EC Accepts, published online ahead of print on 26 June 2009

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Eukaryotic Cell doi:10.1128/EC.00069-09
Copyright (c) 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Functional analysis of cis- and trans- acting elements of the Candida albicans CDR2 promoter with a novel promoter reporter system

Alix T. Coste, Jérôme Crittin, Christopher Bauser, Bettina Rohde, and Dominique Sanglard^*

Institute of Microbiology, University of Lausanne and University Hospital Center, CH-1011 Lausanne, Switzerland; and GATC Biotech, Konstanz (Germany)

^* To whom correspondence should be addressed. Email: Dominique.Sanglard{at}chuv.ch.

Azole resistance in Candida albicans can be mediated by the upregulation of the ATP binding Cassette (ABC)-transporter genes CDR1 and CDR2. Both genes are regulated by a cis-acting element called DRE (for Drug Responsive Element with the consensus sequence: 5'-CGGAWATCGGATATTTTTTT-3') and the transcription factor Tac1p. In order to analyse in details the DRE sequence necessary for the regulation of CDR1/CDR2 and properties of TAC1 alleles, a one-hybrid system was designed. This system is based on a P_(CDR2)-HIS3 reporter system into which complementation of histidine auxotrophy can be monitored by activation of the reporter system to CDR2 inducing drugs such as oestradiol. Our results show that most of the modifications within the DRE, but especially at the level of CGG triplets, strongly reduce CDR2 expression. The CDR2 DRE was replaced by putative DREs deduced from promoters of co-regulated genes (CDR1, RTA3 and IFU5). Surprisingly, even if Tac1p was able to bind these putative DREs as shown by chromatin immunoprecipitation, those from RTA3 and IFU5 did not functionally replace the CDR2 DRE. The one-hybrid system was also used for the identification of gain-of-function (GOF) mutations TAC1 alleles by random mutagenesis and from clinical C. albicans isolates. Taken together, 17 different GOF mutations were identified at 13 distinct positions. Five of them (G980E, N972D, A736V, T225A, N977D) were already described in clinical isolates and four others (G980W, A736T, N972S and N972I) occurred at already described positions, thus suggesting that GOF mutations can occur in a limited number of positions in Tac1p. In conclusion, the one-hybrid system developed here is rapid and powerful and can be used for characterization of cis- and trans-acting elements in C. albicans.