Gain of Function Mutations in CgPDR1of Candida glabrataNot Only Mediate Antifungal Resistance but Also Enhance Virulence Se´le`ne Ferrari1, Franc¸oise Ischer1, David Calabrese1¤, Brunella Posteraro2, Maurizio Sanguinetti2, Giovanni Fadda2, Bettina Rohde3, Christopher Bauser3, Oliver Bader4, Dominique Sanglard1* 1InstituteofMicrobiology,UniversityofLausanneandUniversityHospitalCenter,Lausanne,Switzerland,2InstituteofMicrobiology,Universita`CattolicadelSacroCuore, Rome,Italy,3GATCBiotechAG,Konstanz,Germany,4Institutfu¨rMedizinischeMikrobiologie,Universita¨tsklinikenGo¨ttingen,Go¨ttingen,Germany Abstract CgPdr1p is a Candida glabrata Zn(2)-Cys(6) transcription factor involved in the regulation of the ABC-transporter genes CgCDR1, CgCDR2, and CgSNQ2, which are mediators of azole resistance. Single-point mutations in CgPDR1 are known to increasetheexpressionofatleastCgCDR1andCgCDR2andthustocontributetoazoleresistanceofclinicalisolates.Inthis study,weinvestigatedtheincidenceofCgPDR1mutationsinalargecollectionofclinicalisolatesandtestedtheirrelevance, not only to azole resistance in vitro and in vivo, but also to virulence. The comparison of CgPDR1 alleles from azole- susceptibleandazole-resistantmatchedisolatesenabledtheidentificationof57aminoacidsubstitutions,eachpositioned in distinct CgPDR1 alleles. These substitutions, which could be grouped into three different ‘‘hot spots,’’ were gain of function(GOF)mutationssincetheyconferredhyperactivitytoCgPdr1prevealedbyconstitutivehighexpressionofABC- transporter genes. Interestingly, the major transporters involved in azole resistance (CgCDR1, CgCDR2, and CgSNQ2) were not always coordinately expressed in presence of specific CgPDR1 GOF mutations, thus suggesting that these are rather trans-actingelements(GOFinCgPDR1)thancis-actingelements(promoters)thatleadtoazoleresistancebyupregulating specific combinations of ABC-transporter genes. Moreover, C. glabrata isolates complemented with CgPDR1 hyperactive alleleswerenotonlymorevirulentinmicethanthosewithwildtypealleles,buttheyalsogainedfitnessinthesameanimal model.ThepresenceofCgPDR1hyperactiveallelesalsocontributedtofluconazoletreatmentfailureinthemousemodel.In conclusion, this study shows for the first time that CgPDR1 mutations are not only responsible for in vitro/in vivo azole resistance butthatthey can also conferaselective advantageunder hostconditions. Citation:FerrariS,IscherF,CalabreseD,PosteraroB,SanguinettiM,etal.(2009)GainofFunctionMutationsinCgPDR1ofCandidaglabrataNotOnlyMediate AntifungalResistancebutAlsoEnhanceVirulence.PLoSPathog5(1):e1000268.doi:10.1371/journal.ppat.1000268 Editor:ScottG.Filler,DavidGeffenSchoolofMedicineatUniversityofCaliforniaLosAngeles,UnitedStatesofAmerica ReceivedSeptember18,2008;AcceptedDecember15,2008;PublishedJanuary16,2009 Copyright: (cid:1) 2009 Ferrari et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited. Funding: This study was supported by a grant from the European community under the research program EURESFUN (LSHM-CT-2005-518199). M.S. was supportedbyagrantfromtheIstitutodiRicoveroeCuraaCarattereScientifico(IRCCS)‘‘LazzaroSpallanzani’’(StrategicResearchProgram2006,Italy). CompetingInterests:Theauthorshavedeclaredthatnocompetinginterestsexist. *E-mail:[email protected] ¤Currentaddress:SelexisSA,Plan-les-Ouates/Geneva,Switzerland Introduction andthemajorfacilitatorsuperfamily(MFS),areinvolvedinazole resistance.InC.glabrata,theconstitutiveupregulatedexpressionof Candida glabrata has recently emerged as the second most ABC-transporter genes CgCDR1 and, to a lesser extent, CgCDR2 common cause of invasive candidiasis, and there are increasing (also known as PDH1) plays a dominant role in azole resistance numbers of reports showing its important role in mucosal or [4,8–11]. Each of these genes can be upregulated in C. glabrata bloodstreaminfections[1,2].SystemicinfectionsduetoC.glabrata clinical isolates and disruption of CgCDR1 or CgCDR1/CgCDR2 arecharacterizedbyahighmortalityrate,andtheyaredifficultto leads to hypersusceptibility to fluconazole, cycloheximide, and treat due to the intrinsically low susceptibility of this species to chloramphenicol [4,9,10,12,13]. azole drugs, especially to fluconazole [3]. In addition, C. glabrata The expression of CgCDR genes is regulated by a single Zn(2)- easilydevelopsfluconazoleresistanceinresponsetodrugexposure Cys(6)transcriptionfactor,CgPdr1p,anhomologueofS.cerevisiae during patient treatment [4–6]. Pdr1p/Pdr3p[11].CgPDR1deletionleadstoalossofCgCDR1and Azole antifungals target the cytochrome P-450 lanosterol 14-a CgCDR2 regulation andtoa sharp increasein azolesusceptibility demethylase, encoded by ERG11. Resistance of yeast clinical [14]. Due to the presence of PDRE (pleiotropic drug response isolates to azole antifungal agents can result from either element) sequences in the CgCDR1 and CgCDR2 promoters, overexpression or mutations in ERG11. Alternatively, the cells CgPdr1pactsprobablybybindingtotheseregulatoryelementsas can fail to accumulate azole antifungal agents due to enhanced Pdr1p and Pdr3p in S. cerevisiae. CgPDR1 contains a PDRE in its drug efflux, a consequence of transcriptional activation of drug promoter suggesting an auto-regulation of its transcription. effluxpumps(forreview,see[7]).Atleasttwofamiliesofmultidrug Consistent with this observation, upregulation of CgCDR1 and transporters, the ABC (ATP-binding cassette) transporter family CgCDR2inazole-resistantstrainsiscorrelatedwithanincreaseof PLoSPathogens | www.plospathogens.org 1 January2009 | Volume 5 | Issue 1 | e1000268 AzoleResistanceandImpactonVirulence amino acid substitutions in CgPdr1p enhanced virulence and led Author Summary tofluconazoletreatmentfailureinmousemodels.Takentogether Candida glabrata is a yeast causing several diseases in our data demonstrate a high variability in CgPDR1 mutations, humans and especially in immuno-compromised people. which themselves have differentiated effects on target genes C. glabrata infections are treated with antifungal agents, including ABC-transporters and probably on yet unidentified however the use of some agents, for example azoles, is virulence factors. associated with the development of resistance. In this yeast species, azole resistance is mediated almost exclu- Results sivelybyATPBindingCassette(ABC)multidrugtransport- ers. Their overexpression results in enhanced efflux of Isolation and Characterization of CgPDR1 Alleles from C. azoles and thus generates resistance. Regulation of ABC glabrata Clinical Isolates transporters is therefore of pivotal importance to under- The incidence of CgPDR1 mutations was investigated in a standingazoleresistance.InC.glabrata,theexpressionof collection of C. glabrata clinical isolates (n=122) consisting of 30 ABCtransportersismediatedbyazincfingertranscription factor calledCgPDR1.Gainoffunction (GOF) mutationsin groupsofsequentialisolates(n=66).Eachgroupcontainedatleast CgPDR1 result in high ABC transporter expression. In this one azole-susceptible (MIC fluconazole#16mg/ml) and one study,weinvestigatedtheoccurrenceofGOFmutationsin azole-resistant (MIC fluconazole$32mg/ml) isolates, which were a large collection of azole-resistant isolates and found a showntobehighlyrelatedbygenotypingmethods(Cg6andCg12 high variety of mutations localized in three distinct repetition probes or MLST) (data not shown). There were 36 domains of CgPDR1. We found that these mutations are azole-resistant isolates among the groups of related isolates. The not only associated with resistance but also enhanced 56 remaining isolates were unrelated (41 azole-resistant and 15 virulence and fitness of C. glabrata in animal models. Our azole-susceptible, Table S1). In this collection, azole-resistant study provides for the first time evidence that mutations isolates upregulated at least one of the ABC-transporter genes causing antifungal resistance can also provide a selective including CgCDR1, CgCDR2 or CgSNQ2 (see Figure 1 for ABC- advantage under host conditions and thus highlights the transporter genes expression levels measured by real-time RT- needof carefullymonitoring resistance inthis pathogen. PCR in groups of isolates and Figure S1 for CgCdr1p and CgCdr2p levels determined by western blot). CgPDR1 from each isolate was cloned and sequenced. To determine nucleotide CgPDR1 expression [14,15]. CgPDR1 is also essential in azole polymorphisms, the 122 sequences were aligned and showed 66 resistancecausedbymitochondrialdysfunctioninC.glabratapetite non-synonymous nucleotide substitutions among a total of 70 mutants.SinceenhancedCgPDR1expressionisobservedinsome distinct CgPDR1 alleles. By comparison of CgPDR1 alleles from petitemutants,ithasbeenproposedthatCgPDR1regulatesitsown azole-susceptible and azole-resistant isolates, we identified 12 expression in response to mitochondrial dysfunction [15]. different alleles recovered only from azole-susceptible isolates CgPdr1p acts as nuclear receptor by directly binding to diverse containing combinations of eight different mutations and 58 drugs and xenobiotics, such as azoles, to activate expression of different alleles specific forazole-resistant isolates with58distinct efflux pumps genes resulting in multidrug resistance [16]. The mutations. These mutations yielded 57 single amino acid activation domain of CgPdr1p binds directly tothe Mediator co- substitutions (Table S2) located at 50 locations along the protein