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

巨噬细胞感染性增强子(MIP)蛋白是嗜肺军团菌的主要毒力因子，也是引起军团病的病原。MIP具有多肽-脯氨酰-顺式/反式异构酶(PPIase)活性，与细胞外基质(ECM)成分IV型胶原结合，并以一种未知的机制促进细菌跨肺上皮屏障的迁移。我们先前的分析表明，胶原源多肽P290与MIP的特定疏水空腔结合，从而在体外抑制细菌上皮迁移。此外，我们还证明了嗜肺乳杆菌的分泌组特征受到MIP的强烈影响。因此，我们打算回答三个基本问题。(I)MIP如何影响嗜肺乳杆菌的蛋白质分泌？(Ii)MIP在人类肺部感染中的关键作用是什么？(Iii)MIP最初是否适应线虫等简单环境后生动物的胶原蛋白？为了解决这些问题，我们将利用蛋白质组学、底物酶谱、特异性分泌突变体和不同功能的抑制剂来分析嗜肺乳杆菌依赖MIP的分泌组以及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MIP依赖的细胞外环境变化与组织破坏和细菌复制联系起来，我们将分析肿瘤患者移植的人肺组织切片在体外感染过程中MIP依赖的组织病理学。MIP的直接和间接作用将通过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、原位酶谱和激光共聚焦扫描显微镜(CLSM)的特异性抑制试验来分析。由于初步的环境数据和体外感染强烈表明嗜肺乳杆菌定植于线虫，我们还将分析线虫富含IV型胶原的肠道对MIP的依赖感染。在嗜肺乳杆菌和线虫都很多的水生采样点，我们还将解决线虫是否是嗜肺乳杆菌的真正宿主的问题。军团菌与线虫天然相互作用的证明将开启军团菌生态、进化和毒力研究的新篇章。

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.