¹³C Metabolic Flux Analysis Identifies an Unusual Route for Pyruvate Dissimilation in Mycobacteria which Requires Isocitrate Lyase and Carbon Dioxide Fixation

Abstract

Mycobacterium tuberculosis requires the enzyme isocitrate lyase (ICL) for growth and virulence in vivo. The demonstration that M. tuberculosis also requires ICL for survival during nutrient starvation and has a role during steady state growth in a glycerol limited chemostat indicates a function for this enzyme which extends beyond fat metabolism. As isocitrate lyase is a potential drug target elucidating the role of this enzyme is of importance; however, the role of isocitrate lyase has never been investigated at the level of in vivo fluxes. Here we show that deletion of one of the two icl genes impairs the replication of Mycobacterium bovis BCG at slow growth rate in a carbon limited chemostat. In order to further understand the role of isocitrate lyase in the central metabolism of mycobacteria the effect of growth rate on the in vivo fluxes was studied for the first time using ¹³C-metabolic flux analysis (MFA). Tracer experiments were performed with steady state chemostat cultures of BCG or M. tuberculosis supplied with ¹³C labeled glycerol or sodium bicarbonate. Through measurements of the ¹³C isotopomer labeling patterns in protein-derived amino acids and enzymatic activity assays we have identified the activity of a novel pathway for pyruvate dissimilation. We named this the GAS pathway because it utilizes the Glyoxylate shunt and Anapleurotic reactions for oxidation of pyruvate, and Succinyl CoA synthetase for the generation of succinyl CoA combined with a very low flux through the succinate – oxaloacetate segment of the tricarboxylic acid cycle. We confirm that M. tuberculosis can fix carbon from CO ₂ into biomass. As the human host is abundant in CO ₂ this finding requires further investigation in vivo as CO ₂ fixation may provide a point of vulnerability that could be targeted with novel drugs. This study also provides a platform for further studies into the metabolism of M. tuberculosis using ¹³C-MFA.

Author Summary

The role of the enzyme isocitrate lyase (ICL) in the metabolism of Mycobacterium tuberculosis has been intensively studied since the demonstration that both of the isocitrate lyase genes encoded by this pathogen, icl1 and icl2 (although some strains only have icl1) are essential for the survival of this pathogen in the host. This finding has been generally interpreted as being due to a shift to the consumption of fats in the host. We previously demonstrated increased ICL activity when the vaccine strain of M. tuberculosis (BCG) was growing slowly on carbohydrates, suggesting a more extended role for ICL than just fat metabolism. Here we show that the gene icl1 is actually essential for slow growth on glycerol. By feeding BCG and M. tuberculosis cells isotopically labeled carbon sources and using a combination of experimental and computational analysis we identified a novel metabolic route for carbohydrate metabolism in which ICL is a key enzyme. We also demonstrated that M. tuberculosis is able to acquire biomass carbon from CO ₂. As CO ₂ is abundant in the human host, inhibition of CO ₂ fixation could be exploited in the development of novel drug treatments against tuberculosis.