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.

Keywords: Listeria monocytogenes; bacterial metabolism; bacterial pathogensis; intracellular bacteria; isotopic tracers; isotopologue profiling.

Figure 1

Intracellular replication behavior of L. monocytogenes. L. monocytogenes EGDe and its mutants were used to infect J774A.1 macrophages in the presence of 10 mM glucose (A) or 20 mM glycerol (B). 45 min after infection, extracellular bacteria were removed by washing with PBS and adding gentamycin. At the indicated intervals, intracellular L. monocytogenes were counted by disruption of the monolayer, cell lysis and plating of the supernatant on BHI.

Figure 2

Isotopologue profiling of protein-derived amino acids from L. monocytogenes grown in J774A.1 macrophages labeled with a mix of [U-¹³C]-amino acids. (A) ¹³C-Excess (in %) of amino acids from L. monocytogenes EGDe wild-type. The color map indicates ¹³C excess from 0 to 25% in quasi-logarithmic form. (B) Isotopologue profiles in listerial amino acids from the same experiment with EGDe wild-type. For comparison, the isotopologue profiles of the [U-¹³C]-amino acid mix (¹³C-Powder Growth Mix) which was supplied to the infected host cells are also shown. The colored columns indicate the relative fractions (in %) of the ¹³C-isotopologues carrying one to nine ¹³C-atoms (M+1 to M+9). For the numerical values, see Supplemental Material.

Figure 3

Analysis of protein-derived amino acids from L. monocytogenes grown in J774A.1 macrophages labeled with 10 mM [U-¹³C₆]glucose (Eylert et al., 2008) or 10 mM [U-¹³C₃]glycerol. (A) ¹³C-Excess (in % as a color map) of amino acids from L. monocytogenes EGDe. Column 1: from the labeling experiment with [U-¹³C₆]glucose and EGDe wild-type (Eylert et al., 2008), column 2: from the labeling experiment with [U-¹³C₃]glycerol and EGDe wild-type (mean value of three replicates), column 3: from the labeling experiment with [U-¹³C₃]glycerol and the EGDe ΔC3 mutant, column 4: from the labeling experiment with [U-¹³C₃]glycerol and the EGDe ΔC3ΔuhpT mutant, and column 5: from the labeling experiment with [U-¹³C₃]glycerol and the EGDe ΔldH mutant. Boxes with white asterisks indicate high standard deviations in the measurement of the overall ¹³C-enrichments. (B) Isotopologue profiles in listerial Ala, Asp, and Glu from the same experiments. The colored columns indicate the relative fractions (in %) of the ¹³C-isotopologues (M+1 to M+5). For the numerical values, see Supplemental Material.

Figure 4

Analysis of protein-derived amino acids from L. monocytogenes grown in J774A.1 macrophages labeled with 20 mM [U-¹³C₃]pyruvate or 20 mM [U-¹³C₃]lactate, respectively. (A) ¹³C-Excess (in %) of amino acids from L. monocytogenes wild-type (EGDe) and the Δldh mutant in the experiment with [U-¹³C₃]lactate. The color map again indicates ¹³C excess in quasi-logarithmic form in order to also visualize small values. Boxes with white asterisks indicate high standard deviations in the measurement of the overall ¹³C-enrichments. (B) Isotopologue profiles in listerial Ala, Asp, and Glu, respectively, from the same experiments. The colored columns indicate the relative fractions (in %) of the ¹³C-isotopologues (M+1 to M+5) in the labeled amino acids (left scales). For comparison, the gray bars indicate the ¹³C-excess values with the standard deviations from three technical replicates (right scales). (C),¹³C-Excess (in %) of amino acids from J774A.1 proteins in the infection experiments with [U-¹³C₃]pyruvate or [U-¹³C₃]lactate. (D), Isotopologue profiles of J774A.1 Ala, Asp, and Glu, respectively, from the same experiments. For the numerical values, see Supplemental Material.

Figure 5

Analysis of protein-derived amino acids from L. monocytogenes EGDe in the experiments with [1,2-¹³C₂]glucose. (A) ¹³C-excess (in % as a color map) of L. monocytogenes EGDe grown in minimal medium (MM) in the presence of 10 mM [1,2-¹³C₂]glucose (column 1) of from L. monocytogenes EGDe grown in J774A.1 cells in the presence of 10 mM [1,2-¹³C₂]glucose. Boxes with white asterisks indicate high standard deviations in the measurement of the overall ¹³C-enrichments. Isotopologue profiles in listerial Ser, Ala, Asp, and Glu, respectively, from the same experiments. The colored columns indicate the relative fractions (in %) of the ¹³C-isotopologues (M+1 to M+5) in the labeled amino acids. (B) Reaction scheme displaying the conversion of [1,2-¹³C₂]glucose into the detected isotopologues of Ala, Asp, and Glu. ¹³C-Label is indicated by blue bars. For the numerical values, see Supplemental Material.

Figure 6

Model for a bipartite metabolism for intracellular L. monocytogenes. Gray-framed yellow boxes and yellow arrows mark (by color intensity) the major carbon compounds taken up by the intracellular listeriae from the host cell. Catabolic reactions fed by glucose-6P and glycerol are indicated by blue and black arrows, respectively. Reactions leading to anabolic performances (in red-framed boxes) are indicated by red arrows. Abbreviations: PPP, pentose phosphate pathway; GL, glycolysis; GN, gluconeogenesis; TCA, tricarboxylic acid pathway; PYC, pyruvate carboxylase; ME, malic enzyme (decarboxylating malate dehydrogenase).

Figure 7

Analysis of protein-derived amino acids from J774A.1 cells labeled with [U-¹³C₆]glucose. The columns indicate ¹³C-excess (in %) of Ala, Asp, and Glu in experiments with uninfected cells, or cells infected L. monocytogenes EGDe wild-type, ΔC3, ΔuhpT, and ΔC3ΔuhpT mutants, respectively.