Relative Contributions of the Candida albicans ABC Transporters Cdr1p and Cdr2p to Clinical Azole Resistance

Candida albicans frequently develops resistance to treatmentwith azole drugs due to the acquisition of gain-of-functionmutations in the transcription factor Tac1p. Tac1p hyperactivationin azole-resistant isolates results in the constitutive overexpressionof several genes, including CDR1 and CDR2, which encode twohomologous transporters of the ATP-binding cassette family.Functional studies of Cdr1p and Cdr2p have been carried outso far by heterologous expression in the budding yeast Saccharomycescerevisiae and by gene deletion or overexpression in azole-sensitiveC. albicans strains in which CDR1 expression is low and CDR2expression is undetectable. Thus, the direct demonstration thatCDR1 and CDR2 overexpression causes azole resistance in clinicalstrains is still lacking, as is our knowledge of the relativecontribution of each transporter to clinical azole resistance.In the present study, we used the SAT1 flipper system to deletethe CDR1 and CDR2 genes from clinical isolate 5674. This strainis resistant to several azole derivatives due to a strong hyperactivemutation in Tac1p and expresses high levels of Cdr1p and Cdr2p.We found that deleting CDR1 had a major effect, reducing resistanceto fluconazole (FLC), ketoconazole (KTC), and itraconazole (ITC)by 6-, 4-, and 8-fold, respectively. Deleting CDR2 had a muchweaker effect, reducing FLC or KTC resistance by 1.5-fold, andhad no effect on ITC resistance. These results demonstrate thatCdr1p is a major determinant of azole resistance in strain 5674and potentially in other clinical strains overexpressing Cdr1pand Cdr2p, while Cdr2p plays a more minor role.

INTRODUCTION

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INTRODUCTION

Candida albicans is one of the leading causes of fungal infectionsaffecting immunocompromised individuals. Candida infectionsrange from chronic superficial infections of the skin and mucosalsurfaces to invasive, life-threatening systemic infections (21,38). Many antifungal drugs used to treat Candida infectionstarget the biosynthesis of ergosterol, the major sterol in thefungal cell membranes (24). Polyenes, such as amphotericin B(AMB), directly bind to ergosterol and form pores in the cellmembrane, resulting in low selectivity and high toxicity (24).Azoles, a class of well-tolerated antifungal drugs that includesfluconazole (FLC), ketoconazole (KTC), itraconazole (ITC), andnew-generation derivatives such as voriconazole and posaconazole,target the enzyme lanosterol 14 ${alpha}$ -demethylase (Erg11p), whichis involved in ergosterol biosynthesis, blocking the productionof ergosterol and causing the accumulation of toxic intermediatesterol species (24). As a consequence, the fluidity and permeabilityof the fungal cell membrane are changed and the activity ofmembrane-bound proteins, such as enzymes involved in cell wallsynthesis, is altered (24).

However, the fungistatic rather than fungicidal action of azoledrugs leads to the frequent emergence of azole-resistant (A^r)C. albicans strains (1, 44). One mechanism of azole resistanceconsists of increased levels of ERG11 RNA, resulting in increasedproduction of the Erg11p enzyme, or point mutations in the ERG11gene, producing an enzyme with a reduced binding affinity forazole drugs (1, 44). Also, several A^r clinical isolates overexpressthe CDR1 and CDR2 genes, which encode two homologous transportersof the ATP-binding cassette (ABC) family, and/or the MDR1 gene,which encodes a major facilitator (1, 44). A number of A^r strainsoverexpress CDR1 and CDR2 but not MDR1, whereas other strainsoverexpress only MDR1 (34), suggesting the involvement of twodistinct transcriptional pathways. Also, some A^r strains overexpressthe three genes, probably due to the accumulation of independentmutations in the two pathways, leading to high levels of resistancein response to stepwise drug selection (44). The overexpressionof transporter genes in A^r isolates suggested that a reducedaccumulation of azoles in the cell was responsible for the observedazole resistance phenotype (1, 44). By using a dominant selectablemarker, it was shown that deleting MDR1 from A^r clinical isolatesoverexpressing this gene reduced the resistance of the cellsto FLC, providing a direct demonstration that MDR1 is involvedin clinical FLC resistance (45). However, the direct contributionof CDR1 and CDR2 to clinical azole resistance remained to bedetermined.