activatorsubunitCgGal11pinaxenobiotic-dependentmannerin and encompassing three distinct protein domains: i) the region order toactivate transcription oftarget genes [16]. similar to the transcriptional inhibitory domain of Pdr1p from S. Two studies have identified three separate amino acid cerevisiae,ii)themiddlehomologyregion(MHR)andiii)aputative substitutions (W297S, F575L, P927L) in CgPdr1p of azole- transcriptionalactivationdomain(Figure2).Elevendistinctamino resistant strains that are responsible for constitutive high acids substitutions were found repeatedly in azole-resistant expression of CgCDR1, CgCDR2 and CgPDR1 itself [14,15]. resistant isolates (six found twice and five found three times) and Recently, another Pdr1p-regulated ABC-transporter gene, severalsubstitutionsoccurredatthesamepositioninsixdifferent CgSNQ2,wasshowntoparticipatetoazoleresistanceofC.glabrata cases (Table S2). Overall, CgPdr1p unique substitutions were clinical isolates [17]. In this study, a fourth CgPdr1p amino found in 46 distinct azole-resistant isolates. This suggests that substitution, P822L, was identified. Interestingly, the P822L saturationofCgPDR1mutationsinazole-resistantisolatesmaybe substitution is responsible for the constitutive overexpression of still not reached. Indeed, a parallel and independent CgPDR1 CgSNQ2, but has no effect on the expression of CgCDR1 and sequence analysis of ten azole-resistant C. glabrata isolates still CgCDR2 [17]. revealed one unknown mutation and two additional distinct In the present study, we investigated the incidence of CgPDR1 mutations on one of the 51 nucleotide positions identified in this mutationsinalargecollectionofclinicalisolates.Becausenostudy study (O. Bader, unpublished). We hypothesized that CgPDR1 has yet addressed whether the presence of CgPDR1 mutations is mutations may confer enhanced activity (or hyperactivity) to correlated with fitness costs in C. glabrata, we engineered isogenic CgPdr1p leading to increased expression of the CgCDR and/or strains with individual CgPDR1 mutations and tested their CgSNQ2genes. virulenceintwodifferentanimalmodels.Thestrainsthatacquired Azoleresistancewasgenerallycorrelatedwiththeoccurrenceof in vitro azole resistance were used to test the in vivo response to mutations in CgPDR1, except in four azole-resistant isolates fluconazole. We observed a high diversity among CgPDR1 alleles (DSY717, DSY2282, DSY2325 and BPY41). Mitochondrial and identified 57 distinct single amino acid substitutions, which dysfunction is one of the possible mechanism by which azole may confer hyperactivity to CgPdr1p in order to mediate high resistance can occur during azole treatment of patients [18]. expressionofABCtransportergenes.AlthoughCgCDR1,CgCDR2 Indeed,threeisolates(DSY2282,DSY2325andBPY41)displayed and CgSNQ2 are all regulated by CgPdr1p, they are not always altered mitochondrial respiratory capacity as deduced from their coordinately expressed in azole-resistant isolates indicating that inability to grow on medium containing glycerol as sole carbon ABCtransportergenesweredifferentiallyregulateddependingon source andfrom their defects inmitochondrial DNA (Figure S2). themutationpresentontheCgPDR1allele.Finally,theidentified Although CgCDR1 was upregulated in the remaining isolate PLoSPathogens | www.plospathogens.org 2 January2009 | Volume 5 | Issue 1 | e1000268 AzoleResistanceandImpactonVirulence Figure 1. Expression of CgCDR1, CgCDR2, and CgSNQ2in C.glabrataazole-resistant isolates from groups of sequential clinical isolates.Quantificationwasperformedbyreal-timeRT-PCR.Thevalues,whichareaveragesoffourseparatedexperiments,representtheincreasein geneexpressionrelativetotheazole-susceptibleparentalstrains(setat1.00).Errorbarsshowstandarddeviations. doi:10.1371/journal.ppat.1000268.g001 Figure2. Localisation of 65 non-synonymous substitutions in CgPdr1p.The eightpolymorphismspresentin CgPDR1 alleles from both azole-susceptibleandazole-resistantisolatesareindicatedbygreybars.The57aminoacidpolymorphisms(fromatotalof58mutations)specificfor CgPDR1allelesfromazole-resistantisolatesareindicatedbyblackbars.PutativeDBD(DNAbindingdomain)andMHR(middlehomologyregion) werelocatedbyPfamanalysis.ID(putativeinhibitorydomain)andAD(putativetranscriptionalactivationdomain)werededucedbysimilaritywith Pdr1pandPdr3pfromS.cerevisiae[11].ThelimitsofthesedomainsareindicatedbyaminoacidspositioninCgPdr1p. doi:10.1371/journal.ppat.1000268.g002 PLoSPathogens | www.plospathogens.org 3 January2009 | Volume 5 | Issue 1 | e1000268 AzoleResistanceandImpactonVirulence DSY717, no CgPDR1 mutation could be detected. We are Table1.AzolesusceptibilitiesandCgPDR1mutationsfromC. currently investigating azole resistance in this isolate and these