Characterization of Phosphofructokinase Activity in Mycobacterium tuberculosis Reveals That a Functional Glycolytic Carbon Flow Is Necessary to Limit the Accumulation of Toxic Metabolic Intermediates under Hypoxia

Abstract

Metabolic versatility has been increasingly recognized as a major virulence mechanism that enables Mycobacterium tuberculosis to persist in many microenvironments encountered in its host. Glucose is one of the most abundant carbon sources that is exploited by many pathogenic bacteria in the human host. M. tuberculosis has an intact glycolytic pathway that is highly conserved in all clinical isolates sequenced to date suggesting that glucose may represent a non-negligible source of carbon and energy for this pathogen in vivo. Fructose-6-phosphate phosphorylation represents the key-committing step in glycolysis and is catalyzed by a phosphofructokinase (PFK) activity. Two genes, pfkA and pfkB have been annotated to encode putative PFK in M. tuberculosis. Here, we show that PFKA is the sole PFK enzyme in M. tuberculosis with no functional redundancy with PFKB. PFKA is required for growth on glucose as sole carbon source. In co-metabolism experiments, we report that disruption of the glycolytic pathway at the PFK step results in intracellular accumulation of sugar-phosphates that correlated with significant impairment of the cell viability. Concomitantly, we found that the presence of glucose is highly toxic for the long-term survival of hypoxic non-replicating mycobacteria, suggesting that accumulation of glucose-derived toxic metabolites does occur in the absence of sustained aerobic respiration. The culture medium traditionally used to study the physiology of hypoxic mycobacteria is supplemented with glucose. In this medium, M. tuberculosis can survive for only 7–10 days in a true non-replicating state before death is observed. By omitting glucose in the medium this period could be extended for up to at least 40 days without significant viability loss. Therefore, our study suggests that glycolysis leads to accumulation of glucose-derived toxic metabolites that limits long-term survival of hypoxic mycobacteria. Such toxic effect is exacerbated when the glycolytic pathway is disrupted at the PKF step.

Central Role of Pyruvate Kinase in Carbon Co-catabolism of Mycobacterium tuberculosis

“…Previous studies with a phosphofructokinase A knock-out strain of Mtb showed glucose toxicity under hypoxic but not aerobic conditions (15). The difference in phenotype may arise from the position of the two enzymes in the glycolytic pathway.…”

“…Recent studies with the adenosine triphosphate (ATP) synthase inhibitor, Bedaquiline, showed a delayed cidal effect when Mtb was grown on fermentable carbon sources (13), suggesting that Mtb can produce ATP through substrate level phosphorylation when oxidative-phosphorylation is limited. However, studies in mice with Mtb strains containing knockouts of glycolytic enzymes did not result in strong attenuation, thus leaving the role of glucose metabolism in the virulence and physiology of Mtb unclear (14,15).…”

Multifunctional T Cell Response to DosR and Rpf Antigens Is Associated with Protection in Long-Term Mycobacterium tuberculosis-Infected Individuals in Colombia

“…Furthermore, it has been reported that DosR antigens, including Rv2029c, induce predominant mono-and bifunctional (IFN-␥ and/or TNF-␣) CD4 ϩ and/or CD8 ϩ T cell responses in LTBI (33). Rv2029c (pfkB), a probable phosphofructokinase, is a key enzyme in glycolysis (58). A recent study suggests that, in M. tuberculosis, glycolysis leads to the accumulation of toxic metabolites, limiting M. tuberculosis survival under hypoxic conditions (58).…”

“…Rv2029c (pfkB), a probable phosphofructokinase, is a key enzyme in glycolysis (58). A recent study suggests that, in M. tuberculosis, glycolysis leads to the accumulation of toxic metabolites, limiting M. tuberculosis survival under hypoxic conditions (58). Rv1737c ϩ and TNF-␣ ϩ CD8 ϩ T cells in ltLTBI and PTB.…”

Multiscale Model of Mycobacterium tuberculosis Infection Maps Metabolite and Gene Perturbations to Granuloma Sterilization Predictions

“…Additionally, glycolysis under hypoxic conditions leads to the accumulation of toxic byproducts (65). Therefore, fatty acids are considered to be a major carbon source of M. tuberculosis in vivo.…”