Essential roles of methionine and S-adenosylmethionine in the autarkic lifestyle of Mycobacterium tuberculosis

Abstract

Multidrug resistance, strong side effects, and compliance problems in TB chemotherapy mandate new ways to kill Mycobacterium tuberculosis (Mtb). Here we show that deletion of the gene encoding homoserine transacetylase (metA) inactivates methionine and S-adenosylmethionine (SAM) biosynthesis in Mtb and renders this pathogen exquisitely sensitive to killing in immunocompetent or immunocompromised mice, leading to rapid clearance from host tissues. Mtb ΔmetA is unable to proliferate in primary human macrophages, and in vitro starvation leads to extraordinarily rapid killing with no appearance of suppressor mutants. Cell death of Mtb ΔmetA is faster than that of other auxotrophic mutants (i.e., tryptophan, pantothenate, leucine, biotin), suggesting a particularly potent mechanism of killing. Time-course metabolomics showed complete depletion of intracellular methionine and SAM. SAM depletion was consistent with a significant decrease in methylation at the DNA level (measured by single-molecule real-time sequencing) and with the induction of several essential methyltransferases involved in biotin and menaquinone biosynthesis, both of which are vital biological processes and validated targets of antimycobacterial drugs. Mtb ΔmetA could be partially rescued by biotin supplementation, confirming a multitarget cell death mechanism. The work presented here uncovers a previously unidentified vulnerability of Mtb-the incapacity to scavenge intermediates of SAM and methionine biosynthesis from the host. This vulnerability unveils an entirely new drug target space with the promise of rapid killing of the tubercle bacillus by a new mechanism of action.

Keywords: amino acid biosynthesis; bactericidal auxotrophy; host–pathogen interaction; metabolism.

Fig. 1.

MetA encodes a homoserine transacetylase that is essential for growth in unsupplemented 7H9 medium. (A) Methionine and SAM biosynthetic pathway in Mtb. MetA, homoserine O-acetyltransferase; MetB, cystathionine gamma-synthase; MetC, O-acetylhomoserine sulfhydrylase; MetE/MetH/MmUM, methionine synthase; MetK, S-adenosylmethionine synthetase; Rv0075/Rv2294, cystathionine beta-lyase. (B) Lineweaver–Burk plot of the inverses of initial enzymatic reaction rates versus the inverses of multiple concentrations of homoserine. The Michaelis–Menten constant (K_m) and k_cat were calculated as 5 × 10⁻² mM and 2.4 s⁻¹, respectively. (C) Time courses of the enzymatic reaction with acetyl CoA (Ac-CoA) and succinyl CoA (Succ-CoA). The formation of CoA was monitored by the Ellman’s reagent with absorption at 412 nm. (D and E) Growth of WT (blue), ΔmetA (red), and ΔmetAcomp (black) strains in 7H9 medium (D) or supplemented with 50 µg/mL methionine (E). (F) Survival of ΔmetA in the presence or absence of 3 μg/mL methionine. Cultures were serially diluted and plated on 7H10 plates containing 50 µg/mL methionine. Detection limit, 100 cfu/mL. (G) Survival curves of different auxotrophic mutants in the Mtb CDC1551 background starved in unsupplemented medium. Detection limit, 10⁻⁶. ΔleuCD, leucine auxotroph; ΔpanCD, pantothenate auxotroph; ΔtrpD, tryptophan auxotroph. (H) Rescue of growth of ΔmetA with 125 µM of different pathway intermediates of methionine biosynthesis. Ac-HoSe, O-acetyl homoserine; HoCy, homocysteine; HoSe, homoserine; Met, methionine; SAM, S-adenosylmethionine.

Fig. 2.

Mtb ΔmetA is avirulent in immunocompetent (C57BL/6) mice, immunocompromised (SCID) mice, and human macrophages. Mice were infected with Mtb H37Rv (blue), ΔmetA (red), or ΔmetAcomp (black) via a low-dose aerosol (100 bacilli), and bacterial burdens in lungs and spleens were measured at 1, 7, 21, 56, and 112 d postinfection in C57BL/6 mice and at 1, 7, 21, and 42 d postinfection in SCID mice. (A–C) C57BL/6 mice. (A) The cfu in lung. (B) Gross appearance of lungs demonstrating pathology induced in infected mice on day 56. (C) The cfu in spleen. (D–F) SCID mice. (D) The cfu in lung. (E) Gross lung pathology on day 42. (F) The cfu in spleen. (G) Spleen pathology in SCID mice on day 42. Spleens cross-sectioned at the point of entry of the splenic vasculatures demonstrate varying degrees of splenomegaly as a readout for the severity of infection. (H) Survival of SCID mice (n = 6) after low-dose aerosol infection. (I) Survival of SCID mice (n = 6) after high-dose (10⁶ bacilli) infection via tail vein injection. (J) Lung pathology highlighted by H&E staining. No signs of inflammation were detected with ΔmetA strain infection. (K) Primary human macrophages infected at an MOI of 0.02 were incubated for 2 wk in RPMI medium supplemented with four different methionine concentrations (0, 3, 15, and 50 µg/mL). Macrophages were lysed, serially diluted, and plated for cfu at 0, 7, and 14 d postinfection. Error bars represent the SD of three biological replicates. ***P < 0.001; two-tailed t-test.

Fig. 3.

Time-course metabolic profile of ∆metA compared with WT and ∆metAcomp during 6 d of starvation. Samples from three biologically independent replicates were harvested on days 0, 2, 4, and 6, and metabolites were extracted. Aqueous-phase metabolites were measured by UPLC-MS. Fold changes in metabolite abundance were calculated relative to time 0. (A) Methionine biosynthetic pathway including SAM metabolism and lysine metabolism. In the ∆metA strain metabolites downstream of MetA are depleted, whereas those upstream accumulate. (B) Changes in metabolite abundance of glyoxylate shunt intermediates and cAMP. MetA, homoserine transacetylase; MetC, O-acetylhomoserine sulfhydrylase; MetE/MetH, methionine synthase; MetK, S-adenosylmethionine synthetase; ThrA, homoserine dehydrogenase. Error bars represent the SD of three biologically independent replicates. *P < 0.05; **P < 0.01; ***P < 0.001; two-tailed t-test.

Fig. 4.

Time-course transcriptomic profile of the ∆metA strain during methionine starvation. Samples from three biologically independent replicates were harvested on days 0, 2, 4, and 6, and RNA was extracted. (A) Genes involved in respiration are mainly down-regulated during methionine starvation of ∆metA. As is consistent with SAM depletion (Fig. 3A), the cells up-regulate SAM-dependent methyltransferases, including the essential genes bioA, bioB, and menH. (B) Depletion of SAM (Fig. 3A) leads to the induction of metC, whose expression is controlled by a SAM-IV–dependent riboswitch. Consequently, metC expression increases 30-fold. (C) Genes associated with antioxidant defense during antibiotic treatment (28) are strongly induced. (D) As is consistent with the depletion of methionine (Fig. 3A), we observed a gradual increase in the expression of genes involved in translation initiation. Error bars represent the SD of three biologically independent replicates. *P < 0.05; **P < 0.01; ***P < 0.001; two-tailed t-test. P values for heat maps can be found in the GEO database (accession no. GSE67843).