Host adapted N-metabolism of Listeria monocytogenes

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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.