Methylcitrate cycle defines the bactericidal essentiality of isocitrate lyase for survival of Mycobacterium tuberculosis on fatty acids

Abstract

Few mutations attenuate Mycobacterium tuberculosis (Mtb) more profoundly than deletion of its isocitrate lyases (ICLs). However, the basis for this attenuation remains incompletely defined. Mtb's ICLs are catalytically bifunctional isocitrate and methylisocitrate lyases required for growth on even and odd chain fatty acids. Here, we report that Mtb's ICLs are essential for survival on both acetate and propionate because of its methylisocitrate lyase (MCL) activity. Lack of MCL activity converts Mtb's methylcitrate cycle into a "dead end" pathway that sequesters tricarboxylic acid (TCA) cycle intermediates into methylcitrate cycle intermediates, depletes gluconeogenic precursors, and results in defects of membrane potential and intrabacterial pH. Activation of an alternative vitamin B 12 -dependent pathway of propionate metabolism led to selective corrections of TCA cycle activity, membrane potential, and intrabacterial pH that specifically restored survival, but not growth, of ICL-deficient Mtb metabolizing acetate or propionate. These results thus resolve the biochemical basis of essentiality for Mtb's ICLs and survival on fatty acids. However, our understanding of the pathways and physiologic functions they serve is incomplete. Enzymes catalyze reactions whose identities, rates, and directions are often difficult to determine and, where known, function within the context of complex and extensively interconnected physiologic networks (1). Thus, although recent genetic and biochemical approaches have made it possible to determine the essentiality of a given enzyme, our understanding of the biochemical mechanisms underlying their essentiality remains incomplete.Unlike the case for most microbial pathogens, humans serve as both host and reservoir for Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB). Within humans, Mtb resides within the biochemically stringent environment of the macrophage phagosome. Growing evidence has separately implicated Mtb's metabolic enzymes as a key determinant of its pathogenicity. Mtb's metabolic network thus appears to have evolved to serve interdependent physiologic and pathogenic roles (2, 3).Isocitrate lyase (ICL) is a metabolic enzyme used by bacteria to sustain growth on even-chain fatty acids through an anaplerotic pathway called the glyoxylate shunt. Mtb encodes two paralogous ICLs, each of which also functions as a methylisocitrate lyase (MCL), and serves a parallel pathway involved in the metabolism of propionyl CoA generated by β-oxidation of oddchain fatty acids, called the methylcitrate cycle. Mtb lacking both ICLs were shown to be incapable of either establishing or maintaining infection in a mouse model of pulmonary TB (4, 5). Although loss of ICL activity is expected to result in the inability to incorporate carbon from fatty acids (a form of starvation), simultaneous loss of MCL activity is predicted to result in the potentially toxic accumulation of propionyl-CoA metabolites (a form of intoxication). Here, we applied 13 C-based metabolomic tra...

E1 of α-ketoglutarate dehydrogenase defends Mycobacterium tuberculosis against glutamate anaplerosis and nitroxidative stress

“…Thus, elevation of SSA could reflect the joint impact of two effects of HOAS deficiency: increased activity of the GABA shunt and inhibition of SSADH activity by increased glyoxylate. The toxic potential of SSA illustrates the principle that intermediary metabolism produces not just molecules that sustain life but life-threatening molecules as well, as shown for Mtb, which also accumulates branched chain α-ketoacids, propionate, maltose 1-phosphate, or glycerol phosphate when the relevant metabolic pathways are disrupted by chemical or genetic means (5,8,(43)(44)(45)(46)(47).…”

Mycobacterial Gene cuvA Is Required for Optimal Nutrient Utilization and Virulence

“…Though there is some similarity in the phenotypes of the ⌬cuvA strains and the strains disrupted in glyoxylate or methylcitrate pathways, there are important differences that suggest that CuvA does not participate directly in these pathways. First, the M. tuberculosis strains with defects in the glyoxylate or methylcitrate pathways show minimal growth on cholesterol or propionate in liquid MM (17,45,46). In contrast, the M. tuberculosis ⌬cuvA strain did not have a significant defect in cholesterol-containing liquid medium, and the M. smegmatis ⌬cuvA strain grew more slowly but did achieve sustained growth in MM plus cholesterol and MM plus propionate.…”

Acetate Dissimilation and Assimilation in Mycobacterium tuberculosis Depend on Carbon Availability

“…The inhibition of the Icl1 was achieved by the addition of itaconate. Through inhibition of the glyoxylate shunt, M. tuberculosis loses the ability to grow on C 2 substrates derived from fatty acid catabolism, due to the lack of anaplerotic precursors (34). The reduced growth in the presence of itaconate was accompanied by higher levels of acetate detected in the supernatant (Fig.…”