Recent progress has been made in deciphering the regulatorycircuitry that governs the regulation of CDR1, CDR2, MDR1, andERG11 in C. albicans clinical strains. It was shown that theupregulation of the CDR1 and CDR2 genes in A^r isolates is dueto gain-of-function mutations in the zinc cluster transcriptionfactor Tac1p (5, 6). Most of these mutations consist of C-terminalamino acid substitutions or small in-frame deletions (4, 5).Tac1p was also shown to activate the transcription of CDR1 andCDR2 upon cell treatment with different compounds such as fluphenazine(FPZ) and steroids (estrogen, progesterone) (6), but the mechanismsby which these compounds trigger Tac1p activity are still unknown.Similarly, gain-of-function mutations in two other zinc clustertranscription factors, Mrr1p and Upc2p, have recently been shownto be responsible for the constitutive upregulation of Mdr1pand Erg11p, respectively, in clinical A^r isolates (10, 22).These data confirmed the involvement of different transcriptionalpathways in the upregulation of the CDR1/CDR2, MDR1, and ERG11genes in A^r isolates.

The Cdr1p and Cdr2p transporters show 84% amino acid sequenceidentity and are close homologs of Saccharomyces cerevisiaePdr5p, a major effector of cell tolerance to xenobiotic compoundsin budding yeast (2, 25, 32). These transporters are formedby two similar halves, each with an N-terminal hydrophilic domainthat contains an ATP-binding motif followed by a C-terminalhydrophobic domain with six predicted transmembrane segmentsthat presumably contain the drug binding sites, a structurecharacteristic of the pleiotropic drug resistance subfamilyof ABC transporters found in fungi and plants (41, 43). Becauseof the advanced genetics of S. cerevisiae, most studies of Cdr1pand Cdr2p have been carried out with heterologous expressionsystems in S. cerevisiae, where Cdr1p and Cdr2p were expressedunder the control of a strong promoter, leading to very highlevels of azole resistance ( $~$ 100-fold) (12, 31). It was shownthat the two transporters localize at the plasma membrane (35,37), bind rhodamine 6G (R6G) (12), export their substrates inan energy-dependent manner, and possess ATPase and phospholipidtranslocase activities (15, 39). Expression systems in S. cerevisiaehave also proved useful for structure-function studies of thetransmembrane and ATP-binding domains of Cdr1p (14, 26, 31).

However, there is evidence supporting the importance of studyingthese transporters in a potentially more relevant host, namely,C. albicans A^r cells. First, the function of ABC transportersis influenced by their lipid environment (29, 40) and, sinceS. cerevisiae and C. albicans have different plasma membranelipid compositions (17, 42), the function of Cdr1p and Cdr2pcould be altered in S. cerevisiae. Also, the S. cerevisiae strainsused for these studies carry deletions in (up to seven) geneswhich encode endogenous ABC transporters (12, 15). Since theseABC transporters can function as sterol transporters or phospholipidflippases (8, 27), the deletion of these genes may affect theplasma membrane composition of the recipient yeast and thusCdr1p and Cdr2p function. On the other hand, Cdr1p and Cdr2pfunction has been studied by gene deletion, by using the URA-blastersystem, in C. albicans azole-sensitive (A^s) strains, in whichCDR1 expression is low and CDR2 expression is undetectable (32).Deletion of CDR1 resulted in increased azole susceptibility,whereas deletion of CDR2 did not render the cells more susceptibleto azoles, due to the lack of CDR2 expression in these cells(32). Interestingly, the cdr1 ${Delta}$ /cdr1 ${Delta}$ cdr2 ${Delta}$ /cdr2 ${Delta}$ double mutant wasfound to be more susceptible to azole drugs and other toxiccompounds than the cdr1 ${Delta}$ /cdr1 ${Delta}$ single mutant (32). Another studyshowed that the inducible expression of CDR1 in an A^s straincarrying a homozygous deletion of the gene resulted in resistanceto several antifungal agents, including azole drugs (23). Also,genome-wide expression and location analyses of the Tac1p regulonhave shown that, in A^r strains, CDR1 and CDR2 are coregulatedwith many other genes, some of them predicted to be involvedin membrane protein trafficking and lipid metabolism and potentiallymodulating Cdr1p and Cdr2p function (16). Taken together, theseobservations emphasized the need for directly studying Cdr1pand Cdr2p in A^r C. albicans clinical isolates. In the presentstudy, we addressed this question by deleting the two genes,individually and in combination, from a well-characterized A^rclinical isolate in which the Tac1p pathway is activated (30,46).

MATERIALS AND METHODS

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MATERIALS AND METHODS

Strains and growth media. The C. albicans strains used in this study are listed in Table1; for details of their construction, see the supplemental material.Strains were routinely grown at 30°C in YPD medium containing1% yeast extract (EMD Biosciences, Darmstadt, Germany), 2% Bactopeptone (BD Biosciences, Sparks, MD), and 2% glucose (Sigma,St. Louis, MO). For solid medium, 2% agar (Difco, BD) was added.

