{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,7]],"date-time":"2026-07-07T13:19:41Z","timestamp":1783430381923,"version":"3.54.6"},"reference-count":17,"publisher":"Ovid Technologies (Wolters Kluwer Health)","issue":"2","license":[{"start":{"date-parts":[[2026,4,1]],"date-time":"2026-04-01T00:00:00Z","timestamp":1775001600000},"content-version":"unspecified","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"content-domain":{"domain":["lww.com","ovid.com"],"crossmark-restriction":true},"short-container-title":[],"published-print":{"date-parts":[[2026,4]]},"abstract":"\n Background:<\/jats:title>\n \n Efflux-mediated export of pyrazinoic acid (POA) has been associated with pyrazinamide (PZA) resistance, yet the specific transport components remain incompletely defined.\n Mycobacterium smegmatis<\/jats:italic>\n , which exhibits intrinsically high POA efflux, provides a quantitative model to study PZA\/POA transport mechanisms.\n <\/jats:p>\n <\/jats:sec>\n \n Methods:<\/jats:title>\n \n Using clustered regularly interspaced short palindromic repeats interference, we silenced three efflux pump orthologs of\n Mycobacterium tuberculosis<\/jats:italic>\n in\n M. smegmatis<\/jats:italic>\n :\n MSMEG_5046<\/jats:italic>\n (\n Rv1250c<\/jats:italic>\n ),\n MSMEG_0241<\/jats:italic>\n (\n Rv0202c<\/jats:italic>\n \/MmpL11), and\n MSMEG_0232<\/jats:italic>\n (\n Rv0191c<\/jats:italic>\n ). 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 \u00b5g\/mL) using a colorimetric assay over 0\u201360 min. Efflux rates were calculated from slope values, normalized to intracellular protein content, and compared across biological replicates using analysis of covariance.\n <\/jats:p>\n <\/jats:sec>\n \n Results:<\/jats:title>\n \n All silenced strains showed significant differences in efflux slope compared with controls:\n MSMEG_5046<\/jats:italic>\n (\n P<\/jats:italic>\n = 0.0184),\n MSMEG_0241<\/jats:italic>\n (\n P<\/jats:italic>\n = 0.0497), and\n MSMEG_0232<\/jats:italic>\n (\n P<\/jats:italic>\n < 0.0001). At 60 min, normalized POA export changed by +32% (0.0048 mM POA\/protein) for\n MSMEG_5046<\/jats:italic>\n , \u221289.33% (0.0134 mM POA\/protein) for\n MSMEG_0241<\/jats:italic>\n , and \u221239.33% (0.0059 mM POA\/protein) for\n MSMEG_0232<\/jats:italic>\n relative to wild type.\n MSMEG_0241<\/jats:italic>\n and\n MSMEG_0232<\/jats:italic>\n knockdowns reduced both efflux slope and total export, supporting a direct role in POA transport, whereas\n MSMEG_5046<\/jats:italic>\n repression increased efflux, suggesting compensatory activity.\n <\/jats:p>\n <\/jats:sec>\n \n Conclusion:<\/jats:title>\n \n This protein-normalized, slope-based POA export assay resolves gene-specific contributions within a networked efflux system and prioritizes targeted validation in\n M. tuberculosis.<\/jats:italic>\n <\/jats:p>\n <\/jats:sec>","DOI":"10.4103\/ijmy.ijmy_8_26","type":"journal-article","created":{"date-parts":[[2026,6,30]],"date-time":"2026-06-30T22:15:23Z","timestamp":1782857723000},"page":"147-152","update-policy":"https:\/\/doi.org\/10.1097\/lww.0000000000001000","source":"Crossref","is-referenced-by-count":0,"title":["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"],"prefix":"10.4103","volume":"15","author":[{"given":"Kevin R. Obando","family":"Ballardo","sequence":"first","affiliation":[{"name":"Laboratory 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"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kiara Aricoche-Del","family":"Campo","sequence":"additional","affiliation":[{"name":"Laboratory 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"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"B Carlos Alonso","family":"Flores","sequence":"additional","affiliation":[{"name":"Laboratory 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"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Robert H.","family":"Gilman","sequence":"additional","affiliation":[{"name":"Department of International Health, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mirko","family":"Zimic","sequence":"additional","affiliation":[{"name":"Laboratory 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"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7118-9301","authenticated-orcid":false,"given":"Patricia","family":"Sheen","sequence":"additional","affiliation":[{"name":"Laboratory 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"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"276","published-online":{"date-parts":[[2026,6,30]]},"reference":[{"key":"R4-20260707","doi-asserted-by":"crossref","first-page":"2002013","DOI":"10.1183\/13993003.02013-2020","article-title":"Optimising pyrazinamide for the treatment of tuberculosis","volume":"58","author":"Zhang","year":"2021","journal-title":"Eur Respir J"},{"key":"R5-20260707","doi-asserted-by":"crossref","first-page":"e0309352","DOI":"10.1371\/journal.pone.0309352","article-title":"Studying the dynamics of the drug processing of pyrazinamide in Mycobacterium tuberculosis","volume":"19","author":"Requena","year":"2024","journal-title":"PLoS One"},{"key":"R6-20260707","doi-asserted-by":"crossref","first-page":"dlae037","DOI":"10.1093\/jacamr\/dlae037","article-title":"Prediction of pyrazinamide resistance in Mycobacterium 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