SAGA/ADA Complex Subunit Ada2 Is Required for Cap1- but NotMrr1-Mediated Upregulation of the Candida albicans Multidrug EffluxPump MDR1

Bernardo Ramírez-Zavala,a Selene Mogavero,a,b* Eva Schöller,a Christoph Sasse,a* P. David Rogers,c Joachim Morschhäusera

Institut für Molekulare Infektionsbiologie, Universität Würzburg, Würzburg, Germanya; Department of Biology, University of Pisa, Pisa, Italyb; Department of ClinicalPharmacy, University of Tennessee Health Science Center, Memphis, Tennessee, USAc

Overexpression of the multidrug efflux pump MDR1 is one mechanism by which the pathogenic yeast Candida albicans developsresistance to the antifungal drug fluconazole. The constitutive upregulation of MDR1 in fluconazole-resistant, clinical C. albi-cans isolates is caused by gain-of-function mutations in the zinc cluster transcription factor Mrr1. It has been suggested thatMrr1 activates MDR1 transcription by recruiting Ada2, a subunit of the SAGA/ADA coactivator complex. However, MDR1 ex-pression 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 independentlyof Ada2. In contrast, a C-terminally truncated, hyperactive Cap1 caused MDR1 overexpression in a wild-type strain but onlyweakly 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 ada2� cells. These results indi-cate that Cap1, but not Mrr1, recruits Ada2 to the MDR1 promoter to induce the expression of this multidrug efflux pump andthat Ada2 is not required for MDR1 overexpression in fluconazole-resistant C. albicans strains containing gain-of-function mu-tations in Mrr1.

The Candida albicans MDR1 gene encodes a multidrug effluxpump of the major facilitator superfamily that confers resis-

tance to various toxic compounds, including the antifungal drugfluconazole (1, 2). MDR1 is not significantly expressed understandard growth conditions, but its expression is induced by cer-tain chemicals, like benomyl or H2O2 (3–6). The bZIP transcrip-tion factor Cap1 and the zinc cluster transcription factor Mrr1mediate the upregulation of MDR1 in the presence of these com-pounds. Cap1, which regulates the oxidative stress response, isrequired for the induction of MDR1 transcription by H2O2 andalso contributes to benomyl-induced MDR1 expression (6–8).Similarly, little or no induction of MDR1 expression occurs inmutants lacking MRR1 (7, 9–11). Therefore, Cap1 and Mrr1, bothof which bind to the MDR1 promoter (7, 8), cooperate to promoteMDR1 upregulation in response to inducing chemicals.

Many fluconazole-resistant, clinical C. albicans isolates consti-tutively overexpress MDR1, which contributes to their drug-resis-tant phenotype (2, 12). In all cases investigated so far, MDR1 over-expression is caused by gain-of-function mutations in Mrr1 thatrender the transcription factor constitutively active (9, 13, 14). Incells containing such a hyperactive Mrr1, Cap1 is dispensable forMDR1 expression (7). Vice versa, a C-terminally truncated, hy-peractive form of Cap1 also causes constitutive MDR1 upregula-tion that is partially independent of Mrr1 (7, 15).

In addition to the direct inhibition of efflux pumps, blockingtheir expression is considered a potential strategy to overcomedrug resistance in pathogenic fungi (16, 17). It is currently notknown how transcription factors that mediate drug resistance inC. 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 recruitRNA polymerase II to the promoters of their target genes (18, 19).

Mrr1 may act in a similar fashion, but it has recently been pro-posed to recruit Ada2, a subunit of the SAGA/ADA coactivatorcomplex, to induce the transcription of drug resistance genes (20).The SAGA/ADA complex mediates histone acetylation to allowtranscriptional activation. By chromatin immunoprecipitationexperiments it was shown that Ada2 binds to 200 promoters in theC. albicans genome (20). Among the Ada2 targets were genes in-volved in the response to drugs and oxidative stress, and an ada2�mutant was found to be hypersusceptible to fluconazole and reac-tive oxygen species. Ada2 bound to many genes that are regulatedby Cap1, and binding was reduced in the absence of Cap1. Ada2also bound to many Mrr1 target genes in an Mrr1-dependentmanner, and it was suggested that Ada2 might function as a co-activator of Mrr1 (20). Yet, whether Mrr1 depends on Ada2 toactivate MDR1 expression was not tested in that study. Since bothCap1 and Mrr1 mediate MDR1 expression, recruitment of Ada2to the MDR1 promoter might be accomplished by either of thetwo transcription factors. In order to clarify the role of Ada2 inCap1- and Mrr1-mediated MDR1 expression, we investigated theimportance of Ada2 for MDR1 activation by the inducers benomyl

Received 15 April 2014 Returned for modification 3 May 2014Accepted 7 June 2014

Published ahead of print 16 June 2014

Address correspondence to Joachim Morschhäuser,joachim.morschhaeuser@uni-wuerzburg.de.

