{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,30]],"date-time":"2026-09-30T13:22:03Z","timestamp":1790774523701,"version":"4.1.0"},"reference-count":56,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2025,4,3]],"date-time":"2025-04-03T00:00:00Z","timestamp":1743638400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Microbiol."],"abstract":"<jats:p>Clofazimine (CFZ) is an anti-leprosy drug shown to improve outcomes in treatment of multidrug-resistant tuberculosis (TB) and nontuberculous mycobacterial infections. Studies in <jats:italic>Mycobacterium tuberculosis<\/jats:italic> and <jats:italic>Mycobacterium avium<\/jats:italic> identified CFZ resistance mutations in the gene that encodes the MmpR5\/MmpT5 regulator, which increase expression of the mycobactin (MBT) transporter, MmpS5\/L5. We found exposure of <jats:italic>M. tuberculosis<\/jats:italic> to CFZ induced a pattern of gene expression that mirrored low iron conditions, including strong induction of genes that encode MBT synthesis and transport. We identified a corresponding increase in MBT levels indicating a role in iron homeostasis in CFZ activity. CFZ bactericidal activity against both <jats:italic>Mycobacterium smegmatis<\/jats:italic> and <jats:italic>M. tuberculosis<\/jats:italic> was increased in high iron conditions in which MTB synthesis and transport was limited. We show the presence of MBT correlated with decreased CFZ killing activity while inhibition of MBT synthesis increased killing. Considerable iron efflux was observed during CFZ treatment indicating iron loss may be a feature of CFZ anti-mycobacterial activity. CFZ solubility studies and CFZ-mediated reduction of free iron indicate a potential redox interaction between CFZ and iron. MBT or MBT flux across the cell envelope appears to block CFZ killing in <jats:italic>M. smegmatis<\/jats:italic> and potentially <jats:italic>M. tuberculosis<\/jats:italic>. The specific mechanism by which MBT inhibits CFZ lethality remains unclear but may involve, increased iron acquisition, the MmpS5\/L5 MBT efflux pump, or the CFZ subcellular localization altered by the redox state and solubility of CFZ. CFZ has thus far been proven most effective against <jats:italic>Mycobacterium leprae<\/jats:italic>, which lacks MBT, indicating an understanding of the complex interaction of CFZ with iron acquisition systems may suggest more effective therapeutic applications.<\/jats:p>","DOI":"10.3389\/fmicb.2025.1539139","type":"journal-article","created":{"date-parts":[[2025,4,3]],"date-time":"2025-04-03T07:18:00Z","timestamp":1743664680000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":2,"title":["Mycobactin and clofazimine activity are negatively correlated in mycobacteria"],"prefix":"10.3389","volume":"16","author":[{"given":"Martin I.","family":"Voskuil","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christopher R.","family":"Covey","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Matthew J.","family":"Reichlen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anushila","family":"Chatterjee","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Breck A.","family":"Duerkop","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Surendra","family":"Dawadi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Courtney C.","family":"Aldrich","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alexander","family":"Aaring","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2025,4,3]]},"reference":[{"key":"ref1","doi-asserted-by":"publisher","first-page":"574","DOI":"10.1128\/JCM.02087-16","article-title":"Emergence of mmpT5 variants during bedaquiline treatment of Mycobacterium intracellulare lung disease","volume":"55","author":"Alexander","year":"2017","journal-title":"J. Clin. Microbiol."},{"key":"ref2","doi-asserted-by":"publisher","first-page":"4635","DOI":"10.1016\/0006-2952(88)90332-2","article-title":"Apparent involvement of phospholipase A2, but not protein kinase C, in the pro-oxidative interactions of clofazimine with human phagocytes","volume":"37","author":"Anderson","year":"1988","journal-title":"Biochem. Pharmacol."