Coste Alix, Selmecki Anna, Forche Anja, Diogo Dorothée, Bougnoux Marie-Elisabeth, d'Enfert Christophe, Berman Judith, Sanglard Dominique
Institute of Microbiology, University of Lausanne and University Hospital Center, Rue du Bugnon 48, CH-1011, Lausanne, Switzerland.
Eukaryot Cell. 2007 Oct;6(10):1889-904. doi: 10.1128/EC.00151-07. Epub 2007 Aug 10.
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 the 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 analyzed for the TAC1 and ERG11 alleles and for Chr 5 alterations. While recovered ERG11 alleles contained known mutations, 17 new TAC1 alleles were isolated, including 7 hyperactive alleles with five separate new GOF mutations. Single-nucleotide-polymorphism 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 5L [i(5L)] in two azole-resistant strains. i(5L) leads to increased copy numbers of azole resistance genes present on the left arm of Chr 5, among them TAC1 and ERG11. Our work shows that azole resistance was due not only to the presence of specific mutations 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.
TAC1（CDR基因转录激活因子）对于ABC转运蛋白CDR1和CDR2的上调至关重要，这两种转运蛋白介导白色念珠菌对唑类药物的耐药性。野生型TAC1等位基因在诱导剂作用下会驱动CDR1/2的高表达，而我们之前表明，TAC1可因一个导致CDR1/2组成型高表达的功能获得性（GOF）点突变而变得过度活跃。当白色念珠菌仅因5号染色体（Chr 5）上TAC1位点杂合性缺失（LOH）而携带过度活跃的等位基因时，就会出现高唑类耐药水平，该位点与类交配型（MTL）位点相连。两者与ERG11（唑类药物作用靶点）均位于Chr 5左臂上。在这项研究中，对五组包含唑类药物敏感和耐药菌株的相关分离株进行了TAC1和ERG11等位基因以及Chr 5改变的分析。虽然回收的ERG11等位基因包含已知突变，但分离出了17个新的TAC1等位基因，其中包括7个过度活跃的等位基因，带有5个不同的新GOF突变。Chr 5的单核苷酸多态性分析表明，唑类耐药菌株通过不系统包括MTL位点的LOH事件获得了TAC1过度活跃的等位基因，并且在大多数情况下还获得了ERG11突变等位基因。TAC1的LOH是由Chr 5左臂的有丝分裂重组、TAC1位点内的基因转换或整个Chr 5的丢失和重复导致的。在一个案例中，获得了两个独立的TAC1过度活跃等位基因。比较基因组杂交和核型分析显示，在两个唑类耐药菌株中存在5号等臂染色体[i(5L)]。i(5L)导致Chr 5左臂上存在的唑类耐药基因拷贝数增加，其中包括TAC1和ERG11。我们的研究表明，唑类耐药不仅归因于唑类耐药基因（至少ERG11和TAC1）中存在特定突变，还归因于通过LOH导致的拷贝数增加以及额外Chr 5拷贝的添加。通过这些不同修饰的组合，获得了复杂的基因型。因此，白色念珠菌中唑类耐药的发展是产生遗传多样性的有力手段。
