Abstract
Metabolic adaptation of Mycobacterium tuberculosis (M. tuberculosis) to microbicidal intracellular environment of host macrophages is fundamental to its pathogenicity. However, an in-depth understanding of metabolic adjustments through key reaction pathways and networks is limited. To understand how such changes occur, we measured the cellular metabolome of M. tuberculosis subjected to four microbicidal stresses using liquid chromatography-mass spectrometric multiple reactions monitoring (LC-MRM/MS). Overall, 87 metabolites were identified. The metabolites best describing the separation between stresses were identified through multivariate analysis. The coupling of the metabolite measurements with existing genome-scale metabolic model, and using constraint-based simulation led to several new concepts and unreported observations in M. tuberculosis; such as (i) the high levels of released ammonia as an adaptive response to acidic stress was due to increased flux through L-asparaginase rather than urease activity; (ii) nutrient starvation-induced anaplerotic pathway for generation of TCA intermediates from phosphoenolpyruvate using phosphoenolpyruvate kinase; (iii) quenching of protons through GABA shunt pathway or sugar alcohols as possible mechanisms of early adaptation to acidic and oxidative stresses; and (iv) usage of alternate cofactors by the same enzyme as a possible mechanism of rewiring metabolic pathways to overcome stresses. Besides providing new leads and important nodes that can be used for designing intervention strategies, the study advocates the strength of applying flux balance analyses coupled with metabolomics to get a global picture of complex metabolic adjustments.
Highlights
Mycobacterium tuberculosis (M. tuberculosis), tuberculosis (TB) causing bacteria in human, is a very successful pathogen, that still claims millions of lives worldwide every year (World Health Organization [WHO], 2018)
While maximum segregations were observed in nutrient starvation and acidic stress, iron deprivation showed the least segregation from the control (Figures 1A–D)
We discuss the inferences drawn from flux balance analysis (FBA) simulations using new approaches employed in this study that led to testable hypothesis such as, presence of a probable anaplerotic path for the generation of TCA intermediates from phosphoenolpyruvate using phosphoenolpyruvate kinase under nutrient stress and events of alternate use of cofactors by enzymes during oxidative stress response, showcasing M.tb’s potential to utilize multiple cofactors as stress-adaptation mechanism
Summary
Mycobacterium tuberculosis (M. tuberculosis), tuberculosis (TB) causing bacteria in human, is a very successful pathogen, that still claims millions of lives worldwide every year (World Health Organization [WHO], 2018). Transcriptomics, and proteomics have unraveled a wide array of molecular mechanisms employed by M. tuberculosis to survive this hostile microbicidal milieu of macrophage phagosomes (Forrellad et al, 2013; Gopinath et al, 2015; Liu et al, 2016; Danelishvili et al, 2017; Hoffmann et al, 2018) This included mycobacterial detoxification system of catalase (KatG) (Ng et al, 2004), superoxide dismutases (SodA and SodC) (Wu et al, 1998; Dussurget et al, 2001; Piddington et al, 2001; Sassetti and Rubin, 2003), mycothiol (MSH) (Buchmeier and Fahey, 2006; Buchmeier et al, 2006; Vilcheze et al, 2008), NADH-dependent peroxidase, and peroxynitrite reductase system (Bryk et al, 2000, 2002; Tian et al, 2005a; Shi and Ehrt, 2006). There are reports on pathways involving DNA and protein repair or degradation to play direct or indirect roles in the pathogenesis of M. tuberculosis (Forrellad et al, 2013; Ehrt et al, 2015, and references therein)
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