Glucose Phosphorylation Is Required for <em>Mycobacterium tuberculosis</em> Persistence in Mice

<div><p><em>Mycobacterium tuberculosis</em> (Mtb) is thought to preferentially rely on fatty acid metabolism to both establish and maintain chronic infections. Its metabolic network, however, allows efficient co-catabolism of multiple carbon substrates. To gain insight into the importance of carbohydrate substrates for Mtb pathogenesis we evaluated the role of glucose phosphorylation, the first reaction in glycolysis. We discovered that Mtb expresses two functional glucokinases. Mtb required the polyphosphate glucokinase PPGK for normal growth on glucose, while its second glucokinase GLKA was dispensable. ¹³C-based metabolomic profiling revealed that both enzymes are capable of incorporating glucose into Mtb's central carbon metabolism, with PPGK serving as dominant glucokinase in wild type (wt) Mtb. When both glucokinase genes, <em>ppgK</em> and <em>glkA</em>, were deleted from its genome, Mtb was unable to use external glucose as substrate for growth or metabolism. Characterization of the glucokinase mutants in mouse infections demonstrated that glucose phosphorylation is dispensable for establishing infection in mice. Surprisingly, however, the glucokinase double mutant failed to persist normally in lungs, which suggests that Mtb has access to glucose <em>in vivo</em> and relies on glucose phosphorylation to survive during chronic mouse infections.</p> </div

Rv0650 (GLKA) can mediate growth on glucose.

<p>(A) Growth of <i>ΔppgK</i> and <i>ΔppgK</i> complemented with <i>ppgK</i> on an integrative plasmid or with <i>glkA</i> on an episomal plasmid in carbon defined media with 0.4% glucose. (B, C) Growth of <i>ΔglkA</i> (B) and <i>ΔppgKΔglkA</i> (C) in carbon defined media with 0.4% glycerol (squares), 0.4% glucose (circles) or no carbon (asterisks). Open circles depict growth of the <i>ΔppgKΔglkA</i> mutant complemented with <i>ppgK</i> in glucose containing medium. (D) Glucose dose response in liquid media of wt, <i>ΔglkA</i> and <i>ΔppgK ΔglkA</i>. (E, F) Growth of wt (E) and <i>ΔppgKΔglkA</i> (F) in carbon defined media with 0.2% acetate and 0.2% acetate+0.2% glucose. Data represent two to three independent experiments.</p

Gluokinases are dispensable for trehalose metabolism and survival during starvation.

<p>(A) Growth in carbon defined media with 0.4% trehalose. (B) Survival of wt, <i>ΔglkA</i>, <i>ΔppgK</i>, and <i>ΔppgKΔglkA</i> in PBS. (C) Survival of <i>ΔppgKΔglkA</i> in carbon defined media containing 0.4% glucose or no carbon.</p

The glucokinase double mutant is hypersusceptible to hydrogen peroxide, but attenuated in phagocyte oxidase deficient mice.

<p>(A) Susceptibility to low pH, reactive nitrogen intermediates and hydrogen peroxide. Strains were exposed to pH 4.5 for 6 days, to 3 mM NaNO₂ at pH 5.5 for 3 days and to 5 mM H₂O₂ for 4 hrs and bacterial survival was determined by plating CFU. Data are means ± s.d. of triplicate cultures. Hypersusceptibility of <i>ΔppgKΔglkA</i> to 5 mM H₂O₂ was demonstrated in three independent experiments. (B) Bacterial titers in lungs of gp96^phox−/− mice infected with wt and <i>ΔppgKΔglkA</i>. Data are means ± s.d. from three mice per time point per group and representative of two independent experiments.</p

Both PPGK and GLKA mediate glucose incorporation into central carbon metabolism.

<p>Schematic illustration of the metabolic pathways studied using carbon tracing analysis and isotopic incorporation of U¹³C-glucose into the intracellular pool of selected metabolites. Isotopic labeling is indicated on the y-axis as nmol labeled/mg protein/16 h labeling interval. Each bar represents the mean of three sample replicates and error bars indicate standard deviation from the mean. nd = not detected. * P≤0.05, ** P≤0.01, *** P≤0.001. Data are representative of two independent experiments.</p

PpgK is required for normal growth with glucose as sole carbon source.

