TAC1, transcriptional activator of CDR genes, is a new transcription factor involved in the regulation of Candida albicans ABC transporters CDR1 and CDR2 - PubMed (original) (raw)

TAC1, transcriptional activator of CDR genes, is a new transcription factor involved in the regulation of Candida albicans ABC transporters CDR1 and CDR2

Alix T Coste et al. Eukaryot Cell. 2004 Dec.

Abstract

The ABC transporter genes CDR1 and CDR2 can be upregulated in Candida albicans developing resistance to azoles or can be upregulated by exposing cells transiently to drugs such as fluphenazine. The cis-acting drug-responsive element (DRE) present in the promoters of both genes and necessary for their upregulation contains 5'-CGG-3' triplets that are often recognized by transcriptional activators with Zn(2)-Cys(6) fingers. In order to isolate regulators of CDR1 and CDR2, the C. albicans genome was searched for genes encoding proteins with Zn(2)-Cys(6) fingers. Interestingly, three of these genes were tandemly arranged near the mating locus. Their involvement in CDR1 and CDR2 upregulation was addressed because a previous study demonstrated a link between mating locus homozygosity and azole resistance. The deletion of only one of these genes (orf19.3188) was sufficient to result in a loss of transient CDR1 and CDR2 upregulation by fluphenazine and was therefore named TAC1 (transcriptional activator of CDR genes). Tac1p has a nuclear localization, and a fusion of Tac1p with glutathione S-transferase could bind the cis-acting regulatory DRE in both the CDR1 and the CDR2 promoters. TAC1 is also relevant for azole resistance, since a TAC1 allele (TAC1-2) recovered from an azole-resistant strain could trigger constitutive upregulation of CDR1 and CDR2 in an azole-susceptible laboratory strain. Transcript profiling experiments performed with a TAC1 mutant and a revertant containing TAC1-2 revealed not only CDR1 and CDR2 as targets of TAC1 regulation but also other genes (RTA3, IFU5, and HSP12) that interestingly contained a DRE-like element in their promoters. In conclusion, TAC1 appears to be the first C. albicans transcription factor involved in the control of genes mediating antifungal resistance.

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Figures

FIG. 1.

FIG. 1.

(A) Restriction map of zinc cluster genes. The physical map shows the zinc cluster and the neighboring MTL locus (MTLa). White arrows show the positions of ZNC ORFs, and black arrows show the positions of MTLa locus genes. A second map with a higher scale for ZNC2 (TAC1) focuses on the location of the TAC1 deletion constructed with the _URA3_-blaster cassette. The entire TAC1 ORF was used for cloning and construction of disruption cassettes (see Material and Methods for details). Underlined restriction sites were created by PCR cloning. (B) Southern analysis of the TAC1 disruption. Genomic DNA was digested with BglII. The identity of each band of the expected size is shown at the right side of the Southern blot. The positions of molecular size standards are shown at the left side. The following strains correspond to the indicated genotypes: TAC1/TAC1, CAF2-1; TAC1/tac1Δ, DSY2875; tac1Δ/Δ, DSY2903; and tac1Δ/Δ + TAC1, DSY2937.

FIG. 2.

FIG. 2.

TAC1 functions as a transcriptional activator of CDR1 and CDR2. (A) Immunodetection of Cdr1p and Cdr2p in TAC1 mutant and revertant strains. Protein extracts of each strain were separated by SDS-10% PAGE and immunoblotted with rabbit polyclonal anti-Cdr1p and anti-Cdr2p antibodies as described previously (8). C. albicans strains were grown in YEPD to mid-log phase and exposed (+) or not exposed (−) to fluphenazine (10 μg/ml) for 20 min. See the legend to Fig. 1 for strain and genotype designations. (B) Northern analysis of TAC1 mutant and revertant strains with CDR1 and CDR2 probes.

FIG. 3.

FIG. 3.

