SAGA/ADA Complex Subunit Ada2 Is Required for Cap1- but Not Mrr1-Mediated Upregulation of the Candida albicans Multidrug Efflux Pump MDR1

Abstract

Overexpression of the multidrug efflux pump MDR1 is one mechanism by which the pathogenic yeast Candida albicans develops resistance to the antifungal drug fluconazole. The constitutive upregulation of MDR1 in fluconazole-resistant, clinical C. albicans isolates is caused by gain-of-function mutations in the zinc cluster transcription factor Mrr1. It has been suggested that Mrr1 activates MDR1 transcription by recruiting Ada2, a subunit of the SAGA/ADA coactivator complex. However, MDR1 expression is also regulated by the bZIP transcription factor Cap1, which mediates the oxidative stress response in C. albicans. Here, we show that a hyperactive Mrr1 containing a gain-of-function mutation promotes MDR1 overexpression independently of Ada2. In contrast, a C-terminally truncated, hyperactive Cap1 caused MDR1 overexpression in a wild-type strain but only weakly in mutants lacking ADA2. In the presence of benomyl or H₂O₂, compounds that induce MDR1 expression in an Mrr1- and Cap1-dependent fashion, MDR1 was upregulated with the same efficiency in wild-type and ada2Δ cells. These results indicate that Cap1, but not Mrr1, recruits Ada2 to the MDR1 promoter to induce the expression of this multidrug efflux pump and that Ada2 is not required for MDR1 overexpression in fluconazole-resistant C. albicans strains containing gain-of-function mutations in Mrr1.

INTRODUCTION

The Candida albicans MDR1 gene encodes a multidrug efflux pump of the major facilitator superfamily that confers resistance to various toxic compounds, including the antifungal drug fluconazole (1, 2). MDR1 is not significantly expressed under standard growth conditions, but its expression is induced by certain chemicals, like benomyl or H₂O₂ (3–6). The bZIP transcription factor Cap1 and the zinc cluster transcription factor Mrr1 mediate the upregulation of MDR1 in the presence of these compounds. Cap1, which regulates the oxidative stress response, is required for the induction of MDR1 transcription by H₂O₂ and also contributes to benomyl-induced MDR1 expression (6–8). Similarly, little or no induction of MDR1 expression occurs in mutants lacking MRR1 (7, 9–11). Therefore, Cap1 and Mrr1, both of which bind to the MDR1 promoter (7, 8), cooperate to promote MDR1 upregulation in response to inducing chemicals.

Many fluconazole-resistant, clinical C. albicans isolates constitutively overexpress MDR1, which contributes to their drug-resistant phenotype (2, 12). In all cases investigated so far, MDR1 overexpression is caused by gain-of-function mutations in Mrr1 that render the transcription factor constitutively active (9, 13, 14). In cells containing such a hyperactive Mrr1, Cap1 is dispensable for MDR1 expression (7). Vice versa, a C-terminally truncated, hyperactive form of Cap1 also causes constitutive MDR1 upregulation that is partially independent of Mrr1 (7, 15).

In addition to the direct inhibition of efflux pumps, blocking their expression is considered a potential strategy to overcome drug resistance in pathogenic fungi (16, 17). It is currently not known how transcription factors that mediate drug resistance in C. albicans activate gene expression. In Saccharomyces cerevisiae, the paralogous zinc cluster transcription factors Pdr1 and Pdr3, which control the expression of several multidrug efflux pumps, interact with different subunits of the mediator complex to recruit RNA polymerase II to the promoters of their target genes (18, 19). Mrr1 may act in a similar fashion, but it has recently been proposed to recruit Ada2, a subunit of the SAGA/ADA coactivator complex, to induce the transcription of drug resistance genes (20). The SAGA/ADA complex mediates histone acetylation to allow transcriptional activation. By chromatin immunoprecipitation experiments it was shown that Ada2 binds to 200 promoters in the C. albicans genome (20). Among the Ada2 targets were genes involved in the response to drugs and oxidative stress, and an ada2Δ mutant was found to be hypersusceptible to fluconazole and reactive oxygen species. Ada2 bound to many genes that are regulated by Cap1, and binding was reduced in the absence of Cap1. Ada2 also bound to many Mrr1 target genes in an Mrr1-dependent manner, and it was suggested that Ada2 might function as a coactivator of Mrr1 (20). Yet, whether Mrr1 depends on Ada2 to activate MDR1 expression was not tested in that study. Since both Cap1 and Mrr1 mediate MDR1 expression, recruitment of Ada2 to the MDR1 promoter might be accomplished by either of the two transcription factors. In order to clarify the role of Ada2 in Cap1- and Mrr1-mediated MDR1 expression, we investigated the importance of Ada2 for MDR1 activation by the inducers benomyl and H₂O₂ and for the constitutive MDR1 overexpression caused by hyperactive forms of Mrr1 and Cap1.

MATERIALS AND METHODS

Strains and growth conditions.