glabrata clinicalisolates. data willbereported elsewhere. CgPDR1 Expression Levels Have a Moderate Effect on Strain SiteofisolationCgPDR1mutationb MIC(mgml21)a Azole Resistance FLCc ITCc KTCc SinglepointmutationsinCgPDR1havebeenshowntoincrease the expression of both CgCDR genes and CgPDR1, thus DSY486 Oropharynx - 16 1 1 contributing to azole resistance of clinical isolates [14,15]. To DSY489 Oropharynx L328F(G984T) 128 2 2 evaluate whether theexpression levelof CgPDR1 isimportantfor DSY738 Oropharynx - 8 0.5 1 the upregulation of CgCDR genes in our isolates, the CgPDR1 DSY739 Oropharynx R376W(C1126T) 64 2 2 mRNA levels of 21matched pairs ofazole-susceptible and azole- DSY2253 Oropharynx - 16 1 1 resistant isolates (listed in Table 1) were quantified by slot blot DSY2254 Oropharynx D1082G(A3245G) 128 2 2 analysis and real-time RT-PCR (Figure 3). The comparison between CgPDR1 expression levels from azole-susceptible and DSY2235 Oropharynx - 4 0.125 0.125 azole-resistant matched isolates yielded comparable results as DSY2234 Oropharynx T588A(A1762G) 32 1 1 judgedbysimilarrelativeincreaseofCgPDR1expressionobtained DSY701 Oropharynx 4 0.25 0.25 withthetwomethods(Figure3).Weconcludedfromtheseresults DSY704 Oropharynx T607S(C1820G) 32 1 1 that CgPDR1 upregulation was not correlating with azole DSY529 Oropharynx - 4 0.5 0.5 resistance since CgPDR1 was overexpressed up to two-fold in DSY530 Oropharynx E1083Q(G3247C) 64 2 2 someresistantisolates(DSY2268,DSY565,DSY756)ascompared totheirmatchedazole-susceptibleisolate,whereasitwassimilarly DSY753 Oropharynx - 4 0.125 0.125 expressed inothers (DSY2257,DSY2277, DSY2271)(Figure 3). DSY754 Oropharynx Y584C(A1751G) 32 1 1 To determine whether the two-fold increase in CgPDR1 DSY726 Oropharynx - 8 0.25 0.25 expressionobservedinsomeisolateswassufficienttoinducehigh DSY727 Oropharynx D876Y(G2626T) 64 2 2 levels of CgCdr1p and CgCdr2p and thus azole resistance, DSY562 Oropharynx - 8 0.125 0.125 CgPDR1 alleles from an azole-susceptible and an azole-resistant matched isolate (DSY2235 and DSY2234, respectively) were DSY565 Oropharynx I280F(G840C) 128 2 2 clonedintotheCEN-ARSplasmidpCgACU-5[19]andexpressed DSY2256 Oropharynx - 4 2 1 in a strain lacking CgPDR1. The CgPDR1 alleles from DSY2235 DSY2257 Oropharynx N691D(A2071G) 32 1 1 and DSY2234 only differ by the amino acid substitution T588A. DSY759 Oropharynx - 4 0.5 0.125 Expression of CgPDR1 alleles from the episomal plasmid resulted DSY2268 Oropharynx S316I(G947T) 32 1 1 inathree-tofour-foldincreaseofCgPDR1mRNAintherevertant DSY2270 Oropharynx - 8 1 1 strains as compared to the clinical isolates (Figure 4A), but no significantchangewasobservedinCgCdr1pandCgCdr2plevels DSY2271 Oropharynx D261G(A782G) 128 2 2 andinazolesusceptibility(Figure4Band4C).Similarresultswere DSY2272 Oropharynx - 4 0.5 0.5 obtained by overexpressing other mutated CgPDR1 alleles (data DSY2273 Oropharynx R293I(G878T) 32 2 1 not shown), indicating that the slight increase in CgPDR1 DSY2276 Oropharynx - 8 0.5 0.5 expression observed in some resistant isolates could not account DSY2277 Oropharynx R592S(G1776C) 32 2 1 for azoleresistance. DSY2278 Oropharynx - 4 0.5 0.5 CgPDR1 Mutations Induce Azole Resistance DSY2279 Oropharynx G583S(G1747A) 32 0.5 0.5 The identified CgPDR1 mutations were next investigated for DSY2281 Oropharynx - 8 0.25 0.25 theirabilitytoconferhyperactivitytoCgPdr1pbymediatinghigh DSY2282 Oropharynx - 64 2 1 expressionofABC-transporter genesresultinginazoleresistance. DSY755 Oropharynx - 4 0.25 0.125 For thispurpose, CgPDR1 was first inactivated in a pairof azole- DSY756 Oropharynx S343F(C1028T) 128 2 2 susceptible and azole-resistant isolates (DSY562 and DSY565, DSY2316 Oropharynx - 8 1 1 respectively) and reintroduced at the CgPDR1 genomic locus by DSY2315 Oropharynx R376G(C1126G) 32 1 1 homologousrecombinationintheobtainedpdr1Dstrains.CgPDR1 alleles from these two isolates only differ by the amino acid DSY773 Oropharynx - 16 0.5 1 substitutionL280FintheputativeinhibitorydomainofCgPdr1p. DSY774 Oropharynx R376G(C1126G) 32 1 1 DisruptionofCgPDR1inbothazole-susceptibleandazole-resistant DSY2317 Oropharynx - 4 0.25 0.5 isolates led to a drastic increase of azole susceptibility and to the DSY717 Oropharynx - 64 2 2 complete downregulation of both CgCDR genes (Figure 5A and DSY2324 Oropharynx - 4 0.25 0.125 5B), thus confirming the involvement of CgPdr1p in azole resistance. Each DSY562 and DSY565 pdr1D mutant received DSY2325 Oropharynx - 128 2 2 the CgPDR1 wild type or mutated alleles. Expression of CgCDR1, aMICstotheseantifungalagentsweredeterminedbybrothmicrodilution CgCDR2 and CgSNQ2 in reconstituted strains was restored to methodinaccordancewiththeCLSIM27-A2document(NationalCommittee