},{"key":"ref3","doi-asserted-by":"publisher","first-page":"413","DOI":"10.1038\/s41586-020-2136-9","article-title":"The ABC exporter IrtAB imports and reduces mycobacterial siderophores","volume":"580","author":"Arnold","year":"2020","journal-title":"Nature"},{"key":"ref4","doi-asserted-by":"publisher","first-page":"1180","DOI":"10.5588\/ijtld.14.0100","article-title":"Successful \u20189-month Bangladesh regimen\u2019 for multidrug-resistant tuberculosis among over 500 consecutive patients","volume":"18","author":"Aung","year":"2014","journal-title":"Int. J. Tuberc. Lung Dis."},{"key":"ref5","doi-asserted-by":"publisher","first-page":"1013","DOI":"10.1038\/1791013a0","article-title":"A new series of phenazines (rimino-compounds) with high antituberculosis activity","volume":"179","author":"Barry","year":"1957","journal-title":"Nature"},{"key":"ref6","doi-asserted-by":"publisher","first-page":"4970","DOI":"10.1038\/s41467-019-12956-2","article-title":"Plasticity of the Mycobacterium tuberculosis respiratory chain and its impact on tuberculosis drug development","volume":"10","author":"Beites","year":"2019","journal-title":"Nat. Commun."},{"key":"ref7","doi-asserted-by":"publisher","first-page":"1252","DOI":"10.1073\/pnas.97.3.1252","article-title":"The salicylate-derived mycobactin siderophores of Mycobacterium tuberculosis are essential for growth in macrophages","volume":"97","author":"De Voss","year":"2000","journal-title":"Proc. Natl. Acad. Sci."},{"key":"ref8","doi-asserted-by":"publisher","first-page":"2256","DOI":"10.1128\/AAC.05114-14","article-title":"A novel antimycobacterial compound acts as an intracellular iron chelator","volume":"59","author":"Dragset","year":"2015","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref9","doi-asserted-by":"publisher","first-page":"453","DOI":"10.3109\/00365518309168286","article-title":"Ferene-S as the chromogen for serum iron determinations","volume":"43","author":"Eskelinen","year":"1983","journal-title":"Scand. J. Clin. Lab. Invest."},{"key":"ref10","doi-asserted-by":"publisher","first-page":"1097","DOI":"10.1128\/AAC.02615-15","article-title":"Clofazimine prevents the regrowth of Mycobacterium abscessus and Mycobacterium avium type strains exposed to amikacin and clarithromycin","volume":"60","author":"Ferro","year":"2015","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref11","doi-asserted-by":"publisher","first-page":"557","DOI":"10.1111\/j.1365-2958.1994.tb02189.x","article-title":"Identification of genes involved in the sequestration of iron in mycobacteria: the ferric exochelin biosynthetic and uptake pathways","volume":"14","author":"Fiss","year":"1994","journal-title":"Mol. Microbiol."},{"key":"ref12","doi-asserted-by":"publisher","first-page":"851","DOI":"10.1046\/j.1365-2958.2001.02684.x","article-title":"The Mycobacterium tuberculosis IdeR is a dual functional regulator that controls transcription of genes involved in iron acquisition, iron storage and survival in macrophages","volume":"42","author":"Gold","year":"2001","journal-title":"Mol. Microbiol."},{"key":"ref13","doi-asserted-by":"publisher","first-page":"1001","DOI":"10.5588\/ijtld.12.0144","article-title":"Systematic review of clofazimine for the treatment of drug-resistant tuberculosis","volume":"17","author":"Gopal","year":"2013","journal-title":"Int. J. Tuberc. Lung Dis."},{"key":"ref14","doi-asserted-by":"publisher","first-page":"2979","DOI":"10.1128\/AAC.00037-14","article-title":"Cross-resistance between clofazimine and bedaquiline through upregulation of MmpL5 in Mycobacterium tuberculosis","volume":"58","author":"Hartkoorn","year":"2014","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref15","doi-asserted-by":"publisher","first-page":"4026","DOI":"10.1128\/AAC.02565-14","article-title":"Limited activity of clofazimine as a single drug in a mouse model of tuberculosis exhibiting caseous necrotic granulomas","volume":"58","author":"Irwin","year":"2014","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref16","doi-asserted-by":"publisher","first-page":"e00292","DOI":"10.1128\/AAC.02141-18","article-title":"In