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

10.1371/journal.pone.0056037PLoS ONE82

Nitrate Respiration Protects Hypoxic Mycobacterium tuberculosis Against Acid- and Reactive Nitrogen Species Stresses

There are strong evidences that Mycobacterium tuberculosis survives in a non-replicating state in the absence of oxygen in closed lesions and granuloma in vivo. In addition, M. tuberculosis is acid-resistant, allowing mycobacteria to survive in acidic, inflamed lesions. The ability of M. tuberculosis to resist to acid was recently shown to contribute to the bacillus virulence although the mechanisms involved have yet to be deciphered. In this study, we report that M. tuberculosis resistance to acid is oxygen-dependent; whereas aerobic mycobacteria were resistant to a mild acid challenge (pH 5.5) as previously reported, we found microaerophilic and hypoxic mycobacteria to be more sensitive to acid. In hypoxic conditions, mild-acidity promoted the dissipation of the protonmotive force, rapid ATP depletion and cell death. Exogenous nitrate, the most effective alternate terminal electron acceptor after molecular oxygen, protected hypoxic mycobacteria from acid stress. Nitrate-mediated resistance to acidity was not observed for a respiratory nitrate reductase NarGH knock-out mutant strain. Furthermore, we found that nitrate respiration was equally important in protecting hypoxic non-replicating mycobacteria from radical nitrogen species toxicity. Overall, these data shed light on a new role for nitrate respiration in protecting M. tuberculosis from acidity and reactive nitrogen species, two environmental stresses likely encountered by the pathogen during the course of infection

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

Mycobacterium tuberculosis metabolic versatility has been increasingly recognized as a major virulence mechanism enabling this pathogen to persist in many microenvironments encountered in its host. Glucose is one of the most abundant source of carbon 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, in addition to lipids, 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 phosphofructokinase (PFK). 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. Furthermore, in co-metabolism experiments, a disrupted glycolytic pathway resulted in decreased survival due to the accumulation of glucose-derived toxic intermediate metabolites. Coincidentally we found that glucose metabolism is highly toxic for the long term survival of hypoxic non-replicating M. tuberculosis. Indeed, M. tuberculosis survived several order of magnitudes better in a glucose-depleted culture medium, compared to what is traditionally achieved in the original glucose-supplemented medium. This novel finding improves the potential and relevance of the Wayne model for the study of the mechanisms of persistence in M. tuberculosis. In conclusion, although a functional glycolytic pathway is not required for infection and persistence in the mouse model, we propose that glycolysis is required for regulating the pool of sugar phosphate that may be otherwise toxic for hypoxic mycobacteria

Construction and characterization of a stable subgenomic dengue virus type 2 replicon system for antiviral compound and siRNA testing.

Self-replicating, non-infectious flavivirus subgenomic replicons have been broadly used in the studies of trans-complementation, adaptive mutation, viral assembly and packaging in Kunjin, yellow fever and West Nile viruses. We describe here the construction of subgenomic EGFP- or Renilla luciferase-reporter based dengue replicons of the type 2 New Guinea C (NGC) strain and the establishment of stable BHK21 cell lines harboring the replicons. In replicon cells, viral proteins and RNAs are stably expressed at levels similar to cells transfected with the full length NGC infectious RNA. Furthermore, the replicon can be packaged by separately transfected C (core)-prM (pre-membrane)-E (envelope) polyprotein construct. The replicon cells were subjected to treatment with several antiviral compounds and inhibition of the replicon was observed in treatment with known nucleoside analog inhibitors of NS5 such as 2'-C-methyladenosine (EC(50)=2.42 +/- 0.59 microM), or ribavirin (EC(50)=6.77 +/- 1.33 microM), mycophenolic acid (EC(50)=1.31 +/- 0.27 microM) and siRNA against NS3. The BHK-replicon cells have been stably maintained for about 10 passages without significant loss in reporter intensity and are sufficiently robust for both research and drug discovery

<i>pfkA</i> encodes a functional phosphofructokinase.

<p>Fructose-6-phosphate kinase activity of cell-free extracts from <i>M. tuberculosis</i> strains was measured by coupling fructose-1,6-bisphosphate formation to oxidation of NADH with aldose, triosephosphate isomerase and α-glycerophosphate dehydrogenase. Each biological sample was measured in duplicate. The data represent the values obtained for each duplicate of each biological sample. ∧ Enzymatic assay of purified recombinant His-tagged PFKA and His-tagged PFKB of <i>M. tuberculosis</i> was performed in triplicates and results are expressed as mean ± SD. Each experiment was repeated as least once independently and comparable values and trends were observed. Legend: nd, not detectable.</p

Infection profile of <i>ΔpfkA</i> mutant in mouse.

<p>8-weeks old female BALB/c mice were nasally infected with the wild-type (black circle) or Δ<i>pfkA</i> (open circle) strains. Four animals per time point per group were used. Bacterial loads in lung (A) and spleen (B) were determined by CFU counts. Data are expressed in Log₁₀ CFU per organ as the mean ± SD of four mice per group.</p

Schematic representation of the glycolytic pathway.

<p>Key committing step of glycolysis is catalyzed by <i>pfkA/pfkB</i>.</p