similarlevelsthanthoseoftheoriginalclinicalisolates(Figure5B). forClinicalLaboratoryStandards,2002). ExpressionoftheCgPDR1allelecontainingtheL280Fsubstitution bNumberscorrespondtothepositionsofaminoacidchangeswhilenumbersin parenthesescorrespondtothepositionofchangednucleotideschangedin was sufficient to confer CgCdr1p and CgCdr2p constitutive high CgPDR1relativetotheATGstartcodon. expressionandthusazoleresistanceinC.glabrataindependentlyon cFLC:Fluconazole;ITC:itraconazole;KTC:ketoconazole. thestrain genetic background(Figure 5C). doi:10.1371/journal.ppat.1000268.t001 PLoSPathogens | www.plospathogens.org 4 January2009 | Volume 5 | Issue 1 | e1000268 AzoleResistanceandImpactonVirulence Figure3.ExpressionofCgPDR1inmatchedpairsofazole-susceptible(S)andazole-resistant(R)isolates.RNAwasisolatedfromlog phasecultures,slot-blottedtomembranesandhybridizedwiththeindicatedgeneprobes.CgACT1servedasinternalcontrol.Signalsobtainedin blottedmembraneswerequantifiedbycountingradioactivitybyphosphorimaging.SignalsobtainedforCgPDR1werenormalizedwithCgACT1. ExpressionvaluesrepresenttheincreaseofCgPDR1expressioninazole-resistantisolatesrelativetotheirazole-susceptibleparentalstrains.RNAwas alsousedforreal-timequantitativeRT-PCRasdescribedinMaterialandMethods.Valuesgivenontherightdiagramaremeans(6SEM)ofthree separateexperimentsandexpressCgPDR1relativeexpressioninresistantisolatesascomparedtotheirmatchedsusceptibleparents. doi:10.1371/journal.ppat.1000268.g003 Selected CgPDR1 alleles from eight other pairs of isolates Distinct CgPDR1 GOF Mutations Have Differentiated (Table 1) were reintroduced at the CgPDR1 genomic locus in an Effects on ABC-Transporter Genes Expression azole-susceptible background lacking CgPDR1. CgPDR1 alleles To determine the effect of distinct mutated CgPDR1 alleles on fromeachpairofisolatesonlydifferbyapointmutationleadingto ABC-transporter genes expression, mRNA levels of CgCDR1, a single amino acid substitution in either the inhibitory domain, CgCDR2 and CgSNQ2 were determined by real-time RT-PCR in theMHRortheactivationdomainofCgPdr1p.Thesemutations, 21 matched pairs of azole-susceptible and azole-resistant isolates which were specific for azole-resistant isolates, restored azole clinicalisolateslistedinTable1(Figure1).Aspreviouslyobserved resistanceinapdr1Dmutant(fluconazoleMICsfrom64–128mg/ by Torelli et al. [17], CgCDR1, CgCDR2 and CgSNQ2 were not ml, Figure 6A). Since only alleles containing these mutations always coordinately expressed in azole-resistant isolates. Among conferred CgCDR1 constitutive high expression (from 4- to 150- them,CgSNQ2showedingeneralthelowestexpressionvariations. fold expression increase, Figure 6B), these mutations could be Taking a significant upregulation between two isolates as a assigned as GOF mutations. Moreover, single amino acid thresholdoftwo-foldexpressiondifference,onlynineisolateswere substitutions in either the inhibitory domain, the MHR or the abovethisvalue.CgSNQ2wasmoreupregulated(8.5-fold)thanthe activation domain could confer drug resistance. Once again, otherABCtransportersonlyinDSY2277(Figure1).Upregulation alteredCgPDR1expressioncouldnotaccountforazoleresistance, ofCgCDR1andCgCDR2inazole-resistantisolatesfollowedsimilar since theCgPDR1mRNA levelsweresimilar betweentheclinical patterns but without reaching statistical significance (data not strains and the revertant strains expressing their corresponding shown). In some cases, significant upregulation (from 13- to 170- CgPDR1 alleles (FigureS3). fold) of only CgCDR1 was measured (DSY2268, DSY2273, PLoSPathogens | www.plospathogens.org 5 January2009 | Volume 5 | Issue 1 | e1000268 AzoleResistanceandImpactonVirulence alleles were reintroduced in the same background (DSY562 pdr1D). mRNA levels of CgCDR1, CgCDR2 and CgSNQ2 were measuredbyreal-timeRT-PCRintherevertantstrains(Figure6B) andcomparedwiththoseobtainedintheclinicalisolates(Figure1). For example, the presence of the Y584C substitution (from CgPDR1 of DSY754) led to CgCDR1 upregulation only (17- and 12-foldexpressionincrease,Figure1andFigure6B),whereasthe presence of the T588A substitution (from CgPDR1 of DSY2234) resultedinhighmRNAlevelsofbothCgCDRgenes(4-and8-fold expression increase for CgCDR1, 23- and 30-fold increase for CgCDR2, Figure 1 and Figure 6B). Finally the presence of the P822L substitution (from CgPDR1 of BPY55) in a pdr1D mutant resulted in the upregulation of CgSNQ2 only (15-fold, Figure 6B), whichisconsistentwithpreviousobservationmadeinthisclinical isolate [17]. These results as well as those obtained with other engineeredisolateswereoverallconsistentwithexpressionprofiles fromclinicalisolatescontainingthesameCgPDR1alleles(Figure1) and thus indicate that ABC-transporter