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.
Figures
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.
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.
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.
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.
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.
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.
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.
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.
Similar articles
- 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.
Coste A, Turner V, Ischer F, Morschhäuser J, Forche A, Selmecki A, Berman J, Bille J, Sanglard D. Coste A, et al. Genetics. 2006 Apr;172(4):2139-56. doi: 10.1534/genetics.105.054767. Epub 2006 Feb 1. Genetics. 2006. PMID: 16452151 Free PMC article. - The zinc cluster transcription factor Tac1p regulates PDR16 expression in Candida albicans.
Znaidi S, De Deken X, Weber S, Rigby T, Nantel A, Raymond M. Znaidi S, et al. Mol Microbiol. 2007 Oct;66(2):440-52. doi: 10.1111/j.1365-2958.2007.05931.x. Mol Microbiol. 2007. PMID: 17897373 - Comparison of gene expression profiles of Candida albicans azole-resistant clinical isolates and laboratory strains exposed to drugs inducing multidrug transporters.
Karababa M, Coste AT, Rognon B, Bille J, Sanglard D. Karababa M, et al. Antimicrob Agents Chemother. 2004 Aug;48(8):3064-79. doi: 10.1128/AAC.48.8.3064-3079.2004. Antimicrob Agents Chemother. 2004. PMID: 15273122 Free PMC article. - The TAC1 Gene in Candida albicans: Structure, Function, and Role in Azole Resistance: A Mini-Review.
Mahdizade AH, Hoseinnejad A, Ghazanfari M, Boozhmehrani MJ, Bahreiny SS, Abastabar M, Galbo R, Giuffrè L, Haghani I, Romeo O. Mahdizade AH, et al. Microb Drug Resist. 2024 Jul;30(7):288-296. doi: 10.1089/mdr.2023.0334. Epub 2024 May 21. Microb Drug Resist. 2024. PMID: 38770776 Review. - All about CDR transporters: Past, present, and future.
Prasad R, Balzi E, Banerjee A, Khandelwal NK. Prasad R, et al. Yeast. 2019 Apr;36(4):223-233. doi: 10.1002/yea.3356. Epub 2018 Oct 29. Yeast. 2019. PMID: 30192990 Review.
Cited by
- Comparative genomics of the extremophile Cryomyces antarcticus and other psychrophilic Dothideomycetes.
Gomez-Gutierrrez SV, Sic-Hernandez WR, Haridas S, LaButti K, Eichenberger J, Kaur N, Lipzen A, Barry K, Goodwin SB, Gribskov M, Grigoriev IV. Gomez-Gutierrrez SV, et al. Front Fungal Biol. 2024 Sep 6;5:1418145. doi: 10.3389/ffunb.2024.1418145. eCollection 2024. Front Fungal Biol. 2024. PMID: 39309730 Free PMC article. - Single-cell detection of copy number changes reveals dynamic mechanisms of adaptation to antifungals in Candida albicans.
Zhou X, Hilk A, Solis NV, Scott N, Beach A, Soisangwan N, Billings CL, Burrack LS, Filler SG, Selmecki A. Zhou X, et al. Nat Microbiol. 2024 Sep 3. doi: 10.1038/s41564-024-01795-7. Online ahead of print. Nat Microbiol. 2024. PMID: 39227665 - Step-wise evolution of azole resistance through copy number variation followed by KSR1 loss of heterozygosity in Candida albicans.
Vande Zande P, Gautier C, Kawar N, Maufrais C, Metzner K, Wash E, Beach AK, Bracken R, Maciel EI, Pereira de Sá N, Fernandes CM, Solis NV, Del Poeta M, Filler SG, Berman J, Ene IV, Selmecki A. Vande Zande P, et al. PLoS Pathog. 2024 Aug 30;20(8):e1012497. doi: 10.1371/journal.ppat.1012497. eCollection 2024 Aug. PLoS Pathog. 2024. PMID: 39213436 Free PMC article. - Deletion of the Candida albicans TLO gene family results in alterations in membrane sterol composition and fluconazole tolerance.
O'Connor-Moneley J, Fletcher J, Bean C, Parker J, Kelly SL, Moran GP, Sullivan DJ. O'Connor-Moneley J, et al. PLoS One. 2024 Aug 9;19(8):e0308665. doi: 10.1371/journal.pone.0308665. eCollection 2024. PLoS One. 2024. PMID: 39121069 Free PMC article. - Probing gene function in Candida albicans wild-type strains by Cas9-facilitated one-step integration of two dominant selection markers: a systematic analysis of recombination events at the target locus.
Ramírez-Zavala B, Hoffmann A, Krüger I, Schwanfelder S, Barker KS, Rogers PD, Morschhäuser J. Ramírez-Zavala B, et al. mSphere. 2024 Jul 30;9(7):e0038824. doi: 10.1128/msphere.00388-24. Epub 2024 Jun 28. mSphere. 2024. PMID: 38940507 Free PMC article.
References
- Alarco, A. M., I. Balan, D. Talibi, N. Mainville, and M. Raymond. 1997. AP1-mediated multidrug resistance in Saccharomyces cerevisiae requires FLR1 encoding a transporter of the major facilitator superfamily. J. Biol. Chem. 272:19304-19313. - PubMed
- Balzi, E., and A. Goffeau. 1994. Genetics and biochemistry of yeast multidrug resistance. Biochim. Biophys. Acta 1187:152-162. - PubMed
- Bissinger, P. H., and K. Kuchler. 1994. Molecular cloning and expression of the Saccharomyces cerevisiae STS1 gene product. A yeast ABC transporter conferring mycotoxin resistance. J. Biol. Chem. 269:4180-4186. - PubMed
- Blackwell, C., C. L. Russell, S. Argimon, A. J. Brown, and J. D. Brown. 2003. Protein A-tagging for purification of native macromolecular complexes from Candida albicans. Yeast 20:1235-1241. - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
Molecular Biology Databases