AAC Accepts, published online ahead of print on 2 November 2009

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Antimicrob. Agents Chemother. doi:10.1128/AAC.01102-09
Copyright (c) 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

An A643T mutation in the transcription factor Upc2p causes constitutive ERG11 upregulation and increased fluconazole resistance in Candida albicans

Clemens J. Heilmann, Sabrina Schneider, Katherine S. Barker, P. David Rogers, and Joachim Morschhäuser^*

Institut für Molekulare Infektionsbiologie, Universität Würzburg, Röntgenring 11, D-97070 Würzburg, Germany; 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, TN, USA

^* To whom correspondence should be addressed. Email: joachim.morschhaeuser{at}mail.uni-wuerzburg.de.

The zinc cluster transcription factor Upc2p mediates upregulation of ergosterol biosynthesis genes in response to ergosterol depletion in the fungal pathogen Candida albicans. One mechanism of acquired resistance to the antifungal drug fluconazole, which inhibits ergosterol biosynthesis, is constitutively increased expression of the ERG11 gene encoding the drug target enzyme. A G648D mutation in Upc2p has recently been shown to cause hyperactivity of the transcription factor, resulting in overexpression of ergosterol biosynthesis genes and increased fluconazole resistance. In order to investigate if gain-of-function mutations in Upc2p are a common mechanism of ERG11 upregulation and fluconazole resistance, we sequenced the UPC2 alleles of four ERG11 overexpressing, fluconazole-resistant C. albicans isolates and matched susceptible isolates from the same patients. In three of the isolate pairs no differences in the UPC2 alleles were found, suggesting that other mechanisms than Upc2p mutations can cause ERG11 overexpression. One resistant isolate had become homozygous for a UPC2 allele containing a G1927A substitution that caused an alanine to threonine exchange at amino acid position 643 of Upc2p. Replacement of one of the endogenous UPC2 alleles in a fluconazole-susceptible strain by the UPC2^A643T allele resulted in ERG11 overexpression and increased fluconazole resistance, which was further elevated when the A643T mutation was also introduced into the second UPC2 allele. These results further establish gain-of-function mutations in UPC2, which can be followed by loss of heterozygosity for the mutated allele, as a mechanism of ERG11 overexpression and increased fluconazole resistance in C. albicans, but other mechanisms of ERG11 upregulation also exist.