Drug-resistant mycobacterial infections are a serious global health challenge, leading to high mortality and socioeconomic burdens in developing countries worldwide. New innovative approaches, from identification of new targets to discovery of novel chemical scaffolds, are urgently needed. Recently, energy metabolism in mycobacteria, in particular the oxidative phosphorylation pathway, has emerged as an object of intense microbiological investigation and as a novel target pathway in drug discovery. New classes of antibacterials interfering with elements of the oxidative phosphorylation pathway are highly active in combating dormant or latent mycobacterial infections, with a promise of shortening tuberculosis chemotherapy. The regulatory approval of the ATP synthase inhibitor bedaquiline and the discovery of Q203, a candidate drug targeting the cytochrome bc ₁ complex, have highlighted the central importance of this new target pathway. In this review, we discuss key features and potential applications of inhibiting energy metabolism in our quest for discovering potent novel and sterilizing drug combinations for combating tuberculosis. We believe that the combination of drugs targeting elements of the oxidative phosphorylation pathway can lead to a completely new regimen for drug-susceptible and multidrug-resistant tuberculosis.

Targeting respiration and ATP synthesis has received strong interest as a new strategy for combatting drug-resistant Mycobacterium tuberculosis. Mycobacteria employ a respiratory chain terminating with two branches. One of the branches includes a cytochrome bc₁ complex and an aa₃-type cytochrome c oxidase while the other branch terminates with a cytochrome bd-type quinol oxidase. In this communication we show that genetic inactivation of cytochrome bd, but not of cytochrome bc₁, enhances the susceptibility of Mycobacterium smegmatis to hydrogen peroxide and antibiotic-induced stress. The type-II NADH dehydrogenase effector clofazimine and the ATP synthase inhibitor bedaquiline were bacteriostatic against wild-type M. smegmatis, but strongly bactericidal against a cytochrome bd mutant. We also demonstrated that the quinone-analog aurachin D inhibited mycobacterial cytochrome bd at sub-micromolar concentrations. Our results identify cytochrome bd as a key survival factor in M. smegmatis during antibiotic stress. Targeting the cytochrome bd respiratory branch therefore appears to be a promising strategy that may enhance the bactericidal activity of existing tuberculosis drugs.

Mycobacterial energy metabolism currently attracts strong attention as new target space for development of anti-tuberculosis drugs. The imidazopyridine Q203 targets the cytochrome bcc complex of the respiratory chain, a key component in energy metabolism. Q203 blocks growth of Mycobacterium tuberculosis at nanomolar concentrations, however, it fails to actually kill the bacteria, which may limit the clinical applicability of this candidate drug. In this report we show that inhibition of cytochrome bd, a parallel branch of the mycobacterial respiratory chain, by aurachin D invoked bactericidal activity of Q203. In biochemical assays using inverted membrane vesicles from Mycobacterium tuberculosis and Mycobacterium smegmatis we found that inhibition of respiratory chain activity by Q203 was incomplete, but could be enhanced by inactivation of cytochrome bd, either by genetic knock-out or by inhibition with aurachin D. These results indicate that simultaneously targeting the cytochrome bcc and the cytochrome bd branch of the mycobacterial respiratory chain may turn out as effective strategy for combating M. tuberculosis.

The branched respiratory chain of Mycobacterium tuberculosis has attracted attention as a highly promising target for next-generation antibacterials. This system includes two terminal oxidases of which the exclusively bacterial cytochrome bd represents the less energy-efficient one. Albeit dispensable for growth under standard laboratory conditions, cytochrome bd is important during environmental stress. In this review, we discuss the role of cytochrome bd during infection of the mammalian host and in the defense against antibacterials. Deeper insight into the biochemistry of mycobacterial cytochrome bd is needed to understand the physiological role of this bacteria-specific defense factor. Conversely, cytochrome bd may be utilized to gain information on mycobacterial physiology in vitro and during host infection. Knowledge-based manipulation of cytochrome bd function may assist in designing the next-generation tuberculosis combination chemotherapy.