{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,2,21]],"date-time":"2025-02-21T08:26:11Z","timestamp":1740126371251,"version":"3.37.3"},"reference-count":33,"publisher":"Oxford University Press (OUP)","issue":"8","license":[{"start":{"date-parts":[[2021,2,7]],"date-time":"2021-02-07T00:00:00Z","timestamp":1612656000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/academic.oup.com\/journals\/pages\/open_access\/funder_policies\/chorus\/standard_publication_model"}],"funder":[{"DOI":"10.13039\/501100001711","name":"Swiss National Science Foundation","doi-asserted-by":"publisher","award":["PZ00P3 161140"],"award-info":[{"award-number":["PZ00P3 161140"]}],"id":[{"id":"10.13039\/501100001711","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2021,7,14]]},"abstract":"<jats:title>Abstract<\/jats:title>\n               <jats:sec>\n                  <jats:label\/>\n                  <jats:p>Aspergillus fumigatus is the main cause of invasive aspergillosis, for which azole drugs are the first-line therapy. Emergence of pan-azole resistance among A. fumigatus is concerning and has been mainly attributed to mutations in the target gene (cyp51A). However, azole resistance may also result from other mutations (hmg1, hapE) or other adaptive mechanisms. We performed microevolution experiment exposing an A. fumigatus azole-susceptible strain (Ku80) to sub-minimal inhibitory concentration of voriconazole to analyze emergence of azole resistance. We obtained a strain with pan-azole resistance (Ku80R), which was partially reversible after drug relief, and without mutations in cyp51A, hmg1, and hapE. Transcriptomic analyses revealed overexpression of the transcription factor asg1, several ATP-binding cassette (ABC) and major facilitator superfamily transporters and genes of the ergosterol biosynthesis pathway in Ku80R. Sterol analysis showed a significant decrease of the ergosterol mass under voriconazole exposure in Ku80, but not in Ku80R. However, the proportion of the sterol compounds was similar between both strains. To further assess the role of transporters, we used the ABC transporter inhibitor milbemycine oxime (MLB). MLB inhibited transporter activity in both Ku80 and Ku80R and demonstrated some potentiating effect on azole activity. Criteria for synergism were reached for MLB and posaconazole against Ku80. Finally, deletion of asg1 revealed some role of this transcription factor in controlling drug transporter expression, but had no impact on azole susceptibility.<\/jats:p>\n                  <jats:p>This work provides further insight in mechanisms of azole stress adaptation and suggests that drug transporters inhibition may represent a novel therapeutic target.<\/jats:p>\n               <\/jats:sec>\n               <jats:sec>\n                  <jats:title>Lay Summary<\/jats:title>\n                  <jats:p>A pan-azole-resistant strain was generated in vitro, in which drug transporter overexpression was a major trait. Analyses suggested a role of the transporter inhibitor milbemycin oxime in inhibiting drug transporters and potentiating azole activity.<\/jats:p>\n               <\/jats:sec>","DOI":"10.1093\/mmy\/myaa118","type":"journal-article","created":{"date-parts":[[2021,1,18]],"date-time":"2021-01-18T20:22:57Z","timestamp":1611001377000},"page":"763-772","source":"Crossref","is-referenced-by-count":3,"title":["Insights in the molecular mechanisms of an azole stress adapted laboratory-generated <i>Aspergillus fumigatus<\/i> strain"],"prefix":"10.1093","volume":"59","author":[{"given":"Marion","family":"Aruanno","sequence":"first","affiliation":[{"name":"Institute of Microbiology, Lausanne University Hospital and University of Lausanne, 1011 Lausanne, Switzerland"},{"name":"Infectious Diseases Service, Department of Medicine, Lausanne University Hospital and University of Lausanne, 1011 Lausanne, Switzerland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Samantha","family":"Gozel","sequence":"additional","affiliation":[{"name":"Institute of Microbiology, Lausanne University Hospital and University of Lausanne, 1011 Lausanne, Switzerland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Isabelle","family":"Mouyna","sequence":"additional","affiliation":[{"name":"Unit\u00e9 des Aspergillus, Institut Pasteur, 75015 Paris, France"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Josie E","family":"Parker","sequence":"additional","affiliation":[{"name":"Centre for Cytochrome