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 CO2 into biomass. As the human host is abundant in CO2 this finding requires further investigation in vivo as CO2 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.

Multifunctional essentiality of succinate metabolism in adaptation to hypoxia in Mycobacterium tuberculosis

“…1). This pattern was indicative of an activation of M. tuberculosis isocitrate lyases (ICLs), which serve dual roles in the glyoxylate shunt and methylcitrate cycles, and have previously been reported to be up-regulated in M. tuberculosis in response to hypoxia and essential for survival of bacillus Calmette-Guérin at low growth rates (37,38). Accordingly, we observed a hypoxia-induced increase in icl transcript levels even when M. tuberculosis was already metabolizing acetate ( Fig.…”

“…ICLdeficient M. tuberculosis is one of the most severely attenuated mutants tested in a mouse model of TB. Such attenuation may reflect the loss of multiple functions, some of which were revealed by metabolomic studies and could not be foreseen by genetic or bioinformatic approaches (37,38).…”

Genetic Strategies for Identifying New Drug Targets

“…The glyoxylate shunt enzymes isocitrate lyase (20,21) and malate synthase (22) are good examples and are currently being explored as drug targets. Similarly, Beste et al recently showed that anaplerotic reactions are likely to be important during infection (23). This is corroborated by the fact that inhibitors of carbonic anhydrase were shown to have an antibiotic effect on M. tuberculosis (24).…”

Mycobacterium tuberculosis Phosphoenolpyruvate Carboxykinase Is Regulated by Redox Mechanisms and Interaction with Thioredoxin

“…This suggests that Pck might contribute to regulation of metabolic adaptations of MTb central metabolism in response to changing environments (9,12,14). In this study, we set out to systematically investigate in vitro reaction conditions that influence the activity of the Pck anaplerotic and gluconeogenic reactions and could thus regulate the PEP-OAA node in vivo.…”

“…Furthermore, infection of primary macrophages and mice with a Pck knock-out MTb strain were impaired, demonstrating the gene's pivotal role for MTb infection and for persistence of MTb in mice (12,13). Although the requirement for Pck was linked to the enzyme's gluconeogenic (anabolic) function, in vitro studies under hypoxic and growth-limiting conditions demonstrated its anaplerotic function in MTb (9,14). Additionally, a recent study applying 13 C labeling during THP1 cell infection with a Pck knock-out strain demonstrated that Pck contributes to carbon fixation in MTb (15).…”