Function of Legionella pneumophila Mip in bacterial protein secretion, human lung tissue infection and colonization of nematodes

嗜肺军团菌Mip在细菌蛋白分泌、人肺组织感染和线虫定植中的作用

• 批准号: 232674996
• 负责人: Professor Dr. Michael Steinert
• 金额: --
• 依托单位: Institut für Mikrobiologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2013
• 资助国家: 德国
• 起止时间: 2012-12-31 至 2016-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/232674996?language=en
• 关键词: Function; Legionella; pneumophila; Mip; bacterial

The macrophage infectivity potentiator (Mip) protein is a major virulence factor of Legionella pneumophila, an aquatic bacterium and the causative agent of Legionnaires´ disease. Mip exhibits peptidyl-prolyl-cis/trans-isomerase (PPIase) activity, binds to the extracellular matrix (ECM) component collagen IV and contributes to the bacterial transmigration across the lung epithelial barrier by a yet unknown mechanism. Our previous analyses revealed that the collagen-derived peptide P290 binds to a specific hydrophobic cavity of Mip, thus inhibiting bacterial epithelial transmigration in vitro. Moreover, we demonstrated that the secretome profile of L. pneumophila is strongly influenced by Mip. Therefore, we aim to answer three fundamental questions. (i) How does Mip influence protein secretion of L. pneumophila? (ii) What is the crucial contribution of Mip during human lung infection? (iii) Did Mip initially adapt to collagen of simple environmental metazoans such as nematodes? To address these questions, the Mip-dependent secretome of L. pneumophila and the structural, enzymatic and regulatory functions of Mip during L. pneumophila protein secretion will be analyzed using a proteomic approach, substrate zymography, specific secretion mutants and functionally varied inhibitors. To link Mip-dependent alterations of the extracellular milieu with tissue destruction and bacterial replication, we will analyze the Mip-dependent histopathology during ex vivo infections of explanted human lung tissue sections from tumor patients. Direct and indirect effects of Mip will be analyzed by specific inhibition assays with PPIase inhibitors (FK506, rapamycin), the collagen binding inhibitor P290, specific protease inhibitors, in situ zymography and confocal laser scanning microscopy (CLSM). Since preliminary environmental data and in vitro infections strongly suggest that L. pneumophila colonizes nematodes, we will also analyze the Mip-dependent infection of the collagen IV-rich intestinal tract of Caenorhabditis elegans. At an aquatic sampling site, where L. pneumophila and nematodes are both abundant, we will also address the question whether or not nematodes are real hosts of L. pneumophila. The proof of a natural interaction between Legionella and nematodes would open a new chapter in the research of Legionella ecology, evolution and virulence.

巨噬细胞感染增强因子（Mip）蛋白是嗜肺军团菌的主要毒力因子，嗜肺军团菌是一种水生细菌，也是军团病的病原体。Mip具有肽基脯氨酸顺式/反式异构酶（PPIase）活性，与细胞外基质（ECM）成分胶原IV结合，并以一种未知的机制促进细菌跨越肺上皮屏障的迁移。我们之前的分析表明，胶原来源的肽P290与Mip的特定疏水腔结合，从而在体外抑制细菌上皮的迁移。此外，我们证明嗜肺乳杆菌的分泌组谱受到Mip的强烈影响。因此，我们的目标是回答三个基本问题。(i) Mip如何影响嗜肺乳杆菌的蛋白分泌？（ii） Mip在人类肺部感染中的重要作用是什么？（iii） Mip最初是否适应简单环境后生动物（如线虫）的胶原？为了解决这些问题，我们将使用蛋白质组学方法、底物酶谱学、特异性分泌突变体和功能变化抑制剂来分析嗜肺乳杆菌的Mip依赖性分泌组以及Mip在嗜肺乳杆菌蛋白分泌过程中的结构、酶和调节功能。为了将细胞外环境的mip依赖性改变与组织破坏和细菌复制联系起来，我们将分析肿瘤患者的外植人肺组织切片在离体感染期间的mip依赖性组织病理学。通过PPIase抑制剂（FK506、雷帕霉素）、胶原结合抑制剂P290、特异性蛋白酶抑制剂、原位酶谱法和共聚焦激光扫描显微镜（CLSM）的特异性抑制实验，分析Mip的直接和间接作用。由于初步的环境数据和体外感染强烈表明嗜肺乳杆菌在线虫中定植，我们还将分析秀丽隐杆线虫富含胶原iv的肠道的mip依赖性感染。在嗜肺乳杆菌和线虫都丰富的水生采样点，我们也将解决线虫是否是嗜肺乳杆菌的真正宿主的问题。军团菌与线虫自然相互作用的证明将为军团菌生态学、进化和毒力研究开辟新的篇章。