New pump structure hints at rising bedaquiline resistance

Bedaquiline is a cornerstone drug for treating multidrug-resistant tuberculosis, but resistance is already emerging. Much of the known clinical resistance to bedaquiline comes from genetic changes that increase expression of an efflux pump called MmpS5/MmpL5 (MmpS5L5), which can push the drug out of Mycobacterium tuberculosis cells. To learn how this pump works and how it might contribute to resistance, a team led by Ben F. Luisi determined the structure of the full MmpS5L5 complex. The goal was to move beyond simply knowing that pump upregulation occurs, and instead to see how bedaquiline physically enters and moves through the pump. By revealing the pump’s shape and testing how genetic changes affect its function, the researchers aimed to find whether other kinds of changes — not just higher pump levels — could make bacteria more resistant to bedaquiline. What they found points to a specific architecture and transport route that help explain how MmpS5L5 can remove bedaquiline from bacterial cells.

The investigators used cryo-electron microscopy to determine the structure of the MmpS5L5 complex and combined structure prediction modelling with functional genetic analysis to test how the pump moves molecules. The cryo-electron microscopy structure revealed a novel trimeric architecture that is distinct from the canonical tripartite RND efflux pumps found in Gram-negative bacteria. The data and modelling indicate that MmpS5L5 uses a periplasmic α-helical coiled-coil tube to transport molecules across the cell wall. Guided by the structure, the team performed genetic experiments and identified specific MmpL5 mutations that alter bedaquiline transport. Those mutations cluster in a region of MmpL5 located in the lower portion of the periplasmic cavity, close to the outer leaflet of the inner membrane, which suggests a likely entry route for bedaquiline into the pump. While current clinical resistance is known to arise from pump upregulation, several MmpL5 variants characterized here increase bedaquiline efflux, pointing to alternate resistance mechanisms.

These findings have immediate significance for how scientists and clinicians think about bedaquiline resistance. By revealing the detailed structure of MmpS5L5 and a likely path for bedaquiline entry, the work shows that bacteria could gain stronger resistance not only by making more pump but also by changing the pump itself. The discovery of a trimeric architecture and a unique α-helical coiled-coil transport tube provides clear targets for future surveillance: sequence changes in MmpL5 that fall in the identified cavity region could signal emerging resistance. The structural map can also guide laboratory studies of how mutations affect drug movement and could inform efforts to design bedaquiline analogues or adjunct therapies that avoid or block this efflux route. In short, the study moves understanding from gene-level observations to a mechanistic picture that presages how new forms of clinical resistance might arise.