Comparative transcriptomic profiling of pyrazinamide resistance in Mycobacterium tuberculosis

Pyrazinamide (PZA) resistance in Mycobacterium tuberculosis challenges tuberculosis management, primarily through pncA mutations, while broader transcriptional adaptations remain unclear. This study aimed to characterize the transcriptomic adaptations associated with PZA resistance using a PZA-resistant clinical strain carrying a 10-nucleotide deletion in pncA (positions 118-127) that abolishes PZA activation (PZAR), in comparison with drug susceptible laboratory strain H37Rv under PZA treated (RvT) and untreated (UTRv) conditions.

Strain specific PZA concentrations was established at 200 µg mL-1 for the PZA-resistant strain (PZAR) and 12.5 µg mL-1 for the H37Rv strain (RvT). Untreated H37Rv strain was used as a reference for comparison. RNA-sequencing identified 3413 differentially expressed genes (Padj ≤ 0.05), including 1428 upregulated and 1360 downregulated genes, while the remaining 625 genes showed moderate but statistically significant expression changes. Functional enrichment was most pronounced in PZAR vs RvT comparison, followed by PZAR vs UTRv, whereas no significant enrichment was observed in RvT vs UTRv, indicating a strong association between the pncA mutation and PZA-responsive transcriptomic profiles. Ribosomal machinery genes (rplC, rplD, and rpsH) were significantly enriched and strongly upregulated in the resistant strain under treatment but only mildly regulated in the laboratory strain. Several anti-TB drug targets (katG, ethA, atpE, panD) were downregulated, while efflux pump genes (Rv1258, Rv3008, Rv3756c) were upregulated, reflecting a coordinated transcriptional response across drug targets. Network analysis identified 19 gene clusters, with prominent modules comprising polyketide synthases, phthiocerol dimycocerosate synthesis genes, fatty acid β-oxidation enzymes, and ESAT-6 (ESX) secretion system.

These findings uncover mutation-associated and PZA-responsive transcriptomic signatures that reveal adaptive pathways involved in tolerance under drug pressure and provide a framework for future functional and therapeutic investigations.