Mycobacterium tuberculosis DosS binds H₂S through its Fe³⁺ heme iron to regulate the DosR dormancy regulon

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 (H₂S) is an important signaling molecule in mammals, but its role in Mtb physiology is unclear. We have previously shown that exogenous H₂S can modulate expression of genes in the Dos dormancy regulon via an unknown mechanism(s). Here, we test the hypothesis that Mtb senses and responds to H₂S via the DosS/T/R system. Using UV-Vis and EPR spectroscopy, we show that H₂S binds directly to the ferric (Fe³⁺) heme of DosS (K_D^app = 5.30 μM) but not the ferrous (Fe²⁺) form. No interaction with DosT(Fe²⁺-O₂) was detected. We found that the binding of sulfide can slowly reduce the DosS heme iron to the ferrous form. Steered Molecular Dynamics simulations show that H₂S, and not the charged HS^- species, can enter the DosS heme pocket. We also show that H₂S increases DosS autokinase activity and subsequent phosphorylation of DosR, and H₂S-mediated increases in Dos regulon gene expression is lost in Mtb lacking DosS. Finally, we demonstrate that physiological levels of H₂S in macrophages can induce DosR regulon genes via DosS. Overall, these data reveal a novel mechanism whereby Mtb senses and responds to a third host gasotransmitter, H₂S, 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.