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TABLE 1. C. albicans strains used in this study

Reagents and antifungal compounds. Molecular biology reagents and restriction enzymes were purchasedfrom Invitrogen (Carlsbad, CA) or from New England BioLabs (Ipswich,MA). Hot-Start KOD⁺ DNA polymerase (Novagen, La Jolla, CA) wasused for the amplification and cloning of PCR products. DNAprimers were purchased from Integrated DNA Technologies (SanDiego, CA). PCR and plasmid DNA fragments were purified withDNA purification kits from Qiagen (Mississauga, Ontario, Canada).Acid-washed glass beads (425 to 600 nm) used for genomic DNA,and total protein extracts were purchased from Sigma. All chemicaland antifungal compounds were purchased from Sigma unless otherwisestated.

RNA preparation and Northern blotting. Cell growth, total RNA extraction by the hot phenol method,and Northern blot hybridizations were carried out as describedpreviously (30). The CDR1 and CDR2 probes used in this experimentwere also described previously (30). The resulting blots wereexposed to a Fujifilm Imagine Plate screen and analyzed withthe MultiGauge software (version 2.3; Fujifilm).

Membrane protein preparation and Western blotting. Total membrane extracts from C. albicans cells were preparedas follows. Overnight cultures were diluted into 100 ml of freshYPD medium to an optical density at 600 nm (OD₆₀₀) of 0.1. Logarithmicallygrowing cells (OD₆₀₀ of 1.0) were harvested, washed once withice-cold distilled H₂O, and resuspended in 5 ml of extractionbuffer (10 mM Tris-HCl [pH 7.5]; 400 mM NaCl; 10% glycerol;1 mM sodium orthovanadate; 50 mM sodium fluoride; 50 mM sodiumβ-glycerophosphate; 10 mM β-mercaptoethanol; 1 µMMG132 and protease inhibitors; 1 mM phenylmethylsulfonyl fluoride;leupeptin, pepstatin, and aprotinin at 5 µg/ml each).Cell suspensions were then frozen in liquid nitrogen and storedat –80°C. Frozen cell pellets were disrupted witha Freezer Mill (SPEX CetriPrep, Metuchen, NJ) with four cyclesof successive grinding and cooling periods, each cycle consistsof 2 min of grinding at an impact frequency of 14 times/s followedby a 2-min cooling interval. Total protein extracts were clearedby centrifugation at 1,000 x g for 5 min at 4°C. Total membraneextracts were harvested by centrifuging the total protein extractsat 260,000 x g for 45 min at 4°C. Protein concentrationswere determined with the micro-BCA protein assay kit from Pierce(Rockford, IL), and total protein extracts (20 µg) wereseparated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(7.5% acrylamide, 37.5:1 acrylamide/bisacrylamide ratio). Thegels were either stained with Coomassie or transferred to anitrocellulose membrane with a Trans Blot SD Semi-Dry transferapparatus (Bio-Rad). The membrane was stained with Ponceau reagent(0.1% Ponceau S in 5% acetic acid) prior to immunodetection.Immunodetection of Cdr1p and Cdr2p was performed with anti-Cdrp(1:1,000 dilution), anti-Cdr2p (1:4,000 dilution) (12), or anti-Cdr1p(1: 4,000 dilution) (9) polyclonal antibodies and an ECL chemiluminescencekit (SuperSignal chemiluminescent substrate; Pierce).

Drug susceptibility testing. Liquid microtiter plate assays were performed as described previously(46). The drug concentrations tested were 0.2, 0.4, 1.5, 3.1,6.2, 12.5, 25, 50, 100, 150, and 200 µg/ml for FLC; 0.001,0.002, 0.005, 0.009, 0.019, 0.038, 0.075, 0.15, 0.2, 0.3, 0.4,0.6, and 1.2 µg/ml for KTC; 0.0004, 0.0008, 0.0016, 0.0031,0.0063, 0.0125, 0.025, 0.05, 0.1, 0.2, and 0.4 µg/ml forITC; 0.8, 1.6, 3.1, 6.3, 12.5, 19, 25, 38, 50, 75, and 100 µg/mlfor FPZ; 0.032, 0.063, 0.125, 0.25, 1, 2, 4, 8, and 16 µg/mlfor R6G; and 0.032, 0.063, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16,and 32 µg/ml for AMB. Stock solutions of FLC, FPZ, andR6G were prepared in water at concentrations of 5, 10, and 128mg/ml, respectively; stock solutions of KTC, ITC, and AMB wereprepared in dimethyl sulfoxide at concentrations of 3, 1, and10 mg/ml, respectively. Cell growth was measured spectrophotometricallyby determining the OD₆₂₀ after 48 h of incubation at 30°Cin YPD. The MICs for 50% of the strains tested (MIC₅₀s) weredetermined as the first concentration of drug capable of reducinggrowth by 50% compared to that of control cells grown in theabsence of the drug. The data are presented as the mean of threeindependent experiments performed in duplicate. Spot assayswere performed as described previously (30). Cells grown overnightwere resuspended in YPD to an OD₆₀₀ of 0.1. Tenfold serial dilutionsof each strain were spotted onto YPD plates containing the drugstested.

