Fountain, A. J. | bioRxiv Science

bioRxiv2025

Structural and functional analysis of the Mycobacterium tuberculosis MmpS5L5 efflux pump presages a pathway to increased bedaquiline resistance

Bedaquiline, an antitubercular drug that targets ATP-synthase, is a key component of a new oral drug regimen that has revolutionized the treatment of multi drug resistant tuberculosis. Clinical bedaquiline resistance in Mycobacterium tuberculosis has rapidly emerged, primarily due to mutations in the transcriptional repressor, Rv0678 that result in upregulation of the Resistance-Nodulation-Division (RND) efflux pump MmpS5/MmpL5 (MmpS5L5). Here, to understand how MmpS5L5 effluxes bedaquiline, we determined the structure of the MmpS5L5 complex using cryo-electron microscopy, revealing a novel trimeric architecture distinct from the canonical tripartite RND efflux pumps of Gram-negative bacteria. Structure prediction modelling in conjunction with functional genetic analysis indicates that it uses a periplasmic coiled-coil tube to transport molecules across the cell wall. Structure-guided genetic approaches identify MmpL5 mutations that alter bedaquiline transport; these mutations converge on a region in MmpL5 located in the lower portion of the periplasmic cavity, proximal to the outer leaflet of the inner membrane, suggesting a route for bedaquiline entry into the pump. While currently known clinical resistance to bedaquiline is due to pump upregulation, our findings that several MmpL5 variants increase bedaquiline efflux may presage the emergence of additional modes of clinical resistance. Significance StatementResistance to bedaquiline, a cornerstone drug for treating multidrug-resistant tuberculosis, is rapidly emerging due to mutations that upregulate expression of the MmpS5L5 efflux pump. Here, we reveal the cryo-EM structure of this pump, showing a novel trimeric architecture and a unique -helical coiled-coil tube for drug transport. Structure-guided genetic analysis identifies MmpL5 variants that further increase bedaquiline efflux, suggesting potential resistance mechanisms beyond pump upregulation.

microbiology↗

bioRxiv2025

Verapamil and its metabolite norverapamil inhibit the Mycobacterium tuberculosis MmpS5L5 efflux pump to potentiate the activity bedaquiline and other antitubercular drugs

Bedaquiline is the cornerstone of a new regimen for the treatment of drug-resistant tuberculosis. However, its clinical use is threatened by the emergence of bedaquiline-resistant strains of Mycobacterium tuberculosis. Bedaquiline targets mycobacterial ATP synthase but the predominant route to clinical bedaquiline resistance is via upregulation of the MmpS5L5 efflux pump due to mutations that inactivate the transcriptional repressor Rv0678. Here, we show that the MmpS5L5 efflux pump reduces susceptibility to bedaquiline as well as its new, more potent derivative TBAJ-876 and other antimicrobial substrates, including clofazimine and the DprE1 inhibitors PBTZ-169 and OPC-167832. Furthermore, the increased resistance of Rv0678 mutants stems entirely from increased MmpS5L5 activity. These results highlight the potential of a pharmacological MmpS5L5 inhibitor to increase drug efficacy. Verapamil, primarily used as a calcium channel inhibitor, is known to inhibit diverse efflux pumps and to potentiate bedaquiline and clofazimine activity in M. tuberculosis. Here, we show that verapamil potentiates the activity of multiple diverse MmpS5L5 substrates. Using biochemical approaches, we demonstrate that verapamil does not exert this effect by acting as a disruptor of the protonmotive force used to power MmpS5L5, as previously proposed, suggesting that verapamil inhibits the function of the MmpS5L5 pump. Finally, norverapamil, the major verapamil metabolite, which has greatly reduced calcium channel activity, has equal potency in reducing resistance to MmpS5L5 substrates. Our findings highlight verapamils potential for enhancing bedaquiline TB treatment, for preventing acquired resistance to bedaquiline and other MmpS5L5 substrates, while also providing the impetus to identify additional MmpS5L5 inhibitors. Significance StatementBedaquiline, an antitubercular drug targeting ATP synthase, forms the backbone of highly efficacious treatment regimens for drug-resistant tuberculosis. Bedaquiline resistance is emerging as a significant problem and is most commonly caused by mutations in Rv0678 that result in upregulation of the MmpS5L5 efflux pump. Here we define the contribution of the MmpS5L5 efflux pump to drug resistance in wild-type and Rv0678 mutant strains and show that the commonly used drug verapamil can inhibit MmpS5L5 activity. This suggests that this safe and inexpensive drug may be useful in enhancing bedaquiline treatment of TB and to help prevent bedaquiline acquired resistance.

microbiology↗