Intracellular peroxynitrite perturbs redox balance, bioenergetics, and Fe-S cluster homeostasis in Mycobacterium tuberculosis

The ability of Mycobacterium tuberculosis (Mtb) to tolerate nitric oxide (^•NO) and superoxide (O₂^•-) produced by phagocytes contributes to its success as a human pathogen. Recombination of ^•NO and O₂^•- generates peroxynitrite (ONOO^-), a potent oxidant produced inside activated macrophages causing lethality in diverse organisms. While the response of Mtb toward ^•NO and O₂^•- is well established, how Mtb responds to ONOO^- remains unclear. Filling this knowledge gap is important to understand the persistence mechanisms of Mtb during infection. We synthesized a series of compounds that generate both ^•NO and O₂^•-, which should combine to produce ONOO^-. From this library, we identified CJ067 that permeates Mtb to reliably enhance intracellular ONOO^- levels. CJ067-exposed Mtb strains, including multidrug-resistant (MDR) and extensively drug-resistant (XDR) clinical isolates, exhibited dose-dependent, long-lasting oxidative stress and growth inhibition. In contrast, Mycobacterium smegmatis (Msm), a fast-growing, non-pathogenic mycobacterial species, maintained redox balance and growth in response to intracellular ONOO^-. RNA-sequencing with Mtb revealed that CJ067 induces antioxidant machinery, sulphur metabolism, metal homeostasis, and a 4Fe-4S cluster repair pathway (suf operon). CJ067 impaired the activity of the 4Fe-4S cluster-containing TCA cycle enzyme, aconitase, and diminished bioenergetics of Mtb. Work with Mtb strains defective in SUF and IscS involved in Fe-S cluster biogenesis pathways showed that both systems cooperatively protect Mtb from intracellular ONOO^- in vitro and inducible nitric oxide synthase (iNOS)-dependent growth inhibition during macrophage infection. Thus, Mtb is uniquely sensitive to intracellular ONOO^- and targeting Fe-S cluster homeostasis is expected to promote iNOS-dependent host immunity against tuberculosis (TB).