Mucinases as Emerging Players in Legionella pneumophila Pathogenesis

项目摘要/摘要 嗜肺军团菌是引起军团菌病肺炎的病原，其发病率呈上升趋势。 在此之前，我们证明了由II型分泌系统(T2ss)分泌的几丁质酶(Chia)是 LP在肺内的最佳存活率。由于CHIA突变体对巨噬细胞的细胞内感染没有损害 或者上皮细胞，我们假设CHIA介导了一个被忽视的细胞外事件(S)。因为甲壳素不是由 对于哺乳动物，我们进一步推测，Chia是双功能的，作用于感染肺中的甲壳素样因子。因此， 我们最近确定了Chia的结构，功能分析揭示了C端结构域(CTD)与 几丁质酶活性部位。然而，正如我们假设的那样，CTD也有一种独特的锌依赖的肽酶活性 介导粘蛋白切割的部位，包括MUC5AC，一种粘液凝胶形成的糖蛋白，在 肺部。最重要的是，我们发现：i)chia突变株上清液对粘蛋白的切割能力不足；ii) Chia突变体在体外穿透粘蛋白层的能力受到损害。因此，我们假设，恰亚 促进LP通过肺内的粘蛋白层传播，作为进入其梦寐以求的细胞内的一步 利基市场。虽然已经在各种细菌中研究了粘液酶，但我们的工作是第一次对LP进行记录 粘蛋白酶和为数不多的被定义为肺部病原体或巨噬细胞的细胞内寄生虫的酶和 上皮细胞。扩大我们的研究范围，我们想知道起亚是否也可能降解粘蛋白样糖蛋白，并发现 纯化的Chia-CTD裂解人的c1-INH。由于C1-INH是补语系统的一个组成部分，我们 假设Chia在感染中可能还有另一个作用，即促进LP的长期已知但仍然很差 理解对补体介导的杀伤的抵抗力。据我们所知，贾目前是一个独立的 降解I)甲壳素，II)肺中形成凝胶的粘蛋白，以及III)人类补体的C1-异烟肼的胞外酶 系统，除了帮助肺部的LP。因此，这一建议将区分是否由chia介导的粘蛋白降解。 促进LP进入人类细胞，如果Chia介导的C1-INH裂解使LP对 人的补体，以及如果粘蛋白-肽酶结构域与几丁质酶结构域相比促进LP生长 在肺里。尽管chia和粘蛋白之间有联系，chia突变株确实保留了一些粘蛋白降解。 活性，这表明LP分泌作用于凝胶形成粘蛋白的额外蛋白质。因此，这项提议将 我们还试图确定我们之前发现的其他哪些T2SS依赖型多肽酶可以降解 粘蛋白，也许也是补充成分。除了生成急需的LP粘蛋白酶数据外- 多肽酶和补体抗性，这项工作都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.