RESULTS

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RESULTS

Deletion of the CDR1 and CDR2 genes in C. albicans A^r clinical isolate 5674. To determine the contribution of CDR1 and CDR2 to clinical azoleresistance, we used the SAT1 flipper strategy, which allowsthe dominant selection of transformants with the SAT1 gene,which confers nourseothricin resistance (28), to delete theCDR1 and CDR2 genes, independently or in combination, from theA^r clinical isolate 5674. This strain and A^s strain 5457 wereisolated from the same patient and shown by DNA fingerprintinganalysis to be highly related (30). Compared to strain 5457,strain 5674 is resistant to several azole derivatives and overexpressesthe CDR1 and CDR2 RNAs at high levels due to a strong gain-of-functionmutation in transcription factor Tac1p (N972D) (30, 46). Tac1pappears to be the key determinant of azole resistance in strain5674, since deletion of the TAC1 gene from that strain causesa complete loss of azole resistance, the resulting tac1 ${Delta}$ /tac1 ${Delta}$ mutant being as susceptible to azole drugs as strain 5457 (46).This proposition is also supported by the demonstration thatstrain 5674 does not overexpress the MDR1 or ERG11 gene (30),suggesting that no other transcriptional pathways are activatedin this strain besides the Tac1p pathway, and does not carrymutations in ERG11 (K. S. Barker and P. D. Rogers, personalcommunication). Finally, strain 5674, as well as other A^r strainscarrying an activated Tac1p protein, overexpresses many genesthat encode proteins with predicted functions in lipid metabolism,phospholipid translocation, and protein trafficking which couldpossibly modulate Cdr1p and Cdr2p activity (16). Therefore,the use of strain 5674 to delete CDR1 and CDR2 allows the analysisof Cdr1p and Cdr2p function in a clinically relevant, well-characterizedhost.

We constructed a CDR2 deletion cassette consisting of the SAT1-FLPmarker flanked by approximately 1 kb of CDR2 upstream and downstreamsequences amplified from strain SC5314 (see Fig. S1A in thesupplemental material). The correct integration of the deletioncassette at the CDR2 locus was verified by Southern blotting(see Fig. S1C in the supplemental material). Two independentheterozygous mutants with the expected integration profile werechosen to delete the second allele, yielding two independenthomozygous cdr2 ${Delta}$ /cdr2 ${Delta}$ mutants, STY7 and STY8 (see Fig. S1C inthe supplemental material). Deletion of the CDR1 gene from strain5674 was performed essentially as described for CDR2, with theexception that two different CDR1 deletion constructs were usedto delete the two alleles, generating the two independent homozygouscdr1 ${Delta}$ /cdr1 ${Delta}$ mutants STY19 and STY20 (see Fig. S2 in the supplementalmaterial). CDR1 was also deleted from strain STY7, producingtwo mutant strains lacking both transporter genes, STY31 andSTY32 (cdr1 ${Delta}$ /cdr1 ${Delta}$ cdr2 ${Delta}$ /cdr2 ${Delta}$ ) (see Fig. S2 in the supplementalmaterial). Finally, we constructed strain STY47, in which oneallele of CDR2 was reintegrated at its original locus in strainSTY31 (see Fig. S3 in the supplemental material).

Cdr1p and Cdr2p expression in strains 5457 and 5674 and strain 5674 mutant derivatives. We analyzed the expression of the CDR1 and CDR2 genes in strainsSC5314, 5457, and 5674 and different 5674 mutant derivativesby Northern and Western blotting.

For the Northern blot analysis, total RNA was prepared and themRNA levels of CDR1 and CDR2 were detected with gene-specificprobes derived from the first 340 and 294 bp of the CDR1 orCDR2 open reading frame, respectively, a region where the levelof sequence homology between the two genes is low (30). Lowlevels of CDR1 transcript were detected in A^s strains SC5314and 5457, and high levels were detected in A^r strain 5674 (Fig.1A, top, lanes 1 to 3), whereas CDR2 transcripts were detectedonly in the A^r strain (Fig. 1A, middle, lanes 1 to 3). As previouslyreported, deleting the TAC1 gene from strain 5674 (SZY31) decreasedCDR1 and CDR2 expression to levels similar to those detectedin the A^s strains (Fig. 1A, lane 4) (46). In strain STY7 (cdr2 ${Delta}$ /cdr2 ${Delta}$ ),we detected a signal for CDR1 but not for CDR2, confirming thedeletion of CDR2 in that strain (Fig. 1A, lane 5). Similarly,the absence of a signal for CDR1 in strain STY19 (cdr1 ${Delta}$ /cdr1 ${Delta}$ )was consistent with the deletion of CDR1 in that strain (Fig.1A, lane 6). Furthermore, we did not detect any signal for CDR1or CDR2 in strain STY31 (cdr1 ${Delta}$ /cdr1 ${Delta}$ cdr2 ${Delta}$ /cdr ${Delta}$ ), in line with thedeletion of the two genes from that strain (Fig. 1A, lane 7).The CDR1 and CDR2 RNA levels detected in strains STY7 and STY19,respectively, were comparable to those detected for each genein strain 5674, indicating that the expression of one gene remainedunaffected by the deletion of the other one, ruling out thepresence of a potential compensatory overexpression effect.Finally, detection of a full-length CDR2 transcript confirmedthe expression of CDR2 in CDR2-complemented strain STY47 (Fig.1A, lane 8).

