A mutation in Tac1p, a transcription factor regulating CDR1 and CDR2, is coupled with loss of heterozygosity at chromosome 5 to mediate antifungal resistance in Candida albicans

Abstract

TAC1, a Candida albicans transcription factor situated near the mating-type locus on chromosome 5, is necessary for the upregulation of the ABC-transporter genes CDR1 and CDR2, which mediate azole resistance. We showed previously the existence of both wild-type and hyperactive TAC1 alleles. Wild-type alleles mediate upregulation of CDR1 and CDR2 upon exposure to inducers such as fluphenazine, while hyperactive alleles result in constitutive high expression of CDR1 and CDR2. Here we recovered TAC1 alleles from two pairs of matched azole-susceptible (DSY294; FH1: heterozygous at mating-type locus) and azole-resistant isolates (DSY296; FH3: homozygous at mating-type locus). Two different TAC1 wild-type alleles were recovered from DSY294 (TAC1-3 and TAC1-4) while a single hyperactive allele (TAC1-5) was isolated from DSY296. A single amino acid (aa) difference between TAC1-4 and TAC1-5 (Asn977 to Asp or N977D) was observed in a region corresponding to the predicted activation domain of Tac1p. Two TAC1 alleles were recovered from FH1 (TAC1-6 and TAC1-7) and a single hyperactive allele (TAC1-7) was recovered from FH3. The N977D change was seen in TAC1-7 in addition to several other aa differences. The importance of N977D in conferring hyperactivity to TAC1 was confirmed by site-directed mutagenesis. Both hyperactive alleles TAC1-5 and TAC1-7 were codominant with wild-type alleles and conferred hyperactive phenotypes only when homozygous. The mechanisms by which hyperactive alleles become homozygous was addressed by comparative genome hybridization and single nucleotide polymorphism arrays and indicated that loss of TAC1 heterozygosity can occur by recombination between portions of chromosome 5 or by chromosome 5 duplication.

Figure 1.

Analysis of drug resistance properties dependent on TAC1 alleles. (A) Drug susceptibility testing of C. albicans tac1Δ/Δ mutant and TAC1 revertant strains with different TAC1 alleles. Drug susceptibility assays were carried out by plating serial dilutions of overnight cultures onto YEPD agar plates containing different drugs as indicated. Plates were incubated for 48 hr at 35°. MIC assays were performed as described in materials and methods. (B) Immunodetection of Cdr1p and Cdr2p in tac1Δ/Δ mutant and TAC1 revertant strains with different TAC1 alleles. Protein extracts of each strain were separated on SDS-10% polyacrylamide gels and immunoblotted with rabbit polyclonal anti-Cdr1p and anti-Cdr2p as described previously (de Micheli et al. 2002). C. albicans strains were grown in liquid YEPD to midlog phase and exposed (+) or not (−) to fluphenazine (10 μg/ml) for 20 min. The following Ura⁺ strains correspond to the following genotypes: CAF2-1, TAC1-1/TAC1-2; DSY2903, tac1-1Δ/tac1-2Δ; DSY2925-47, tac1Δ/Δ + TAC1-3; DSY2925-18, tac1Δ/Δ + TAC1-4; DSY2984, tac1Δ/Δ + TAC1-5; DSY3010-80, tac1Δ/Δ + TAC1-6; DSY3010-113, tac1Δ/Δ + TAC1-7-FH1; DSY3013, tac1Δ/Δ + TAC1-7-FH3. For phenotypes and genotypes of the different strains of this study refer to supplemental Table S1 at http://www.genetics.org/supplemental/.

Figure 2.

Effect of the mutation N977D on the properties of TAC1. (A) Drug susceptibility testing of C. albicans tac1Δ/Δ mutant and TAC1 revertant strains with different TAC1 alleles containing the N977D substitution. Drug susceptibility assays were carried out as described in Figure 1. MIC assays were performed as described in materials and methods. (B) Immunodetection of Cdr1p and Cdr2p in C. albicans tac1Δ/Δ mutant and TAC1 revertant strains containing different TAC1 alleles with the N977D substitution. See legend of Figure 1B for other details. The following strains correspond to the following genotypes: DSY2937-35, tac1Δ/Δ + TAC1-1; VTY21, tac1Δ/Δ + TAC1-1^N977D; VTY28, tac1Δ/Δ + TAC1-4^N977D; ACY11, tac1Δ/Δ + TAC1-6^N977D; ACY12, tac1Δ/Δ + TAC1-7^D977N; VTY9, tac1Δ/Δ + TAC1-5^D977N. See legend of Figure 1 for other strains and genotype designations.

