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

项目摘要 嗜肺军团菌(LP)是军团病的病原体，军团病是一种经常致命的肺炎，在 发病率。LP在水系统中普遍存在，在吸入受污染的飞沫后感染肺部。在……里面 Waters，LP作为阿米巴的一种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，模仿阿米巴的功能。了解LP如何在水中生存并在阿米巴虫中生长 生物膜对于理解并可能预防人类疾病至关重要。在过去，Cianciotto实验室 Lp编码一种II型分泌系统(T2SS)，该系统调节&gt；25蛋白底物的分泌。 从突变分析来看，至少4种阿米巴、巨噬细胞和 小鼠的肺。实验室还确定了感染卡氏棘阿米巴所需的8种底物。 和其他阿米巴虫。然而，考虑到缺乏整个T2SS的突变体所表现出的巨大缺陷，与更温和的 缺乏单独底物的突变体的缺陷，我推测有更重要的T2SS底物是 找到了。当实验室使用蛋白质组学来定义最初的25种底物时，计算机分析表明 还有60多个底物，最近LP上清液的蛋白质组学表明，预测的47/60 底物实际上是分泌出来的。因为T2SS底物之间存在正相关 军团菌在整个属中的流行率以及该底物对阿米巴感染的要求 缺乏9/47“新”底物的突变体出现在93%的军团菌种中。然后我决定 新底物Lpw20501主要促进卡氏拟青霉的侵染。在免疫印迹确认后 对Lpw20501的T2SS依赖，生物信息学显示该蛋白代表一种未鉴定的 多糖脱乙酰酶家族，被预测作用于I)含N-乙酰氨基葡萄糖 化合物，可包括甲壳素，ii)含有乙酰基的木聚糖或醋酸纤维素，或iii)其他(新的)乙酰基- 含有底物的。我进一步观察到，lpw20501突变体的聚集速度比wt更快， 提示分泌的Lpw20501可能唯一地使LP的外表面脱乙酰化，从而影响 生物膜的形成。因此，我推测Lpw20501是一种新的分泌蛋白，可以增强LP在体内的存活。 多个细胞内和细胞外的利基。本方案将：1)纯化Lpw20501并鉴定其酶活性；2) 进一步确定与Lpw20501连锁的表面性状，并判断Lpw20501‘S对不同品种Lp生长的影响 阿米巴，在生物膜中，以及在含有乙酰基的多糖上。这项工作将I)增加我们对一个 关键病原体，II)定义一种新类型的胞外酶，III)对使用T2SS或 是细胞内的寄生虫，以及iv)可能定义了在建筑环境中控制LP的新靶点。

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.