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Antimicrobial Agents and Chemotherapy, December 2008, p. 4274-4280, Vol. 52, No. 12
0066-4804/08/$08.00+0     doi:10.1128/AAC.00740-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Gain-of-Function Mutations in the Transcription Factor MRR1 Are Responsible for Overexpression of the MDR1 Efflux Pump in Fluconazole-Resistant Candida dubliniensis Strains {triangledown} ,{dagger}

Sabrina Schubert,1 P. David Rogers,2 and Joachim Morschhäuser1*

Institut für Molekulare Infektionsbiologie, Universität Würzburg, Röntgenring 11, Würzburg D-97070, Germany,1 Department of Pharmacy and Pharmaceutical Sciences, College of Pharmacy, and Department of Pediatrics, College of Medicine, University of Tennessee Health Science Center, Children's Foundation Research Center at Le Bonheur Children's Medical Center, Memphis, Tennessee2

Received 5 June 2008/ Returned for modification 20 August 2008/ Accepted 14 September 2008

Candida dubliniensis, a yeast that is closely related to Candida albicans, can rapidly develop resistance to the commonly used antifungal agent fluconazole in vitro and in vivo during antimycotic therapy. Fluconazole resistance in C. dubliniensis is usually caused by constitutive overexpression of the MDR1 gene, which encodes a multidrug efflux pump of the major facilitator superfamily. The zinc cluster transcription factor Mrr1p has recently been shown to control MDR1 expression in C. albicans in response to inducing stimuli, and gain-of-function mutations in the MRR1 gene result in constitutive upregulation of the MDR1 efflux pump. We identified a gene with a high degree of similarity to C. albicans MRR1 (CaMRR1) in the C. dubliniensis genome sequence. When C. dubliniensis MRR1 (CdMRR1) was expressed in C. albicans mrr1{Delta} mutants, it restored benomyl-inducible MDR1 expression, demonstrating that CdMRR1 is the ortholog of CaMRR1. To investigate whether MDR1 overexpression in C. dubliniensis is caused by mutations in MRR1, we sequenced the MRR1 alleles from a fluconazole-resistant, clinical C. dubliniensis isolate and a matched, fluconazole-susceptible isolate from the same patient as well as those from four in vitro-generated, fluconazole-resistant C. dubliniensis strains derived from two different C. dubliniensis isolates. We found that all five resistant strains contained single nucleotide substitutions or small in-frame deletions that resulted in amino acid changes in Mrr1p. Expression of these mutated alleles in C. albicans resulted in the constitutive activation of the MDR1 promoter and multidrug resistance. Therefore, mutations in MRR1 are the major cause of MDR1 upregulation in both C. albicans and C. dubliniensis, demonstrating that the transcription factor Mrr1p plays a central role in the development of drug resistance in these human fungal pathogens.

* Corresponding author. Mailing address: Institut für Molekulare Infektionsbiologie, Universität Würzburg, Röntgenring 11, Würzburg D-97070, Germany. Phone: 49-931-31 21 52. Fax: 49-931-31 25 78. E-mail: joachim.morschhaeuser{at}mail.uni-wuerzburg.de

{triangledown} Published ahead of print on 22 September 2008.

{dagger} Supplemental material for this article may be found at http://aac.asm.org/.

Antimicrobial Agents and Chemotherapy, December 2008, p. 4274-4280, Vol. 52, No. 12
0066-4804/08/$08.00+0     doi:10.1128/AAC.00740-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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