The C. albicans strains used in this study are listed in Table 1. All strains were stored as frozen stocks with 15% glycerol at −80°C and subcultured on yeast extract-peptone-dextrose (YPD) agar plates (10 g yeast extract, 20 g peptone, 20 g glucose, and 15 g agar per liter) at 30°C. Strains were routinely grown in YPD liquid medium at 30°C in a shaking incubator. For selection of nourseothricin-resistant transformants, 200 μg/ml nourseothricin (Werner Bioagents, Jena, Germany) was added to YPD agar plates. To obtain nourseothricin-sensitive derivatives in which the SAT1 flipper cassette was excised by FLP-mediated recombination, transformants were grown overnight in yeast carbon base-bovine serum albumin-yeast extract (YCB-BSA-YE) medium (23.4 g yeast carbon base, 4 g bovine serum albumin, and 2 g yeast extract per liter [pH 4.0]) without selective pressure to induce the SAP2 promoter controlling caFLP expression (for strains containing the SAT1 flipper cassette from pADA2M2 and pADA2K1). Alternatively, strains containing a SAT1 flipper cassette in which the caFLP gene is expressed from the MAL2 promoter (as in plasmids pADA2M4, pCAP1R1, and pMRR1R3) were grown overnight in YPM medium (10 g yeast extract, 20 g peptone, and 20 g maltose per liter) instead of YCB-BSA-YE to induce the MAL2 promoter. One hundred to 200 cells were then spread on YPD plates containing 10 μg/ml nourseothricin and grown for 2 days at 30°C. Nourseothricin-sensitive clones were identified by their small colony size and confirmed by restreaking on YPD plates containing 200 μg/ml nourseothricin, as described previously (21). In the case of the homozygous ada2Δ mutants, which were hypersensitive to nourseothricin, cells from the YPM cultures were grown on YPD plates without nourseothricin. Single colonies were picked and restreaked on YPD plates with and without nourseothricin to identify those in which the SAT1 flipper cassette was excised.

TABLE 1.

