Gene-specific Functional Roles of MSMEG_5046, MSMEG_0241, and MSMEG_0232 in Pyrazinoic Acid Efflux Identified through Clustered Regularly Interspaced Short Palindromic Repeat Interference

Background: Efflux-mediated export of pyrazinoic acid (POA) has been associated with pyrazinamide (PZA) resistance, yet the specific transport components remain incompletely defined. Mycobacterium smegmatis, which exhibits intrinsically high POA efflux, provides a quantitative model to study PZA/POA transport mechanisms. Methods: Using clustered regularly interspaced short palindromic repeats interference, we silenced three efflux pump orthologs of Mycobacterium tuberculosis in M. smegmatis: MSMEG_5046 (Rv1250c), MSMEG_0241 (Rv0202c/MmpL11), and MSMEG_0232 (Rv0191c). Gene knockdown was validated by quantitative reverse transcription polymerase chain reaction, achieving 45.1-, 14.6-, and 4.18-fold repression, respectively. POA export kinetics were assessed after PZA loading (final concentration 6.5 mM; 800 µg/mL) using a colorimetric assay over 0–60 min. Efflux rates were calculated from slope values, normalized to intracellular protein content, and compared across biological replicates using analysis of covariance. Results: All silenced strains showed significant differences in efflux slope compared with controls: MSMEG_5046 (P = 0.0184), MSMEG_0241 (P = 0.0497), and MSMEG_0232 (P < 0.0001). At 60 min, normalized POA export changed by +32% (0.0048 mM POA/protein) for MSMEG_5046, −89.33% (0.0134 mM POA/protein) for MSMEG_0241, and −39.33% (0.0059 mM POA/protein) for MSMEG_0232 relative to wild type. MSMEG_0241 and MSMEG_0232 knockdowns reduced both efflux slope and total export, supporting a direct role in POA transport, whereas MSMEG_5046 repression increased efflux, suggesting compensatory activity. Conclusion: This protein-normalized, slope-based POA export assay resolves gene-specific contributions within a networked efflux system and prioritizes targeted validation in M. tuberculosis.