词: ADP ribosylation; AGFG1 gene; Address; Autophagocytosis; Bacteria; Bacterial Translocation; Biogenesis; Biological Process; Biotinylation; Cells; Communicable Diseases; Complex; Critical Thinking; Cytoplasm; Data; Defect; Degradation Pathway; Development; Disease; Endosomes; Family; Genetic Epistasis; Grant; Growth; Immune; Immunofluorescence Immunologic; Immunoprecipitation; Infection; Institution; Invaded; Knowledge; Laboratories; Legionella; Legionella pneumophila; Libraries; Lysosomes; Mammalian Cell; Mass Spectrum Analysis; Membrane; Mentors; Mono(ADP-Ribose) Transferases; N-terminal; Nature; Nerve Degeneration; Organelles; Pathogenesis; Pathway interactions; Phosphorylation; Phosphotransferases; Physiological; Plasmids; Play; Process; Protein Family; Proteins; Proteomics; Reagent; Regulation; Reporter; Research; Role; Scientist; Shapes; Side; Signal Transduction; Students; Technical Expertise; Testing; Training; Vacuole; Virulence; Visualization; Work; Yeasts; collaborative environment; combinatorial; design; fluorescence microscope; human disease; inhibition of autophagy; inhibitor; microbial; mutant; novel; pathogen; pathogenic bacteria; prevent; protein aggregation; protein degradation; protein function; receptor; recruit; seal; skills; trafficking

PROJECT SUMMARY Autophagy is a highly conserved, primarily degradative pathway defined by the growth of a cup-shaped membrane that envelopes and delivers cytosolic cargo (such as bacteria) to the lysosome for degradation. Legionella pneumophila is a species of facultative intracellular bacteria that secretes over 300 effector proteins that subvert host pathways such as the degradative endosomal and autophagy pathways to promote intracellular survival. Legionella lacking currently known autophagy-inhibiting effectors are still capable of evading this pathway, indicating that there are additional, undiscovered autophagy-inhibiting effectors. Taking advantage of the highly conserved nature of this pathway, a recent screen identified several Legionella effectors that blocked autophagy in yeast. One of those effectors, Lem26, was confirmed to inhibit autophagy in mammalian cells. With the central hypothesis that Lem26 inhibits autophagy to prevent the capture and lysosomal degradation of Legionella, the proposed research is designed to identify the mechanism by which Lem26 inhibits autophagy (Aim 1) and determine its physiological relevance in the context of infection (Aim 2). To discover the mechanism by which Lem26 inhibits autophagy, Aim 1 utilizes both an unbiased mass spectrometry-based approach to identify the host targets of Lem26 as well as targeted experimentation on putative targets based on epistasis data identifying the autophagic step inhibited by Lem26. To determine the physiological relevance of Lem26 in the context of infection, Aim 2 utilizes multiple approaches to identify the localization and interactome of bacterially translocated Lem26 as well as assess the impact of Lem26 on the progression of autophagy and the intracellular growth of Legionella. The research and training plans laid out in this proposal will be executed in a highly collaborative environment that is suited for the development of the critical thinking skills and technical expertise required for a future research group leader. Given the broad implication of autophagy in various diseases and the common virulence strategies employed by intracellular pathogens, this proposed work will have broad implications in human disease.

项目概要 自噬是一种高度保守的、主要是降解的途径，由杯状细胞的生长所定义 包裹胞质货物（例如细菌）并将其输送至溶酶体进行降解的膜。 嗜肺军团菌是一种兼性胞内细菌，可分泌 300 多种效应蛋白 颠覆宿主途径，例如降解内体和自噬途径，以促进 细胞内存活。缺乏目前已知的自噬抑制效应器的军团菌仍然能够 逃避这个途径，表明还有其他未被发现的自噬抑制效应器。服用 利用该途径的高度保守性，最近的一项筛选鉴定出了几种军团菌 阻止酵母自噬的效应器。其中一种效应子 Lem26 被证实可以抑制自噬 在哺乳动物细胞中。中心假设是 Lem26 抑制自噬以防止捕获 和军团菌的溶酶体降解，拟议的研究旨在通过以下方式确定其机制： Lem26 抑制自噬（目标 1）并确定其在感染情况下的生理相关性 （目标 2）。为了发现 Lem26 抑制自噬的机制，Aim 1 利用了无偏质量 基于光谱测定的方法来识别 Lem26 的宿主靶标以及针对 基于确定 Lem26 抑制的自噬步骤的上位数据的推定目标。确定 Lem26 在感染情况下的生理相关性，目标 2 利用多种方法来识别 细菌易位 Lem26 的定位和相互作用组，并评估 Lem26 对细菌易位的影响 自噬的进展和军团菌的细胞内生长。中列出的研究和培训计划 该提案将在适合开发的高度协作环境中执行 未来研究小组领导者所需的批判性思维技能和技术专长。鉴于广泛 自噬在各种疾病中的意义以及细胞内自噬所采用的常见毒力策略 病原体，这项拟议的工作将对人类疾病产生广泛的影响。