vitro study of stepwise acquisition of rv0678 and atpE mutations conferring bedaquiline resistance","volume":"63","author":"Ismail","year":"","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref17","doi-asserted-by":"publisher","first-page":"e02141","DOI":"10.1128\/AAC.00292-19","article-title":"Clofazimine exposure in vitro selects efflux pump mutants and bedaquiline resistance","volume":"63","author":"Ismail","year":"","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref18","doi-asserted-by":"publisher","first-page":"1767","DOI":"10.1128\/JB.01312-10","article-title":"Mycobacterium tuberculosis can utilize heme as an iron source","volume":"193","author":"Jones","year":"2011","journal-title":"J. Bacteriol."},{"key":"ref19","doi-asserted-by":"publisher","first-page":"1945","DOI":"10.1073\/pnas.1311402111","article-title":"Self-poisoning of Mycobacterium tuberculosis by interrupting siderophore recycling","volume":"111","author":"Jones","year":"2014","journal-title":"Proc. Natl. Acad. Sci."},{"key":"ref20","first-page":"58","article-title":"Absence of mycobactin in Mycobacterium leprae; probably a microbe dependent microorganism implications","volume":"57","author":"Kato","year":"1985","journal-title":"Indian J. Lepr."},{"key":"ref21","doi-asserted-by":"publisher","first-page":"e02349","DOI":"10.1128\/AAC.02349-19","article-title":"Synergistic activity of clofazimine and clarithromycin in an aerosol mouse model of Mycobacterium avium infection","volume":"64","author":"Lanoix","year":"2020","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref22","doi-asserted-by":"publisher","first-page":"10A. 2.1","DOI":"10.1002\/9780471729259.mc10a02s6","article-title":"Genetic manipulation of Mycobacterium tuberculosis","volume":"6","author":"Larsen","year":"2007","journal-title":"Curr. Protoc. Microbiol."},{"key":"ref23","doi-asserted-by":"publisher","first-page":"1662","DOI":"10.1128\/JB.00926-09","article-title":"The Mycobacterium tuberculosis DosR regulon assists in metabolic homeostasis and enables rapid recovery from nonrespiring dormancy","volume":"192","author":"Leistikow","year":"2010","journal-title":"J. Bacteriol."},{"key":"ref24","doi-asserted-by":"publisher","first-page":"10333","DOI":"10.1038\/srep10333","article-title":"The cytochrome bd-type quinol oxidase is important for survival of Mycobacterium smegmatis under peroxide and antibiotic-induced stress","volume":"5","author":"Lu","year":"2015","journal-title":"Sci. Rep."},{"key":"ref25","doi-asserted-by":"publisher","first-page":"5138","DOI":"10.1128\/AAC.00918-13","article-title":"Pharmacokinetic and in vivo efficacy studies of the mycobactin biosynthesis inhibitor salicyl-AMS in mice","volume":"57","author":"Lun","year":"2013","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref26","doi-asserted-by":"publisher","first-page":"2809","DOI":"10.1128\/JB.00088-12","article-title":"Analyses of MbtB, MbtE, and MbtF suggest revisions to the mycobactin biosynthesis pathway in Mycobacterium tuberculosis","volume":"194","author":"McMahon","year":"2012","journal-title":"J. Bacteriol."},{"key":"ref27","doi-asserted-by":"publisher","first-page":"e0162922","DOI":"10.1128\/aac.01629-22","article-title":"Intricate link between siderophore secretion and drug efflux in Mycobacterium tuberculosis","volume":"67","author":"Meikle","year":"2023","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref28","doi-asserted-by":"publisher","first-page":"84","DOI":"10.1016\/j.tube.2008.08.003","article-title":"Azole resistance in Mycobacterium tuberculosis is mediated by the MmpS5-MmpL5 efflux system","volume":"89","author":"Milano","year":"2009","journal-title":"Tuberculosis (Edinb.)"},{"key":"ref29","doi-asserted-by":"publisher","first-page":"2770","DOI":"10.1128\/JB.00353-12","article-title":"Complete annotated genome sequence of Mycobacterium tuberculosis Erdman","volume":"194","author":"Miyoshi-Akiyama","year":"2012","journal-title":"J. Bacteriol."},{"key":"ref30","doi-asserted-by":"publisher","first-page":"1465","DOI":"10.1080\/01932691.2010.513314","article-title":"Interaction of clofazimine with divalent metal ions: a fluorescence quenching study","volume":"32","author":"Mohd","year":"2011","journal-title":"J. Dispers. Sci. Technol."