genes might be differen- tially regulated depending on the CgPDR1 GOF mutation and independently on thestrain background. CgPDR1 GOF Mutations Are Responsible for Increased Virulence of C. glabrata The C. glabrata isolates analysed in this study are of clinical origin.Theseisolatesmayhaveadapted tothehostconditionsin order to cause disease. Moreover, azole therapy in these patients selectedforazole-resistantisolatesandultimatelyledtotreatment failure. One interesting and unresolved question is whether CgPDR1 mutations responsible for high transporter expression have an impact on virulence and fitness of C. glabrata under host conditions. We therefore first measured tissue fungal burdens in animal models using tail-vein injections in groups of immuno- Figure 4. Effect ofCgPDR1expression on azole resistance. (A) competent and immuno-suppressed mice according to previously ExpressionofCgPDR1inamatchedpairofazole-susceptibleandazole- establishedprotocols[20–22].Fungalloadsinkidneys,spleenand resistant isolates (DSY2235 and DSY2234), in revertant strains (‘‘rev’’) liverofallmiceweremeasuredsevendaysafterinfection.Wenext overexpressing CgPDR1 alleles from an episomal plasmid in a pdr1D performed virulence assays by measuring mice survival after mutant derived from DSY562 (SFY53). CgPDR1 alleles present in each infection with several C. glabrata isolates to challenge correlations strain (‘‘DSY’’ for clinical strains and ‘‘rev’’ for revertant strains) were between virulenceand possibledifferences intissuefungal loads. namedaccordingtotheirstrainnumberorigin.RNAwasisolatedfrom logphasecultures,slot-blottedtomembranes,andhybridizedwiththe Using first DSY562 and DSY565 in immuno-competent or indicated gene probes. CgACT1 served as internal control. Signals immuno-suppressed mice(Figure 7Aand 7B),our resultsshowed obtainedinblottedmembraneswerequantifiedbycountingradioac- that fungal loads in kidneys were significantly increased in both tivity by phosphor imaging. Signals obtained for CgPDR1 were mouse models infected with DSY565 as compared to DSY562 normalizedwithCgACT1andsubstractedfrombackground.Expression (P,0.0001 and P=0.0007, respectively; see details in Table S3). valuesrepresenttheincreaseofCgPDR1expressioninrevertantstrains relative to the clinical isolates expressing the same CgPDR1 allele. (B) The same trend was observed in fungal loads of spleen and liver FluconazolesusceptibilitytestingofC.glabrataclinicalisolatesDSY2235 (FigureS4).Higherfungalloadsintissuesofmiceinfectedwiththe andDSY2234,revertantstrains(rev)overexpressingCgPDR1allelesand azole-resistant isolate DSY565 correlated with a significant of a pdr1D mutant (SFY53). Isolates were grown on YPD medium increased virulence as compared to DSY562 (Figure 8A). Taken containing the drug at the indicated concentration at 30uC for two together, these results demonstrate that the azole-resistant isolate days. (C) Immunodetection of CgCdr1p and CgCdr2p in C. glabrata (DSY565) was more virulent than its azole-susceptible parent, clinical isolates DSY2235 and DSY2234, in revertant strains (rev) overexpressing CgPDR1 alleles in a pdr1D mutant (SFY53). Proteins DSY562. Since DSY562 and DSY565 differ at least by the extract were separated by SDS-10% PAGE and immunoblotted with presenceoftheL280FsubstitutioninCgPDR1,itwastemptingto rabbit polyclonal anti-CgCdr1p and anti-CgCdr2p antibodies as testwhetherthissubstitutionwasresponsibleforthisbehavior.We described previously [9]. MICs to fluconazole were determined by therefore replaced the mutated allele with the CgPDR1 wild type broth microdilution method in accordance with the CLSI M27-A2 alleleinDSY565(namedasDSY565pdr1D-PDR1;SFY118)and document(NationalCommitteeforClinicalLaboratoryStandards,2002). alsoreplacedtheCgPDR1 wildtypeallele withthemutatedallele doi:10.1371/journal.ppat.1000268.g004 inDSY562(namedasDSY562pdr1D-L280F;SFY115).CgPDR1 alleles were also reconstituted in their original backgrounds DSY754, DSY2315, DSY565 and DSY2746). In two cases (DSY562 pdr1D-PDR1; SFY114 and DSY565 pdr1D-L280F; (DSY2254andDSY2234),CgCDR2upregulationoutreachedthat SFY119). Mice infected with DSY562 pdr1D-L280F or DSY565 ofCgCDR1(Figure1).SincethethreeABC-transportergenesare pdr1D-L280F had significant higher fungal loads in their kidneys regulated by CgPDR1, their uncoordinated expression might be as compared toDSY565 pdr1D-PDR1or DSY562 pdr1D-PDR1 explained byeitherdifferencesintheirpromotersequencesorby (Figure 7A, P,0.0001). The same was observed in immuno- differences in the transcriptional capacity of CgPdr1p. To avoid suppressedmice(Figure7B,P,0.0001).Nosignificantchangesin differences due to strain genetic backgrounds, pairs of CgPDR1 kidneys fungal loads occurred between immuno-competent mice PLoSPathogens | www.plospathogens.org 6 January2009 | Volume 5 | Issue 1 | e1000268 AzoleResistanceandImpactonVirulence Figure5.QuantitativeanalysisofazoleresistanceinselectedC.glabrataisolates.