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FIG. 1. Expression of CDR1 and CDR2 in strains SC5314, 5457, and 5674 and in 5674 mutant derivatives. (A) Northern blot analysis. Total RNA extracts were prepared from the strains indicated at the top and analyzed by Northern blotting with gene-specific probes for CDR1 (top) or CDR2 (middle). rRNAs are shown as loading controls (bottom). (B) Western blot analysis. For the immunodetection of Cdr1p and Cdr2p, total membrane protein extracts were prepared from the strains and analyzed by Western blotting with the anti-Cdr1p, anti-Cdr2p, and anti-Cdrp antibodies. A Coomassie-stained gel of the protein extracts is shown at the bottom, with the positions of the molecular mass standards and the predicted positions of Cdr1p and Cdr2p indicated on the left and right, respectively. The values on the left are molecular sizes in kilodaltons.

Immunodetection of Cdr1p and Cdr2p was performed with threedifferent polyclonal antibodies, two antibodies specific forCdr1p or Cdr2p that were raised against the NH₂-terminal domainof the Cdr1p (anti-Cdr1p) or Cdr2p (anti-Cdr2p) protein, respectively,and one antibody that was raised against a short peptide sequenceperfectly conserved between the two proteins and that recognizesboth transporters (anti-Cdrp) (9, 12). By using the anti-Cdr1pantibody, we found that the pattern of Cdr1p expression wasconsistent with the one observed by Northern blotting, namely,low levels in strains SC5314, 5457, and SZY31 and high levelsin strain 5674 (Fig. 1B, lanes 1 to 4). The Cdr1p levels instrain STY7 were similar to those in strain 5674, confirmingthat Cdr1p expression was unaffected by the deletion of CDR2(Fig. 1B, lanes 3 and 5). Finally, Cdr1p was undetectable instrains STY19, STY31, and STY47, consistent with the deletionof the CDR1 gene from these strains (Fig. 1B, lanes 6 to 8).Western blotting with the anti-Cdr2p antibody also showed thatCdr2p expression in these strains was consistent with the Northernblot results. Cdr2p levels were similar in strains 5674 andSTY19, indicating that Cdr2p expression remained unchanged bythe deletion of CDR1 (Fig. 1B, lanes 3 and 6). Cdr2p expressionwas lower in strain STY47 than in strain 5674 or STY19, consistentwith the presence of only one allele of CDR2 in STY47 versustwo alleles in 5674 and STY19 (Fig. 1B, lane 8). Finally, Westernblotting with the generic anti-Cdrp antibody confirmed the resultsobtained with the anti-Cdr1p and anti-Cdr2p specific antibodies.The absence of a signal in strain STY31 demonstrated that theanti-Cdrp antibody, which was previously characterized withS. cerevisiae strains expressing Cdr1p or Cdr2p (12), does notrecognize any other proteins in C. albicans besides Cdr1p andCdr2p and is therefore specific for these two transporters.The results obtained with the generic antibody also showed thatCdr2p appears to be expressed at slightly (approximately twofold)lower levels than Cdr1p, an observation corroborated by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis and Coomassiestaining (Fig. 1B, compare lanes 5 and 6). In addition, theWestern blot assay with the generic antibody and the Coomassiegel showed that Cdr2p migrates slightly faster than Cdr1p (Fig.1B, compare lanes 5 and 6). Since the two transporters havevery similar predicted lengths (1,501 residues for Cdr1p and1,499 for Cdr2p), this difference in migration may potentiallyreflect distinct posttranslational modifications.

