A simple assay for inhibitors of mycobacterial oxidative phosphorylation

Oxidative phosphorylation, the combined activities of the electron transport chain (ETC) and ATP synthase, has emerged as a valuable target for antibiotics to treat infection with Mycobacterium tuberculosis and related pathogens. In oxidative phosphorylation, the ETC establishes a transmembrane electrochemical proton gradient that powers ATP synthesis. Monitoring oxidative phosphorylation with luciferase-based detection of ATP synthesis or measurement of oxygen consumption can be technically challenging and expensive. These limitations reduce the utility of these methods for characterization of mycobacterial oxidative phosphorylation inhibitors. Here, we show that fluorescence-based measurement of acidification of inverted membrane vesicles (IMVs) can detect and distinguish between inhibition of the ETC, inhibition of ATP synthase, and nonspecific membrane uncoupling. In this assay, IMVs from Mycobacterium smegmatis are acidified either through the activity of the ETC or ATP synthase, the latter modified genetically to allow it to serve as an ATP-driven proton pump. Acidification is monitored by fluorescence from 9-amino-6-chloro-2-methoxyacridine, which accumulates and quenches in acidified IMVs. Nonspecific membrane uncouplers prevent both succinate- and ATP-driven IMV acidification. In contrast, the ETC Complex III₂IV₂ inhibitor telacebec (Q203) prevents succinate-driven acidification but not ATP-driven acidification, and the ATP synthase inhibitor bedaquiline prevents ATP-driven acidification but not succinate-driven acidification. We use the assay to show that, as proposed previously, lansoprazole sulfide is an inhibitor of Complex III₂IV₂, whereas thioridazine uncouples the mycobacterial membrane nonspecifically. Overall, the assay is simple, low cost, and scalable, which will make it useful for identifying and characterizing new mycobacterial oxidative phosphorylation inhibitors.