P450 Biodiversity, Institute of Life Science, Swansea University Medical School, Swansea SA2 8PP, UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Daniel","family":"Bachmann","sequence":"additional","affiliation":[{"name":"Institute of Microbiology, Lausanne University Hospital and University of Lausanne, 1011 Lausanne, Switzerland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Patricia","family":"Flamant","sequence":"additional","affiliation":[{"name":"Unit\u00e9 des Aspergillus, Institut Pasteur, 75015 Paris, France"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Alix T","family":"Coste","sequence":"additional","affiliation":[{"name":"Institute of Microbiology, Lausanne University Hospital and University of Lausanne, 1011 Lausanne, Switzerland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Dominique","family":"Sanglard","sequence":"additional","affiliation":[{"name":"Institute of Microbiology, Lausanne University Hospital and University of Lausanne, 1011 Lausanne, Switzerland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Frederic","family":"Lamoth","sequence":"additional","affiliation":[{"name":"Institute of Microbiology, Lausanne University Hospital and University of Lausanne, 1011 Lausanne, Switzerland"},{"name":"Infectious Diseases Service, Department of Medicine, Lausanne University Hospital and University of Lausanne, 1011 Lausanne, Switzerland"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"286","published-online":{"date-parts":[[2021,2,7]]},"reference":[{"key":"2021080323595952900_bib1","doi-asserted-by":"crossref","first-page":"e1","DOI":"10.1016\/j.cmi.2018.01.002","article-title":"Diagnosis and management of Aspergillus diseases: executive summary of the 2017 ESCMID-ECMM-ERS guideline","volume":"24","author":"Ullmann","year":"2018","journal-title":"Clin Microbiol Infect"},{"issue":"6","key":"2021080323595952900_bib2","doi-asserted-by":"crossref","first-page":"1041","DOI":"10.3201\/eid2106.140717","article-title":"Prospective multicenter international surveillance of azole resistance in Aspergillus fumigatus","volume":"21","author":"van der Linden","year":"2015","journal-title":"Emerg Infect Dis"},{"key":"2021080323595952900_bib3","doi-asserted-by":"crossref","first-page":"493","DOI":"10.1097\/QCO.0000000000000005","article-title":"Azole resistance in Aspergillus fumigatus: a growing public health concern","volume":"26","author":"Vermeulen","year":"2013","journal-title":"Curr Opin Infect Dis"},{"issue":"Suppl_3","key":"2021080323595952900_bib4","doi-asserted-by":"crossref","first-page":"S436","DOI":"10.1093\/infdis\/jix210","article-title":"Azole-Resistant aspergillosis: epidemiology, molecular mechanisms, and treatment","volume":"216","author":"Chowdhary","year":"2017","journal-title":"J Infect Dis"},{"key":"2021080323595952900_bib5","doi-asserted-by":"crossref","first-page":"e1005775","DOI":"10.1371\/journal.ppat.1005775","article-title":"Sterol biosynthesis and azole tolerance is governed by the opposing actions of SrbA and the CCAAT binding complex","volume":"12","author":"Gsaller","year":"2016","journal-title":"PLoS Pathog"},{"key":"2021080323595952900_bib6","doi-asserted-by":"crossref","first-page":"e00437","DOI":"10.1128\/mBio.00437-19","article-title":"Mutations in hmg1, challenging the paradigm of clinical triazole resistance in Aspergillus fumigatus","volume":"10","author":"Rybak","year":"2019","journal-title":"mBio"},{"key":"2021080323595952900_bib7","doi-asserted-by":"crossref","first-page":"1486","DOI":"10.1093\/jac\/dkt075","article-title":"The cdr1B efflux transporter is associated with non-cyp51a-mediated itraconazole resistance in Aspergillus fumigatus","volume":"68","author":"Fraczek","year":"2013","journal-title":"J Antimicrob Chemother"},{"key":"2021080323595952900_bib8","doi-asserted-by":"crossref","first-page":"e1006096","DOI":"10.1371\/journal.ppat.1006096","article-title":"A Novel Zn2-Cys6 transcription factor AtrR plays a key role in an azole resistance mechanism of aspergillus fumigatus by co-regulating cyp51A and cdr1B expressions","volume":"13","author":"Hagiwara","year":"2017","journal-title":"PLoS Pathog"},{"key":"2021080323595952900_bib9","doi-asserted-by":"crossref","first-page":"70","DOI":"10.3389\/fmicb.2015.00070","article-title":"Multiple mechanisms contribute to the development of clinically significant azole resistance in Aspergillus fumigatus","volume":"6","author":"Moye-Rowley","year":"2015","journal-title":"Front