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Analysis of carbon substrates used by Listeria monocytogenes during growth in J774A.1 macrophages suggests a bipartite intracellular metabolism.

ABSTRACT: Intracellular bacterial pathogens (IBPs) are dependent on various nutrients provided by the host cells. Different strategies may therefore be necessary to adapt the intracellular metabolism of IBPs to the host cells. The specific carbon sources, the catabolic pathways participating in their degradation, and the biosynthetic performances of IBPs are still poorly understood. In this report, we have exploited the technique of (13)C-isotopologue profiling to further study the carbon metabolism of Listeria monocytogenes by using the EGDe wild-type strain and mutants (defective in the uptake and/or catabolism of various carbon compounds) replicating in J774A.1 macrophages. For this goal, the infected macrophages were cultivated in the presence of [1,2-(13)C2]glucose, [U-(13)C3]glycerol, [U-(13)C3]pyruvate, [U-(13)C3]lactate, or a mix of [U-(13)C]amino acids. GC/MS-based isotopologue profiling showed efficient utilization of amino acids, glucose 6-phosphate, glycerol, and (at a low extent) also of lactate but not of pyruvate by the IBPs. Most amino acids imported from the host cells were directly used for bacterial protein biosynthesis and hardly catabolized. However, Asp was de novo synthesized by the IBPs and not imported from the host cell. As expected, glycerol was catabolized via the ATP-generating lower part of the glycolytic pathway, but apparently not used for gluconeogenesis. The intermediates generated from glucose 6-phosphate in the upper part of the glycolytic pathway and the pentose phosphate shunt likely serve primarily for anabolic purposes (probably for the biosynthesis of cell wall components and nucleotides). This bipartite bacterial metabolism which involves at least two major carbon substrates-glycerol mainly for energy supply and glucose 6-phosphate mainly for indispensible anabolic performances-may put less nutritional stress on the infected host cells, thereby extending the lifespan of the host cells to the benefit of the IBPs.

SUBMITTER: Grubmuller S

PROVIDER: S-EPMC4217532 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Analysis of carbon substrates used by Listeria monocytogenes during growth in J774A.1 macrophages suggests a bipartite intracellular metabolism.

Grubmüller Stephanie S Schauer Kristina K Goebel Werner W Fuchs Thilo M TM Eisenreich Wolfgang W

Frontiers in cellular and infection microbiology 20141103

Intracellular bacterial pathogens (IBPs) are dependent on various nutrients provided by the host cells. Different strategies may therefore be necessary to adapt the intracellular metabolism of IBPs to the host cells. The specific carbon sources, the catabolic pathways participating in their degradation, and the biosynthetic performances of IBPs are still poorly understood. In this report, we have exploited the technique of (13)C-isotopologue profiling to further study the carbon metabolism of Li ...[more]

PMID: 25405102

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Project description:MiRNAs are important post-transcriptional regulators of gene expression. MiRNA expression is a crucial part of host responses to bacterial infection, however there is limited knowledge of their impact on the outcome of infections. We investigated the influence of miR-21 on macrophage responses during infection with Listeria monocytogenes, which establishes an intracellular niche within macrophages. MiR-21 is induced following infection of bone marrow-derived macrophages (BMDMs) with Listeria. MiR-21-/- macrophages display an increased bacterial burden with Listeria at 30 min and 2 h post-infection. This phenotype was reversed by the addition of synthetic miR-21 mimics to the system. To assess the immune response of wildtype (WT) and miR-21-/- macrophages, BMDMs were treated with bacterial LPS or infected with Listeria. There was no difference in IL-10 and IL-6 between WT and miR-21-/- BMDMs in response to LPS or Listeria. TNF-? was increased in miR-21-/- BMDMs stimulated with LPS or Listeria compared to WT macrophages. We next assessed the production of nitric oxide (NO), a key bactericidal factor in Listeria infection. There was no significant difference in NO production between WT and miR-21-/- cells, indicating that the increased bacterial burden may not be due to impaired killing. As the increased bacterial load was observed early following infection (30 min), we questioned whether this is due to differences in uptake of Listeria by WT and miR-21-/- macrophages. We show that miR-21-deficiency enhances uptake of FITC-dextran and FITC-Escherichia coli bioparticles by macrophages. The previously observed Listeria burden phenotype was ablated by pre-treatment of cells with the actin polymerization inhibitor cytochalasin-D. From analysis of miR-21 targets, we selected the pro-phagocytic regulators myristoylated alanine-rich C-kinase substrate (MARCKS) and Ras homolog gene family, member B (RhoB) for further investigation. MARCKS and RhoB are increased in miR-21-/- BMDMs, correlating with increased uptake of Listeria. Finally, intra-peritoneal infection of mice with Listeria led to increased bacterial burden in livers of miR-21-/- mice compared to WT mice. These findings suggest a possible role for miR-21 in regulation of phagocytosis during infection, potentially by repression of MARCKS and RhoB, thus serving to limit the availability of the intracellular niche of pathogens like L. monocytogenes.

Dataset Information

Analysis of carbon substrates used by Listeria monocytogenes during growth in J774A.1 macrophages suggests a bipartite intracellular metabolism.

Publications

Analysis of carbon substrates used by Listeria monocytogenes during growth in J774A.1 macrophages suggests a bipartite intracellular metabolism.

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