* Present address: Selene Mogavero, Department of Microbial PathogenicityMechanisms, Leibniz Institute for Natural Product Research and Infection Biology,Hans Knöll Institute (HKI), Jena, Germany; Christoph Sasse, Institut fürMikrobiologie und Genetik, Universität Göttingen, Göttingen, Germany.

Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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and H2O2 and for the constitutive MDR1 overexpression causedby hyperactive forms of Mrr1 and Cap1.

MATERIALS AND METHODSStrains and growth conditions. The C. albicans strains used in this studyare 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 gagar per liter) at 30°C. Strains were routinely grown in YPD liquid me-dium at 30°C in a shaking incubator. For selection of nourseothricin-resistant transformants, 200 �g/ml nourseothricin (Werner Bioagents,Jena, Germany) was added to YPD agar plates. To obtain nourseothricin-

TABLE 1 C. albicans strains used in this study

Strain(s) Parent Relevant characteristic or genotypea

Referenceor source

SC5314 Wild-type reference strain 31

ada2� mutantsSCADA2M1A and -B SC5314 ADA2/ada2�::SAT1-FLIP This studySCADA2M2A SCADA2M1A ADA2/ada2�::FRT This studySCADA2M2B SCADA2M1B ADA2/ada2�::FRT This studySCADA2M3A SCADA2M2A ada2�::SAT1-FLIP/ada2�::FRT This studySCADA2M3B SCADA2M2B ada2�::SAT1-FLIP/ada2�::FRT This studySCADA2M4A SCADA2M3A ada2�::FRT/ada2�::FRT This studySCADA2M4B SCADA2M3B ada2�::FRT/ada2�::FRT This studySCADA2MK1A SCADA2M4A ada2�::FRT/ADA2-SAT1-FLIP This studySCADA2MK1B SCADA2M4B ada2�::FRT/ADA2-SAT1-FLIP This studySCADA2MK2A SCADA2MK1A ada2�::FRT/ADA2-FRT This studySCADA2MK2B SCADA2MK1B ada2�::FRT/ADA2-FRT This study

Strains with hyperactive CAP1 andMRR1 alleles

SCCAP1R14A and -B SC5314 CAP1�C333-FRT/CAP1�C333-FRT 7SCMRR1R34A and -B SC5314 MRR1P683S-FRT/MRR1P683S-FRT 7SC�ada2CAP1R11A SCADA2M4A ada2�::FRT/ada2�::FRT CAP1�C333-SAT1-FLIP/CAP1-2 This studySC�ada2CAP1R11B SCADA2M4B ada2�::FRT/ada2�::FRT CAP1–1/CAP1�C333-SAT1-FLIP This studySC�ada2CAP1R12A SC�ada2CAP1R11A ada2�::FRT/ada2�::FRT CAP1�C333-FRT/CAP1-2 This studySC�ada2CAP1R12B SC�ada2CAP1R11B ada2�::FRT/ada2�::FRT CAP1–1/CAP1�C333-FRT This studySC�ada2CAP1R13A SC�ada2CAP1R12A ada2�::FRT/ada2�::FRT CAP1�C333-FRT/CAP1�C333-SAT1-FLIP This studySC�ada2CAP1R13B SC�ada2CAP1R12B ada2�::FRT/ada2�::FRT CAP1�C333-SAT1-FLIP/CAP1�C333-FRT This studySC�ada2CAP1R14A SC�ada2CAP1R13A ada2�::FRT/ada2�::FRT CAP1�C333-FRT/CAP1�C333-FRT This studySC�ada2CAP1R14B SC�ada2CAP1R13B ada2�::FRT/ada2�::FRT CAP1�C333-FRT/CAP1�C333-FRT This studySC�ada2MRR1R31A SCADA2M4A ada2�::FRT/ada2�::FRT MRR1/MRR1P683S-SAT1-FLIP This studySC�ada2MRR1R31B SCADA2M4B ada2�::FRT/ada2�::FRT MRR1/MRR1P683S-SAT1-FLIP This studySC�ada2MRR1R32A SC�ada2MRR1R31A ada2�::FRT/ada2�::FRT MRR1/MRR1P683S-FRT This studySC�ada2MRR1R32B SC�ada2MRR1R31B ada2�::FRT/ada2�::FRT MRR1/MRR1P683S-FRT This studySC�ada2MRR1R33A SC�ada2MRR1R32A ada2�::FRT/ada2�::FRT MRR1P683S-SAT1-FLIP/MRR1P683S-FRT This studySC�ada2MRR1R33B SC�ada2MRR1R32B ada2�::FRT/ada2�::FRT MRR1P683S-SAT1-FLIP/MRR1P683S-FRT This studySC�ada2MRR1R34A SC�ada2MRR1R33A ada2�::FRT/ada2�::FRT MRR1P683S-FRT/MRR1P683S-FRT This studySC�ada2MRR1R34B SC�ada2MRR1R33B ada2�::FRT/ada2�::FRT MRR1P683S-FRT/MRR1P683S-FRT This study

