Nitric Oxide-Dependent Electron Transport Chain Inhibition by the Cytochrome bc1 Inhibitor and Pretomanid Combination Kills Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of human tuberculosis, harbors a branched electron transport chain, preventing the bactericidal action of cytochrome bc(1) inhibitors (e.g., TB47). Here, we investigated, using luminescent mycobacterial strains, the in vitro combination activity of cytochrome bc(1) inhibitors and nitric oxide (NO) donors including pretomanid (PMD) and explored the mechanisms of combination activity. The TB47 and PMD combination quickly abolished the light emission of luminescent bacilli, as was the case for the combination of TB47 and aurachin D, a putative cytochrome bd inhibitor. The TB47 and PMD combination inhibited M. tuberculosis oxygen consumption, decreased ATP levels, and had a delayed bactericidal effect. The NO scavenger carboxy-PTIO prevented the bactericidal activity of the drug combination, suggesting the requirement for NO. In addition, cytochrome bc(1) inhibitors were largely bactericidal when administered with DETA NONOate, another NO donor. Proteomic analysis revealed that the cotreated bacilli had a compromised expression of the dormancy regulon proteins, PE/PPE proteins, and proteins required for the biosynthesis of several cofactors, including mycofactocin. Some of these proteomic changes, e.g., the impaired dormancy regulon induction, were attributed to PMD. In conclusion, combination of cytochrome bc(1) inhibitors with PMD inhibited M. tuberculosis respiration and killed the bacilli. The activity of cytochrome bc(1) inhibitors can be greatly enhanced by NO donors. Monitoring of luminescence may be further exploited to screen cytochrome bd inhibitors.