(A)FluconazolesusceptibilitytestingofC.glabrataclinical isolatesDSY562andDSY565andtheirderivativemutants.IsolatesweregrownonYPDmediumcontainingthedrugattheindicatedconcentrationat 30uCfortwodays.Theindicatedgenotypescorrespondtothefollowingstrains:DSY562pdr1D:SFY92;DSY565pdr1D:SFY94;DSY562pdr1D+562 (standingforre-introductionofCgPDR1fromDSY562):SFY114;DSY562pdr1D+565L280F(standingforre-introductionofCgPDR1fromDSY565with L280F substitution): SFY115; DSY565pdr1D+562: SFY118; DSY565 pdr1D+565L280F: SFY119. (B) Expression of CgCDR1, CgCDR2, and CgSNQ2 in C. glabrataclinicalisolatesDSY562andDSY565andtheirderivativemutants.Quantificationwasperformedbyreal-timeRT-PCR.Thevalues,whichare averages of four separated experiments, represent the increase in gene expression relative to DSY562 (set at 1.00). Error bars show standard deviations.(C)ImmunodetectionofCgCdr1pandCgCdr2pinC.glabrataclinicalisolatesDSY562andDSY565andtheirderivativemutants.Proteins extract were separated by SDS-10% PAGE and immunoblotted with rabbit polyclonal anti-CgCdr1p and anti-CgCdr2p antibodies as described previously[9].MICstofluconazoleweredeterminedbybrothmicrodilutionmethodinaccordancewiththeCLSIM27-A2document. doi:10.1371/journal.ppat.1000268.g005 infected with DSY562 and DSY562 pdr1D-PDR1 (P=0.07) or virulence assays obtained with the first tested CgPDR1 mutant withDSY562pdr1D-PDR1andDSY565pdr1D-PDR1(P=0.62). allele(seeresultsobtainedwithDSY562pdr1D-T588A,-Q1083Q, Virulenceassayswiththesamestrainsconfirmedtheassociationof -P822LandDSY562pdr1D-P822L,Figure7andFigure8B)and increased virulence with increased fungal loads of infected tissues thus one can predict that increased virulence may be caused by (Figure 8A). Thus, increased virulence was associated with the any of the GOF mutations so far identified in CgPDR1. presence of a GOF mutation in CgPDR1 independently on the Interestingly,theabsenceofCgPDR1inDSY562(DSY562pdr1D; strainbackground.Introducingothermutationsinthebackground SFY92) did not significantly alter fungal tissue burdens and of DSY562 mimicked results of fungal tissue burdens and of virulenceinanimalmodels(Figure7andFigure8A).Thisstrongly PLoSPathogens | www.plospathogens.org 7 January2009 | Volume 5 | Issue 1 | e1000268 AzoleResistanceandImpactonVirulence Figure6.ReconstitutionofCgPDR1GOFallelesinC.glabrata.(A)FluconazolesusceptibilitytestingofDSY562pdr1Dmutantstrain(SFY93) expressingdifferentCgPDR1alleles,whichwerenamedaccordingtotheirstrainnumberoriginandbyindicatingtheaminoacidsubstitution(in superscript) associated with a specific strain number. The following strains correspond to the indicated genotypes: DSY562 pdr1D+486:SFY98; 489L328F:SFY99;738:SFY100;739R376W:SFY101;2253:SFY102;2254D1082G:SFY103;2235:SFY104;2234T588A:SFY105;701:SFY106;704T607S:SFY107; 529:SFY108;530E1083Q:SFY109;753:SFY110;754Y584C:SFY111;726:SFY112;727D876Y:SFY113;BPY55P882L:SFY116.(B)ExpressionofCgCDR1,CgCDR2, and CgSNQ2 in the DSY562 pdr1D mutant strain (SFY93) expressing different CgPDR1 alleles, named according to their strain number origin. Quantificationwasperformedbyreal-timeRT-PCR.Thevalues,whichareaveragesoffourseparatedexperiments,representtheincreaseingene expressionrelativetoDSY562(setat1.00).Errorbarsshowstandarddeviations. doi:10.1371/journal.ppat.1000268.g006 suggeststhatitisratherthepresenceofaGOFmutationthanthe a 1:1 population ratio remained in equivalent population presence ofCgPDR1 that directly affects virulence. proportions (Figure 9A). It is only in vivo that the azole-resistant This assumption isalso based on fitness tests performed invitro population displayed a selective advantage over susceptible and in vivo with two selected isolates, each containing a wild type isolates. After inoculating mice with a 1:1 population ratio, a (DSY562 pdr1D-PDR1; SFY114) or a mutated CgPDR1 allele progressivedisappearanceofthesusceptiblepopulationwasvisible (DSY562 pdr1D-L280F; SFY115). We observed that a GOF over the time lapse of the animal experiment (Figure 9B). These mutation had no selective advantage in vitro for C. glabrata over a results demonstrate that a GOF in CgPDR1 is associated with a susceptibleparentisolatesincebothstrainscultivatedover24hat gain infitness invivoeven intheabsence ofdrug selection. PLoSPathogens | www.plospathogens.org 8 January2009 | Volume 5 | Issue 1 | e1000268 AzoleResistanceandImpactonVirulence Figure7.C.glabratatissueburdensinmurineinfectionmodels.