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.

项目摘要 自噬是一种高度保守的，主要是由杯状细胞生长所定义的降解途径。 包裹并将细胞溶质货物（如细菌）递送到溶酶体进行降解的膜。 嗜肺军团菌是一种兼性胞内细菌，分泌超过300种效应蛋白 破坏宿主途径，如降解内体和自噬途径， 细胞内存活缺乏目前已知的自噬抑制效应物的军团菌仍然能够 逃避这一途径，表明有额外的，未发现的自噬抑制效应。以 利用这一途径的高度保守性，最近的筛选鉴定出了几种军团菌， 在酵母中阻断自噬的效应子。其中一个效应子Lem26被证实可以抑制自噬 在哺乳动物细胞中。Lem26抑制自噬以防止捕获的中心假设 和军团菌的溶酶体降解，拟议的研究旨在确定机制， Lem26抑制自噬（Aim 1）并确定其在感染背景下的生理相关性 (Aim 2）的情况。为了发现Lem26抑制自噬的机制，Aim 1利用了无偏质量 基于光谱的方法来识别Lem26的宿主靶标，以及针对Lem26的靶向实验。 基于上位性数据的推定靶点，鉴定了Lem26抑制的自噬步骤。确定 在感染的背景下，Lem26的生理相关性，目的2利用多种方法来确定Lem26的生理相关性。 细菌易位Lem26的定位和相互作用组，以及评估Lem26对 自噬的进展和军团菌的细胞内生长。研究和培训计划载于 该提案将在一个高度协作的环境中执行，该环境适合开发 批判性思维能力和技术专长所需的未来研究小组的领导者。鉴于广泛的 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，这项拟议的工作将在人类疾病中产生广泛的影响。