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.host-pathogen interaction | bactericidal auxotrophy | amino acid biosynthesis | metabolism U nderstanding the metabolic interactions between an invading microbe and its host is becoming a new cornerstone of host-pathogen research (1-3). Many intracellular pathogens modulate the host response to satisfy their nutritional needs and as a result have become auxotrophic for several essential amino acids and cofactors (4-6). Mycobacterium tuberculosis (Mtb), arguably the most deadly bacterial pathogen in the world (7), adopted a different strategy. This ultra-slow-growing bacterium is prototrophic for all essential cofactors and amino acids, suggesting that it either dwells in host compartments where such metabolites are unavailable or actively chooses this autarkic lifestyle to retain metabolic flexibility and remain invisible to the host. Indeed, much of Mtb's long-term success as a human pathogen is ascribed to its extraordinary stealth in the face of host immunity (8, 9); Mtb's ability to evade detection by the host might explain why devising an efficient vaccine has failed thus far and why drug therapy is difficult. Therefore, understanding Mtb's in vivo metabolic requirements could help in the development of much-needed new strategies for antimycobacterial therapy.Methionine and S-adenosylmethionine (SAM) are essential metabolites that have gained considerable scientific attenti...

Central Role of Pyruvate Kinase in Carbon Co-catabolism of Mycobacterium tuberculosis

“…Metabolite extraction was performed as described previously (36). Briefly, 5 ml of cell culture was grown to A 0.5 and quenched in 10 ml of 100% methanol at 4°C, spun down, and re-suspended in acetonitrile:methanol:water (2:2:1).…”

Biochemical Characterization of Isoniazid-resistant Mycobacterium tuberculosis: Can the Analysis of Clonal Strains Reveal Novel Targetable Pathways?

“…Methionine and SAM metabolism are expected to be a promising drug target in Mtb, as metA mutants are unable to grow in vitro and are rapidly cleared in vivo either in immunocompetent or immunocompromised mice [52]. Although we did not find significant differences in the levels of MetA, we did find significantly reduced abundances of MetK, in addition to SahH, both of which are involved in these metabolic pathways (supplemental Fig S4).…”

One of These is Not Like the Others

“…This enzyme is part of a pathway that branches off of the main aspartate family pathway that generates methionine, threonine, lysine, and diaminopimelate, the latter of which is the direct precursor to lysine and also a component of the peptidoglycan layer in the cell envelope. As expected, the mutant required methionine for growth and could be fed with intermediates downstream of the metA reaction, or S-adenosylmethionine [10]. As par for the course for an M. tuberculosis auxotrophic strain, the metA mutant failed to replicate in human tissue cell culture, and was rapidly cleared from both immunocompetent and immunocompromised mice [10].…”

“…A recent paper by Berney et al, published in the Proceedings of the National Academy of Sciences [10], describes the novel phenotype of a M. tuberculosis methionine auxotroph made by targeted deletion of the metA gene, encoding homoserine transacetylase. This enzyme is part of a pathway that branches off of the main aspartate family pathway that generates methionine, threonine, lysine, and diaminopimelate, the latter of which is the direct precursor to lysine and also a component of the peptidoglycan layer in the cell envelope.…”