Microbiol"},{"key":"2021080323595952900_bib10","doi-asserted-by":"crossref","first-page":"e02563","DOI":"10.1128\/mBio.02563-18","article-title":"AtrR is an essential determinant of azole resistance in Aspergillus fumigatus","volume":"10","author":"Paul","year":"2019","journal-title":"mBio"},{"key":"2021080323595952900_bib11","doi-asserted-by":"crossref","first-page":"207","DOI":"10.1128\/EC.5.1.207-211.2006","article-title":"The akuB(KU80) mutant deficient for nonhomologous end joining is a powerful tool for analyzing pathogenicity in Aspergillus fumigatus","volume":"5","author":"da Silva Ferreira","year":"2006","journal-title":"Eukaryotic Cell"},{"key":"2021080323595952900_bib12","doi-asserted-by":"crossref","first-page":"1091","DOI":"10.1128\/EC.00139-06","article-title":"Calcineurin controls growth, morphology, and pathogenicity in Aspergillus fumigatus","volume":"5","author":"Steinbach","year":"2006","journal-title":"Eukaryotic Cell"},{"key":"2021080323595952900_bib13","doi-asserted-by":"crossref","first-page":"2386","DOI":"10.1074\/jbc.M109.077545","article-title":"Characterization of a new beta(1-3)-glucan branching activity of Aspergillus fumigatus","volume":"285","author":"Gastebois","year":"2010","journal-title":"J Biol Chem"},{"key":"2021080323595952900_bib14","doi-asserted-by":"crossref","first-page":"6411","DOI":"10.1128\/IAI.69.10.6411-6418.2001","article-title":"Differential expression of the Aspergillus fumigatus pksP gene detected in vitro and in vivo with green fluorescent protein","volume":"69","author":"Langfelder","year":"2001","journal-title":"Infect Immun"},{"volume-title":"Reference method for broth dilution antifungal susceptibiluty testing of filamentous fungi: second edition (M38-A3)","year":"2017","author":"Clinical and Laboratory Standards Institute (CLSI)","key":"2021080323595952900_bib15"},{"key":"2021080323595952900_bib16","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1093\/jac\/dkg301","article-title":"Synergy, antagonism, and what the chequerboard puts between them","volume":"52","author":"Odds","year":"2003","journal-title":"J Antimicrob Chemother"},{"key":"2021080323595952900_bib17","doi-asserted-by":"crossref","first-page":"e00942","DOI":"10.1128\/mBio.00942-15","article-title":"RNA enrichment method for quantitative transcriptional analysis of pathogens in vivo applied to the fungus Candida albicans","volume":"6","author":"Amorim-Vaz","year":"2015","journal-title":"mBio"},{"key":"2021080323595952900_bib18","doi-asserted-by":"crossref","DOI":"10.1128\/AAC.00208-19","article-title":"Link between heat shock protein 90 and the mitochondrial respiratory chain in the caspofungin stress response of Aspergillus fumigatus","volume":"63","author":"Aruanno","year":"2019","journal-title":"Antimicrob Agents Chemother"},{"key":"2021080323595952900_bib19","doi-asserted-by":"crossref","first-page":"139","DOI":"10.1093\/bioinformatics\/btp616","article-title":"edgeR: a bioconductor package for differential expression analysis of digital gene expression data","volume":"26","author":"Robinson","year":"2010","journal-title":"Bioinformatics"},{"key":"2021080323595952900_bib20","doi-asserted-by":"crossref","first-page":"321","DOI":"10.1093\/biostatistics\/kxm030","article-title":"Small-sample estimation of negative binomial dispersion, with applications to SAGE data","volume":"9","author":"Robinson","year":"2007","journal-title":"Biostatistics"},{"key":"2021080323595952900_bib21","doi-asserted-by":"crossref","first-page":"402","DOI":"10.1006\/meth.2001.1262","article-title":"Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method","volume":"25","author":"Livak","year":"2001","journal-title":"Methods"},{"key":"2021080323595952900_bib22","doi-asserted-by":"crossref","first-page":"1639","DOI":"10.1128\/AEM.03246-12","article-title":"Prothioconazole and prothioconazole-desthio activities against Candida albicans sterol 14-alpha-demethylase","volume":"79","author":"Parker","year":"2013","journal-title":"Appl Environ Microbiol"},{"key":"2021080323595952900_bib23","doi-asserted-by":"crossref","first-page":"873","DOI":"10.1128\/AAC.02040-12","article-title":"Milbemycins: more than efflux inhibitors for fungal pathogens","volume":"57","author":"Silva","year":"2013","journal-title":"Antimicrob Agents Chemother"},{"key":"2021080323595952900_bib24","doi-asserted-by":"crossref","first-page":"1008","DOI":"10.1099\/jmm.0.000120","article-title":"An in vitro and in vivo study on