Genotypic evolution of azole resistance mechanisms in sequential Candida albicans isolates

Abstract

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.

FIG. 1.

Expression levels of Cdr1p and Cdr2p in five groups of C. albicans isolates. Protein extracts of each strain were separated on sodium dodecyl sulfate-10% polyacrylamide gels and immunoblotted with rabbit polyclonal anti-Cdr1p and anti-Cdr2p as described previously (10). C. albicans strains were grown in liquid YEPD to mid-log phase and treated (+) or not treated (−) with fluphenazine (10 μg/ml) for 20 min. The origin of each strain is indicated with its corresponding FLC susceptibility and MTL locus status.

FIG. 2.

Characteristics of TAC1 alleles from individual C. albicans isolates. Each TAC1 allele was introduced in DSY2906 (tac1Δ/Δ) by the integration of pDS178-derived plasmids (Table 2) at the LEU2 locus by SalI digestion. The FLC MIC of each strain containing the individual TAC1 allele is indicated. The different TAC1 alleles were grouped according to their origin (group A to group E). In group E, TAC1-25 was introduced in single (TAC1-25) or double (2× TAC1-25) copies. Hyperactive alleles are underlined. Experimental conditions were identical to those described in the legend to Fig. 1.

FIG. 3.

Hyperactivity of TAC1 is mediated by distinct GOF mutations. (A) Drug susceptibility testing of C. albicans tac1Δ/Δ mutant and TAC1 revertant strains with different TAC1-1-derived alleles carrying single point mutations or deletions. Drug susceptibility testing was carried out by plating serial dilutions of overnight cultures on YEPD agar plates containing different drugs as indicated. Plates were incubated for 48 h at 35°C in the absence or presence of FLC and cyclosporine at a concentration of 1 μg/ml. MIC assays were performed as described in Materials and Methods. (B) Expression levels of Cdr1p and Cdr2p of C. albicans tac1Δ/Δ mutant and TAC1 revertant strains with different TAC1-1-derived alleles carrying single point mutations or deletions. Experimental conditions were identical to those described in the legend to Fig. 1. WT, wild type.

FIG. 4.

ERG11 expression levels in C. albicans isolates sequential. ERG11 expression was quantified by Northern blot analysis using a Typhoon Trio phosphorimager (GE Healthcare, Otelfingen, Switzerland). ERG11 expression was normalized using ACT1 expression (ACT1 is considered a housekeeping gene). ERG11 expression is given as the relative increase of expression compared to that in the most azole-susceptible strain of each group.

FIG. 5.

Mapping of SNP on Chr 5 of C. albicans sequential isolates. (A to E) SNP on Chr 5 of isolates from groups A to E. Circles indicate SNP of Chr 5 observed by the use of an SNP microarray. The following markers were described by Forche et al. (16): 102, 1855/2172; 103, HST3; 104, SNF1; 109, 1899/2008; 110, 1445/2395; 111, 1922/2344; 112, PDE1; 113, 1969/2162; 114, DPH5; 117, 1817/2082; 118, 1341/2493; 120, 2340/2493. Triangles indicate additional markers of Chr 5 determined by multilocus sequence typing: B, orf19.1926; D, orf19.4225; E, orf19.4251; F, orf19.4288; G, orf19.429; I, orf19.6680; J, CRH12; K, orf19.971; L, ERG11; M, orf19.921; N, orf19.1971; O, orf19.3178. Empty and filled symbols indicate heterozygosity and homozygosity for the indicated markers, respectively. MTL and TAC1 loci in gray and black indicate heterozygosity and homozygosity, respectively. The gray region on Chr 5 delimitates the maximal region of LOH. (B) Schematic representation of the origin of Tac1-10p. The area in gray delimits a region spanning from codon 558 to 776 which originates from Tac1-9p and Tac1-10p.

FIG. 6.

Mapping of gene copy number on Chr 5 by CGH. The genomes of the tested strains were hybridized against the SC5314 genome according to the protocol published by Selmecki et al. (46). Each gene on Chr 5 is represented by its relative intensity compared to signals obtained in SC5314.

FIG. 7.

Detection of i(5L) in C. albicans isolates. (A and C) Southern blot of EcoNI-digested DNA probed with CSE4. A 10-kb fragment reveals an i(5L) structure. Uns., unspecific signal. (B) Karyotype analysis. Whole-chromosome CHEF analysis was carried out as described in Material and Methods, with ethidium bromide staining or hybridization with TAC1, MTLa, or MTLα1 probes as indicated under each panel. Arrows indicate the position of i(5L) on each blot.

FIG. 8.

Schematic representation of Chr 5 and azole resistance gene modifications in the groups of investigated strains. After acquisition of GOF mutations on one TAC1 allele and also possible acquisition of mutations in ERG11 (step I), different LOH events occurred at these loci, including loss of Chr 5 and duplication, mitotic recombination of the left arm of Chr 5, and gene conversion in TAC1 (step II). The formation of i(5L) is an additional event (step III) leading to the increase of the copy numbers of azole resistance genes and possibly yet unidentified resistance genes on the 5L arm. For simplification, the acquisition of the TAC1 mutation (step I) is shown linked with MTLα in group E, although it is linked to MTLa. It is possible that steps I to III can be sequentially arranged in a different manner. MR, mitotic recombination. In group A, sequence analysis allow the deduction that TAC1-18 originated from TAC1-17 by acquisition of only one nonsynonymous substitution (ΔM677). In group B, TAC1-11 arose from TAC1-9 by acquisition of the A736V substitution. TAC1-10 is the result of gene conversions between TAC1-8 and TAC1-11. In group C, it is not possible to determine whether or not TAC1-15 arose from TAC1-12 or TAC1-13. In group D, TAC1-21 and TAC1-22 originated from TAC1-19 and TAC1-20, respectively, by acquisition of two distinct GOF mutations, A736V and ΔL962-ΔN969. Finally, in group E, TAC1-25 originated from TAC1-23 by acquisition of the A225T substitution.