C. albicans strains used in this study

Strain(s)	Parent	Relevant characteristic or genotypea	Reference or source
SC5314		Wild-type reference strain	31
ada2Δ mutants
SCADA2M1A and -B	SC5314	ADA2/ada2Δ::SAT1-FLIP	This study
SCADA2M2A	SCADA2M1A	ADA2/ada2Δ::FRT	This study
SCADA2M2B	SCADA2M1B	ADA2/ada2Δ::FRT	This study
SCADA2M3A	SCADA2M2A	ada2Δ::SAT1-FLIP/ada2Δ::FRT	This study
SCADA2M3B	SCADA2M2B	ada2Δ::SAT1-FLIP/ada2Δ::FRT	This study
SCADA2M4A	SCADA2M3A	ada2Δ::FRT/ada2Δ::FRT	This study
SCADA2M4B	SCADA2M3B	ada2Δ::FRT/ada2Δ::FRT	This study
SCADA2MK1A	SCADA2M4A	ada2Δ::FRT/ADA2-SAT1-FLIP	This study
SCADA2MK1B	SCADA2M4B	ada2Δ::FRT/ADA2-SAT1-FLIP	This study
SCADA2MK2A	SCADA2MK1A	ada2Δ::FRT/ADA2-FRT	This study
SCADA2MK2B	SCADA2MK1B	ada2Δ::FRT/ADA2-FRT	This study
Strains with hyperactive CAP1 and MRR1 alleles
SCCAP1R14A and -B	SC5314	CAP1ΔC333-FRT/CAP1ΔC333-FRT	7
SCMRR1R34A and -B	SC5314	MRR1P683S-FRT/MRR1P683S-FRT	7
SCΔada2CAP1R11A	SCADA2M4A	ada2Δ::FRT/ada2Δ::FRT CAP1ΔC333-SAT1-FLIP/CAP1-2	This study
SCΔada2CAP1R11B	SCADA2M4B	ada2Δ::FRT/ada2Δ::FRT CAP1–1/CAP1ΔC333-SAT1-FLIP	This study
SCΔada2CAP1R12A	SCΔada2CAP1R11A	ada2Δ::FRT/ada2Δ::FRT CAP1ΔC333-FRT/CAP1-2	This study
SCΔada2CAP1R12B	SCΔada2CAP1R11B	ada2Δ::FRT/ada2Δ::FRT CAP1–1/CAP1ΔC333-FRT	This study
SCΔada2CAP1R13A	SCΔada2CAP1R12A	ada2Δ::FRT/ada2Δ::FRT CAP1ΔC333-FRT/CAP1ΔC333-SAT1-FLIP	This study
SCΔada2CAP1R13B	SCΔada2CAP1R12B	ada2Δ::FRT/ada2Δ::FRT CAP1ΔC333-SAT1-FLIP/CAP1ΔC333-FRT	This study
SCΔada2CAP1R14A	SCΔada2CAP1R13A	ada2Δ::FRT/ada2Δ::FRT CAP1ΔC333-FRT/CAP1ΔC333-FRT	This study
SCΔada2CAP1R14B	SCΔada2CAP1R13B	ada2Δ::FRT/ada2Δ::FRT CAP1ΔC333-FRT/CAP1ΔC333-FRT	This study
SCΔada2MRR1R31A	SCADA2M4A	ada2Δ::FRT/ada2Δ::FRT MRR1/MRR1P683S-SAT1-FLIP	This study
SCΔada2MRR1R31B	SCADA2M4B	ada2Δ::FRT/ada2Δ::FRT MRR1/MRR1P683S-SAT1-FLIP	This study
SCΔada2MRR1R32A	SCΔada2MRR1R31A	ada2Δ::FRT/ada2Δ::FRT MRR1/MRR1P683S-FRT	This study
SCΔada2MRR1R32B	SCΔada2MRR1R31B	ada2Δ::FRT/ada2Δ::FRT MRR1/MRR1P683S-FRT	This study
SCΔada2MRR1R33A	SCΔada2MRR1R32A	ada2Δ::FRT/ada2Δ::FRT MRR1P683S-SAT1-FLIP/MRR1P683S-FRT	This study
SCΔada2MRR1R33B	SCΔada2MRR1R32B	ada2Δ::FRT/ada2Δ::FRT MRR1P683S-SAT1-FLIP/MRR1P683S-FRT	This study
SCΔada2MRR1R34A	SCΔada2MRR1R33A	ada2Δ::FRT/ada2Δ::FRT MRR1P683S-FRT/MRR1P683S-FRT	This study
SCΔada2MRR1R34B	SCΔada2MRR1R33B	ada2Δ::FRT/ada2Δ::FRT MRR1P683S-FRT/MRR1P683S-FRT	This study
PMDR1-GFP reporter strains
SCMG3A and -B	SC5314	MDR1/mdr1::PMDR1-GFP-caSAT1	10
SCΔada2MG3A	SCADA2M4A	ada2Δ::FRT/ada2Δ::FRT MDR1/mdr1::PMDR1-GFP-caSAT1	This study
SCΔada2MG3B	SCADA2M4B	ada2Δ::FRT/ada2Δ::FRT MDR1/mdr1::PMDR1-GFP-caSAT1	This study
SCCAP1R14MG3A	SCCAP1R14A	CAP1ΔC333-FRT/CAP1ΔC333-FRT MDR1/mdr1::PMDR1-GFP-caSAT1	This study
SCCAP1R14MG3B	SCCAP1R14B	CAP1ΔC333-FRT/CAP1ΔC333-FRT MDR1/mdr1::PMDR1-GFP-caSAT1	This study
SCMRR1R34MG3A	SCMRR1R34A	MRR1P683S-FRT/MRR1P683S-FRT MDR1/mdr1::PMDR1-GFP-caSAT1	10
SCMRR1R34MG3B	SCMRR1R34B	MRR1P683S-FRT/MRR1P683S-FRT MDR1/mdr1::PMDR1-GFP-caSAT1	10
SCΔada2CAP1R14MG3A	SCΔada2CAP1R14A	ada2Δ::FRT/ada2Δ::FRT CAP1ΔC333-FRT/CAP1ΔC333-FRT MDR1/mdr1::PMDR1-GFP-caSAT1	This study
SCΔada2CAP1R14MG3B	SCΔada2CAP1R14B	ada2Δ::FRT/ada2Δ::FRT CAP1ΔC333-FRT/CAP1ΔC333-FRT MDR1/mdr1::PMDR1-GFP-caSAT1	This study
SCΔada2MRR1R34MG3A	SCΔada2MRR1R34A	ada2Δ::FRT/ada2Δ::FRT MRR1P683S-FRT/MRR1P683S-FRT MDR1/mdr1::PMDR1-GFP-caSAT1	This study
SCΔada2MRR1R34MG3B	SCΔada2MRR1R34B	ada2Δ::FRT/ada2Δ::FRT MRR1P683S-FRT/MRR1P683S-FRT MDR1/mdr1::PMDR1-GFP-caSAT1	This study

^aSAT1-FLIP denotes the SAT1 flipper cassette; FRT is the FLP recombination target sequence, one copy of which remains in the genome after recycling of the SAT1 flipper cassette; caSAT1 is the Candida-adapted SAT1 gene. The two CAP1 alleles in strain SC5314 were distinguished by a BglII restriction site polymorphism; the CAP1 allele containing the variable upstream BglII site was arbitrarily designated CAP1-2.

Plasmid constructions.

