A Comprehensive Functional Validation of pncA Variants in Mycobacterium tuberculosis Clinical Isolates.

OBJECTIVES: Pyrazinamide (PZA) resistance is attributed to mutations in pncA and complete functional evidence is lacking. We aimed to functionally assess mutations in pncA and its upstream regulatory region (URR) in clinical isolates. METHODS: 405 Mycobacterium tuberculosis (Mtb) clinical isolates from Chongqing, China were analyzed for pncA mutations. After deduplication, 64 variants and 113 additional pncA mutations were chosen from global datasets. 177 mutants were tested for pyrazinamidase activity. Additionally, 34 URR mutations from published data were selected for functional validation. Each construct was assessed for promoter activity and PZA MICs in M. smegmatis and Mtb H37Ra. RESULTS: Among 177 pncA mutations assessed for PZase activity, 31.1% were PZase positive. We tested 35 clinical isolates, 11 were PZA-resistant and all lacked PZase activity, while the 24 sensitive isolates retained PZase activity. For the 34 URR mutations, 17 significantly reduced gfp expression, while 12 increased and 5 had no effect. The 17 promoter-down regulating variants raised PZA MICs to resistant ranges, but the other 17 variants did not. CONCLUSIONS: Our integrated database combining PZase activity, promoter activity and PZA MIC, provides an evidence-based platform for clinicians to interpret pncA variants detected by molecular diagnostics and guide pyrazinamide use in individualized TB therapy.