PMDR1-GFP reporter strainsSCMG3A and -B SC5314 MDR1/mdr1::PMDR1-GFP-caSAT1 10SC�ada2MG3A SCADA2M4A ada2�::FRT/ada2�::FRT MDR1/mdr1::PMDR1-GFP-caSAT1 This studySC�ada2MG3B SCADA2M4B ada2�::FRT/ada2�::FRT MDR1/mdr1::PMDR1-GFP-caSAT1 This studySCCAP1R14MG3A SCCAP1R14A CAP1�C333-FRT/CAP1�C333-FRT MDR1/mdr1::PMDR1-GFP-caSAT1 This studySCCAP1R14MG3B SCCAP1R14B CAP1�C333-FRT/CAP1�C333-FRT MDR1/mdr1::PMDR1-GFP-caSAT1 This studySCMRR1R34MG3A SCMRR1R34A MRR1P683S-FRT/MRR1P683S-FRT MDR1/mdr1::PMDR1-GFP-caSAT1 10SCMRR1R34MG3B SCMRR1R34B MRR1P683S-FRT/MRR1P683S-FRT MDR1/mdr1::PMDR1-GFP-caSAT1 10SC�ada2CAP1R14MG3A SC�ada2CAP1R14A ada2�::FRT/ada2�::FRT CAP1�C333-FRT/CAP1�C333-FRT

MDR1/mdr1::PMDR1-GFP-caSAT1This study

SC�ada2CAP1R14MG3B SC�ada2CAP1R14B ada2�::FRT/ada2�::FRT CAP1�C333-FRT/CAP1�C333-FRTMDR1/mdr1::PMDR1-GFP-caSAT1

This study

SC�ada2MRR1R34MG3A SC�ada2MRR1R34A ada2�::FRT/ada2�::FRT MRR1P683S-FRT/MRR1P683S-FRTMDR1/mdr1::PMDR1-GFP-caSAT1

This study

SC�ada2MRR1R34MG3B SC�ada2MRR1R34B ada2�::FRT/ada2�::FRT MRR1P683S-FRT/MRR1P683S-FRTMDR1/mdr1::PMDR1-GFP-caSAT1

This study

a SAT1-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 thevariable upstream BglII site was arbitrarily designated CAP1-2.

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sensitive derivatives in which the SAT1 flipper cassette was excised byFLP-mediated recombination, transformants were grown overnight inyeast carbon base-bovine serum albumin-yeast extract (YCB-BSA-YE)medium (23.4 g yeast carbon base, 4 g bovine serum albumin, and 2 gyeast extract per liter [pH 4.0]) without selective pressure to induce theSAP2 promoter controlling caFLP expression (for strains containing theSAT1 flipper cassette from pADA2M2 and pADA2K1). Alternatively,strains containing a SAT1 flipper cassette in which the caFLP gene is ex-pressed from the MAL2 promoter (as in plasmids pADA2M4, pCAP1R1,and pMRR1R3) were grown overnight in YPM medium (10 g yeast ex-tract, 20 g peptone, and 20 g maltose per liter) instead of YCB-BSA-YE toinduce the MAL2 promoter. One hundred to 200 cells were then spread onYPD plates containing 10 �g/ml nourseothricin and grown for 2 days at30°C. Nourseothricin-sensitive clones were identified by their small col-ony size and confirmed by restreaking on YPD plates containing 200�g/ml nourseothricin, as described previously (21). In the case of thehomozygous ada2� mutants, which were hypersensitive to nourseothri-cin, cells from the YPM cultures were grown on YPD plates withoutnourseothricin. Single colonies were picked and restreaked on YPD plateswith and without nourseothricin to identify those in which the SAT1flipper cassette was excised.

Plasmid constructions. Two different deletion constructs were gen-erated for the inactivation of the ADA2 alleles in strain SC5314. The ADA2upstream and downstream regions were amplified with the primer pairADA2-5 (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 theprimers are underlined), and cloned on both sides of the modified SAT1flipper cassette of plasmid pSFS5 (10) to generate pADA2M2, in which theADA2 coding region from positions �147 to �1330 (6 bp in front of thestop codon) is replaced by the SAT1 flipper cassette. The N-terminal andC-terminal parts of the ADA2 open reading frame (ORF) were amplifiedwith 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 weredigested with SacI-SacII and XhoI-ApaI, respectively, and cloned on bothsides of the SAT1 flipper cassette of plasmid pSFS2 (21) to generatepADA2M4, in which the ADA2 coding region from positions �430 to�1003 is replaced by the SAT1 flipper cassette. For reintroduction of afunctional ADA2 copy into ada2� mutants, the ADA2 coding region and�0.5 kb of upstream and downstream sequences were amplified with theprimers ADA2-11 (5=-ATATGAGCTCGGGCCCACTCACGTGATGTAAGTGC-3=) (SacI and ApaI sites are underlined) and ADA2-12 (5=-CTCCATCTCAACCGCGGCCAGAAGTATTGCC-3=). The PCR product wasdigested with SacI-XhoI or XhoI-SacII to obtain the 5= and 3= parts of thegene and used to replace the ADA2 upstream region in pADA2M2, result-ing in pADA2K1.

