Abstract
Enzymes at the phosphoenolpyruvate (PEP)–pyruvate–oxaloacetate or anaplerotic (ANA) node control the metabolic flux to glycolysis, gluconeogenesis, and anaplerosis. Here we used genetic, biochemical, and 13C isotopomer analysis to characterize the role of the enzymes at the ANA node in intracellular survival of the world's most successful bacterial pathogen, Mycobacterium tuberculosis (Mtb). We show that each of the four ANA enzymes, pyruvate carboxylase (PCA), PEP carboxykinase (PCK), malic enzyme (MEZ), and pyruvate phosphate dikinase (PPDK), performs a unique and essential metabolic function during the intracellular survival of Mtb. We show that in addition to PCK, intracellular Mtb requires PPDK as an alternative gateway into gluconeogenesis. Propionate and cholesterol detoxification was also identified as an essential function of PPDK revealing an unexpected role for the ANA node in the metabolism of these physiologically important intracellular substrates and highlighting this enzyme as a tuberculosis (TB)-specific drug target. We show that anaplerotic fixation of CO2 through the ANA node is essential for intracellular survival of Mtb and that Mtb possesses three enzymes (PCA, PCK, and MEZ) capable of fulfilling this function. In addition to providing a back-up role in anaplerosis we show that MEZ also has a role in lipid biosynthesis. MEZ knockout strains have an altered cell wall and were deficient in the initial entry into macrophages. This work reveals that the ANA node is a focal point for controlling the intracellular replication of Mtb, which goes beyond canonical gluconeogenesis and represents a promising target for designing novel anti-TB drugs.
Highlights
Enzymes at the phosphoenolpyruvate (PEP)–pyruvate– oxaloacetate or anaplerotic (ANA) node control the metabolic flux to glycolysis, gluconeogenesis, and anaplerosis
The ANA node of Mycobacterium tuberculosis (Mtb) consists of the enzymes pyruvate carboxylase (PCA), PEP carboxykinase (PCK), malic enzyme (MEZ), and pyruvate phosphate dikinase (PPDK) (Fig. 1)
This phenotype could be complemented by reintroducing the gene into the genome under control of the constitutive hsp60 promoter into the attB site; but could be complemented by addition of aspartate (as proxy for oxaloacetate, which is labile [14] to the media or by growing the cultures in the presence of 5% CO2 (Table S1). These results demonstrated that PCA is performing an anaplerotic function replenishing oxaloacetate into the TCA cycle when growing on these substrates; but that this role can be complemented when sufficient CO2 is present presumably by the actions of either MEZ or PCK
Summary
Enzymes at the phosphoenolpyruvate (PEP)–pyruvate– oxaloacetate or anaplerotic (ANA) node control the metabolic flux to glycolysis, gluconeogenesis, and anaplerosis. We show that each of the four ANA enzymes, pyruvate carboxylase (PCA), PEP carboxykinase (PCK), malic enzyme (MEZ), and pyruvate phosphate dikinase (PPDK), performs a unique and essential metabolic function during the intracellular survival of Mtb. We show that in addition to PCK, intracellular Mtb requires PPDK as an alternative gateway into gluconeogenesis. The ANA node of Mtb consists of the enzymes pyruvate carboxylase (PCA), PEP carboxykinase (PCK), malic enzyme (MEZ), and pyruvate phosphate dikinase (PPDK) (Fig. 1) Of these enzymes, only PCK has been shown to have an essential role in gluconeogenesis when Mtb is growing on lipid substrates in vitro and for survival in macrophage and murine models of TB [4] or hypoxia in a chemostat [10].
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