Susceptibilities of the cdr mutants to azole drugs and other antifungal agents. We used MIC assays to determine the functional consequencesof deleting CDR1 and/or CDR2 on the azole resistance phenotypeof strain 5674 (Fig. 2). All of the experiments were performedwith two independently generated mutant strains per knockoutconstruction (Table 1) and yielded similar results; thus, onlyone set of strains is shown in Fig. 2 for clarity. As expected,strain 5674 was more resistant to the three azoles tested thanwas strain 5457, whereas deleting TAC1 from strain 5674 (SZY31)decreased the resistance of the cells to levels similar to thoseobserved in strain 5457 (Fig. 2A, B, and C). Deleting CDR1 fromstrain 5674 had a major effect, reducing resistance to FLC,KTC, and ITC by 6-, 4-, and 8-fold, respectively, while deletingCDR2 had a smaller effect (1.5-, 1.5-, and 1.0-fold) (Fig. 2A, B, and C;Table 2). These results demonstrate that Cdr1p is the majordeterminant of azole resistance in strain 5674 while Cdr2p playsa more minor role, even when normalized for the slightly lowerabundance of Cdr2p than of Cdr1p (Fig. 1B). Interestingly, thedeletion of both genes had a drastic effect and caused a reductionof the resistance to FLC, KTC, and ITC by 375-, 300-, and 500-fold,respectively, highlighting a strong synergism between the twotransporters (Table 2). Reintroducing one allele of CDR2 underthe control of its own promoter at its original locus in strainSTY31 (yielding strain STY47) partially restored FLC resistance,causing a 16-fold increase in FLC MIC₅₀s (6.3 µg/ml forstrain STY47 versus 0.4 µg/ml for strain STY31; Fig. 3).This experiment confirmed that the strong azole hypersusceptibilityof strain STY31 was due to the simultaneous inactivation ofthe Cdr1p and Cdr2p functions (Fig. 3). The level of FLC resistanceof strain STY47 was lower than that of strain STY19, consistentwith the lower levels of Cdr2p expression in that strain versusSTY19 (Fig. 1).

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FIG. 2. Profiles of resistance of strains 5457 and 5674 and strain 5674-derived tac1 and cdr mutants to azole drugs and different compounds with antifungal activity, as determined by microtiter plate liquid assays. (A) FLC resistance. Cells were incubated for 48 h at 30°C in liquid YPD medium with the indicated concentrations of FLC. The data are presented as the relative growth of cells in FLC-containing medium compared to the growth of the same strain in FLC-free medium, which was set at 100%. The data are the mean of three independent experiments performed in duplicate. (B) KTC resistance. (C) ITC resistance. (D) FPZ resistance. (E) R6G resistance. (F) AMB resistance. For panels B to F, the experiments were performed as described for panel A.

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TABLE 2. Drug susceptibilities of the strains used in this study

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FIG. 3. Phenotypic analysis of the CDR2 revertant. The FLC susceptibilities of strains 5457, 5674, STY19, STY31, and STY47 were analyzed by spot (A) and MIC (B) assays as described in the legends to Fig. 2 and 4.

We also tested FPZ, a calmodulin inhibitor with antifungal propertiespreviously shown to be a substrate of Cdr1p and Cdr2p and astrong inducer of Tac1p activity (6, 32, 46). We found thatstrain 5674 was not more resistant to FPZ than was strain 5457(Fig. 2D and Table 2), possibly because this compound can directlyinduce Tac1p function. In line with this, we found that strainSZY31 (tac1 ${Delta}$ /tac1 ${Delta}$ ) was twofold more susceptible to FPZ than wasstrain 5674. Deleting CDR1 or CDR2 from strain 5674 had onlya marginal or no effect on the resistance of the cells to FPZ(1.5- and 1-fold, respectively). When comparing strains STY7(cdr2 ${Delta}$ /cdr2 ${Delta}$ ; Cdr1p-expressing strain) and STY19 (cdr1 ${Delta}$ /cdr1 ${Delta}$ ; Cdr2p-expressingstrain) to strain STY31 (cdr1 ${Delta}$ /cdr1 ${Delta}$ cdr2 ${Delta}$ /cdr2 ${Delta}$ ; no Cdr1p and Cdr2p),we found that the presence of CDR1 or CDR2 contributed to three-and twofold increased FPZ resistance, respectively. Finally,the decrease in FPZ susceptibility was about 1.5-fold betweenstrains 5674 and STY19 (due to the deletion of CDR1) and 2-foldbetween strains STY19 and STY31 (due to the deletion of CDR2).Based upon these small differences, it can be concluded thatCdr1p and Cdr2p display similarly low activities toward FPZ.These results also suggest that FPZ is a poorer substrate forCdr1p and Cdr2p than are azole drugs and/or that it possessesa narrower window of antifungal activity.

