Functional characterization of a vitamin B12-dependent methylmalonyl pathway in Mycobacterium tuberculosis: implications for propionate metabolism during growth on fatty acids

Abstract

Mycobacterium tuberculosis is predicted to subsist on alternative carbon sources during persistence within the human host. Catabolism of odd- and branched-chain fatty acids, branched-chain amino acids, and cholesterol generates propionyl-coenzyme A (CoA) as a terminal, three-carbon (C(3)) product. Propionate constitutes a key precursor in lipid biosynthesis but is toxic if accumulated, potentially implicating its metabolism in M. tuberculosis pathogenesis. In addition to the well-characterized methylcitrate cycle, the M. tuberculosis genome contains a complete methylmalonyl pathway, including a mutAB-encoded methylmalonyl-CoA mutase (MCM) that requires a vitamin B(12)-derived cofactor for activity. Here, we demonstrate the ability of M. tuberculosis to utilize propionate as the sole carbon source in the absence of a functional methylcitrate cycle, provided that vitamin B(12) is supplied exogenously. We show that this ability is dependent on mutAB and, furthermore, that an active methylmalonyl pathway allows the bypass of the glyoxylate cycle during growth on propionate in vitro. Importantly, although the glyoxylate and methylcitrate cycles supported robust growth of M. tuberculosis on the C(17) fatty acid heptadecanoate, growth on valerate (C(5)) was significantly enhanced through vitamin B(12) supplementation. Moreover, both wild-type and methylcitrate cycle mutant strains grew on B(12)-supplemented valerate in the presence of 3-nitropropionate, an inhibitor of the glyoxylate cycle enzyme isocitrate lyase, indicating an anaplerotic role for the methylmalonyl pathway. The demonstrated functionality of MCM reinforces the potential relevance of vitamin B(12) to mycobacterial pathogenesis and suggests that vitamin B(12) availability in vivo might resolve the paradoxical dispensability of the methylcitrate cycle for the growth and persistence of M. tuberculosis in mice.

FIG. 1.

Predicted pathways of propionate metabolism in M. tuberculosis. Catabolism of alternative carbon sources including odd- and branched-chain fatty acids (FA), branched-chain amino acids (BCAA), and cholesterol generates propionyl-CoA as a three-carbon (C₃) terminal product. Previous studies have established the importance of the glyoxylate and methylcitrate cycles for anaplerosis and propionyl-CoA metabolism, respectively, during fatty acid catabolism by M. tuberculosis (36, 37). Glyoxylate cycle enzymes are the isocitrate lyases (ICL1/ICL2) and malate synthase (MLS); ICL1 and ICL2 also provide MCL activity in M. tuberculosis (18, 37). Other enzymes of the methylcitrate cycle include MCS and MCD. Methylmalonyl pathway enzymes are PCC, MMCE, and MCM. Pyruvate is produced from malate by malic enzyme (MEZ) or from oxaloacetate by the sequential action of pyruvate carboxykinase (PCK) and pyruvate kinase (PYK); the coupled decarboxylation of pyruvate by the pyruvate dehydrogenase complex (PDHC) yields acetyl-CoA. Anaplerosis during carbohydrate catabolism is by carboxylation of pyruvate to oxaloacetate by pyruvate carboxylase (PCA). ACN, aconitase; CIT, citrate synthase; FUM, fumarase; ICD, isocitrate dehydrogenase; MDH, malate dehydrogenase; MQO, malate:quinine oxidoreductase; PEP, phosphoenolpyruvate; SCS, succinate synthase; SDH, succinate dehydrogenase.

FIG. 2.

Vitamin B₁₂ supplementation enables a prpDC deletion mutant of M. tuberculosis H37Rv to grow on propionate through the action of the mutAB-encoded MCM. (A) Growth on propionate. (B) Growth on propionate supplemented with 10 μg/ml vitamin B₁₂. ⧫, H37Rv; □, ΔprpDC; ▴, complemented ΔprpDC mutant (ΔprpDC::prpDC). (C) Vitamin B₁₂ supplementation enables mutAB-dependent growth of a prpDC-deficient mutant of H37Rv on propionate. ▪, ΔprpDC; ⋄, ΔmutAB ΔprpDC; ▴, ΔmutAB ΔprpDC double mutant containing the reverted mutAB allele (ΔmutAB::mutAB ΔprpDC). (D) Effect of loss of mutAB function on growth of H37Rv on propionate supplemented with vitamin B₁₂. Shown are data for H37Rv with (□) and without (▪) vitamin B₁₂ and the ΔmutAB mutant with (⋄) and without (⧫) vitamin B₁₂. The growth of the complemented ΔmutAB mutant was equivalent to that of the wild type in the presence of vitamin B₁₂ (data not shown). Data are OD₆₀₀ values for a single representative experiment from three independent biological replicates.

FIG. 3.

The methylmalonyl pathway enables bypass of the glyoxylate shunt during growth of M. tuberculosis on propionate. (A) Growth of H37Rv on propionate in the presence of 3NP with (□) or without (▪) vitamin B₁₂ supplementation versus the growth of the ΔprpDC mutant on vitamin B₁₂-supplemented propionate with (▵) or without (▴) 3NP. (B) Growth of the ΔmutAB mutant on vitamin B₁₂-supplemented propionate with (⋄) or without (⧫) 3NP. Data are OD₆₀₀ values for a single representative experiment from three independent biological replicates.

FIG. 4.

Growth of M. tuberculosis on longer odd-chain fatty acids. (A) Stimulatory effect of vitamin B₁₂ on the growth of H37Rv and the ΔprpDC mutant on valerate (C₅). Shown are data for H37Rv with (⋄) or without (⧫) vitamin B₁₂ and the ΔprpDC mutant with (□) or without (▪) vitamin B₁₂. (B) Effect of vitamin B₁₂ on growth of H37Rv and the ΔprpDC mutant on heptadecanoate (C₁₇). Shown are data for H37Rv with (⋄) or without (⧫) vitamin B₁₂ and the ΔprpDC mutant with (□) or without (▪) vitamin B₁₂. The growth curves for H37Rv with or without vitamin B₁₂ and ΔprpDC with vitamin B₁₂ are indistinguishable. Data are OD₆₀₀ values for a single representative experiment from three independent biological replicates.

FIG. 5.

Anaplerotic role for MCM revealed by growth of M. tuberculosis on valerate with vitamin B₁₂ supplementation. (A) Growth of H37Rv on valerate in the presence of 3NP with (□) or without (▪) vitamin B₁₂ supplementation versus growth of the ΔprpDC mutant on vitamin B₁₂-supplemented valerate with (▵) or without (▴) 3NP. (B) Growth of H37Rv on vitamin B₁₂-supplemented valerate with (□) or without (▪) 3NP. Data are OD₆₀₀ for a single representative experiment from three independent biological replicates.

FIG. 6.

Expression of the prpD gene of H37Rv cultured in various carbon sources. H37Rv was grown on propionate (Prop.) or valerate (Val.) in the presence or absence of vitamin B₁₂. Growth on valerate alone was insufficient to support any further downstream analysis, so in this case, prpD expression could be assessed only in the presence of vitamin B₁₂ supplementation. Levels of prpD transcript were determined by real-time qRT-PCR and normalized against the values obtained from bacteria grown in Middlebrook 7H9 medium supplemented with 0.2% glycerol, oleic acid-albumin-dextrose-catalase enrichment, and 0.05% Tween 80 (7H9) to assess any differential regulation of prpD as a function of the carbon source. Significant differences in the expression of prpD in fatty acid carbon sources relative to that in the 7H9 control are indicated by an asterisk (P < 0.0001).