Gluconeogenic carbon flow of tricarboxylic acid cycle intermediates is critical for Mycobacterium tuberculosis to establish and maintain infection

Abstract

Metabolic adaptation to the host niche is a defining feature of the pathogenicity of Mycobacterium tuberculosis (Mtb). In vitro, Mtb is able to grow on a variety of carbon sources, but mounting evidence has implicated fatty acids as the major source of carbon and energy for Mtb during infection. When bacterial metabolism is primarily fueled by fatty acids, biosynthesis of sugars from intermediates of the tricarboxylic acid cycle is essential for growth. The role of gluconeogenesis in the pathogenesis of Mtb however remains unaddressed. Phosphoenolpyruvate carboxykinase (PEPCK) catalyzes the first committed step of gluconeogenesis. We applied genetic analyses and (13)C carbon tracing to confirm that PEPCK is essential for growth of Mtb on fatty acids and catalyzes carbon flow from tricarboxylic acid cycle-derived metabolites to gluconeogenic intermediates. We further show that PEPCK is required for growth of Mtb in isolated bone marrow-derived murine macrophages and in mice. Importantly, Mtb lacking PEPCK not only failed to replicate in mouse lungs but also failed to survive, and PEPCK depletion during the chronic phase of infection resulted in mycobacterial clearance. Mtb thus relies on gluconeogenesis throughout the infection. PEPCK depletion also attenuated Mtb in IFNgamma-deficient mice, suggesting that this enzyme represents an attractive target for chemotherapy.

Fig. 1.

PEPCK is necessary for growth with a fatty acid as sole carbon source. Growth in carbon-defined media of WT Mtb (□), ΔpckA (●), ΔpckA complemented using its native promoter (△), and complemented under TetR control (pckA-TetON, ⋄). (A) Growth in media with 0.1% glycerol (black), glucose (gray), or no carbon (white). (B) Growth in media with 0.1% acetate or (C) 0.1% valeric acid. (D) Growth in media with 0.1% glycerol and 0.1% acetate (gray) or 0.1% glycerol and 0.1% valeric acid (black). Data represent one of three independent experiments.

Fig. 2.

Gluconeogensis is blocked in the absence of PEPCK. Schematic illustration of metabolic pathways studied using carbon tracing analysis and relative extent of incorporation of U-¹³C acetate or U-¹³C glucose into the intracellular pool of selected metabolites in WT, ΔpckA, and complemented mutant. Strains were grown to log-phase on permissive media and then exposed to U-¹³C acetate or U-¹³C glucose for 16 h. U-¹³C–labeled carbon sources are indicated in green, and analyzed metabolites are highlighted in blue. Dashed arrows represent more than one enzymatic step. PEPCK, phoshoenolpyruvate carboxykinase; MEZ, malic enzyme; OAA, oxaloacetate; and PEP, phoshoenolpyruvate. Each bar represents the mean of three sample replicates; error bars indicate the SD. Data are representative of two independent experiments.

Fig. 3.

PEPCK is necessary for replication in macrophages. CFU of WT (□), ΔpckA (●), complemented mutant (△), and pckA-TetON mutant (⋄) in bone marrow–derived macrophages. (A) Infection of resting macrophages. (B) Infection of IFNγ-activated macrophages. (C) Infection of resting macrophages with ΔpckA and pckA-TetON. One set of macrophages infected with pckA-TetON received atc at day 2. Data represent the mean of triplicate cultures; error bars indicate the SD. Data shown in A and B are representative of three independent experiments.

Fig. 4.

PEPEC is essential for growth and survival in mice. Bacterial loads in lungs from mice infected with WT (□), ΔpckA (●), or pckA-TetON (⋄). PckA expression in pckA-TetON was induced by feeding mice the inducer doxycycline (doxy) and silenced by removal of doxy from food. Mice infected with pckA-TetON were fed with doxy for the entire experiment (green diamond), for 10 days (blue diamond), for 21 days (gray diamond), or for 40 days (orange diamond). In mice kept without doxy food (red diamonds), bacteria could be detected only in two (day 56) or one (day 112) of four animals (entire lungs were plated for CFU). Dashed lines indicate that no CFU were detected in whole lungs at the next time point. Data represents the mean of four mice per group; error bars indicate the SD.

Fig. 5.

In vivo killing of ΔpckA occurs independently of IFNγ-mediated immune responses. Bacterial loads in lungs from IFNγ^−/− mice infected with pckA-TetON. PckA expression was induced (■), silenced during the entire experiment (●) or silenced at day 10 (□). Data represent the mean of data from four mice per group; error bars indicate the SD.