Two different deletion constructs were generated for the inactivation of the ADA2 alleles in strain SC5314. The ADA2 upstream and downstream regions were amplified with the primer pair ADA2-5 (5′-AACCTAGCAAACGAGCTCACGTGATGTAAGTG-3′) and ADA2-6 (5′-GATAATCATGCCGCGGTTTATGATCTCCAG-3′) and the primer pair ADA2-3 (5′-GGGATGGTGTTCTCGAGGGGTAAATGTAG-3′) and ADA2-4 (5′-GATCGGGCCCTACTTGATCGGTCATACTGGAGC-3′), respectively. The PCR products were digested with SacI-SacII and XhoI-ApaI, respectively (restriction sites introduced into the primers are underlined), and cloned on both sides of the modified SAT1 flipper cassette of plasmid pSFS5 (10) to generate pADA2M2, in which the ADA2 coding region from positions +147 to +1330 (6 bp in front of the stop codon) is replaced by the SAT1 flipper cassette. The N-terminal and C-terminal parts of the ADA2 open reading frame (ORF) were amplified with the primer pair ADA2-7 (5′-GCAGGATTGACTACTGGAGCTCATAAACCATG-3′) and ADA2-8 (5′-GGTAATGGTATATTTTTCCGCGGTTCTAATCGTTC-3′) and the primer pair ADA2-9 (5′-CCAAACACCAGGATTCTCGAGTGGTAATTCTT-3′) and ADA2-10 (5′-CTGAGAACACGGGCCCATATGCACAAA-3′), respectively. The PCR products were digested with SacI-SacII and XhoI-ApaI, respectively, and cloned on both sides of the SAT1 flipper cassette of plasmid pSFS2 (21) to generate pADA2M4, in which the ADA2 coding region from positions +430 to +1003 is replaced by the SAT1 flipper cassette. For reintroduction of a functional ADA2 copy into ada2Δ mutants, the ADA2 coding region and ∼0.5 kb of upstream and downstream sequences were amplified with the primers ADA2-11 (5′-ATATGAGCTCGGGCCCACTCACGTGATGTAAGTGC-3′) (SacI and ApaI sites are underlined) and ADA2-12 (5′-CTCCATCTCAACCGCGGCCAGAAGTATTGCC-3′). The PCR product was digested with SacI-XhoI or XhoI-SacII to obtain the 5′ and 3′ parts of the gene and used to replace the ADA2 upstream region in pADA2M2, resulting in pADA2K1.

Strain constructions.

C. albicans strains were transformed by electroporation (22) with the following gel-purified linear DNA fragments. The SacI-ApaI fragments from pADA2M2 and pADA2M4 were used to delete the first and second ADA2 allele, respectively, in strain SC5314. The ApaI-ApaI fragment from pADA2K1 was used to reintroduce a functional ADA2 copy into ada2Δ mutants. The SacI-ApaI fragments from pMRR1R3 and pCAP1R1 (7) were used to substitute the hyperactive MRR1^P683S and CAP1^ΔC333 alleles, respectively, for the corresponding wild-type alleles in the ada2Δ mutants. The XhoI-SacII fragment from pMDR1G3 (10) was used to integrate the P_MDR1-GFP reporter fusion into the endogenous MDR1 locus in different strains (see Table 1). The correct integration of each construct and the excision of the SAT1 flipper cassettes were confirmed by Southern hybridization using the flanking sequences as probes. The introduction of the P683S mutation into the first and second MRR1 allele of the transformants was confirmed by reamplification and direct sequencing of the PCR products.

Isolation of genomic DNA and Southern hybridization.

Genomic DNA from C. albicans strains was isolated as described previously (21). The DNA was digested with appropriate restriction enzymes, separated on a 1% agarose gel, transferred by vacuum blotting onto a nylon membrane, and fixed by UV cross-linking. Southern hybridization with enhanced chemiluminescence-labeled probes was performed with the Amersham ECL direct nucleic acid labeling and detection system (GE Healthcare UK Limited, Little Chalfont, Buckinghamshire, United Kingdom) according to the instructions of the manufacturer.

Northern hybridization analysis.

Overnight cultures of the strains were diluted 10⁻² in fresh YPD medium and grown for 4 h at 30°C. For the induction of the MDR1 promoter, 50 μg/ml benomyl or 0.005% H₂O₂ was added after 3 h and the cultures were incubated for an additional 15 min. Total RNA was extracted by the hot acidic phenol method (23) combined with a purification step with the RNeasy minikit (Qiagen, Hilden, Germany). RNA samples were separated on a 1.2% agarose gel, transferred by capillary blotting onto a nylon membrane, fixed by UV cross-linking, and hybridized with a digoxigenin-labeled MDR1 probe (positions 657 to 1688 in the MDR1 coding sequence). Bound probe was detected with a peroxidase-labeled anti-digoxigenin alkaline phosphatase (AP) conjugate (Roche, Basel, Switzerland).

Flow cytometry.

Overnight cultures of the GFP reporter and parental strains were diluted 10⁻² in 3 ml fresh YPD medium in glass tubes and grown for 4 h at 30°C. For the induction of the MDR1 promoter, 50 μg/ml benomyl or 0.005% H₂O₂ was added after 3 h and the cultures were incubated for an additional hour. The cell suspensions were 10-fold diluted in 1 ml cold phosphate-buffered saline (PBS) and flow cytometry was performed using the MACSQuantAnalyzer (Miltenyi Biotec; Bergisch Gladbach, Germany) equipped with an argon laser emitting at 488 nm. Fluorescence was detected using the B1 fluorescence channel equipped with a 525-nm band-pass filter (bandwidth 50 nm). Twenty thousand cells were analyzed per sample and counted at a flow rate of approximately 500 cells per second. Fluorescence data were collected by using logarithmic amplifiers. The mean fluorescence (arbitrary units) was determined with MACSQuantify (version 2.4; Miltenyi Biotec) software.

Western immunoblotting.

