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).