Figure 3.

Analysis of TAC1 alleles from the clinical strains DSY294 and DSY296. (A) Drug susceptibility testing of C. albicans tac1Δ/Δ mutant and TAC1 revertant clinical strains containing specific TAC1 alleles. Drug susceptibility assays were carried out onto YEPD medium containing 2.5 μg/ml of fluconazole and 1 μg/ml cyclosporin A for the DSY294-derived strains and 5 μg/ml of fluconazole and 1 μg/ml cyclosporin A for the DSY296-derived strains. Cyclosporin A alone had no effect on the growth of these strains. All strains were spotted onto agar medium containing 20 μg/ml of terbinafine. Plates were incubated for 48 hr at 35°. MIC assays were performed as described in materials and methods. (B) Immunodetection of Cdr1p and Cdr2p in C. albicans tac1Δ/Δ mutant and TAC1 revertant clinical strains. See legend of Figure 1 for other details. The following strains correspond to the following genotypes: DSY294, TAC1-3/TAC1-4; DSY296, TAC1-5/TAC1-5; DSY3058, TAC1-4/tac1-3Δ; DSY3082; tac1-3Δ/tac1-4Δ; DSY3287-1, tac1-3Δ/tac1-4Δ + TAC1-3; DSY3288-3, tac1-3Δ/tac1-4Δ + TAC1-4; DSY3102-2, tac1-3Δ/tac1-4Δ + TAC1-5; DSY3059, TAC1-5/tac1-5Δ; DSY3083, tac1-5Δ/Δ; DSY3285-1, tac1-5Δ/Δ + TAC1-3; DSY3286-2, tac1-5Δ/Δ + TAC1-4; DSY33284-1, tac1-5Δ/Δ + TAC1-5.

Figure 4.

Codominance of the TAC1-5 hyperactive allele. (A) Drug susceptibility testing of the C. albicans clinical isolates DSY294 and DSY296, heterozygous (TAC1-5/tac1-5Δ) and homozygous (tac1-5Δ/Δ) mutants derived from DSY296, and transformants of the heterozygous mutant in which a TAC1-3, TAC1-4, or TAC1-5 allele was reintroduced. See legend of Figure 1 for other details. (B) Immunodetection of Cdr1p and Cdr2p in the strains listed above. See legend of Figure 1 for other details. The following strains correspond to the following genotypes: DSY3211-4, TAC1-5/tac1-5Δ + TAC1-3; DSY3215-1, TAC1-5/tac1-5Δ + TAC1-4; DSY3210-1, TAC1-5/tac1-5Δ + TAC1-5. See the legend of Figure 3 for other strain and genotype designations.

Figure 5.

Analysis of the TAC1 alleles from clinical strains FH1 and FH3. (A) Drug susceptibility testing of C. albicans FH1 and FH3 derivatives lacking one specific TAC1 allele. See legend of Figure 1 for other details. (B) Immunodetection of Cdr1p and Cdr2p in C. albicans FH1 and FH3 derivatives lacking one specific TAC1 allele. See legend of Figure1 for other details. The following strains correspond to the following genotypes: DSY3132-11, TAC1-6/tac1-7Δ; DSY3132-14, TAC1-7/tac1-6Δ; DSY3133-15, TAC1-7/tac1-7Δ; FH1, TAC1-6/TAC1-7; FH3, TAC1-7/TAC1-7.

Figure 6.

