Analysis of the Patho-Metabolism of Legionella pneumophila during Intracellular Replication

病原菌在人类宿主内的复制是建立感染的关键事件。尽管每种宿主-微生物的相互作用都是独一无二的，但即使是系统发育上相距遥远的细菌也制定了类似的进入和逃离宿主细胞的策略。然而，对于主要的人类病原体来说，宿主细胞内的复制生态位是非常不同的。例如，军团菌、沙门氏菌和分枝杆菌存在于专门的空泡中，而弗朗西塞氏菌、李斯特氏菌和志贺氏菌则选择胞浆作为它们的复制生态位。因此，胞内细菌必须根据各自环境中可获得的营养物质和身体状况来调整自己的新陈代谢。具体地说，泡状细菌必须应对低pH值、自由基、营养缺乏和抗菌化合物，而胞浆似乎是一个更宽松的环境。直到最近，才有新的证据表明，胞内细菌使用多种底物来有效地利用其宿主细胞不同和不断变化的环境中的营养供应。也有越来越多的证据表明，细菌入侵会引发宿主细胞新陈代谢的重大重组，特别是在非癌症原代细胞中。因此，病原体和寄主之间的相互作用是高度动态的，导致在感染期间两种生物的营养和代谢途径都不同。寄主和病原菌之间这种微妙的代谢相互作用最近被称为病理代谢。虽然对于细胞内细菌的成功复制至关重要，但病理代谢是细菌感染的一个相当未被探索和利用的特征。这也适用于本提案的特定主题，即嗜肺军团菌(LP)与其宿主细胞之间的代谢关系。为了阐明细胞内LP的病理代谢，首次采用基于13C同位素图谱、GC-MS和核磁共振代谢组学、全细胞FT-IR和生长/适合性特征的综合方法研究LP及其宿主细胞，如巨噬细胞的代谢组成、途径和通量。在这一综合方法的基础上，应将感染期间的基本代谢模式和反应确定为未来抗菌药物开发的潜在目标。关于预防和控制细菌感染和耐药性的基础研究是2015年最新一届七国集团首脑会议的主要目标之一，强调了这项提案的一般议题的相关性。

The replication of pathogenic bacteria inside the human host is a crucial event for the establishment of an infection. Despite the uniqueness of each host-microbe interaction, even phylogenetically distant bacteria developed comparable strategies to enter and to escape the host cell. However, the replication niche within host cells is quite different for major human pathogens. Whereas for example Legionella, Salmonella and Mycobacterium reside in specialized vacuoles, Francisella, Listeria and Shigella have chosen the cytosol as their replicative niche. As a consequence, intracellular bacteria have to adapt their metabolism to the available nutrients and physical conditions encountered in the respective environments. Specifically, vesicular bacteria have to cope with low pH, free-radicals, nutrient deprivation and antimicrobial compounds, whereas the cytosol seems to be a more permissive milieu. Only recently, there is emerging evidence that intracellular bacteria use multiple substrates to efficiently exploit the nutrient supply from the different and changing environments of their host cells. There is also increasing evidence that the bacterial invaders thereby trigger a major re-organization of the host cell's metabolism, especially in non-cancer primary cells. Consequently, the interactions between pathogens and hosts are highly dynamic resulting in variable nutrient and metabolic pathway usages of both organisms during the infection. This delicate metabolic interplay between host and pathogen was recently termed as patho-metabolism. Although crucial for the successful replication of intracellular bacteria, patho-metabolism is a quite unexplored and unexploited feature of bacterial infections. This also holds true for the specific topic of this proposal, namely the metabolic relationship between Legionella pneumophila (Lp) and its host cells. In order to elucidate the patho-metabolism of intracellular Lp, the metabolic compositions, pathways and fluxes of Lp and its host cells, e.g. macrophages, shall be studied for the first time by an integrative approach based on 13C-isotopologue profiling, GC-MS- and NMR-based metabolomics, whole-cell FT-IR, and growth/fitness characterization of the strains under study. On the basis of this comprehensive approach, essential metabolic patterns and reactions during the infection shall be identified as potential future targets for antimicrobial drug development. Highlighting the relevance of the general topic of this proposal, basic research on the prevention and control of bacterial infections and resistance was among the major goals of the latest G7 Summit in 2015.