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.

H ₂ S is a potent gasotransmitter in eukaryotes and bacteria. Host-derived H ₂ S has been shown to profoundly alter M. tuberculosis ( Mtb ) energy metabolism and growth. However, compelling evidence for endogenous production of H ₂ S and its role in Mtb physiology is lacking. We show that multidrug-resistant and drug-susceptible clinical Mtb strains produce H ₂ S, whereas H ₂ S production in non-pathogenic M. smegmatis is barely detectable. We identified Rv3684 (Cds1) as an H ₂ S-producing enzyme in Mtb and show that cds1 disruption reduces, but does not eliminate, H ₂ S production, suggesting the involvement of multiple genes in H ₂ S production. We identified endogenous H ₂ S to be an effector molecule that maintains bioenergetic homeostasis by stimulating respiration primarily via cytochrome bd . Importantly, H ₂ S plays a key role in central metabolism by modulating the balance between oxidative phosphorylation and glycolysis, and functions as a sink to recycle sulfur atoms back to cysteine to maintain sulfur homeostasis. Lastly, Mtb -generated H ₂ S 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.

Mycobacterium tuberculosis ( Mtb ) senses and responds to host-derived gasotransmitters NO and CO via heme-containing sensor kinases DosS and DosT and the response regulator DosR. Hydrogen sulfide (H ₂ S) is an important signaling molecule in mammals, but its role in Mtb physiology is unclear. We have previously shown that exogenous H ₂ S can modulate expression of genes in the Dos dormancy regulon via an unknown mechanism(s). Here, we tested the hypothesis that Mtb senses and responds to H ₂ S via the DosS/T/R system. Using UV-Vis and EPR spectroscopy, we show that H ₂ S binds directly to the ferric (Fe ³⁺ ) heme of DosS (K _D = 5.64 µM) but not the ferrous (Fe ²⁺ ) form. No interaction with DosT was detected. Thus, the mechanism by which DosS senses H ₂ S is different from that for sensing NO and CO, which bind only the ferrous forms of DosS and DosT. Steered Molecular Dynamics simulations show that H ₂ S, and not the charged HS ^- species, can enter the DosS heme pocket. We also show that H ₂ S increases DosS autokinase activity and subsequent phosphorylation of DosR, and H ₂ S-mediated increases in Dos regulon gene expression is lost in Mtb lacking DosS. Finally, we demonstrate that physiological levels of H ₂ S in macrophages can induce Dos regulon genes via DosS. Overall, these data reveal a novel mechanism whereby Mtb senses and responds to a third host gasotransmitter, H ₂ S, via DosS-Fe3 ⁺ . These findings highlight the remarkable plasticity of DosS and establish a new paradigm for how bacteria can sense multiple gasotransmitters through a single heme sensor kinase. Significance Statement: Hydrogen sulfide (H ₂ S) is an important signaling molecule in eukaryotes and bacteria, and along with CO and NO, is an important part of host defense against Mycobacterium tuberculosis ( Mtb ). However, the mechanism(s) by which Mtb senses and responds to H ₂ S is unknown. Here, we report that the Mtb heme sensor kinase DosS, a known sensor of CO and NO, is also a sensor of H ₂ S. We found that H ₂ S binds DosS in its ferric (Fe ³⁺ ) state, which is considered as its inactive state, to induce the Dos dormancy regulon during infection. These data highlight the unusual capacity of Mtb to sense multiple gasotransmitters through a single sensing protein.