Strain constructions. C. albicans strains were transformed by electro-poration (22) with the following gel-purified linear DNA fragments. TheSacI-ApaI fragments from pADA2M2 and pADA2M4 were used to deletethe first and second ADA2 allele, respectively, in strain SC5314. The ApaI-ApaI fragment from pADA2K1 was used to reintroduce a functionalADA2 copy into ada2� mutants. The SacI-ApaI fragments frompMRR1R3 and pCAP1R1 (7) were used to substitute the hyperactiveMRR1P683S and CAP1�C333 alleles, respectively, for the correspondingwild-type alleles in the ada2� mutants. The XhoI-SacII fragment frompMDR1G3 (10) was used to integrate the PMDR1-GFP reporter fusion intothe endogenous MDR1 locus in different strains (see Table 1). The correctintegration of each construct and the excision of the SAT1 flipper cassetteswere confirmed by Southern hybridization using the flanking sequences

as probes. The introduction of the P683S mutation into the first andsecond MRR1 allele of the transformants was confirmed by reamplifica-tion and direct sequencing of the PCR products.

Isolation of genomic DNA and Southern hybridization. GenomicDNA from C. albicans strains was isolated as described previously (21).The DNA was digested with appropriate restriction enzymes, separated ona 1% agarose gel, transferred by vacuum blotting onto a nylon membrane,and fixed by UV cross-linking. Southern hybridization with enhancedchemiluminescence-labeled probes was performed with the AmershamECL direct nucleic acid labeling and detection system (GE Healthcare UKLimited, Little Chalfont, Buckinghamshire, United Kingdom) accordingto the instructions of the manufacturer.

Northern hybridization analysis. Overnight cultures of the strainswere diluted 10�2 in fresh YPD medium and grown for 4 h at 30°C. For theinduction of the MDR1 promoter, 50 �g/ml benomyl or 0.005% H2O2

was added after 3 h and the cultures were incubated for an additional 15min. 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 UVcross-linking, and hybridized with a digoxigenin-labeled MDR1 probe(positions 657 to 1688 in the MDR1 coding sequence). Bound probe wasdetected with a peroxidase-labeled anti-digoxigenin alkaline phosphatase(AP) conjugate (Roche, Basel, Switzerland).

Flow cytometry. Overnight cultures of the GFP reporter and parentalstrains were diluted 10�2 in 3 ml fresh YPD medium in glass tubes andgrown for 4 h at 30°C. For the induction of the MDR1 promoter, 50 �g/mlbenomyl or 0.005% H2O2 was added after 3 h and the cultures wereincubated for an additional hour. The cell suspensions were 10-fold di-luted in 1 ml cold phosphate-buffered saline (PBS) and flow cytometrywas performed using the MACSQuantAnalyzer (Miltenyi Biotec; BergischGladbach, Germany) equipped with an argon laser emitting at 488 nm.Fluorescence was detected using the B1 fluorescence channel equippedwith a 525-nm band-pass filter (bandwidth 50 nm). Twenty thousandcells were analyzed per sample and counted at a flow rate of approximately500 cells per second. Fluorescence data were collected by using logarith-mic amplifiers. The mean fluorescence (arbitrary units) was determinedwith MACSQuantify (version 2.4; Miltenyi Biotec) software.

Western immunoblotting. For the detection of green fluorescent pro-tein (GFP) by Western immunoblotting, the reporter strains were grownas described above for the flow cytometry analyses, except that the cul-tures were grown in 25-ml volumes in Erlenmeyer flasks. Cells were col-lected 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), andbroken by vortexing for 10 min at 4°C with 500 �l 0.5-mm glass beads in500 �l breaking buffer (100 mM Tris-HCl [pH 7.5], 200 mM NaCl, 20%glycerol, 5 mM EDTA, 4% complete, EDTA-free protease inhibitor cock-tail [Roche Diagnostics GmbH, Mannheim, Germany], and 0.1% �-mer-captoethanol). Samples were centrifuged at 13,000 rpm for 10 min at 4°C,the supernatant was collected, and the protein concentration was quanti-fied using the Bradford protein assay. Extracts were heated for 10 min at65°C, and equal amounts of protein of each sample were separated on anSDS 12% polyacrylamide gel. Proteins were transferred onto a nitrocellu-lose membrane with a Mini-Protean Tetra system (Bio-Rad, Munich,Germany) and stained with Ponceau S to control for equal loading. GFPwas detected using rabbit monoclonal GFP antibody ab32146 (Abcam,Cambridge, United Kingdom) and anti-rabbit HRP G-21234 (InvitrogenGmbH, Darmstadt, Germany) as first and secondary antibodies, respec-tively. A chemiluminescence detection system (GE Healthcare) was usedfor signal detection.

