Hydrogen sulfide (H2S) production by mycobacteria.

Abstract

The gasotransmitter hydrogen sulfide (H2S) has been recognized as a physiological mediator with a variety of functions across all domains of life. Many prokaryotic bacterial species endogenously generate H2S in their natural environments. However, to date, it is not known whether Mycobacterium tuberculosis (Mtb) is an endogenous producer of H2S. In this study, we tested the hypothesis that Mtb endogenously produces H2S to modulate respiration, central metabolism, oxidative stress, and drug susceptibility. We demonstrated that fast-growing non-pathogenic, slow-growing pathogenic mycobacterial species, as well as drug resistant clinical strains of Mtb species produce H2S. Here we demonstrate that fast-growing non-pathogenic M. smegmatis produces barely detectable quantities of H2S, whereas MDR Mtb produces large quantities of H2S. We have also developed a native PAGEbased assay for the rapid screening of H2S producing enzymes in the lysates of mycobacterial species. Using LC-MS/MS, we identified the protein, Rv3684 as an H2S-producing enzyme in Mtb. Disruption of rv3684, demonstrated using the genetic knock out of rv3684, reduces, but does not eliminate, H2S production, suggesting the involvement of multiple genes in H2S production. Whole Mtb cell-based and lysate assays showed reduced levels of H2S production in the Mtb knockout strain compared to the wild-type strain. Noticeably, we demonstrated that the Mtb mutant is resistant to oxidative stress and the anti-TB drugs clofazimine and rifampicin. We also found that endogenous H2S is an effector molecule that maintains bioenergetic homeostasis by regulating Mtb respiration, and that H2S also plays a key role in central metabolism by modulating the balance between oxidative phosphorylation (OXPHOS) and glycolysis. In summary, our findings reveal previously unknown concepts of Mtb physiology with respect to Mtb-derived H2S and energy metabolism which has significant implication for routine laboratory culturing, understanding susceptibility and TB disease.