the synergistic effect and mechanism of itraconazole or voriconazole alone and in combination with tetrandrine against Aspergillus fumigatus","volume":"64","author":"Li","year":"2015","journal-title":"J Med Microbiol"},{"key":"2021080323595952900_bib25","doi-asserted-by":"crossref","first-page":"1266","DOI":"10.3389\/fmicb.2019.01266","article-title":"In vitro and in vivo efficacy of a synergistic combination of itraconazole and verapamil against Aspergillus fumigatus","volume":"10","author":"Zeng","year":"2019","journal-title":"Front Microbiol"},{"key":"2021080323595952900_bib26","doi-asserted-by":"crossref","first-page":"141","DOI":"10.1016\/j.drup.2009.09.002","article-title":"Azole-resistance in Aspergillus: proposed nomenclature and breakpoints","volume":"12","author":"Verweij","year":"2009","journal-title":"Drug Resistance Updates"},{"key":"2021080323595952900_bib27","doi-asserted-by":"crossref","first-page":"176","DOI":"10.1016\/j.rvsc.2014.05.013","article-title":"Effect of the inhibitors phenylalanine arginyl ss-naphthylamide (PAssN) and 1-(1-naphthylmethyl)-piperazine (NMP) on expression of genes in multidrug efflux systems of Escherichia coli isolates from bovine mastitis","volume":"97","author":"Barrero","year":"2014","journal-title":"Res Vet Sci"},{"key":"2021080323595952900_bib28","doi-asserted-by":"crossref","first-page":"826","DOI":"10.1111\/jam.12737","article-title":"Synergistic activity of magnolol with azoles and its possible antifungal mechanism against Candida albicans","volume":"118","author":"Sun","year":"2015","journal-title":"J Appl Microbiol"},{"key":"2021080323595952900_bib29","doi-asserted-by":"crossref","first-page":"1508","DOI":"10.1128\/AAC.05706-11","article-title":"The monoamine oxidase A inhibitor clorgyline is a broad-spectrum inhibitor of fungal ABC and MFS transporter efflux pump activities which reverses the azole resistance of Candida albicans and Candida glabrata clinical isolates","volume":"56","author":"Holmes","year":"2012","journal-title":"Antimicrob Agents Chemother"},{"key":"2021080323595952900_bib30","doi-asserted-by":"crossref","first-page":"1491","DOI":"10.1099\/mic.0.2007\/016063-0","article-title":"Divergent functions of three Candida albicans zinc-cluster transcription factors (CTA4, ASG1 and CTF1) complementing pleiotropic drug resistance in Saccharomyces cerevisiae","volume":"154","author":"Coste","year":"2008","journal-title":"Microbiology (Reading)"},{"key":"2021080323595952900_bib31","doi-asserted-by":"crossref","first-page":"616","DOI":"10.1093\/mmy\/myw005","article-title":"Identification of Aspergillus fumigatus multidrug transporter genes and their potential involvement in antifungal resistance","volume":"54","author":"Meneau","year":"2016","journal-title":"Med Myco"},{"key":"2021080323595952900_bib32","doi-asserted-by":"crossref","first-page":"1719","DOI":"10.1128\/AAC.47.5.1719-1726.2003","article-title":"Multiple resistance mechanisms among Aspergillus fumigatus mutants with high-level resistance to itraconazole","volume":"47","author":"Nascimento","year":"2003","journal-title":"AAC"},{"key":"2021080323595952900_bib33","doi-asserted-by":"crossref","first-page":"103265","DOI":"10.1016\/j.fgb.2019.103265","article-title":"Genomic perspective of triazole resistance in clinical and environmental Aspergillus fumigatus isolates without cyp51A mutations","volume":"132","author":"Sharma","year":"2019","journal-title":"Fungal Genet Biol"}],"container-title":["Medical Mycology"],"original-title":[],"language":"en","link":[{"URL":"http:\/\/academic.oup.com\/mmy\/article-pdf\/59\/8\/763\/38982263\/myaa118.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"http:\/\/academic.oup.com\/mmy\/article-pdf\/59\/8\/763\/38982263\/myaa118.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2021,8,4]],"date-time":"2021-08-04T00:00:53Z","timestamp":1628035253000},"score":1,"resource":{"primary":{"URL":"https:\/\/academic.oup.com\/mmy\/article\/59\/8\/763\/6130100"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,2,7]]},"references-count":33,"journal-issue":{"issue":"8","published-online":{"date-parts":[[2021,2,7]]},"published-print":{"date-parts":[[2021,7,14]]}},"URL":"https:\/\/doi.org\/10.1093\/mmy\/myaa118","relation":{},"ISSN":["1369-3786","1460-2709"],"issn-type":[{"type":"print","value":"1369-3786"},{"type":"electronic","value":"1460-2709"}],"subject":[],"published-other":{"date-parts":[[2021,8]]},"published":{"date-parts":[[2021,2,7]]}}}