For the detection of green fluorescent protein (GFP) by Western immunoblotting, the reporter strains were grown as described above for the flow cytometry analyses, except that the cultures were grown in 25-ml volumes in Erlenmeyer flasks. Cells were collected by centrifugation, washed in 2.5 ml breaking buffer (100 mM Tris-HCl [pH 7.5], 200 mM NaCl, 20% glycerol, and 5 mM EDTA), and broken by vortexing for 10 min at 4°C with 500 μl 0.5-mm glass beads in 500 μl breaking buffer (100 mM Tris-HCl [pH 7.5], 200 mM NaCl, 20% glycerol, 5 mM EDTA, 4% complete, EDTA-free protease inhibitor cocktail [Roche Diagnostics GmbH, Mannheim, Germany], and 0.1% β-mercaptoethanol). Samples were centrifuged at 13,000 rpm for 10 min at 4°C, the supernatant was collected, and the protein concentration was quantified using the Bradford protein assay. Extracts were heated for 10 min at 65°C, and equal amounts of protein of each sample were separated on an SDS 12% polyacrylamide gel. Proteins were transferred onto a nitrocellulose membrane with a Mini-Protean Tetra system (Bio-Rad, Munich, Germany) and stained with Ponceau S to control for equal loading. GFP was detected using rabbit monoclonal GFP antibody ab32146 (Abcam, Cambridge, United Kingdom) and anti-rabbit HRP G-21234 (Invitrogen GmbH, Darmstadt, Germany) as first and secondary antibodies, respectively. A chemiluminescence detection system (GE Healthcare) was used for signal detection.

Fluconazole susceptibility testing.

To determine the fluconazole susceptibilities of the strains, a 2-fold dilution series of fluconazole (Sigma GmbH, Deisenhofen, Germany) was prepared in the assay medium, starting from an initial concentration of 128 μg/ml. Susceptibility tests were carried out using a previously described microdilution method (24), except that the assays were performed in synthetic dextrose (SD) medium (6.7 g yeast nitrogen base [YNB] without amino acids [BIO 101, Vista, CA], 20 g glucose, and 0.77 g of complete supplement medium [CSM] [BIO101]) instead of high-resolution (HR) medium (10). MICs of fluconazole were determined after 48 h of growth at 37°C.

RESULTS

Generation of ada2Δ mutants of the C. albicans wild-type strain SC5314.

To directly compare inducible and constitutive MDR1 expression levels in cells lacking Ada2 with those in previously constructed wild-type strains, we deleted the ADA2 gene in the C. albicans reference strain SC5314. Two independent series of heterozygous and homozygous ada2Δ mutants were generated using the SAT1-flipping strategy (21), and an intact ADA2 copy was reinserted into both homozygous mutants to obtain complemented strains. Phenotypic analysis (Fig. 1) showed that the ada2Δ mutants displayed the previously described hypersensitivity to fluconazole and H₂O₂ (20). The mutants were also highly sensitive to nourseothricin, a phenotype that was first noted when the SAT1 flipper cassette was recycled after inactivation of the second ADA2 allele. We did not recover cells in which the resistance cassette was excised on plates with a low concentration of nourseothricin, which is a convenient screening procedure (21). In addition, we found that the ada2Δ mutants also displayed somewhat reduced growth on media without any inhibitor. All mutant phenotypes were reverted to wild type after reinsertion of an intact ADA2 copy, confirming that they were caused by the deletion of ADA2 (Fig. 1).

FIG 1.

Sensitivity of the wild type, heterozygous and homozygous ada2Δ mutants, and complemented strains to fluconazole, H₂O₂, and nourseothricin. Serial 10-fold dilutions of the strains were spotted onto agar plates containing the indicated compounds. Plates were incubated for 1 day (YPD plates, bottom row) or 2 days (SD plates, top row) at 30°C. The following strains were used: SC5314 (wild type), SCADA2M2A and -B (ADA2/ada2Δ), SCADA2M4A and -B (ada2Δ/ada2Δ), and SCADA2MK2A and -B (ada2Δ/ada2Δ + ADA2).

Ada2 is not required for benomyl- and H₂O₂-induced MDR1 expression.

To compare the inducibility of MDR1 expression in the presence or absence of Ada2, the wild-type strain SC5314 and the two homozygous ada2Δ mutant strains were grown to log phase in liquid YPD medium and treated with the inducers benomyl and H₂O₂. Total RNA was isolated from the cultures and MDR1 mRNA detected by Northern hybridization. No MDR1 transcripts were detected in untreated cells of the wild type and the ada2Δ mutants (Fig. 2A, lanes 1, 4, and 5). MDR1 expression was upregulated by benomyl in both wild-type and ada2Δ cells (Fig. 2A, lanes 2, 6, and 7) and H₂O₂ (Fig. 2A, lanes 3, 8, and 9). The induction of MDR1 expression occurred with similar efficiency in the wild-type and the ada2Δ mutant cells, demonstrating that Ada2 is not required for the induction of MDR1 by these compounds.

FIG 2.

