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 嗜肺军团菌（Lp）是军团菌病的病原体，军团菌病是一种经常致命的肺炎， 发病率。脂蛋白在水系统中无处不在，吸入受污染的飞沫后会感染肺部。在 在沃茨中，Lp作为阿米巴原虫的细胞内寄生虫和生物膜的组成部分而存在。在肺部，它生长在 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 而生物膜对于理解和预防人类疾病至关重要。在过去，Cianciotto实验室 显示Lp编码II型分泌系统（T2SS），其介导> 25种蛋白质底物的分泌。 从突变体分析来看，T2SS是感染至少4种类型的阿米巴原虫、巨噬细胞和 鼠肺该实验室还确定了感染卡氏阿米巴所需的8种基质 和其他变形虫然而，考虑到缺乏整个T2SS的突变体与更温和的突变体所显示的大缺陷， 突变体缺乏单个底物的缺陷，我认为还有更重要的T2 SS底物需要解决 found.当实验室使用蛋白质组学来定义前25种底物时，计算机分析表明， 有约60多个底物，最近的Lp上清液蛋白质组学显示，47/60的预测 底物实际上是分泌的。由于T2SS底物的浓度与T2SS底物的浓度之间存在正相关性， 在整个军团菌属的流行率和阿米巴感染的底物的要求，我做了 突变体缺乏出现在> 93%的军团菌属物种中的9/47种"新"底物中的每一种。然后我决定 新底物L20501主要促进A. castellanii。在免疫印迹证实 L20501的T2SS依赖性，生物信息学显示该蛋白代表了一种未表征的 预计作用于i）含N-乙酰葡萄糖胺的多糖脱乙酰酶家族 化合物，其可包括几丁质，ii）含乙酰基的木聚糖或乙酸纤维素，或iii）其它（新的）乙酰基- 含有底物。我进一步观察到，IAP20501突变体比WT更快地聚集， 这表明分泌的Leptin 20501可以独特地使Lp的外表面脱乙酰基， 生物膜形成因此，我认为LP20501是一种新的分泌蛋白，它能增强Lp在大肠杆菌中的存活。 多个细胞内外的小生境该方案将i）纯化LP20501并鉴定其酶活性，ii） 进一步定义与LP20501相关的表面性状，以及iii）判断LP20501在各种情况下对Lp生长的影响。 变形虫、生物膜和含乙酰基的多糖。这项工作将i）增加我们的知识， 关键病原体，ii）定义了一种新型胞外酶，iii）对使用T2SS其他病原体有影响，或 是细胞内寄生物，和iv）可能定义用于在构建环境中控制Lp的新靶标。