Eukaryotic Cell doi:10.1128/EC.00151-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
Genotypic evolution of azole resistance mechanisms in sequential Candida albicans isolates
Alix Coste,
Anna Selmecki,
Anja Forche,
Dorothée Diogo,
Marie-Elisabeth Bougnoux,
Christophe d'Enfert,
Judith Berman,
and
Dominique Sanglard*
Institute of Microbiology, University of Lausanne and University Hospital Center, Switzerland, Department of Microbiology, University of Minnesota, MN, USA, Unité Biologie et Pathogénicité Fongiques, INRA USC2019, Département Génomes et Génétique, Institut Pasteur, Paris, France
* To whom correspondence should be addressed. Email:
Dominique.Sanglard{at}chuv.ch.
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Abstract |
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TAC1 (for Transcriptional Activator of CDR genes) is critical for the upregulation of the ABC-transporters CDR1 and CDR2, which mediate azole resistance in Candida albicans. While a wild-type TAC1 allele drives high expression of CDR1/2 in response to inducers, we showed previously that TAC1 can be hyperactive by a gain-of-function (GOF) point mutation responsible for constitutive high expression of CDR1/2. High azole resistance levels are achieved when C. albicans carries hyperactive alleles only as a consequence of loss of heterozygosity (LOH) at the TAC1 locus on chromosome 5 (Chr. 5), which is linked to the mating-type like (MTL) locus. Both are located on Chr. 5 left arm along with ERG11 (target of azoles). In this work, five groups of related isolates containing azole-susceptible and -resistant strains were analysed for the TAC1 and ERG11 alleles and for Chr. 5 alterations. While recovered ERG11 alleles contained known mutations, seventeen new TAC1 alleles were isolated, including seven hyperactive alleles with five separate new GOF mutations. Single nucleotide polymorphism (SNP) analysis of Chr. 5 revealed that azole-resistant strains acquired TAC1 hyperactive alleles and, in most cases, ERG11 mutant alleles by LOH events not systematically including the MTL locus. TAC1 LOH resulted from mitotic recombination of the left arm of Chr. 5, gene conversion within the TAC1 locus or the loss and reduplication of the entire Chr. 5. In one case, two independent TAC1 hyperactive alleles were acquired. Comparative genome hybridization and karyotype analysis revealed the presence of isochromosome i(5L) in two azole-resistant strains. i(5L) leads to increased copy number of azole-resistance genes present on the left arm of Chr. 5, among them TAC1 and ERG11. Our work shows that azole resistance was not only due to the presence of specific mutation(s) in azole resistance genes (at least ERG11 and TAC1) but also to their increase in copy number by LOH and to the addition of extra Chr.5 copies. With the combination of these different modifications, sophisticated genotypes were obtained. The development of azole resistance in C. albicans is therefore a powerful instrument for generating genetic diversity.
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