Inducibility of MDR1 expression by benomyl and H₂O₂ in the wild type and ada2Δ mutants. (A) Analysis of MDR1 expression in the wild-type strain SC5314 and two independently constructed ada2Δ mutants (SCADA2M4A and -B) grown in the absence (−) or presence (+) of benomyl or H₂O₂ by Northern hybridization. The 18S RNA bands in the ethidium bromide-stained gel served as loading control. (B) Strains carrying a P_MDR1-GFP reporter fusion in wild-type and ada2Δ backgrounds were grown in the absence or presence of inducers as indicated. The mean fluorescence of the cells was determined by flow cytometry. The results obtained with two independently generated reporter strains are shown in each case (means and standard deviations from three experiments). The following strains were used: SCMG3A and -B (wild type), SCΔada2MG3A and -B (ada2Δ). The background fluorescence of the parental strains, which do not contain the GFP gene, is indicated by the black part of each column. (C) GFP expression in the same strains was detected by Western immunoblotting with an anti-GFP antibody.

To corroborate the results of the Northern hybridization experiments with an independent method, we introduced a P_MDR1-GFP reporter gene fusion into the ada2Δ mutants and compared the activity of the MDR1 promoter in wild-type and mutant cells by flow cytometry. In agreement with the Northern hybridization analysis, MDR1 promoter activity remained below the detection limit in uninduced cells, as the fluorescence of the reporter strains was not increased above the background fluorescence levels of their parental strains that did not contain the GFP gene (Fig. 2B). The MDR1 promoter was strongly induced in wild-type cells treated with benomyl and, more weakly, in the presence of H₂O₂. The fluorescence of the ada2Δ reporter strains was also increased by both inducers, but a direct comparison with the wild-type cells was complicated by the fact that the ada2Δ mutants exhibited a 4-fold higher autofluorescence. We therefore compared GFP levels in the cells by Western immunoblotting with an anti-GFP antibody. The GFP amounts observed in wild-type cells treated with benomyl and H₂O₂ corresponded well with the results of the flow cytometry and Northern hybridization experiments (Fig. 2C, lanes 1 to 6). The Western blot analysis confirmed that MDR1 expression was also induced by these compounds in the ada2Δ mutant cells (Fig. 2C, lanes 7 to 12) and with the same efficiency as in the wild-type cells. Collectively, these results show that, under the conditions used in our experiments, Ada2 is dispensable for benomyl- and H₂O₂-induced MDR1 expression.

Requirement of Ada2 for MDR1 upregulation by hyperactive MRR1 and CAP1 alleles.

As explained in the introduction, hyperactive forms of Mrr1 and Cap1 can independently upregulate MDR1 expression in the absence of inducers, and gain-of-function mutations in MRR1 are the cause of MDR1 overexpression in fluconazole-resistant, clinical C. albicans isolates. In order to assess a potential role of Ada2 in the constitutive MDR1 upregulation in such strains, we introduced the P683S gain-of-function mutation into both resident MRR1 alleles of the ada2Δ mutants, in the same way as in previously constructed derivatives of the parental wild-type strain SC5314 (7). Similarly, both wild-type CAP1 alleles were replaced by the C-terminally truncated, hyperactive CAP1^ΔC333 allele. Northern hybridization analysis demonstrated the constitutive upregulation of MDR1 by the hyperactive Mrr1 and, at a lower level, by the hyperactive Cap1 in the wild-type background (Fig. 3A, lanes 1 to 5). MDR1 was similarly overexpressed in ada2Δ mutants containing the hyperactive MRR1^P683S allele (Fig. 3A, lanes 6 to 9), demonstrating that Ada2 is dispensable for MDR1 upregulation by a constitutively active Mrr1. In contrast, the hyperactive Cap1 caused only a minor upregulation of MDR1 expression in the absence of Ada2 (Fig. 3A, lanes 10 and 11), indicating that Cap1 requires Ada2 to induce MDR1 expression when Mrr1 is not simultaneously activated by inducing compounds.

FIG 3.

MDR1 expression in wild-type and ada2Δ strains carrying hyperactive MRR1 or CAP1 alleles. (A) Detection of MDR1 mRNA in strains containing wild-type, MRR1^P683S or CAP1^ΔC333 alleles by Northern hybridization; 18S RNA is shown as loading control. The following strains were used: SC5314 (wild type, −), SCMRR1R34A and -B (wild type, MRR1^P683S), SCCAP1R14A and -B (wild type, CAP1^ΔC333), SCADA2M4A and -B (ada2Δ, −), SCΔada2MRR1R34A and -B (ada2Δ, MRR1^P683S), and SCΔada2CAP1R14A and -B (ada2Δ, CAP1^ΔC333). (B) Reporter strains containing wild-type or hyperactive MRR1 and CAP1 alleles and expressing GFP under the control of the MDR1 promoter were grown to log phase in YPD medium. The mean fluorescence of the cells was determined by flow cytometry. The results obtained with two independently generated reporter strains are shown in each case (means and standard deviations from three experiments). The following strains were used: SCMG3A and -B (wild type, −), SCMRR1R34MG3A and -B (wild type, MRR1^P683S), SCCAP1R14MG3A and -B (wild type, CAP1^ΔC333), SCΔada2MG3A and -B (ada2Δ, −), SCΔada2MRR1R34MG3A and -B (ada2Δ, MRR1^P683S), and SCΔada2CAP1R14MG3A and -B (ada2Δ, CAP1^ΔC333). The background fluorescence of the parental strains, which do not contain the GFP gene, is indicated by the black part of each column. (C) GFP expression in the same strains was detected by Western immunoblotting with an anti-GFP antibody.

