Host adapted N-metabolism of Listeria monocytogenes

单核细胞增多性乳杆菌是一种兼性的人类细胞内病原体，13C-同位素图谱分析(IPA)已被应用于巨噬细胞和上皮细胞复制过程中的碳代谢研究。对于第二个资助期，提出了一个项目，将研究这种革兰氏阳性模式生物的体内氮(N)代谢。具体地说，将确定主要的氮源(S)，并将研究氮代谢的途径，包括各自的碳链脱氨后的命运。这些结果有望加深我们对李斯特菌N在适应宿主生态位过程中代谢的理解。通过我们小组的分析和微生物学工具和专业知识，将解决三个核心问题：(I)巨噬细胞和秀丽线虫感染期间，单核细胞增多性李斯特菌的N来源是什么？(2)在单核细胞增多性李斯特菌的中心代谢中，这些来源是如何降解和利用的？(Iii)哪些基因缺失特别中断了这些代谢流？通过定量核磁共振(NMR)和质谱仪(MS)，将在体外测定从底物到李斯特菌的代谢通量。这种方法将扩展到巨噬细胞和线虫在15N-和15N/13C-双标记化合物存在下的感染实验。15N/13C-预先标记的细胞或线虫将被感染，以确定在这些条件下从宿主到细菌的代谢途径和通量。IPA将应用于从第一个资助期开始的选定的单核细胞增多性李斯特菌突变株或在乙醇胺和其他氮源的利用方面存在缺陷的突变株。在这里确定的在关键氮代谢反应中有缺陷的突变株的毒力将在小鼠感染模型中进行测试。

L. monocytogenes is a facultative intracellular human pathogen which 13C-isotopologue profiling analysis (IPA) has been applied to with a focus on carbon metabolism during replication in macrophages and epithelial cells. For the 2nd funding period, a project is proposed that will study the in vivo nitrogen (N) metabolism of this Gram-positive model organism. Specifically, main nitrogen source(s) will be identified, and the pathways of N-metabolism including the fate of the respective carbon backbones after deamination will be studied. The results are expected to improve our understanding of the listerial N metabolism during its adaptation to host niches. By the analytical and microbiological tools and expertise of our groups, three central questions will be addressed: (i) Which are the N-sources for L. monocytogenes during infection of macrophages and Caenorhabditis elegans? (ii) How are these sources degraded and utilized in the central metabolism of L. monocytogenes? (iii) Which gene deletions specifically interrupt these metabolic fluxes? By quantitative magnetic resonance (NMR) spectroscopy and mass spectrometry (MS), metabolic fluxes from substrates to listeriae will be determined in vitro. This approach will be extended to infection experiments with macrophages and nematodes in the presence of 15N- and 15N/13C-doubly labeled compounds. 15N/13C-prelabeled cells or nematodes will be infected to determine the metabolic pathways and fluxes from the host to the bacterium under these conditions. IPA will be applied to selected mutants of L. monocytogenes from the 1st funding period or those defective in the utilization of ethanolamine and other N-sources. The virulence of mutants defective in key nitrogen metabolic reactions as determined here will be tested in a mouse infection model.