},{"key":"ref31","first-page":"475","article-title":"Clofazimine binding studies with deoxyribonucleic acid","volume":"44","author":"Morrison","year":"1976","journal-title":"Int. J. Lepr. Other Mycobact. Dis."},{"key":"ref32","doi-asserted-by":"publisher","first-page":"677","DOI":"10.1080\/10715760290029128","article-title":"A simple and rapid method for the determination of \u201cfree\u201d iron in biological fluids","volume":"36","author":"Nilsson","year":"2002","journal-title":"Free Radic. Res."},{"key":"ref33","doi-asserted-by":"publisher","first-page":"631","DOI":"10.1016\/S1074-5521(98)90291-5","article-title":"Identification of a Mycobacterium tuberculosis gene cluster encoding the biosynthetic enzymes for assembly of the virulence-conferring siderophore mycobactin","volume":"5","author":"Quadri","year":"1998","journal-title":"Chem. Biol."},{"key":"ref34","doi-asserted-by":"publisher","first-page":"16526","DOI":"10.1074\/jbc.M113.538959","article-title":"Crystal structure of the transcriptional regulator Rv0678 of Mycobacterium tuberculosis","volume":"289","author":"Radhakrishnan","year":"2014","journal-title":"J. Biol. Chem."},{"key":"ref35","doi-asserted-by":"publisher","first-page":"2207","DOI":"10.1099\/13500872-142-8-2207","article-title":"The occurrence of carboxymycobactin, the siderophore of pathogenic mycobacteria, as a second extracellular siderophore in Mycobacterium smegmatis","volume":"142","author":"Ratledge","year":"1996","journal-title":"Microbiology"},{"key":"ref36","doi-asserted-by":"publisher","first-page":"615","DOI":"10.1093\/jac\/43.5.615","article-title":"Antimycobacterial activities of riminophenazines","volume":"43","author":"Reddy","year":"1999","journal-title":"J. Antimicrob. Chemother."},{"key":"ref37","first-page":"785","article-title":"New prospects for the study of leprosy in the laboratory","volume":"40","author":"Rees","year":"1969","journal-title":"Bull. World Health Organ."},{"key":"ref38","doi-asserted-by":"publisher","first-page":"e01316","DOI":"10.1128\/AAC.01316-18","article-title":"Mutations in the MAB_2299c TetR regulator confer cross-resistance to clofazimine and bedaquiline in Mycobacterium abscessus","volume":"63","author":"Richard","year":"2019","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref39","doi-asserted-by":"publisher","first-page":"424","DOI":"10.1128\/JB.188.2.424-430.2006","article-title":"Identification of an ABC transporter required for iron acquisition and virulence in Mycobacterium tuberculosis","volume":"188","author":"Rodriguez","year":"2006","journal-title":"J. Bacteriol."},{"key":"ref40","doi-asserted-by":"publisher","first-page":"3371","DOI":"10.1128\/IAI.70.7.3371-3381.2002","article-title":"IdeR, an essential gene in Mycobacterium tuberculosis: role of IdeR in iron-dependent gene expression, iron metabolism, and oxidative stress response","volume":"70","author":"Rodriguez","year":"2002","journal-title":"Infect. Immun."},{"key":"ref41","doi-asserted-by":"publisher","first-page":"935","DOI":"10.1093\/jac\/dky526","article-title":"A bedaquiline\/clofazimine combination regimen might add activity to the treatment of clinically relevant non-tuberculous mycobacteria","volume":"74","author":"Ruth","year":"2019","journal-title":"J. Antimicrob. Chemother."},{"key":"ref42","first-page":"226","article-title":"Induced pulmonary tuberculosis in the rhesus monkey: its usefulness in evaluating chemotherapeutic agents","volume-title":"Transactions of the fourteenth conference on the chemotherapy of tuberculosis. Vol. 14","author":"Schmidt","year":"1955"},{"key":"ref43","doi-asserted-by":"publisher","first-page":"3653","DOI":"10.1016\/S0040-4039(01)00531-7","article-title":"Structure and absolute configuration of mycobactin J","volume":"42","author":"Schwartz","year":"2001","journal-title":"Tetrahedron Lett."