Mycobacterium tuberculosis H₂S Functions as a Sink to Modulate Central Metabolism, Bioenergetics, and Drug...

H₂S is a potent gasotransmitter in eukaryotes and bacteria. Host-derived H₂S has been shown to profoundly alter M. tuberculosis (Mtb) energy metabolism and growth. However, compelling evidence for endogenous production of H₂S and its role in Mtb physiology is lacking. We show that multidrug-resistant and drug-susceptible clinical Mtb strains produce H₂S, whereas H₂S production in non-pathogenic M. smegmatis is barely detectable. We identified Rv3684 (Cds1) as an H₂S-producing enzyme in Mtb and show that cds1 disruption reduces, but does not eliminate, H₂S production, suggesting the involvement of multiple genes in H₂S production. We identified endogenous H₂S to be an effector molecule that maintains bioenergetic homeostasis by stimulating respiration primarily via cytochrome bd. Importantly, H₂S plays a key role in central metabolism by modulating the balance between oxidative phosphorylation and glycolysis, and it functions as a sink to recycle sulfur atoms back to cysteine to maintain sulfur homeostasis. Lastly, Mtb-generated H₂S regulates redox homeostasis and susceptibility to anti-TB drugs clofazimine and rifampicin. These findings reveal previously unknown facets of Mtb physiology and have implications for routine laboratory culturing, understanding drug susceptibility, and improved diagnostics.