Deciphering the transcriptomic landscape of pyrazinamide resistance in Mycobacterium tuberculosis

Pyrazinamide resistance in Mycobacterium tuberculosis poses a major challenge to tuberculosis management and is primarily linked to pncA mutations, while the broader transcriptional adaptations underlying this resistance remain unclear. In this study, we performed a comparative analysis of the transcriptomic response of a mutant clinical strain and the drug-susceptible laboratory strain H37Rv under PZA exposure and non-exposure conditions. The clinical strain used for this analysis carried a 10-nucleotide deletion mutation in pncA (positions 118-127) that abolished PZA activation, identified in our previous study. The critical drug concentrations were established at 200 {micro}g/mL for the clinical strain (CST) and 12.5 {micro}g/mL for the H37Rv strain (RvT), with the untreated H37Rv strain (UTRv) used as a reference. RNA-sequence profiles from treated and untreated conditions were analyzed to identify differentially expressed genes, followed by functional enrichment, KEGG pathway mapping, and protein-protein interaction network analysis. Analysis revealed 3,413 differentially expressed genes (padj [≤] 0.05), including 1,428 upregulated and 1,360 downregulated genes. Functional enrichment was predominantly detected in CST vs. RvT followed by CST vs. UTRv, whereas no significant enrichment emerged in RvT vs. UTRv, underscoring the mutations dominant influence on the pyrazinamide response. The ribosomal machinery genes rplC, rplD, and rpsH were significantly enriched and strongly upregulated in the mutant strain under treatment but only mildly regulated in the laboratory strain. We observed that several anti-TB drug targets (katG, ethA, atpE, panD) were downregulated, and a few efflux pumps (Rv1258, Rv3008, Rv3756c) were upregulated, reflecting cross-resistance mechanisms. Network analysis identified 19 clusters, and prominent modules comprised polyketide synthases, PDIM synthesis genes, fatty acid {beta}-oxidation enzymes and ESX secretion system. These interconnected modules highlight the metabolic and structural strategies that support persistence under drug pressure. Collectively, our findings link mutation-driven and PZA-induced transcriptomic alterations to adaptive pathways and provide insights into the mechanisms underlying tolerance and potential therapeutic opportunities. Author summaryPyrazinamide is a vital anti-tuberculosis drug, which helps to shorten the treatment duration and while resistance is common, it is not completely understood. Our previous work identified a clinical strain of Mycobacterium tuberculosis that was resistant to pyrazinamide due to a mutation in target gene responsible for drug activity. In this study, we compared the gene expression pattern of this resistant strain with a standard laboratory strain under drug exposure. The resistant strain exhibited distinct gene expression patterns, specifically, the activated genes associated with cell damage repair, lipid biosynthesis for the cell envelope, and ATP maintenance for energy production while it repressed genes that promote dormancy and virulence. Our findings suggest that the resistant strain is actively and metabolically adapts to the drug stress, rather than simply becoming dormant. We also found notable gene alterations in the other anti-tuberculosis drug targets, indicating possible cross resistance with pyrazinamide. Collectively, our findings provide insights into how genetic mutations alter the gene expression of resistant strains that adapt to survive drug exposure.