Development and use of non-invasive techniques to study the mechanism of an anti-tuberculosis drug in live mycobacteria

Tuberculosis, caused by Mycobacterium tuberculosis, is responsible for over 1 million deaths per year. Bedaquiline is a key antibiotic in the treatment of drug-resistant tuberculosis and its discovery stirred an interest in developing therapies targeting mycobacterial bioenergetics. Despite bedaquiline’s importance, several incompatible models for its mode of action have developed: that it acts as a direct inhibitor of mycobacterial ATP synthase or that it disrupts the proton motive force through protonophore or H+/K+ ionophore action.

Non-invasive techniques applied to living cells offer the potential of monitoring real-time changes in bioenergetic systems. To this end, visible-wavelength remission spectroscopy and 31P NMR are applied here to better understand mycobacterial bioenergetics and unravel bedaquiline’s mode of action.

Visible-wavelength remission spectroscopy was used to examine the effects of bedaquiline on the mycobacterial oxidative phosphorylation system in Mycobacterium smegmatis and Mycobacterium tuberculosis. Comparison between these effects and those of established bioenergetic inhibitors and protonophores/ionophores show bedaquiline acts as a direct inhibitor of ATP synthase. Experiments with cytochrome bd oxidase knockout strains demonstrate the oxygen consumption increase observed on bedaquiline addition is due to an increase in activity of cytochrome bd oxidase.

The use of 31P NMR to study metabolism in live cells was explored: experiments on unoxygenated Mycobacterium smegmatis cultures demonstrate that this method can be used to monitor levels of phosphorylated metabolites. A system for oxygenating and mixing bacterial cultures during NMR experiments was developed for future use.

Establishing that bedaquiline acts as a direct inhibitor of ATP synthase allows rationalisation of its synergy with other compounds and for its role in future combination regimes to be evaluated. The NMR data sets the scene for others to measure the effect bedaquiline has on the ATP/ADP ratio in live mycobacteria to further understand how inhibition of ATP synthase eventually leads to cell death.