We similarly investigated R6G, a fluorescent substrate of manyABC transporters that has been used to study the functions ofCdr1p and Cdr2p in S. cerevisiae (12, 15, 31) and C. albicans(19, 20). Our results showed that strain 5674 was only twofoldmore resistant to R6G than was strain 5457 (Fig. 2E and Table2). Deleting CDR1 from strain 5674 reduced the resistance ofthe cells to R6G by twofold, to levels similar to those observedin strain 5457, while deletion of CDR2 clearly had no effect.These results suggest that R6G is not a good substrate to studyCdr1p and Cdr2p in A^r C. albicans cells. Interestingly, we foundthat the tac1 ${Delta}$ /tac1 ${Delta}$ mutant is eightfold more susceptible thanstrain 5674, indicating that Tac1p possesses another, as-yet-unidentified,target affecting cell susceptibility to R6G.

Finally, we also tested AMB, which targets ergosterol. We foundthat all of the strains had the same profile, with the exceptionof 5457 and the tac1 ${Delta}$ /tac1 ${Delta}$ mutant, which were slightly hypersusceptible(Fig. 2F and Table 2). These results demonstrate that Tac1pplays a minor role in regulating cell tolerance to AMB but througha target other than Cdr1p and Cdr2p.

To assess the phenotypic consequences of deleting the CDR genesby an independent method, the strains were also analyzed byspot assay (Fig. 4). The results obtained with this method wereconsistent with those obtained with the MIC assays, confirmingthe results described above and indicating that the observedphenotypes are similar on solid and liquid media.

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FIG. 4. Profiles of resistance of strains 5457 and 5674 and strain 5674-derived tac1 and cdr mutants to azole drugs and different compounds with antifungal activity, as determined by spot assays. Serial 10-fold dilutions of cells, starting at an OD₆₀₀ of 0.1, were spotted onto YPD plates containing FLC, KTC, ITC, FPZ, R6G, or AMB at the indicated concentrations (micrograms per milliliter) or no drug (CTL). The plates were incubated for 48 h at 30°C.

DISCUSSION

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DISCUSSION

Deletion of the CDR1 and CDR2 genes from A^r clinical isolate5674 with the SAT1 flipper system, coupled to the characterizationof the resulting mutants by quantitative MIC assays, allowedus to directly determine the relative contributions of the Cdr1pand Cdr2p transporters to clinical azole resistance. We showthat deleting CDR1 reduced resistance levels 4- to 8-fold, dependingon the different azoles tested (FLC, KTC, and ITC), while deletingCDR2 only slightly affected FLC and KTC resistance (by 1.5-fold)and did not affect ITC resistance. Thus, Cdr1p plays a majorrole in FLC, KTC, and ITC resistance whereas Cdr2p plays a moreminor role. It is possible that removing Cdr1p or Cdr2p affectsazole resistance directly, by diminishing active azole export,and/or indirectly, by altering the activity of other proteinsable to modulate intracellular azole accumulation (includingCdr1p or Cdr2p) or the phospholipid composition of the membrane.Assuming that transporter-mediated resistance to an antifungalcompound reflects the ability of the transporter to export thiscompound, as previously proposed (11, 15, 33), our data couldbe interpreted as meaning that Cdr1p is a better transporterof azoles than is Cdr2p, although it cannot be ruled out thatthe effect of CDR2 deletion might be masked by Cdr1p activityor by other, as-yet-unknown, compensatory mechanisms.

Our results show that deleting CDR2 from the cdr1 ${Delta}$ /cdr1 ${Delta}$ mutanthas a much stronger effect than deleting the gene from strain5674. Since Cdr1p and Cdr2p have some substrates in common,this could potentially be due to a masking effect of the activityof Cdr2p by Cdr1p, as previously proposed in a similar studyof ABC transporters in S. cerevisiae (8). In that study, deletingPDR5 from a wild-type S. cerevisiae strain did not affect oligomycinsusceptibility whereas deleting PDR5 from a yor1 ${Delta}$ mutant strain(carrying a deletion of the YOR1 gene, which encodes anotherABC transporter) led to pronounced oligomycin sensitivity, strongerthan that observed in the yor1 ${Delta}$ mutant strain (8). Similarly,in the case of S. cerevisiae PDR16 and PDR17, which encode twohomologous phospholipid transfer proteins, deleting PDR16 froma wild-type strain resulted in increased susceptibility of thecells to KTC and miconazole by 10- and 20-fold, respectively,and deleting PDR17 had no effect, whereas deleting both genesfrom the same strain led to 40- and 80-fold increased susceptibility(42). In the present study, however, the double deletion ofCDR1 and CDR2 was performed in an already resistant strain.We found that deleting both genes not only abolished azole resistancebut further reduced it to levels lower than those observed inA^s strain 5457 and the tac1 ${Delta}$ /tac1 ${Delta}$ mutant (Fig. 2A to C). Thiscould be potentially due, at least in part, to the Tac1p-independentlow level of Cdr1p expression in these strains (Fig. 1), whichhas been eliminated from strain STY31 as a result of the CDR1deletion. In fact, the deletion of both genes had a strikingeffect as it reduced azole resistance by 300- to 500-fold (bycomparison to 4- to 8-fold for cdr1 ${Delta}$ /cdr1 ${Delta}$ and 1.0- to 1.5-foldfor cdr2 ${Delta}$ /cdr2 ${Delta}$ ), uncovering a strong synergism between the twotransporters. Synergism between transporters has previouslybeen reported for bacterial multidrug resistance pumps (18)and also members of the P-type ATPase and ABC transporter familiesinvolved in regulating the phospholipid composition of the membraneof erythrocytes in mammals (7). Cdr1p and Cdr2p have been shownto function as phospholipid floppases, being able to translocatefluorescent phospholipids across the membrane lipid bilayerin an ATP-dependent fashion (36, 39), and it is possible thatthe abrogation of Cdr1p and Cdr2p activity may cause a majoralteration of the asymmetrical distribution of phospholipidsin the cell membrane which would exacerbate the cell responseto drugs. Since it was not observed with AMB (Fig. 2F and 4),which directly targets ergosterol, this synergism appears tobe specific for azoles and possibly involves the accumulationin the already perturbed plasma membrane of toxic ergosterolintermediates as a result of Erg11p inhibition.

