Resolving decades of ambiguity around a cornerstone TB drug, this paper establishes that pyrazinamide kills via cytoplasmic acidification amplified by α-ketoglutarate efflux through Rv2571c, uncovering a clinically prevalent, pncA-independent resistance mechanism that transforms diagnostics and therapy.
We discovered that Rv2571c, a previously uncharacterized mycobacterial ion channel, controls Mycobacterium tuberculosis(Mtb) susceptibility to pyrazinamide by facilitating the efflux of the TCA-cycle metabolite α-ketoglutarate, which amplifies the drug's ability to acidify the bacterial cytoplasm. This is, to our knowledge, the first demonstration that an ion channel governs antibiotic resistance through metabolite transport — loss of channel function confers resistance in vitro, in macrophages, and in mice, while gain of function causes hypersusceptibility. Critically, loss-of-function mutations are under positive selection in clinical Mtb isolates, establishing Rv2571c as a bona fide resistance determinant in patients and providing a compelling mechanism for the PZA-resistant cases that lack canonical pncA mutations. The broader importance is conceptual: it reveals that an ion channel can dictate drug susceptibility by linking metabolite flux to cytoplasmic pH homeostasis, opening new avenues for resistance diagnostics and for designing PZA-like drugs that shorten TB treatment.
Loss-of-function mutations in the mycobacterial ion channel Rv2571c constitute a clinically prevalent, pncA-independent mechanism of pyrazinamide resistance that operates by blocking α-ketoglutarate export, thereby protecting Mycobacterium tuberculosis from pyrazinamide-induced cytoplasmic acidification under the acidic conditions encountered during host infection.
Pyrazinamide (PZA) is a cornerstone of modern tuberculosis therapy, yet its context-dependent activity has obscured both its mode of action and resistance mechanisms. Using a host-mimicking culture system integrated with genome-wide CRISPRi profiling, metabolomics, and comparative genomics, we identify a previously unrecognized driver of PZA resistance in humans: loss of the ion channel Rv2571c. Rv2571c mediates α-ketoglutarate efflux, amplifying PZA-induced cytoplasmic acidification under host-relevant acidic conditions. Loss-of-function mutations confer resistance in vitro and in vivo and are under positive selection in clinical isolates, establishing this pathway as a resistance determinant in patients. Together, these findings define a novel, ion channel–mediated resistance mechanism, establish cytoplasmic acidification as the basis of PZA killing, and inform resistance detection and treatment-shortening drug development.