Fluconazole susceptibility testing. To determine the fluconazole sus-ceptibilities of the strains, a 2-fold dilution series of fluconazole (SigmaGmbH, Deisenhofen, Germany) was prepared in the assay medium, start-ing from an initial concentration of 128 �g/ml. Susceptibility tests werecarried out using a previously described microdilution method (24), ex-

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cept 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 flu-conazole were determined after 48 h of growth at 37°C.

RESULTSGeneration of ada2� mutants of the C. albicans wild-type strainSC5314. To directly compare inducible and constitutive MDR1expression levels in cells lacking Ada2 with those in previouslyconstructed wild-type strains, we deleted the ADA2 gene in the C.albicans reference strain SC5314. Two independent series ofheterozygous and homozygous ada2� mutants were generatedusing the SAT1-flipping strategy (21), and an intact ADA2 copywas reinserted into both homozygous mutants to obtain comple-mented strains. Phenotypic analysis (Fig. 1) showed that theada2� mutants displayed the previously described hypersensitiv-ity to fluconazole and H2O2 (20). The mutants were also highlysensitive to nourseothricin, a phenotype that was first noted whenthe SAT1 flipper cassette was recycled after inactivation of thesecond ADA2 allele. We did not recover cells in which the resis-tance cassette was excised on plates with a low concentration ofnourseothricin, which is a convenient screening procedure (21).In addition, we found that the ada2� mutants also displayedsomewhat reduced growth on media without any inhibitor. Allmutant phenotypes were reverted to wild type after reinsertion of

an intact ADA2 copy, confirming that they were caused by thedeletion of ADA2 (Fig. 1).

Ada2 is not required for benomyl- and H2O2-induced MDR1expression. To compare the inducibility of MDR1 expression inthe presence or absence of Ada2, the wild-type strain SC5314 andthe two homozygous ada2� mutant strains were grown to logphase in liquid YPD medium and treated with the inducers beno-myl and H2O2. Total RNA was isolated from the cultures andMDR1 mRNA detected by Northern hybridization. No MDR1transcripts were detected in untreated cells of the wild type and theada2� mutants (Fig. 2A, lanes 1, 4, and 5). MDR1 expression wasupregulated by benomyl in both wild-type and ada2� cells (Fig.2A, lanes 2, 6, and 7) and H2O2 (Fig. 2A, lanes 3, 8, and 9). Theinduction of MDR1 expression occurred with similar efficiency inthe wild-type and the ada2� mutant cells, demonstrating thatAda2 is not required for the induction of MDR1 by these com-pounds.

To corroborate the results of the Northern hybridization ex-periments with an independent method, we introduced a PMDR1-GFP reporter gene fusion into the ada2� mutants and comparedthe activity of the MDR1 promoter in wild-type and mutant cellsby flow cytometry. In agreement with the Northern hybridizationanalysis, MDR1 promoter activity remained below the detectionlimit in uninduced cells, as the fluorescence of the reporter strainswas not increased above the background fluorescence levels of

FIG 1 Sensitivity of the wild type, heterozygous and homozygous ada2� mutants, and complemented strains to fluconazole, H2O2, and nourseothricin. Serial10-fold dilutions of the strains were spotted onto agar plates containing the indicated compounds. Plates were incubated for 1 day (YPD plates, bottom row) or2 days (SD plates, top row) at 30°C. The following strains were used: SC5314 (wild type), SCADA2M2A and -B (ADA2/ada2�), SCADA2M4A and -B(ada2�/ada2�), and SCADA2MK2A and -B (ada2�/ada2� � ADA2).

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their parental strains that did not contain the GFP gene (Fig. 2B).The MDR1 promoter was strongly induced in wild-type cellstreated with benomyl and, more weakly, in the presence of H2O2.The fluorescence of the ada2� reporter strains was also increasedby both inducers, but a direct comparison with the wild-type cellswas complicated by the fact that the ada2� mutants exhibited a4-fold higher autofluorescence. We therefore compared GFP lev-els in the cells by Western immunoblotting with an anti-GFP an-tibody. The GFP amounts observed in wild-type cells treated withbenomyl and H2O2 corresponded well with the results of the flow

FIG 2 Inducibility of MDR1 expression by benomyl and H2O2 in the wild typeand ada2� mutants. (A) Analysis of MDR1 expression in the wild-type strainSC5314 and two independently constructed ada2� mutants (SCADA2M4Aand -B) grown in the absence (�) or presence (�) of benomyl or H2O2 byNorthern hybridization. The 18S RNA bands in the ethidium bromide-stainedgel served as loading control. (B) Strains carrying a PMDR1-GFP reporter fusionin wild-type and ada2� backgrounds were grown in the absence or presence ofinducers as indicated. The mean fluorescence of the cells was determined byflow cytometry. The results obtained with two independently generated re-porter strains are shown in each case (means and standard deviations fromthree experiments). The following strains were used: SCMG3A and -B (wildtype), SC�ada2MG3A and -B (ada2�). The background fluorescence of theparental strains, which do not contain the GFP gene, is indicated by the blackpart of each column. (C) GFP expression in the same strains was detected byWestern immunoblotting with an anti-GFP antibody.