To corroborate the results of the Northern hybridization experiments, we compared MDR1 promoter activity in the same strains. For this purpose, the P_MDR1-GFP reporter gene fusion was also introduced into the ada2Δ mutants containing the hyperactive MRR1^P683S and CAP1^ΔC333 alleles, and the fluorescence of the cells was quantified by flow cytometry. Figure 3B shows that the MDR1 promoter was strongly activated by the hyperactive Mrr1 in both the wild type and the ada2Δ mutants. The hyperactive Cap1 caused MDR1 upregulation in the wild type, whereas the fluorescence of the ada2Δ mutants was only slightly increased above background levels. Western blot analysis demonstrated that comparable amounts of GFP were produced in wild-type and ada2Δ cells containing the MRR1^P683S allele (Fig. 3C, compare lanes 3 and 4 with lanes 9 and 10). The presence of the hyperactive Cap1 also resulted in strongly increased GFP levels in the wild-type background (Fig. 3C, lanes 5 and 6), while only a faint signal was detected in the ada2Δ mutants (Fig. 3C, lanes 11 and 12). Collectively, these results argue that Cap1, but not Mrr1, requires Ada2 to promote MDR1 expression.

Fluconazole susceptibility of strains expressing hyperactive MRR1 and CAP1 alleles in the presence and absence of Ada2.

The hyperactive MRR1^P683S and CAP1^ΔC333 alleles confer increased fluconazole resistance that only partially depends on the Mdr1 efflux pump (7). We therefore compared the fluconazole susceptibilities of strains expressing these alleles in wild-type and ada2Δ backgrounds by a dilution spot assay on solid medium and a broth microdilution assay. As can be seen in Fig. 4, the hyperactive Mrr1 also caused enhanced fluconazole resistance in the ada2Δ mutants, although the increase in the MIC of fluconazole was slightly lower (16-fold, from 0.125 μg/ml to 2 μg/ml) than in the wild type (32-fold, from 0.5 μg/ml to 16 μg/ml), presumably due to the generally decreased fitness of the ada2Δ mutants. The hyperactive Cap1 conferred enhanced fluconazole resistance in the wild type, whereas no increase in fluconazole resistance was observed in the absence of Ada2 on agar plates containing the drug. However, the MIC of fluconazole was also still slightly increased for the ada2Δ mutants containing the hyperactive CAP1 allele, indicating that the hyperactive Cap1 has residual activity in the absence of Ada2. Overall, the differential requirement of Ada2 for Mrr1- and Cap1-mediated fluconazole resistance reflects the capacity of these transcription factors to activate the MDR1 promoter in the absence of Ada2.

FIG 4.

Fluconazole sensitivity of strains expressing hyperactive MRR1 and CAP1 alleles in wild-type or ada2Δ backgrounds. Serial 10-fold dilutions of the strains were spotted onto SD agar plates with or without 5 μg/ml fluconazole and incubated for 2 days at 30°C. The following strains were used: SC5314 (wild type, −), SCMRR1R34A and -B (wild type, MRR1^P683S), SCCAP1R14A and -B (wild type, CAP1^ΔC333), SCADA2M4A and -B (ada2Δ, −), SCΔada2MRR1R34A and -B (ada2Δ, MRR1^P683S), and SCΔada2CAP1R14A and -B (ada2Δ, CAP1^ΔC333). The MIC of fluconazole for each strain, as determined in broth microdilution assays, is given to the right.

DISCUSSION

The results of our study show that a requirement of Ada2 for the transcriptional activation of MDR1 depends on how MDR1 expression is induced. Ada2 was completely dispensable for the upregulation of MDR1 by a hyperactive form of the transcription factor Mrr1 containing the P683S gain-of-function mutation (Fig. 3). Gain-of-function mutations in Mrr1 are responsible for the constitutive MDR1 overexpression in fluconazole-resistant clinical C. albicans isolates (12). It is therefore likely that Ada2 does not play a role in Mrr1-mediated MDR1 overexpression and fluconazole resistance in such strains. In line with this expectation, Sellam et al. reported that deletion of ADA2 in an MDR1-overexpressing clinical isolate did not reduce its fluconazole resistance (20). In contrast, a C-terminally truncated, hyperactive Cap1, which also causes MDR1 overexpression, was largely unable to upregulate MDR1 transcription in the absence of Ada2 (Fig. 3). These findings suggest that Cap1, and not Mrr1, recruits Ada2 to induce MDR1 expression. In the presence of the inducers benomyl and H₂O₂, Cap1 cooperates with Mrr1 to upregulate MDR1 expression (7), explaining why no recruitment of Ada2 was necessary under these conditions (Fig. 2).

