Glucose phosphorylation is required for Mycobacterium tuberculosis persistence in mice

Abstract

Mycobacterium tuberculosis (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. (13)C-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, ppgK and glkA, 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 in vivo and relies on glucose phosphorylation to survive during chronic mouse infections.

Figure 1. PpgK is required for normal growth with glucose as sole carbon source.

Growth of wt Mtb (A) and ΔppgK (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 ΔppgK in response to increasing glucose concentration. Data represent two to three independent experiments.

Figure 2. Rv0650 (GLKA) can mediate growth on glucose.

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

Figure 3. Both PPGK and GLKA mediate glucose incorporation into central carbon metabolism.

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.

Figure 4. Glucose phosphorylation is required for mycobacterial persistence in lungs of chronically infected mice.

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.

Figure 5. Gluokinases are dispensable for trehalose metabolism and survival during starvation.

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

Figure 6. The glucokinase double mutant is hypersusceptible to hydrogen peroxide, but attenuated in phagocyte oxidase deficient mice.

(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 ΔppgKΔglkA to 5 mM H₂O₂ was demonstrated in three independent experiments. (B) Bacterial titers in lungs of gp96^phox−/− mice infected with wt and ΔppgKΔglkA. Data are means ± s.d. from three mice per time point per group and representative of two independent experiments.