(A)Fungaltissueburdensinkidneysfromimmuno-competentBALB/cmice infectedintravenouslywith46107viablecellsofC.glabratastrains.Miceweresacrificedatday7post-infection;andresults,whichareexpressedas CFUs per gram of tissue, represent values recorded separately for each of the ten mice. Geometric means are indicated by horizontal bars and asterisksindicatestatisticallysignificantdifferences(*:P,0.05;**:P,0.01;***:P,0.001).NSindicatesnosignificance(P.0.05).Theoriginofeach strain is indicated. Strain background (DSY562 or DSY565) is indicatedby filled or empty symbols, respectively. The pdr1D mutants from strains DSY562andDSY565correspondtoSFY92andSFY94,respectively.Revertantsconstructedfrompdr1Dmutantsareindicatedbythere-introduced GOFmutationorbythere-introducedwildtypeCgPDR1allelefromDSY562.Prism5.0wasusedforstatisticalanalysisanddatawereprocessedwith non-parametricWilocoxonRanksumtests.ComparisonsareindicatedintheFigure(seeTableS3fordetails)andassociateselecteddatapoints.(B) Fungaltissueburdensinkidneysfromimmuno-suppressedmiceinfectedintravenouslywith46107viablecellsofC.glabratastrains.BALB/cmice were rendered neutropenic by intraperitoneal administration of cyclophosphamide (200mgkg21 of body weight per day) three days before challengeandonthedayofinfection.Miceweresacrificedatday7post-infection. doi:10.1371/journal.ppat.1000268.g007 PLoSPathogens | www.plospathogens.org 9 January2009 | Volume 5 | Issue 1 | e1000268 AzoleResistanceandImpactonVirulence Figure8.VirulenceofC.glabrataisdependentonCgPDR1GOFmutations.(A)SurvivalcurvesofmiceinfectedwithDSY562andDSY565and derivedmutantsandrevertants.StatisticaldifferenceswereperformedusingtheLog-rankMantel-Coxtest(Prism5.0)bycomparingsurvivalcurvesof miceinfectedbyDSY562andbyotherstrainsasindicated.TherangeofsignificantPvaluesobtainedisindicatedforDSY565andstrainscontaining theCgPDR1allelewithaL280Fsubstitution.(B)SurvivalcurvesofmiceinfectedwithDSY562andDSY565andstrainsreconstitutedwithdifferent CgPDR1alleles.TherangeofsignificantPvaluesobtainedisindicatedforDSY565andstrainscontainingGOFmutations. doi:10.1371/journal.ppat.1000268.g008 CgPDR1 GOF Mutations Affect the In Vivo Response to thoseobtainedwiththestartingclinicalisolateDSY562(Figure10). Fluconazole With the exception of DSY562 pdr1D-T588A (P=0.02), the reconstitution of GOF mutations in CgPDR1 in both DSY562 Since DSY565 is resistant to azoles in vitro, we expected little (DSY562 pdr1D–Q1083Q, -P822L) and DSY565 (DSY565 changes between tissue burden of mice infected with this clinical pdr1D-L280F, -P822L) gave by CFU counts in kidneys no isolate in fluconazole-treated or -untreated conditions. The data significant differences between fluconazole-treated and untreated obtained after measurement of colony forming units (CFU) from animals. However, CFU counts of spleen and liver of animals tissuesoftreatedversusuntreatedanimalsinfectedwiththisstrain infected with DSY562 pdr1D-T588A were not significantly confirmed this hypothesis only in spleen and liver (Figure S5, different from untreated animals upon fluconazole treatment P=0.39and0.82),whilepartiallyinkidneys(Figure10,P=0.04). (P=0.28 and 0.16), thus suggesting that this GOF was still These results contrasted with those obtained with the azole- responsiblefortreatmentfailure.Takentogether,ourresultsarein susceptible isolate DSY562: a sharp and significant decrease of agreement with the idea that high fluconazole MIC values are fungal load was observed in all organs when comparing treated mirroredbytreatmentfailureinthisanimalmodel.Moreover,our versus untreated animals (P,0.0001–0.03). The absence of results demonstrate the critical role of CgPDR1 for the adequate CgPDR1 in both DSY562 and DSY565 backgrounds resulted in responseofC.glabratatofluconazolechallenge.Ontheotherhand, this experimental setting in a three-log decrease of fungal loads our results also highlight that CgPDR1 GOF mutations alone are when animal were treated with fluconazole. This result could be responsible for fluconazole treatment failure in the experimental expected from in vitro susceptibility data that yielded for these model tested here. mutants the lowest fluconazole MIC values (Figure 5C). This suggests thatCgPDR1 isessential fortheresponseofC.glabrata to Discussion fluconazolechallengeinvivo.Reconstitutingwildtypeisolatesfrom DSY562 and DSY565 backgrounds with a CgPDR1 wild type CgPdr1p is a Zn(2)-Cys(6) transcription factor involved in the allele (DSY562 pdr1D-PDR1: SFY114; DSY565 pdr1D-PDR1: regulation of the ABC-transporter genes CgCDR1, CgCDR2 and SFY118) gave in terms of fluconazole efficacy similar results to CgSNQ2, which are responsible for azole resistance in C. glabrata. PLoSPathogens | www.plospathogens.org 10 January2009 | Volume 5 | Issue 1 | e1000268