Importance and function of highly conserved substrates of the Legionella pneumophila Type II Secretion System for Infection

嗜肺军团菌 II 型感染分泌系统高度保守底物的重要性和功能

• 批准号: 10678523
• 负责人: Carlton Adams
• 金额: $ 4.77万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10678523
• 关键词: Acanthameba infection; Acanthamoeba castellanii; Acetates; Amoeba genus; Bioinformatics; Cellulose; Chitin; Complement; Deacetylase; Deacetylation; Defect; Dependence; Doctor of Philosophy; Education; Family; Fellowship; Goals; Growth; Health; Human; Incidence; Individual; Individual National Research Service Award; Infection; Inhalation; Knowledge; Learning; Legionella; Legionella pneumophila; Legionnaires' Disease; Life Cycle Stages; Link; Locales; Lung; Macrophage; Mediating; Microbial Biofilms; Muramidase; Mus; Nature; Parasites; Pathogenesis; Pathway interactions; Peptide Signal Sequences; Peptidoglycan; Phase; Pneumonia; Polysaccharides; Prevalence; Protein Secretion; Proteins; Proteomics; Research; Role; Scientist; Surface; System; Training; Type II Secretion System Pathway; Water; Western Blotting; Work; Xylans; built environment; contaminated water; disease diagnosis; disorder prevention; enzyme activity; exoenzyme; extracellular; human disease; in silico; mutant; novel; pathogen; polysaccharide deacetylase; pre-doctoral; prevent; trait

Project Summary Legionella pneumophila (Lp) is the agent of Legionnaires' disease, an oft-fatal pneumonia that is increasing in incidence. Lp is ubiquitous in water systems, infecting the lungs after inhalation of contaminated droplets. In waters, Lp persists as an intracellular parasite of amoebae and constituent of biofilms. In the lungs, it grows in macrophages, mimicking what it does in amoebae. Learning how Lp survives in water and grows in amoebae and biofilms is critical to understanding and possibly preventing human disease. In past, the Cianciotto lab showed that Lp encodes a type II secretion system (T2SS) which mediates secretion of >25 protein substrates. From mutant analysis, the T2SS was required for infection of at least 4 types of amoebae, macrophages, and the murine lung. The lab also identified 8 substrates that are required for infection of Acanthamoeba castellanii and other amoebae. Yet, given the large defect shown by mutants lacking the entire T2SS vs the more modest defects of mutants lacking individual substrates, I posited there are more important T2SS substrates to be found. When the lab had used proteomics to define the first 25 substrates, in silico analysis suggested that there are ~60 more substrates, and recent proteomics on Lp supernatants showed that 47/60 of the predicted substrates are in fact secreted. Since there had been a positive correlation between a T2SS substrate’s prevalence across the Legionella genus and the requirement of that substrate for infection of amoebae, I made mutants lacking each of the 9/47 “new” substrates that occur in > 93% of Legionella species. I then determined that the new substrate Lpw20501 majorly promotes infection of A. castellanii. After immunoblot confirmation of the T2SS-dependency of Lpw20501, bioinformatics revealed that the protein represents an uncharacterized family of polysaccharide deacetylases that is predicted to act on i) N-acetylglucosamine-containing compounds, which may include chitin, ii) acetyl-containing xylan or cellulose acetate, or iii) other (novel) acetyl- containing substrates. I further observed that the lpw20501 mutant aggregated more rapidly than WT did, suggesting that secreted Lpw20501 may uniquely deacetylate the outer surface of Lp and as a result impact biofilm formation. Thus, I posit that Lpw20501 is a novel secreted protein that enhances the survival of Lp in multiple intra- and extracellular niches. This proposal will i) purify Lpw20501 and discern its enzyme activity, ii) further define surface traits tied to Lpw20501, and iii) judge Lpw20501’s impact on Lp growth in various amoebae, in biofilms, and on acetyl-containing polysaccharides. This work will i) increase our knowledge of a key pathogen, ii) define a new type of exoenzyme, iii) have implications for other pathogens that use T2SS or are intracellular parasites, and iv) possibly define a new target for controlling Lp in the built environment.

项目摘要 肺炎军团（LP）是军团疾病的药物，这是一种通常致命的肺炎，正在增加 发病率。 LP在水系统中无处不在，在吸入受污染的液滴后感染了肺部。 LP Waters，作为变形虫的细胞内寄生虫，构成生物膜。在肺中，它长大了 巨噬细胞，模仿它在变形虫中的作用。了解LP如何在水中生存并在变形虫中生长 生物膜对于理解和可能预防人类疾病至关重要。过去，Cianciotto实验室 表明LP编码II型分泌系统（T2SS），该系统介导> 25蛋白底物的分泌。 从突变分析中，T2SS至少感染了4种类型的变形虫，巨噬细胞和 鼠肺。该实验室还鉴定了8个梯形的基材 和其他变形虫。但是，鉴于缺乏整个T2SS与更谦虚的突变体显示的大缺陷 缺乏个体底物的突变体的缺陷，我认为有更多重要的T2SS基板为 成立。当实验室使用蛋白质组学定义前25个底物时，在计算机分析中表明， LP上清液上的近期蛋白质组学更多的底物表明，有47/60的预测 底物实际上是分泌的。由于T2SS基板之间存在正相关 整个军团菌属的患病率以及该基材对变形虫感染的需求，我使 缺少9/47个“新”底物的突变体发生在> 93％的军团菌种类中。然后我确定 新的底物LPW20501主要促进了Castellanii的感染。免疫印迹确认后 LPW20501的T2SS依赖性，生物信息学表明该蛋白质代表一种未表征 多糖脱乙酰基酶家族，预测对I）含N-乙酰葡萄糖的作用 化合物，其中可能包括几丁质，ii）含有乙酰基的木聚糖或乙酸纤维素，或iii）其他（新颖的）乙酰基 - 包含底物。我进一步观察到，LPW20501突变体汇总比WT更快， 表明分泌的LPW20501可能会唯一地脱乙酰化LP的外表面，因此影响 生物膜形成。我认为LPW20501是一种新型的分泌蛋白，可增强LP在 多个细胞内和细胞外壁ni。该建议将i）净化LPW20501并辨别其酶活性，ii） 进一步定义了与LPW20501相关的表面特征，以及III）LPW20501法官对各种LP增长的影响 在生物膜中和含乙酰基多糖中的变形虫。这项工作我会）提高我们对 关键病原体，ii）定义一种新型的外酶，iii）对使用T2SS或 是细胞内寄生虫，iv）可能定义一个在建筑环境中控制LP的新目标。