Analysis of FH1-derived strains after in vitro fluconazole exposure. (A) Drug susceptibility testing of C. albicans FH3-, FH1-, and FH1-derived strains selected for their fluconazole resistance. DSY3157-2 was derived from a single colony of FH1 that arose after spotting onto YEPD medium with 10 μg/ml fluconazole. DSY3301-4 was derived from DSY3157-2 as a fast-growing colony onto medium with 10 μg/ml fluconazole. Drug susceptibility assays were carried out as described in Figure 1 onto YEPD medium containing 10 μg/ml fluconazole and 1 μg/ml cyclosporin A. Cyclosporin A alone had no effect on the growth of these strains. Plates were incubated for 48 hr at 35°. MIC assays were performed as described in materials and methods. (B) Immunodetection of Cdr1p and Cdr2p in C. albicans strains FH1, FH3, DSY3157-2, and DSY3301-4. See legend of Figure 1 for other details. (C) PCR analysis of the mating-type locus. PCR was performed as described (Rustad et al. 2002). “a” and “α” denote analysis performed to detect MTLa and MTLα loci, respectively. (D) Southern blot analysis of the TAC1 alleles in C. albicans FH-derivative strains. Genomic DNA of each strain was digested by EcoRI and Southern blot was performed as described in materials and methods. Radioactivity of signals was quantified as discussed in materials and methods. Restriction maps of both TAC1-6 and TAC1-7 alleles indicate restriction site polymorphism for EcoRI. The solid bar indicates the position of the labeled probe, which corresponded to the region located between the first TAC1 initiation codon and the PstI restriction site. Digestion of genomic DNA with EcoRI is expected to yield positive signals at 1.8 and 3.2 kb for TAC1-6 and TAC1-7, respectively.

Figure 7.

CGH of FH-derived strains. The genomes of the tested strains were hybridized against the SC5314 genome according to the protocol published by Selmecki et al. (2005). Each gene on chromosome 5 is represented by its relative intensity as compared to signals obtained in SC5314.

Figure 8.

SNP analysis of chromosome 5. The map of chromosome 5 was obtained by assembly of the different contig sequences shown by arrows. An asterisk (*) indicates SNPs of chromosome 5 as measured on the SNP microarray: 102, 1855/2172; 103, HST3; 104, SNF1; 109, 1899/2008; 110, 1445/2395; 111, 1922/2344; 112, PDE1; 113, 1969/2162; 114, DPH5; 115, HEX1; 116, 2093/2390; 117, 1817/2082; 118, 1341/2493; 119, F16n1; 120, 2340/2493. “▴” indicates additional markers of chromosome 5: A, orf19.1976 (TRX1); B, orf19.1926; C, ZNC3; D, orf19.4225; E, orf19.4251; F, orf19.4288; G, orf19.1942 (TRR1); H, orf19.2646; I, orf19.6680; J, CRH12. Color codes indicate modifications in SNPs for individual strains. For the strains DSY294 and DSY296, sequences of the C, F, and H markers were not available. The hatched regions on chromosome 5 delimitate the maximal region of a recombination. MRS, major repeat sequence.

Figure 9.

Schematic of chromosome 5 alterations in matched azole-susceptible and azole-resistant strains obtained from patients or developed in vitro. (Top) Chromosome 5 alterations in FH1-derived strains both in vitro and in vivo. Chromosome 5 containing TAC1-7 and MTLα was duplicated after in vitro fluconazole exposure in DSY3157-2; a chromosome 5 copy with TAC1-6 and MTLa was lost in DSY3301-4 and replaced by another chromosome 5 copy containing TAC1-7 and MTLα after a second fluconazole exposure; in FH3, a portion of chromosome 5 underwent mitotic recombination between markers B and E, resulting in TAC1-7 and MTLα homozygosity. (Bottom) Chromosome 5 alterations in DSY294 and DSY296 in vivo. In DSY296, a portion of chromosome 5 underwent mitotic recombination between markers 109 and D, resulting in TAC1-5 and MTLα homozygosity. An intermediate strain between DSY294 and DSY296 in which a single nucleotide change in TAC1-4 yielded TAC1-5 resulting in Asn⁹⁷⁷ to Asp could have existed. TAC1-3, -4, and -6 are defined as wild-type alleles; TAC1-5 and -7 are defined as hyperactive alleles. “a” and “α”designate MTL types. Position of markers 109, B, D, and E are indicated in Figure 8.