Pyrazinamide kills Mycobacterium tuberculosis via pH-driven weak-acid permeation and cytosolic acidification

Pyrazinamide (PZA) is a cornerstone drug in tuberculosis (TB) treatment with a strong bactericidal activity in vivo on both actively and non-replicating bacterial subpopulations. Yet the precise mode of action of its active form, pyrazinoic acid (HPOA), remains unclear. In this study, we comprehensively explore and challenge the two major and conflicted models of PZA mode of action. The pH-dependent model, where the drug is mostly effective at acidic pH by acidifying Mycobacterium tuberculosis (Mtb) cytosol, and the PanD-dependent model where PZA's active form targets the aspartate decarboxylase PanD, therefore depleting pantothenate and subsequently coenzyme A (CoA) levels regardless of the surrounding pH. By combining standard antimicrobial susceptibility testing at various pH with fluorescence-based live recording of Mtb intrabacterial pH, we demonstrate that PZA kills Mtb by decreasing intrabacterial pH, independently of pantothenate levels. Comparative studies between a prototrophic Mtb strain and a pantothenate auxotrophic mutant lacking the panCD locus confirmed that PZA bactericidal activity is primarily driven by pH and its ability to acidify Mtb cytosol, independently of the aspartate decarboxylase PanD. Bio-electrophysiology experiments revealed that acidic pH promotes the conversion of the pyrazinoate anion POA^- into HPOA which in turn acts as conventional weak acid that facilitates membrane permeation and cytosolic acidification. Finally, using custom-based culture media, we demonstrate that PZA displays heterogeneous efficacy according to the media composition, therefore proposing a revisited biological model that might explain the discrepancies around PZA's unique mode of action. Overall, this work constitutes the first comprehensive side-by-side investigation of the two models and univocally supports a pH-dependent mechanism of action underlying PZA sterilizing activity, providing new insights for the development of more effective PZA-like drugs.