Mucinases as Emerging Players in Legionella pneumophila Pathogenesis

粘蛋白酶作为嗜肺军团菌发病机制中的新兴参与者

• 批准号: 10643053
• 负责人: NICHOLAS P CIANCIOTTO
• 金额: $ 23.03万
• 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: 2023-02-01 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10643053
• 关键词: Active Sites; Address; Alveolar; Bacteria; Binding; Biological; Biological Assay; C-terminal; CD46 Antigen; Carbohydrates; Cells; Chitin; Chitinase; Complement; Data; Development; Devices; Disease; Documentation; Epithelial Cells; Epithelium; Event; Exopeptidase; Fostering; Gel; Glycoproteins; Gram-Negative Bacteria; Growth; Human; Impairment; In Vitro; Incidence; Infection; Inhalation; Intervention; Legionella pneumophila; Legionnaires' Disease; Link; Lung; Lung infections; MUC5AC gene; Macrophage; Mammals; Mediating; Modeling; Mucins; Mucous body substance; Mus; N-terminal; Parasites; Pathogenesis; Penetration; Peptide Hydrolases; Pneumonia; Proteins; Reporting; Research Project Grants; Resistance; Role; Serum; Structure; Testing; Time; Type II Secretion System Pathway; United States National Institutes of Health; Water; Work; aerosolized; complement system; exoenzyme; extracellular; improved; in vivo; insight; intracellular parasitism; lung pathogen; monolayer; mucinase; mutant; pathogen; programs

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.

项目概要/摘要 嗜肺军团菌 (Lp) 是军团病性肺炎的病原体，该病的发病率正在不断增加。 之前，我们证明 II 型分泌系统（T2SS）分泌的几丁质酶（ChiA）是 Lp 在肺中的最佳存活率。由于 chiA 突变体不会因巨噬细胞的细胞内感染而受损 或上皮细胞，我们假设 ChiA 介导被忽视的细胞外事件。由于甲壳素不是由 哺乳动物中，我们进一步推测 ChiA 具有双功能，可作用于受感染肺部中的几丁质样因子。因此， 我们最近确定了 ChiA 的结构，功能分析揭示了 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 还具有独特的 Zn 依赖性肽酶活性 介导粘蛋白裂解的位点，包括 MUC5AC，一种粘液凝胶形成糖蛋白，表达于 肺。最重要的是，我们发现 i) chiA 突变体上清液缺乏粘蛋白的裂解和 ii) chiA突变体穿透体外粘蛋白层的能力受到损害。因此，我们假设 ChiA 促进 Lp 通过肺部粘蛋白层传播，作为进入其令人垂涎的细胞内的一步 利基市场。尽管粘蛋白酶已在多种细菌中进行了研究，但我们的工作是 Lp 的第一个记录 粘蛋白酶，是少数定义为肺部病原体或巨噬细胞胞内寄生虫的酶之一 上皮细胞。扩展我们的研究，我们想知道 ChiA 是否也可能降解粘蛋白样糖蛋白，并发现 纯化的 ChiA-CTD 可裂解人 C1-INH。由于 C1-INH 是补体系统的一个组成部分，我们 假设 ChiA 可能在感染中还有另一种作用，即促进 Lp 的作用，这一作用早已为人所知，但仍知之甚少。 了解对补体介导的杀伤的抵抗力。据我们所知，ChiA 目前是唯一一家 降解 i) 几丁质、ii) 肺部凝胶形成粘蛋白和 iii) 人类补体的 C1-INH 的外酶 系统，除了帮助肺部的 Lp 之外。因此，该提案将辨别 ChiA 介导的粘蛋白降解是否 如果 ChiA 介导的 C1-INH 裂解赋予 Lp 对杀伤的抵抗力，则促进 Lp 进入人体细胞 人类补体，并且 ChiA 的粘蛋白酶-肽酶结构域与几丁质酶结构域是否促进 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功能 活性，表明 Lp 分泌额外的蛋白质，作用于形成凝胶的粘蛋白。因此，该提案将 还试图确定我们之前发现的哪些其他 T2SS 依赖性肽酶会降解 粘蛋白也许还有补充成分。除了生成急需的 Lp 粘蛋白酶数据外， 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过程中，可能揭示疾病干预的新目标。