FIG 3 MDR1 expression in wild-type and ada2� strains carrying hyperactiveMRR1 or CAP1 alleles. (A) Detection of MDR1 mRNA in strains containingwild-type, MRR1P683S or CAP1�C333 alleles by Northern hybridization; 18SRNA is shown as loading control. The following strains were used: SC5314(wild type, �), SCMRR1R34A and -B (wild type, MRR1P683S), SCCAP1R14Aand -B (wild type, CAP1�C333), SCADA2M4A and -B (ada2�, �),SC�ada2MRR1R34A and -B (ada2�, MRR1P683S), and SC�ada2CAP1R14Aand -B (ada2�, CAP1�C333). (B) Reporter strains containing wild-type or hy-peractive MRR1 and CAP1 alleles and expressing GFP under the control of theMDR1 promoter were grown to log phase in YPD medium. The mean fluores-cence of the cells was determined by flow cytometry. The results obtained withtwo independently generated reporter strains are shown in each case (meansand standard deviations from three experiments). The following strains wereused: SCMG3A and -B (wild type, �), SCMRR1R34MG3A and -B (wildtype, MRR1P683S), SCCAP1R14MG3A and -B (wild type, CAP1�C333),SC�ada2MG3A and -B (ada2�, �), SC�ada2MRR1R34MG3A and -B(ada2�, MRR1P683S), and SC�ada2CAP1R14MG3A and -B (ada2�,CAP1�C333). The background fluorescence of the parental strains, which donot contain the GFP gene, is indicated by the black part of each column. (C)GFP expression in the same strains was detected by Western immunoblottingwith an anti-GFP antibody.

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cytometry and Northern hybridization experiments (Fig. 2C,lanes 1 to 6). The Western blot analysis confirmed that MDR1expression was also induced by these compounds in the ada2�mutant cells (Fig. 2C, lanes 7 to 12) and with the same efficiency asin the wild-type cells. Collectively, these results show that, underthe conditions used in our experiments, Ada2 is dispensable forbenomyl- and H2O2-induced MDR1 expression.

Requirement of Ada2 for MDR1 upregulation by hyperactiveMRR1 and CAP1 alleles. As explained in the introduction, hyper-active forms of Mrr1 and Cap1 can independently upregulateMDR1 expression in the absence of inducers, and gain-of-func-tion mutations in MRR1 are the cause of MDR1 overexpression influconazole-resistant, clinical C. albicans isolates. In order to as-sess a potential role of Ada2 in the constitutive MDR1 upregula-tion in such strains, we introduced the P683S gain-of-functionmutation into both resident MRR1 alleles of the ada2� mutants,in the same way as in previously constructed derivatives of theparental wild-type strain SC5314 (7). Similarly, both wild-typeCAP1 alleles were replaced by the C-terminally truncated, hyper-active CAP1�C333 allele. Northern hybridization analysis demon-strated the constitutive upregulation of MDR1 by the hyperactiveMrr1 and, at a lower level, by the hyperactive Cap1 in the wild-type background (Fig. 3A, lanes 1 to 5). MDR1 was similarly over-expressed in ada2� mutants containing the hyperactiveMRR1P683S allele (Fig. 3A, lanes 6 to 9), demonstrating that Ada2 isdispensable for MDR1 upregulation by a constitutively activeMrr1. In contrast, the hyperactive Cap1 caused only a minor up-regulation of MDR1 expression in the absence of Ada2 (Fig. 3A,lanes 10 and 11), indicating that Cap1 requires Ada2 to induceMDR1 expression when Mrr1 is not simultaneously activated byinducing compounds.

To corroborate the results of the Northern hybridization ex-

periments, we compared MDR1 promoter activity in the samestrains. For this purpose, the PMDR1-GFP reporter gene fusion wasalso introduced into the ada2� mutants containing the hyperac-tive MRR1P683S and CAP1�C333 alleles, and the fluorescence of thecells was quantified by flow cytometry. Figure 3B shows that theMDR1 promoter was strongly activated by the hyperactive Mrr1in both the wild type and the ada2� mutants. The hyperactiveCap1 caused MDR1 upregulation in the wild type, whereas thefluorescence of the ada2� mutants was only slightly increasedabove background levels. Western blot analysis demonstrated thatcomparable amounts of GFP were produced in wild-type andada2� cells containing the MRR1P683S allele (Fig. 3C, comparelanes 3 and 4 with lanes 9 and 10). The presence of the hyperactiveCap1 also resulted in strongly increased GFP levels in the wild-type background (Fig. 3C, lanes 5 and 6), while only a faint signalwas detected in the ada2� mutants (Fig. 3C, lanes 11 and 12).Collectively, these results argue that Cap1, but not Mrr1, requiresAda2 to promote MDR1 expression.

