Metabolic fluxes for nutritional flexibility of Mycobacterium tuberculosis

Abstract

The co-catabolism of multiple host-derived carbon substrates is required by Mycobacterium tuberculosis (Mtb) to successfully sustain a tuberculosis infection. However, the metabolic plasticity of this pathogen and the complexity of the metabolic networks present a major obstacle in identifying those nodes most amenable to therapeutic interventions. It is therefore critical that we define the metabolic phenotypes of Mtb in different conditions. We applied metabolic flux analysis using stable isotopes and lipid fingerprinting to investigate the metabolic network of Mtb growing slowly in our steady-state chemostat system. We demonstrate that Mtb efficiently co-metabolises either cholesterol or glycerol, in combination with two-carbon generating substrates without any compartmentalisation of metabolism. We discovered that partitioning of flux between the TCA cycle and the glyoxylate shunt combined with a reversible methyl citrate cycle is the critical metabolic nodes which underlie the nutritional flexibility of Mtb. These findings provide novel insights into the metabolic architecture that affords adaptability of bacteria to divergent carbon substrates and expand our fundamental knowledge about the methyl citrate cycle and the glyoxylate shunt.

Dissecting Host-Pathogen Interactions in TB Using Systems-Based Omic Approaches

“…Similar isotopologue analyses have been successfully applied across various host-pathogen models to measure intracellular metabolism of these pathogens, such as 13 C glucose profiles of M. leprae growing in Schwann cells (70) and Trypanosoma brucei in bloodstream form (71) and 13 C glycerol profiles in legionella pneumophila replicating in macrophages (72). 13 C-isotopic analysis of Mtb in vitro cultures in a chemostat setup at controlled growth rate demonstrated efficient isotope incorporation and co-catabolism of 13 C-labelled acetate and cholesterol, which are intracellular nutrient sources for Mtb (73). Recent advances in MS technologies have enabled the use of several stable isotopes including both single and mixed labelled species such as 15 N and 2 H. 2 H cholesterol labelling of Mtb in vitro cultures confirmed that Mtb utilized cholesterol to synthesize amino acids (73).…”

“…13 C-isotopic analysis of Mtb in vitro cultures in a chemostat setup at controlled growth rate demonstrated efficient isotope incorporation and co-catabolism of 13 C-labelled acetate and cholesterol, which are intracellular nutrient sources for Mtb (73). Recent advances in MS technologies have enabled the use of several stable isotopes including both single and mixed labelled species such as 15 N and 2 H. 2 H cholesterol labelling of Mtb in vitro cultures confirmed that Mtb utilized cholesterol to synthesize amino acids (73). 15 N-asparagine was used to measure the uptake and assimilation of nitrogen in murine macrophages using NANO-Secondary ion mass spectrometry (SIMS) (74).…”

“…13 C-MFA of Mtb in vitro cultures cultivated at slow and fast growth rate using 13 C-glycerol as the labelled carbon substrate identified a novel pathway for pyruvate dissimilation through glyoxylate shunt and anaplerotic reactions and demonstrated the ability of Mtb to fix CO 2 for synthesizing biomass (86). Recently, the intracellular carbon fluxes that support co-catabolism of multiple carbon substrates by Mtb were identified using 13 C-MFA (73). This study demonstrated that flux partitioning between the TCA cycle and glyoxylate shunt with a reversible methyl citrate cycle enables Mtb to co-catabolise cholesterol and acetate.…”

“…The experimental part of MFA involves cultivation of microbial or eukaryotic cells in a growth medium containing isotopically labelled substrates. Majority of the MFA studies are conducted on steady state cultures where the cells are grown at a controlled growth rate using a bioreactor/chemostat setup ( 73 , 85 , 86 ) or assuming a pseudo-steady state during exponential growth mainly for eukaryotic cells ( 87 , 88 ). Metabolic steady state of the system is confirmed through measurement of substrate uptake, CO 2, and biomass production rates ( 73 ).…”

“…Majority of the MFA studies are conducted on steady state cultures where the cells are grown at a controlled growth rate using a bioreactor/chemostat setup ( 73 , 85 , 86 ) or assuming a pseudo-steady state during exponential growth mainly for eukaryotic cells ( 87 , 88 ). Metabolic steady state of the system is confirmed through measurement of substrate uptake, CO 2, and biomass production rates ( 73 ). Metabolically steady state cultures are next checked for isotopic steady state through addition of isotopic substrates and their incorporation into the biomass at various time points of cultivation.…”