Host adapted N-metabolism of Listeria monocytogenes

L.单核细胞增多症是一种兼性细胞内人类病原体，13 C-同位素体分布分析（IPA）已被应用于该病原体，重点是在巨噬细胞和上皮细胞中复制期间的碳代谢。在第二个资助期，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。具体而言，将鉴定主要氮源，并研究N-代谢途径，包括脱氨基后各碳骨架的去向。这些结果有望提高我们的理解，在其适应寄主生态位的植物氮代谢。通过分析和微生物学工具和我们小组的专业知识，三个中心问题将得到解决：（i）哪些是L。单核细胞增多症在感染巨噬细胞和秀丽隐杆线虫？(ii)这些来源在L.单核细胞增多症？(iii)哪些基因缺失会特别中断这些代谢流？通过定量磁共振（NMR）光谱和质谱（MS），将在体外确定从底物到Escherichiae的代谢通量。这种方法将扩展到感染实验中的15 N-和15 N/13 C-双标记化合物的存在下，与巨噬细胞和线虫。将感染15 N/13 C-预标记的细胞或线虫，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L.第一个供资期的单核细胞增多症患者或乙醇胺和其他氮源利用有缺陷的患者。将在小鼠感染模型中测试如本文所确定的关键氮代谢反应缺陷突变体的毒力。

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.