},{"key":"ref44","doi-asserted-by":"publisher","first-page":"187","DOI":"10.1042\/bj3050187","article-title":"Isolation, purification and structure of exochelin MS, the extracellular siderophore from Mycobacterium smegmatis","volume":"305","author":"Sharman","year":"1995","journal-title":"Biochem. J."},{"key":"ref45","doi-asserted-by":"publisher","first-page":"18792","DOI":"10.1073\/pnas.0900589106","article-title":"Mycobacterial Esx-3 is required for mycobactin-mediated iron acquisition","volume":"106","author":"Siegrist","year":"2009","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref46","doi-asserted-by":"publisher","first-page":"31","DOI":"10.1021\/jm051060o","article-title":"Rationally designed nucleoside antibiotics that inhibit siderophore biosynthesis of Mycobacterium tuberculosis","volume":"49","author":"Somu","year":"2006","journal-title":"J. Med. Chem."},{"key":"ref47","doi-asserted-by":"publisher","first-page":"2399","DOI":"10.1128\/JB.00359-16","article-title":"Iron homeostasis in Mycobacterium tuberculosis: mechanistic insights into siderophore-mediated iron uptake","volume":"198","author":"Sritharan","year":"2016","journal-title":"J. Bacteriol."},{"key":"ref48","doi-asserted-by":"publisher","first-page":"1361","DOI":"10.1093\/cid\/civ027","article-title":"Clofazimine for the treatment of multidrug-resistant tuberculosis: prospective, multicenter, randomized controlled study in China","volume":"60","author":"Tang","year":"2015","journal-title":"Clin. Infect. Dis."},{"key":"ref49","doi-asserted-by":"publisher","first-page":"684","DOI":"10.1164\/rccm.201001-0077OC","article-title":"Short, highly effective, and inexpensive standardized treatment of multidrug-resistant tuberculosis","volume":"182","author":"Van Deun","year":"2010","journal-title":"Am. J. Respir. Crit. Care Med."},{"key":"ref50","doi-asserted-by":"publisher","first-page":"684","DOI":"10.1093\/jac\/dkw502","article-title":"Unexpected high prevalence of resistance-associated Rv0678 variants in MDR-TB patients without documented prior use of clofazimine or bedaquiline","volume":"72","author":"Villellas","year":"2017","journal-title":"J. Antimicrob. Chemother."},{"key":"ref51","doi-asserted-by":"publisher","first-page":"e1003120","DOI":"10.1371\/journal.ppat.1003120","article-title":"Discovery of a siderophore export system essential for virulence of Mycobacterium tuberculosis","volume":"9","author":"Wells","year":"2013","journal-title":"PLoS Pathog."},{"key":"ref52","volume-title":"WHO consolidated guideline on drug-resistant tuberculosis treatment","year":"2022"},{"key":"ref53","volume-title":"Global tuberculosis report 2023","year":"2023"},{"key":"ref54","doi-asserted-by":"publisher","first-page":"10276","DOI":"10.1074\/jbc.M110.200501","article-title":"Reduction of clofazimine by mycobacterial type 2 NADH:quinone oxidoreductase: a pathway for the generation of bactericidal levels of reactive oxygen species","volume":"286","author":"Yano","year":"2011","journal-title":"J. Biol. Chem."},{"key":"ref55","doi-asserted-by":"publisher","first-page":"e1008337","DOI":"10.1371\/journal.ppat.1008337","article-title":"Comprehensive analysis of iron utilization by Mycobacterium tuberculosis","volume":"16","author":"Zhang","year":"2020","journal-title":"PLoS Pathog."},{"key":"ref56","doi-asserted-by":"publisher","first-page":"629","DOI":"10.1046\/j.1365-2958.1998.00961.x","article-title":"Exochelin genes in Mycobacterium smegmatis: identification of an ABC transporter and two non-ribosomal peptide synthetase genes","volume":"29","author":"Zhu","year":"1998","journal-title":"Mol. Microbiol."}],"container-title":["Frontiers in Microbiology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fmicb.2025.1539139\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,4,3]],"date-time":"2025-04-03T07:18:27Z","timestamp":1743664707000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fmicb.2025.1539139\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,4,3]]},"references-count":56,"alternative-id":["10.3389\/fmicb.2025.1539139"],"URL":"https:\/\/doi.org\/10.3389\/fmicb.2025.1539139","relation":{},"ISSN":["1664-302X"],"issn-type":[{"value":"1664-302X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,4,3]]},"article-number":"1539139"}}