Mucinases as Emerging Players in Legionella pneumophila Pathogenesis

项目总结/摘要 嗜肺军团菌（Legionella pneumophila，Lp）是军团菌性肺炎的病原体，其发病率呈上升趋势。 以前，我们表明，需要由II型分泌系统（T2 SS）分泌的几丁质酶（ChiA）， Lp在肺中的最佳存活。由于chiA突变体对巨噬细胞的细胞内感染没有损害， 或上皮细胞，我们假设ChiA介导了一个被忽视的细胞外事件。由于甲壳素不是由 在哺乳动物中，我们进一步推测ChiA是双功能的，并在感染的肺中作用于几丁质样因子。因此，在本发明中， 我们最近确定了ChiA的结构，功能分析揭示了一个C末端结构域（CTD）， 几丁质酶活性位点然而，正如我们所假设的，CTD也有一个独特的锌依赖性，肽酶活性 一个介导粘蛋白裂解的位点，包括MUC 5AC，一种在大肠杆菌中表达的粘液凝胶形成糖蛋白， 肺最重要的是，我们发现i）chiA突变体上清液缺乏粘蛋白的切割，和ii） chiA突变体穿透体外粘蛋白层的能力受损。因此，我们假设， 促进Lp通过肺中的粘蛋白层传播，作为进入其梦寐以求的细胞内的一步。 壁龛虽然粘蛋白酶已经在各种细菌中进行了研究，但我们的工作是第一次证明Lp 粘蛋白酶和少数定义为肺病原体或巨噬细胞的细胞内寄生虫之一， 上皮细胞扩展我们的研究，我们想知道ChiA是否也可以降解粘蛋白样糖蛋白，并发现 纯化的ChiA-CTD切割人C1-INH。由于C1-INH是补体系统的组分，我们 假设ChiA在感染中可能还有另一个作用，即，促进LP的长期知名，但仍然很差， 了解对补体介导的杀伤的抗性。据我们所知，中国目前是一个独立的 外切酶，其降解i）几丁质，ii）肺的凝胶形成粘蛋白，和iii）人补体的C1-INH 系统，除了帮助LP在肺部。因此，该提议将辨别ChiA介导的粘蛋白降解是否 如果ChiA介导的C1-INH裂解赋予Lp对通过以下途径杀伤的抗性，则促进Lp进入人细胞 如果粘蛋白酶-肽酶结构域与ChiA的几丁质酶结构域促进Lp生长， 在肺部。尽管ChiA和粘蛋白之间存在联系，但ChiA突变体确实保留了一些粘蛋白降解 活性，表明Lp分泌作用于凝胶形成粘蛋白的额外蛋白质。因此，该提案将 我还试图确定我们以前发现的其他T2 SS依赖性肽酶中的哪一种降解 粘蛋白，也许还有补体成分。除了产生急需的关于Lp粘蛋白酶的数据外， 肽酶和补体抗性，这项工作将提高我们对粘蛋白酶的广泛理解 在肺部感染和细胞内寄生期间，并可能揭示疾病干预的新靶点。

PROJECT SUMMARY / ABSTRACT Legionella pneumophila (Lp) is the agent of Legionnaires disease pneumonia, which is increasing in incidence. Previously, we showed that a chitinase (ChiA) secreted by the type II secretion system (T2SS) is needed for optimal survival of Lp in lungs. Since chiA mutants were not impaired for intracellular infection of macrophages or epithelia, we posited that ChiA mediates an overlooked extracellular event(s). Since chitin is not made by mammals, we further surmised that ChiA is bi-functional and acts on a chitin-like factor in infected lungs. Thus, we recently determined ChiA's structure, and functional analyses revealed a C-terminal domain (CTD) with the chitinase active site. Yet, as we had hypothesized, the CTD also had a unique Zn-dependent, peptidase active site that mediates the cleavage of mucins, including MUC5AC, a mucus gel-forming glycoprotein expressed in lungs. Most significantly, we found that i) chiA mutant supernatants are deficient for cleavage of mucins and ii) the chiA mutant is impaired for the ability to penetrate an in vitro mucin layer. Thus, we hypothesize that ChiA promotes Lp dissemination through mucin layers in lungs as a step toward accessing its coveted intracellular niches. Though mucinases have been studied in various bacteria, our work was the first documentation of a Lp mucinase and one of the few defined for a lung pathogen or an intracellular parasite of macrophages and epithelia. Extending our inquiry, we wondered if ChiA might also degrade mucin-like glycoproteins and found that purified ChiA-CTD cleaves human C1-INH. Since C1-INH is a component of the complement system, we hypothesize that ChiA may have yet another role in infection, i.e., promoting Lp's long-known but still poorly understood resistance to complement-mediated killing. To our knowledge, ChiA currently stands alone as an exoenzyme that degrades i) chitin, ii) gel-forming mucins of the lung, and iii) C1-INH of the human complement system, besides aiding Lp in the lungs. Thus, this proposal will discern if ChiA-mediated degradation of mucin promotes Lp access into human cells, if ChiA-mediated cleavage of C1-INH confers Lp resistance to killing by human complement, and if the mucinase-peptidase domain vs the chitinase domain of ChiA fosters Lp growth in lungs. Despite the link between ChiA and mucin, the chiA mutant did retain some mucin-degradation activity, suggesting that Lp secretes additional proteins that act on gel-forming mucins. Thus, this proposal will also seek to identify which of the other T2SS-dependent peptidases that we previously identified degrades mucin and perhaps also complement components. Besides generating much-needed data on Lp mucinase- peptidases and complement-resistance, this work will both improve our broad understanding of mucinases during lung infection and intracellular parasitism and potentially reveal new targets for disease intervention.