R6G is a heterocyclic, lipophilic, and cationic fluorescentcompound that is a substrate for many ABC transporters. Becauseof its antifungal properties and the ease of its detection,R6G has been used extensively to study yeast ABC transportersfor their ability to confer R6G resistance and transport, thesetwo parameters being closely correlated (11, 15). In particular,it was shown that expression of Pdr5p, Cdr1p, and Cdr2p in S.cerevisiae confers high levels of R6G resistance and efflux(11, 15, 31; our unpublished observation). In one of these studies,R6G resistance was quantified by MIC assay and shown to be morethan 1,000-fold greater than that of the negative control strain(31). Based on these findings, it was expected that deletionof the CDR1 and CDR2 genes from strain 5674 would affect cellsusceptibility to R6G. Instead, we found that CDR1 and CDR2deletion only marginally reduced R6G resistance in strain 5674(Fig. 2E). A similar situation has been observed in other A^rstrains, TIMM3165 and GU5, in which elevated levels of the CDR1and CDR2 RNAs were accompanied by a 128-fold increase in resistanceto FLC but only by a 4-fold increase in resistance to R6G, comparedto A^s strains (3, 19). Interestingly, we found that deletionof TAC1 from strain 5674 significantly decreased the resistanceof the cells to R6G (by eightfold), uncovering the existenceof another, as-yet-unidentified, Tac1p target that modulatesR6G resistance. Our recent analysis of the Tac1p regulon identifieda gene, orf19.4531, whose protein product may have this function.This gene encodes a putative ABC transporter of the pleiotropicdrug resistance subfamily homologous to Cdr1p and Cdr2p (16).orf19.4531 was found to be upregulated together with CDR1 andCDR2 in a TAC1-dependent manner in four independent A^r clinicalstrains, including strain 5674 (16). Alternatively, the decreasedresistance of the cells to R6G may be due to another Tac1p targetor to a combination of other Tac1p targets, since many of thesegenes are predicted to regulate the lipid or phospholipid compositionof the plasma membrane in C. albicans. We are currently investigatingthe role of these genes in azole and R6G resistance by deletingthem from strains 5674 and STY31. This approach may allow usto identify new genes that affect drug resistance either directlyor indirectly by regulating Cdr1p or Cdr2p function.

ACKNOWLEDGMENTS

We thank Sadri Znaidi for critical reading of the manuscript,Sébastien Lemieux for advice, Joachim Morschhäuserfor the gift of pSFS2A, Dominique Sanglard for providing theanti-Cdr1p antibody, the Institute for Research in Immunologyand Cancer Genomic Platform for DNA sequencing, and the CandidaGenome Database for sequence information.

This work was supported by a research grant to M.R. from theCanadian Institutes of Health Research (MT-15679). The Institutefor Research in Immunology and Cancer is supported by the CanadianCenter of Excellence in Commercialization and Research, theCanadian Foundation for Innovation, and the Fonds de Rechercheen Santé du Québec.

FOOTNOTES

* Corresponding author. Mailing address: Institute for Research in Immunology and Cancer, Université de Montréal, P.O. Box 6128, Station Centre-Ville, Montreal, Quebec, Canada H3C 3J7. Phone: (514) 343-6746. Fax: (514) 343-6843. E-mail: martine.raymond{at}umontreal.ca

Published ahead of print on 17 February 2009.

Supplemental material for this article may be found at http://aac.asm.org/.

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