Metabolic control of drug resistance by a mycobacterial ion channel

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 -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.