Dynamic exometabolome analysis reveals active metabolic pathways in non‐replicating mycobacteria

Abstract

An organism's metabolic activity leaves an extracellular footprint and dynamic changes in this exometabolome inform about nutrient uptake, waste disposal and signalling activities. Using non-targeted mass spectrometry, we report exometabolome dynamics of hypoxia-induced, non-replicating mycobacteria that are thought to play a role in latent tuberculosis. Despite evidence of active metabolism, little is known about the mechanisms enabling obligate aerobic mycobacteria to cope with hypoxia, resulting in long-term survival and increased chemotherapeutic tolerance. The dynamics of 379 extracellular compounds of Mycobacterium smegmatis were deconvoluted with a genome-scale metabolic reaction-pair network to generate hypotheses about intracellular pathway usage. Time-resolved (13) C-tracing and mutant experiments then demonstrated a crucial, energy-generating role of asparagine utilization and non-generic usage of the glyoxylate shunt for hypoxic fitness. Experiments with M. bovis and M. tuberculosis revealed the general relevance of asparagine fermentation and a variable contribution of the glyoxylate shunt to non-replicative, hypoxic survival between the three species.

The bacillary and macrophage response to hypoxia in tuberculosis and the consequences for T cell antigen recognition

“…However, under hypoxia Icl appears to have multiple roles, as Icl null mutants grown on glycolytic carbon sources are significantly growth impaired at low oxygen concentrations relative to WT strains [35]. Icl may be involved in conservation of carbon units and/or maintaining optimal NADH/NAD + ratios under the reducing conditions of hypoxia by bypassing the two oxidative and CO 2 releasing TCA cycle steps, or alternatively in the maintenance of the membrane potential and/or the proton motive force (PMF) via secretion of Icl-produced succinate through a succinate/H + symport system [35], [36]. Indeed, large amounts of succinate are found to accumulate extracellularly in Mtb grown anaerobically and Icl contributes significantly to this effect [35], [36], [37].…”

“…Icl may be involved in conservation of carbon units and/or maintaining optimal NADH/NAD + ratios under the reducing conditions of hypoxia by bypassing the two oxidative and CO 2 releasing TCA cycle steps, or alternatively in the maintenance of the membrane potential and/or the proton motive force (PMF) via secretion of Icl-produced succinate through a succinate/H + symport system [35], [36]. Indeed, large amounts of succinate are found to accumulate extracellularly in Mtb grown anaerobically and Icl contributes significantly to this effect [35], [36], [37]. Elsewhere, upregulation of other fatty-acid biosynthetic and catabolic genes have been observed in Mtb grown under hypoxia ( fas , kasA , cholesterol catabolism regulation, gluconeogenic pckA ) [26], [33], [37].…”

“…The relative contributions of Icl and fumarate reduction to the production of succinate under hypoxia is debated, but is likely influenced by the available carbon source (i.e. glycolytic vs fatty acid [35], [36], [37]). …”

“…The canonical metabolic fate of glycoxylate is condensation with acetyl-CoA to form malate catalyzed by GlcB, however GlcB activity is down-regulated in hypoxic Mtb [33]. Instead, glycine levels are seen to increase in an Icl-dependent manner [35], [36], inferring subsequent reduction of glyoxylate to glycine as an alternative detoxification step under these conditions. Consistent with this hypothesis, expression and activity of glycine dehydrogenase increases substantially in hypoxic Mtb [14].…”

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

“…It also ensures a feasible CBM solution when low levels of nutrients constrain the ATP hydrolysis rate. However, GSMN-TB does not capture metabolism in the absence of oxygen (via anaerobic respiration [74,75]). This is a limitation of our model, though we do not expect it to significantly impact our results, as the metabolism of M. tuberculosis is very slow during hypoxia.…”