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. 1999 Feb;181(3):700-8.

The bZip Transcription Factor Cap1p Is Involved in Multidrug Resistance and Oxidative Stress Response in Candida Albicans

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Free PMC article

The bZip Transcription Factor Cap1p Is Involved in Multidrug Resistance and Oxidative Stress Response in Candida Albicans

A M Alarco et al. J Bacteriol. .
Free PMC article

Abstract

Candida albicans is an opportunistic pathogenic yeast which frequently develops resistance to the antifungal agent fluconazole (FCZ) in patients undergoing long-term therapy. FCZ-resistant strains often display a reduced intracellular FCZ accumulation which correlates with the overexpression of the ATP-binding cassette transporters CDR1 and CDR2 or the major facilitator (MF) MDR1. We have recently cloned a C. albicans gene, named CAP1, which codes for a bZip transcription factor of the AP-1 family homologous to the Yap1 protein involved in multidrug resistance and response to oxidative stress in Saccharomyces cerevisiae. CAP1 was found to confer FCZ resistance in S. cerevisiae by transcriptionally activating FLR1, a gene coding for an MF homologous to the C. albicans MDR1 gene product (A.-M. Alarco, I. Balan, D. Talibi, N. Mainville, and M. Raymond, J. Biol. Chem. 272:19304-19313, 1997). To study the role of CAP1 in C. albicans, we constructed a CAI4-derived mutant strain carrying a homozygous deletion of the CAP1 gene (CJD21). We found that deletion of CAP1 did not affect the susceptibility of CJD21 cells to FCZ, cerulenin, brefeldin A, and diamide but caused hypersensitivity to cadmium, 4-nitroquinoline N-oxide, 1,10-phenanthroline, and hydrogen peroxide, an effect which was reverted by reintroduction of the CAP1 gene in these cells. Introduction of a hyperactive truncated allele of CAP1 (CAP1-TR) in CJD21 resulted in resistance of the cells to all of the above compounds except hydrogen peroxide. The hyperresistant phenotype displayed by the CJD21 CAP1-TR transformants was found to correlate with the overexpression of a number of potential CAP1 transcriptional targets such as MDR1, CaYCF1, CaGLR1, and CaTRR1. Taken together, our results demonstrate that CAP1 is involved in multidrug resistance and oxidative stress response in C. albicans. Finally, disruption of CAP1 in strain FR2, selected in vitro for FCZ resistance and constitutively overexpressing MDR1, did not suppress but rather increased the levels of MDR1 expression, demonstrating that CAP1 acts as a negative transcriptional regulator of the MDR1 gene in FR2 and is not responsible for MDR1 overexpression in this strain.

Figures

FIG. 1
FIG. 1
Chromosomal deletion of CAP1 in CAI4. (A) Schematic representation of the disruption strategy. The CAP1 locus is contained within a 3.2-kb BglII fragment (top). The start (arrow) and stop (asterisk) codons of the CAP1 ORF are indicated. The disruption cassette (middle) was generated by replacing a 1.35-kb ClaI-BsaBI CAP1 fragment by the 4-kb hisG-URA3-hisG cassette. After counterselection on 5-FOA, recombination between the two hisG direct repeats should generate a 2.8-kb BglII fragment (bottom). Southern blot analysis was used to characterize the different steps of the disruption (B to D). Genomic DNA was extracted from strains CAI4 CAP1/CAP1 (lanes 1), CJD10 CAP1/cap1Δ::hisG-URA3-hisG (lanes 2), CJD11 CAP1/cap1Δ::hisG (lanes 3), CJD20 cap1Δ::hisG-URA3-hisG/cap1Δ::hisG (lanes 4), and CJD21 cap1Δ::hisG/cap1Δ::hisG (lanes 5). DNA samples (2 μg) were digested in triplicate with BglII, separated by electrophoresis on agarose gels, and transferred to nylon membranes. The blots were then probed with either the 3.2-kb BglII fragment comprising the entire wild-type CAP1 gene (B), a 0.9-kb BamHI-BglII hisG fragment (C), or a 0.6-kb XbaI-HincII CAP1 internal fragment deleted in the cap1Δ::hisG-URA3-hisG allele (D). Positions of molecular size markers (in kilobases) are indicated on the left. Membranes were exposed for 6 h at −80°C with two intensifying screens.
FIG. 2
FIG. 2
Expression of the Cap1p and Cap1p-TR proteins in C. albicans. (A) Schematic representation of the full-length (Cap1p) and the truncated (Cap1p-TR) proteins. Positions of the bZip domain (grey) and the CRD (stippled) are indicated. The region used to generate a GST-Cap1p fusion protein (Cap1-350) is indicated. This fusion protein was used to raise an anti-Cap1p-350 polyclonal antibody. (B) Western blot analysis of Cap1p and Cap1p-TR expression. Total proteins were extracted from CAI4, CJD21, CJD21/PMK, CJD21/PMK-CAP1, and CJD21/PMK-CAP1TR cells. Protein samples (50 μg) were separated by electrophoreses on an SDS–12% polyacrylamide gel, transferred to a nitrocellulose membrane, and analyzed with the anti-Cap1p-350 polyclonal antibody. Arrows on the right indicate positions of the full-length (Cap1p) and truncated (Cap1p-TR) proteins. The position of a nonspecific cross-reacting protein is also indicated (★). Positions of molecular size standards are shown on the left.
FIG. 3
FIG. 3
Involvement of CAP1 in C. albicans MDR and OSR. Strains CAI4/PMK, CJD21/PMK, CJD21/PMK-CAP1, and CJD21/PMK-CAP1TR were analyzed by spot assay (as described in Materials and Methods) for the ability to grow on YPD plates containing different antifungal (A) and prooxidant (B) agents. Growth differences were monitored after 2 days at 30°C. A representative plate of control growth on YPD medium is shown for each set of experiments (YPD). Abbreviations for substances are FCZ (fluconazole), Cer (cerulenin), Brf (brefeldin A), Cd (cadmium), 4-NQO (4-nitroquinoline N-oxide), and Phe (1,10-phenanthroline).
FIG. 4
FIG. 4
Northern blot analysis of CAP1 transcriptional targets in C. albicans. Total RNA was extracted from strains CAI4, CJD21, CJD21/PMK, CJD21/PMK-CAP1, and CJD21/PMK-CAP1TR. RNA samples (20 μg) were separated by electrophoresis on a 1% agarose gel, transferred onto nylon membranes, and probed with a CAP1, MDR1, CaYCF1, CaGLR1, or CaTRR1 probe. Positions of rRNAs are indicated on the left; the position of the major CAP1 transcript is indicated by an arrow (top panel). The membranes were exposed for either 24 h (CAP1), 3 days (CaYCF1, CaGLR1, and CaTRR1), or 12 days (MDR1) at −80°C with two intensifying screens.
FIG. 5
FIG. 5
Northern blot analysis of CAP1 and MDR1 in FR2 cap1 disruptants. Total RNA was extracted from strains SGY243, FR2, FJD11 (CAP1/cap1Δ::hisG), and FJD21 (cap1Δ::hisG/cap1Δ::hisG). RNA samples (10 μg) were separated by electrophoresis in duplicate on a 1% agarose gel and transferred onto nylon membranes. The membranes were probed with a CAP1 (A) or MDR1 (B) probe. Both blots were subsequently hybridized with an ACT1 probe as a control for RNA loading and transfer. Membranes were exposed for 10 days (MDR1 and CAP1) or 24 h (ACT1) at −80°C with two intensifying screens. Positions of rRNAs are indicated on the left.

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