Sellam et al. used reverse transcription-quantitative PCR (RT-qPCR) to compare MDR1 mRNA levels in wild-type and ada2Δ cells treated with fluconazole (20). They found that the induction of MDR1 expression by fluconazole was lower in the ada2Δ mutant (6-fold) than in the wild type (18-fold). In our lab, we did not detect induction of MDR1 expression by fluconazole in Northern hybridization experiments (25) or when using GFP as a reporter gene (unpublished results), in agreement with findings by other researchers (3, 4, 26). The comparatively mild MDR1 induction by fluconazole (MDR1 is upregulated several hundredfold in strains with hyperactive MRR1 alleles [7, 9]) is insufficient to confer increased drug resistance, as MDR1 deletion in fluconazole-susceptible strains does not result in hypersusceptibility to the drug (7, 27, 28). Therefore, the increased susceptibility of ada2Δ mutants to fluconazole cannot be due to decreased MDR1 expression. The fact that ada2Δ mutants are hypersusceptible to various unrelated toxic compounds and also grew somewhat more slowly than the wild type in the absence of inhibitors (Fig. 1) indicates that cells lacking Ada2 have a general fitness defect, which may be exacerbated under stress conditions.

Altogether, our findings argue that binding of Ada2 to the MDR1 promoter is most likely mediated by Cap1, but not by Mrr1. Consistent with this assumption, Sellam et al. found that the binding of Ada2 to the MDR1 promoter was abolished in a cap1Δ mutant (20). Binding of Ada2 to the MDR1 promoter was also reduced, but not eliminated, in an mrr1Δ mutant. The latter observation can be explained by the fact that Cap1 requires Mrr1 to induce MDR1 expression in response to inducing signals; the ability of Cap1 to recruit Ada2 to the MDR1 promoter may be compromised in the absence of Mrr1. Although Sellam et al. noted that Ada2 recruitment to a subset of its target genes is mediated by Cap1, Ada2 was proposed to be a coactivator of Mrr1 in the expression of drug resistance genes like MDR1 because of the strong overlap between promoters bound by Mrr1 and Ada2 (20). However, it is possible that the binding of Ada2 to other Mrr1 target genes also does not occur via Mrr1. Like MDR1, three of the other five tested genes that were bound by both Mrr1 and Ada2 are known Cap1 target genes (HSP31, GRP2, and OYE32), and the others are induced by benomyl, which also causes oxidative stress and activates Cap1 (7, 8, 29). Therefore, Ada2 may also be recruited by Cap1, and not by Mrr1, to these common target genes.

In a previous study (30), we found that the MADS box transcription factor Mcm1 is also differentially required for the induction of MDR1 expression by Cap1 and Mrr1. The requirement of Mcm1 was inverse to that observed in the present study for Ada2. A hyperactive Mrr1 did not upregulate MDR1 when Mcm1 was depleted from the cells, whereas Mcm1 was dispensable for the induction of MDR1 expression by a hyperactive Cap1. Figure 5 summarizes our current understanding of the regulation of MDR1 expression by Cap1, Mrr1, Mcm1, and Ada2 under various inducing conditions. In the presence of H₂O₂ (Fig. 5A), Cap1 is activated and cooperates with Mrr1 to induce MDR1 expression. Mrr1 is also required, although some residual MDR1 expression may occur without Mrr1, while Mcm1 is dispensable under these conditions. Cap1 probably recruits Ada2 to the MDR1 promoter, but this was not necessary for MDR1 induction by H₂O₂ under our experimental conditions. When benomyl serves as the inducer (Fig. 5B), Mrr1 becomes activated by a still unknown mechanism and, together with Mcm1, causes MDR1 upregulation. Benomyl also causes oxidative stress and activates Cap1, which maximizes MDR1 expression in an Ada2-independent fashion. While Mrr1 is essential, a partial activation of the MDR1 promoter can occur without either Mcm1 or Cap1, but all three transcription factors are required for fully induced MDR1 expression levels. A constitutively active Cap1 (Fig. 5C) can upregulate MDR1 expression even in the absence of Mrr1, although less efficiently than in its presence. MDR1 induction by the hyperactive Cap1 strongly depends on Ada2, whereas Mcm1 is dispensable. Finally, in strains that have acquired a gain-of-function mutation in Mrr1 (Fig. 5D), Cap1 (which remains mostly in the cytoplasm in the absence of oxidative stress) and Ada2 are not required for MDR1 overexpression, but the hyperactive Mrr1 depends on Mcm1 to cause MDR1 upregulation. Central remaining questions are how Mrr1 achieves its activated state, either in the presence of inducers like benomyl or by gain-of-function mutations, and by which mechanism(s) it recruits the core transcriptional machinery to allow MDR1 expression in a Cap1- and Ada2-independent manner.

FIG 5.

Model of the roles of Cap1, Mrr1, Mcm1, and Ada2 in MDR1 upregulation in the presence of the inducing compounds H₂O₂ (A) and benomyl (B) or by hyperactive forms (*) of Cap1 (C) and Mrr1 (D). The transcription factors Cap1, Mrr1, and Mcm1 directly bind to the MDR1 promoter, while Ada2 is recruited by Cap1. The bent arrow indicates transcription of MDR1. Missing proteins are not required for MDR1 expression under the particular condition. Gray color indicates that the respective regulatory protein contributes to full MDR1 expression levels, but a partial activation of the MDR1 promoter also occurs in its absence. Detailed explanations are given in the text.