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.