Fluconazole susceptibility of strains expressing hyperactiveMRR1 and CAP1 alleles in the presence and absence of Ada2.The hyperactive MRR1P683S and CAP1�C333 alleles confer in-creased fluconazole resistance that only partially depends on theMdr1 efflux pump (7). We therefore compared the fluconazolesusceptibilities of strains expressing these alleles in wild-type andada2� backgrounds by a dilution spot assay on solid medium anda broth microdilution assay. As can be seen in Fig. 4, the hyperac-tive Mrr1 also caused enhanced fluconazole resistance in theada2� mutants, although the increase in the MIC of fluconazolewas slightly lower (16-fold, from 0.125 �g/ml to 2 �g/ml) than inthe wild type (32-fold, from 0.5 �g/ml to 16 �g/ml), presumablydue to the generally decreased fitness of the ada2� mutants. Thehyperactive Cap1 conferred enhanced fluconazole resistance in

FIG 4 Fluconazole sensitivity of strains expressing hyperactive MRR1 and CAP1 alleles in wild-type or ada2� backgrounds. Serial 10-fold dilutions of the strains werespotted onto SD agar plates with or without 5�g/ml fluconazole and incubated for 2 days at 30°C. The following strains were used: SC5314 (wild type,�), SCMRR1R34Aand -B (wild type, MRR1P683S), SCCAP1R14A and -B (wild type, CAP1�C333), SCADA2M4A and -B (ada2�, �), SC�ada2MRR1R34A and -B (ada2�, MRR1P683S), andSC�ada2CAP1R14A and -B (ada2�, CAP1�C333). The MIC of fluconazole for each strain, as determined in broth microdilution assays, is given to the right.

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the wild type, whereas no increase in fluconazole resistance wasobserved in the absence of Ada2 on agar plates containing thedrug. However, the MIC of fluconazole was also still slightly in-creased for the ada2� mutants containing the hyperactive CAP1allele, indicating that the hyperactive Cap1 has residual activity inthe absence of Ada2. Overall, the differential requirement of Ada2for Mrr1- and Cap1-mediated fluconazole resistance reflects thecapacity of these transcription factors to activate the MDR1 pro-moter in the absence of Ada2.

DISCUSSION

The results of our study show that a requirement of Ada2 for thetranscriptional activation of MDR1 depends on how MDR1 ex-pression is induced. Ada2 was completely dispensable for the up-regulation of MDR1 by a hyperactive form of the transcriptionfactor Mrr1 containing the P683S gain-of-function mutation (Fig.3). Gain-of-function mutations in Mrr1 are responsible for theconstitutive MDR1 overexpression in fluconazole-resistant clini-cal C. albicans isolates (12). It is therefore likely that Ada2 does notplay a role in Mrr1-mediated MDR1 overexpression and flucona-zole resistance in such strains. In line with this expectation, Sellamet al. reported that deletion of ADA2 in an MDR1-overexpressingclinical isolate did not reduce its fluconazole resistance (20). Incontrast, a C-terminally truncated, hyperactive Cap1, which alsocauses MDR1 overexpression, was largely unable to upregulateMDR1 transcription in the absence of Ada2 (Fig. 3). These find-ings suggest that Cap1, and not Mrr1, recruits Ada2 to induceMDR1 expression. In the presence of the inducers benomyl andH2O2, Cap1 cooperates with Mrr1 to upregulate MDR1 expres-sion (7), explaining why no recruitment of Ada2 was necessaryunder these conditions (Fig. 2).

Sellam et al. used reverse transcription-quantitative PCR (RT-qPCR) to compare MDR1 mRNA levels in wild-type and ada2�cells treated with fluconazole (20). They found that the inductionof MDR1 expression by fluconazole was lower in the ada2� mu-tant (6-fold) than in the wild type (18-fold). In our lab, we did notdetect induction of MDR1 expression by fluconazole in Northernhybridization experiments (25) or when using GFP as a reportergene (unpublished results), in agreement with findings by otherresearchers (3, 4, 26). The comparatively mild MDR1 induction byfluconazole (MDR1 is upregulated several hundredfold in strainswith hyperactive MRR1 alleles [7, 9]) is insufficient to confer in-creased drug resistance, as MDR1 deletion in fluconazole-suscep-tible strains does not result in hypersusceptibility to the drug (7,27, 28). Therefore, the increased susceptibility of ada2� mutantsto fluconazole cannot be due to decreased MDR1 expression. Thefact that ada2� mutants are hypersusceptible to various unrelatedtoxic compounds and also grew somewhat more slowly than thewild type in the absence of inhibitors (Fig. 1) indicates that cellslacking Ada2 have a general fitness defect, which may be exacer-bated under stress conditions.

