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Gene-specific Functional Roles of MSMEG_5046 , MSMEG_0241 , and MSMEG_0232 in Pyrazinoic Acid Efflux Identified
Kevin R Obando Ballardo1, Kiara Aricoche-Del Campo1, B Carlos Alonso Flores1
1Laboratory of Bioinformatics, Molecular Biology, and Technological Developments, Research and Development Laboratories, Faculty of Sciences and Engineering, Universidad Peruana Cayetano Heredia, San Martin de Porres, Lima, Peru.
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.
Insights
This study identifies key efflux pumps involved in pyrazinoic acid export, a mechanism linked to pyrazinamide resistance. Silencing specific pumps in Mycobacterium smegmatis revealed their direct roles in transport and potential compensatory mechanisms.
Area of Science:
- Microbiology
- Molecular Biology
- Drug Resistance
Background:
- Efflux of pyrazinoic acid (POA) is implicated in pyrazinamide (PZA) resistance.
- The precise transport mechanisms underlying POA efflux remain unclear.
- Mycobacterium smegmatis serves as a model organism for studying POA efflux due to its high intrinsic efflux activity.
Purpose of the Study:
- To elucidate the roles of specific efflux pump orthologs in POA transport.
- To quantify the contribution of individual efflux pumps to POA efflux kinetics.
- To establish a method for resolving gene-specific contributions in networked efflux systems.
Main Methods:
- Utilized CRISPR interference (CRISPRi) to silence three efflux pump orthologs (MSMEG_5046, MSMEG_0241, MSMEG_0232) in Mycobacterium smegmatis.
- Validated gene knockdown using quantitative reverse transcription polymerase chain reaction (qRT-PCR).
- Assessed pyrazinoic acid (POA) export kinetics using a colorimetric assay and calculated normalized efflux rates.
Main Results:
- Significant alterations in POA efflux rates were observed in all silenced strains compared to controls.
- Knockdown of MSMEG_0241 and MSMEG_0232 markedly reduced POA efflux, indicating their direct involvement in transport.
- Silencing of MSMEG_5046 led to increased POA efflux, suggesting a compensatory role in the efflux system.
Conclusions:
- The developed protein-normalized, slope-based assay effectively resolves gene-specific roles in complex efflux systems.
- Findings prioritize MSMEG_0241 and MSMEG_0232 for further investigation as direct POA transporters.
- This approach facilitates targeted validation of efflux mechanisms in Mycobacterium tuberculosis, aiding in understanding PZA resistance.
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