Figure 1.
Growth of L. monocytogenes EGDe in BMM (triangles) and J774A.1 cells (circles
). Bacterial growth was determined over a period of 8 h post infection (pi). Indicated are the colony forming units (cfu) after infection of 2x105 host cells with 5 bacteria per host cell. Each experiment was done three times in triplicate. Values are means ± SD of one representative experiment. The generation times given in the text were calculated from time point 1h pi to 8h pi for BMM and from 1h pi to 3h pi for J774A.1 host cells. Arrow indicates addition of gentamicin.
Figure 2.
13C-Profiles of amino acids from host cells cultivated in the presence of [U-13C6]glucose.
(A) 13C-Excess (mol %; displayed as a color code) of amino acids derived from BMM cells; for each condition, two independent labeling experiments were performed. The color for each amino acid correlates with the mean value of three MS determinations with the same sample. (B) 13C-Excess (mol %; displayed as a color code) of amino acids derived from J774A.1 cells; for each condition, two independent labeling experiments were performed. The color for each amino acid correlates with the mean value of three MS determinations with the same sample. (C) Relative isotopologue composition of amino acids derived from BMM. Colored columns indicate the % amounts of isotopologues comprising 1, 2, 3, 4, or 5 13C-atoms (M+1 to M+5; left scale; * indicate values from only one labeling experiment; otherwise, the values represent means from two labeling experiments). 13C-Excess values are shown as grey columns (mol %, right scale; error bars indicate SD for two independent labeling experiments). (D) Relative isotopologue composition of amino acids derived from J774A.1 cells. Colored columns indicate the % amounts of isotopologues comprising 1, 2, 3, 4, or 5 13C-atoms (M+1 to M+5; left scale; the values represent means from two labeling experiments). 13C-Excess values are shown as grey columns (mol %, right scale; means from two independent labeling experiments; error bars indicate SD for the two labeling experiments). Experiments were done with and without infection by Listeria monocytogenes (L.m.) and with (+) and without (-) activation by interferon-γ (IFN-γ). The color maps indicate 13C-excess in semi-logarithmic form to show even relatively small 13C excess values; for numerical values, see also Table S1.
Figure 3.
13C-Profiles of amino acids from host cells cultivated in the presence of [U-13C5]glutamine.
(A) 13C-Excess (mol %) of amino acids derived from BMM cells, (B) 13C-Excess (mol %) of amino acids derived from J774A.1 cells, (C) Relative isotopologue composition of amino acids derived from BMM. (D) Relative isotopologue composition of amino acids derived from J774A.1 cells. For more details, see legend to Figure 2; for numerical values, see also Table S2.
Figure 4.
13C-Profiles of amino acids from Listeria monocytogenes replicating in BMM or J774A.1 host cells.
(A) Experiment with [U-13C6]glucose. 13C-Excess (mol %) displayed as a color map. (B) Experiment with [U-13C5]glutamine. 13C-Excess (mol %) displayed as a color map. (C) Relative isotopologue compositions. Colored columns indicate the % amounts of isotopologues comprising 1, 2, 3, 4, or 5 13C-atoms (M+1 to M+5; left scale). 13C-Excess values are shown as grey columns (mol %, right scale); for numerical values, see also Tables S1 and S2.
Figure 5.
Reconstructed metabolite fluxes originating from glucose or glutamine in BMM (A) and J774A.1 cells (B) with or without infection by L. monocytogenes.
Black arrows indicate glucose-derived fluxes. Line widths indicate the approximate activities through these reactions. Green arrows indicate fluxes via glutaminolysis. Red arrows display flux modulations due to infection with L. monocytogenes. Bacterial pathways are highlighted in orange. The listerial metabolism is mainly fed by host cell derived [U-13C3]glycerol (deriving from [U-13C6]glucose) and [U-13C6]glucose 6-phosphate as described before [32]. The 13C-labelled intermediates for the formation of the major 13C-amino acid isotopologues derive from glycolytic reactions to pyruvate which is converted by PDH to Ac-CoA. The latter intermediate is channeled into the TCA cycle yielding together with OAA citrate and further to α-KG. OAA is produced by PYC-catalyzed carboxylation of pyruvate due to the disrupted listerial TCA cycle [47] which is also the reason for the inability of L. monocytogenes to catabolize [U-13C5]glutamine.