Drug susceptibility testing of C. albicans TAC1 mutant and revertant strains. Spotting assays were performed with serial dilutions of overnight cultures on YEPD containing the indicated drugs. Plates were incubated for 48 h at 35°C. The following strains correspond to the indicated genotypes: tac1Δ/Δ + TAC1-1, DSY2925; tac1Δ/Δ + TAC1-2, DSY2926; and cdr1Δ/Δ cdr2Δ/Δ, DSY654. See the legend to Fig. 1 for other strain and genotype designations.

FIG. 4.

FIG. 4.

Tac1p binds to the CDR DRE. Labeled probes were separated as described in Materials and Methods. Arrows indicate the positions of specific complexes and of free labeled probes. (A) Binding saturation of the DRE by Tac1p-GST. Increasing amounts of Tac1p-GST were added to the reaction mixtures before loading. (B) Competition experiments with unlabeled DRE. A total of 1 μg of Tac1p-GST was added to the labeled probe along with increasing amounts of unlabeled probe. Probe 1 corresponding to the DRE region (DRE consensus sequence boxed with two CGG triplets highlighted) of the CDR2 promoter was used for the band shifts shown in panels A and B. (C) CGG triplet-dependent binding of Tac1p-GST. Probes 1, 2 (first CGG triplet changed), and 3 (first and second CGG triplets changed) correspond to the DRE region of the CDR2 promoter. Probes 4 and 5 correspond to the DRE region (DRE consensus sequence boxed with two CGG triplets highlighted) of the CDR1 promoter and to the PDRE (consensus sequence highlighted) of PDR5, respectively. Each probe was designed with two complementary oligonucleotides.

FIG. 5.

FIG. 5.

Nuclear localization of Tac1p. DSY2906 transformed with pDS1202 was grown in liquid selective medium to a cell density of 107 cells/ml with constant agitation. Culture aliquots were sampled for nuclear staining as described in Materials and Methods and visualized by microscopy. Digital images were further processed with the computer program Adobe Photoshop 7.0 (Adobe Systems Incorporated, Mountain View, Calif.). Bar, 10 μm.

FIG. 6.

FIG. 6.

The TAC1-2 allele functions as a constitutive transcriptional activator of CDR1 and CDR2. Protein extracts of each strain were separated by SDS-10% PAGE and immunoblotted with rabbit polyclonal anti-Cdr1p and anti-Cdr2p antibodies as described previously (8). C. albicans strains were grown in YEPD to mid-log phase and exposed (+) or not exposed (−) to fluphenazine (10 μg/ml) for 20 min. See the legends to Fig. 1 and 3 for strain and genotype designations.

FIG. 7.

FIG. 7.

Codominance of azole resistance in C. albicans. (A) Strain DSY296, which is homozygous at the _MTL_α locus, was mutagenized to obtain gal1 strains (numbered 1 to 5). These strains were crossed with mating test strains CHY477 (_MTL_α) and CHY439 (MTLa) (26) and replica plated on YNB containing galactose. (B) Fluconazole (Fluco) susceptibility of strain CHY439, strain DSY296-3, and fusion product DSY2781 tested by diffusion disk assays and by corresponding Western blotting with anti-Cdr1p and anti-Cdr2p antibodies.

FIG. 8.

FIG. 8.

Microarray analysis reveals a subset of _TAC1_-dependent genes. (A) Venn diagram analysis of genes upregulated by fluphenazine in strain CAF2-1 and of genes no more upregulated under the same conditions in a TAC1 mutant strain (left side) and of genes commonly upregulated in strain DSY2926 expressing the TAC1-2 allele and in azole-resistant strain DSY296 (right side). DSY296 upregulates CDR1 and CDR2 and is the strain from which TAC1-2 originates. Genes common to these experiments are listed. A threshold of twofold was used to determine genes that were significantly upregulated. (B) Northern analysis of _TAC1_-regulated genes. DRE-like elements are shown for each gene investigated by Northern analysis. Lowercase letters show nucleotides different from those in the DRE of CDR1 and CDR2. See the legends to Fig. 1 and 3 for strain and genotype designations. −, no fluphenazine; +, fluphenazine.

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