Altogether, our findings argue that binding of Ada2 to theMDR1 promoter is most likely mediated by Cap1, but not byMrr1. Consistent with this assumption, Sellam et al. found thatthe binding of Ada2 to the MDR1 promoter was abolished in acap1� mutant (20). Binding of Ada2 to the MDR1 promoter wasalso reduced, but not eliminated, in an mrr1� mutant. The latterobservation can be explained by the fact that Cap1 requires Mrr1to induce MDR1 expression in response to inducing signals; theability of Cap1 to recruit Ada2 to the MDR1 promoter may be

compromised in the absence of Mrr1. Although Sellam et al. notedthat Ada2 recruitment to a subset of its target genes is mediated byCap1, Ada2 was proposed to be a coactivator of Mrr1 in the ex-pression of drug resistance genes like MDR1 because of the strongoverlap between promoters bound by Mrr1 and Ada2 (20). How-ever, it is possible that the binding of Ada2 to other Mrr1 targetgenes also does not occur via Mrr1. Like MDR1, three of the otherfive tested genes that were bound by both Mrr1 and Ada2 areknown Cap1 target genes (HSP31, GRP2, and OYE32), and theothers are induced by benomyl, which also causes oxidative stressand activates Cap1 (7, 8, 29). Therefore, Ada2 may also be re-cruited by Cap1, and not by Mrr1, to these common target genes.

In a previous study (30), we found that the MADS box tran-scription factor Mcm1 is also differentially required for the induc-tion of MDR1 expression by Cap1 and Mrr1. The requirement ofMcm1 was inverse to that observed in the present study for Ada2.A hyperactive Mrr1 did not upregulate MDR1 when Mcm1 wasdepleted from the cells, whereas Mcm1 was dispensable for theinduction of MDR1 expression by a hyperactive Cap1. Figure 5summarizes our current understanding of the regulation ofMDR1 expression by Cap1, Mrr1, Mcm1, and Ada2 under variousinducing conditions. In the presence of H2O2 (Fig. 5A), Cap1 is

Mrr1* Mcm1

MDR1

D Induction by hyperactive Mrr1

A Induction by H2O2

Cap1 Mrr1

MDR1

B Induction by benomyl

Cap1 Mrr1

MDR1

Mcm1

C Induction by hyperactive Cap1

Cap1* Mrr1

MDR1

Ada2

FIG 5 Model of the roles of Cap1, Mrr1, Mcm1, and Ada2 in MDR1 upregu-lation in the presence of the inducing compounds H2O2 (A) and benomyl (B)or by hyperactive forms (*) of Cap1 (C) and Mrr1 (D). The transcriptionfactors Cap1, Mrr1, and Mcm1 directly bind to the MDR1 promoter, whileAda2 is recruited by Cap1. The bent arrow indicates transcription of MDR1.Missing proteins are not required for MDR1 expression under the particularcondition. Gray color indicates that the respective regulatory protein contrib-utes to full MDR1 expression levels, but a partial activation of the MDR1promoter also occurs in its absence. Detailed explanations are given in the text.

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activated and cooperates with Mrr1 to induce MDR1 expres-sion. Mrr1 is also required, although some residual MDR1 ex-pression may occur without Mrr1, while Mcm1 is dispensableunder these conditions. Cap1 probably recruits Ada2 to theMDR1 promoter, but this was not necessary for MDR1 induc-tion by H2O2 under our experimental conditions. When beno-myl serves as the inducer (Fig. 5B), Mrr1 becomes activated bya still unknown mechanism and, together with Mcm1, causesMDR1 upregulation. Benomyl also causes oxidative stress andactivates Cap1, which maximizes MDR1 expression in anAda2-independent fashion. While Mrr1 is essential, a partialactivation of the MDR1 promoter can occur without eitherMcm1 or Cap1, but all three transcription factors are requiredfor fully induced MDR1 expression levels. A constitutively ac-tive Cap1 (Fig. 5C) can upregulate MDR1 expression even inthe absence of Mrr1, although less efficiently than in its pres-ence. MDR1 induction by the hyperactive Cap1 strongly de-pends on Ada2, whereas Mcm1 is dispensable. Finally, instrains that have acquired a gain-of-function mutation in Mrr1(Fig. 5D), Cap1 (which remains mostly in the cytoplasm in theabsence of oxidative stress) and Ada2 are not required forMDR1 overexpression, but the hyperactive Mrr1 depends onMcm1 to cause MDR1 upregulation. Central remaining ques-tions are how Mrr1 achieves its activated state, either in thepresence of inducers like benomyl or by gain-of-function mu-tations, and by which mechanism(s) it recruits the core tran-scriptional machinery to allow MDR1 expression in a Cap1-and Ada2-independent manner.

ACKNOWLEDGMENTS

This study was supported by the Deutsche Forschungsgemeinschaft (SFB630) and the National Institutes of Health (grant AI058145). SeleneMogavero was supported by the University of Pisa.

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