<p>Growth of wt Mtb (A) and <i>ΔppgK</i> (B) in carbon defined media with 0.4% glycerol (squares), 0.4% glucose (circles) or no carbon (asterisks). Open circles depict growth of the complemented mutant in glucose containing medium. (C) Growth of wt and <i>ΔppgK</i> in response to increasing glucose concentration. Data represent two to three independent experiments.</p

Glucose phosphorylation is required for mycobacterial persistence in lungs of chronically infected mice.

<p>Bacterial titers in lungs (A) and spleens (B) from mice infected with wt, glucokinase mutants and complemented mutant. Data are means ± s.d. from four mice per time point per group and represent two independent experiments.</p

Design and characterization of leaky TrxB2-TetON mutants.

<p>(A) Repression of luciferase activity of leaky tet promoters by TetR in <i>M</i>. <i>smegmatis</i>. The x-axis specifies the promoter that was used to express luciferase and its tetO. Mutated nucleotides are shown in red. The <i>kanR</i> luciferase and <i>hygR</i> TetR plasmids were integrated into the <i>M</i>. <i>smegmatis</i> chromosome at the att-L5 and att-Tweety sites, respectively. Integers on the right indicate fold change in RLUs between bacteria without (gray bars) and with TetR (white bars). Data are means ± SD of eight replicates from at least two independent experiments. (B) Growth of H37Rv and TrxB2-TetON-tetO mutants in 7H9 medium in the presence or absence of atc. (C) Kinetics of TrxB2 depletion in TrxB2-TetON-tetO mutants in the absence of atc. TrxB2 in TrxB2-TetON-tetO mutants is of increased molecular weight due to the C-terminal DAS tag. Results in (B) and (C) are representative of three independent experiments.</p

TrxB2 depletion perturbs growth-essential pathways.

<p>(A) Heat-map representation of selected genes with mean expression fold changes >2 in TrxB2-DUC at 6 h post atc treatment (adjusted p<0.02 by one-way ANOVA). (B) Heat-map representation of selected genes with mean expression fold change >3 in TrxB2-DUC at 24 h post atc treatment (adjusted p<0.02 by one-way ANOVA). (C) Impact of extracellular cysteine and methionine on TrxB2 depletion-induced death. Atc-treated TrxB2-DUC cultures were supplemented with 2 mM cysteine, 2 mM methionine or both. CFU were determined at the indicated time points. (D) Impact of partial TrxB2 depletion on susceptibility of <i>Mtb</i> to mitomycin C. TrxB2-TetON-tetO-1C was cultured in 7H9 medium without atc for 3 days to decrease TrxB2 expression before treatment with mitomycin C. Data in (C) and (D) are means ± SD (n = 3 per group) and are representative of three independent experiments.</p

Impact of partial TrxB2 depletion on susceptibility of <i>Mtb</i> to antimicrobial compounds.

<p>(A and B) Impact of partial TrxB2 depletion on susceptibility of <i>Mtb</i> to TrxB2 inhibitors ebselen (A) and auranofin (B). TrxB2-TetON-tetO-1C was washed and suspended in 7H9 medium without atc, then cultured for 3 days to decrease TrxB2 expression before treatment with ebselen or auranofin. OD₅₈₀ was recorded and normalized to the corresponding strain without drug treatment. (C) Survival of strains after exposure to 0.65 μg/ml or 1.5 μg/ml auranofin. (D and E) Impact of partial TrxB2 depletion on susceptibility of <i>Mtb</i> to vancomycin (D) and rifampicin (E). (F) Heat-map representation of MIC₉₀ shift of partially TrxB2 depleted <i>Mtb</i> to antimicrobial compounds. The MIC₉₀ shifts are shown as the ratio of the MIC₉₀ for H37Rv to the MIC₉₀ for TrxB2-TetON-tetO-1C in the absence of atc. Data in (A) to (E) are means ± SD of three replicates and are representative of three independent experiments. Data shown in (F) are representative of at least two independent experiments.</p