UPC2 Is Universally Essential for Azole Antifungal Resistance in Candida albicans ErinM.Vasicek,aElizabethL.Berkow,aStephanieA.Flowers,aKatherineS.Barker,aP.DavidRogersa,b,c DepartmentofClinicalPharmacy,CollegeofPharmacy,aandDepartmentofPediatrics,CollegeofMedicine,bUniversityofTennesseeHealthScienceCenter,and Children’sFoundationResearchCenteratLeBonheurChildren’sMedicalCenter,cMemphis,Tennessee,USA InCandidaalbicans,thetranscriptionfactorUpc2iscentraltotheregulationofergosterolbiosynthesis.UPC2-activatingmuta- tionscontributetoazoleresistance,whereasdisruptionincreasesazolesusceptibility.Inthepresentstudy,weinvestigatedthe relationshipofUPC2tofluconazolesusceptibility,particularlyinazole-resistantstrains.Inadditiontothereducedfluconazole MICpreviouslyobservedwithUPC2disruption,weobservedalowerminimumfungicidalconcentration(MFC)foraupc2(cid:2)/(cid:2) D mutantthanforitsazole-susceptibleparent,SC5314.Moreover,theupc2(cid:2)/(cid:2)mutantwasunabletogrowonasolidmedium o containing10(cid:3)g/mlfluconazoleandexhibitedincreasedsusceptibilityandaclearzoneofinhibitionbyEtest.Time-killanalysis w showedhigherfungistaticactivityagainsttheupc2(cid:2)/(cid:2)mutantthanagainstSC5314.UPC2disruptioninstrainscarryingspecific nlo resistancemutationsalsoresultedinreducedMICsandMFCs.UPC2disruptioninahighlyazoleresistantclinicalisolatecon- a d tainingmultipleresistancemechanismslikewiseresultedinareducedMICandMFC.Thismutantwasunabletogrowonasolid e mediumcontaining10(cid:3)g/mlfluconazoleandexhibitedincreasedsusceptibilityandaclearzoneofinhibitionbyEtest.Time-kill d analysisshowedincreasedfungistaticactivityagainsttheupc2(cid:2)/(cid:2)mutantintheresistantbackground.Microarrayanalysis fro showedattenuatedinductionbyfluconazoleofgenesinvolvedinsterolbiosynthesis,irontransport,orironhomeostasisinthe m absenceofUPC2.Takentogether,thesedatademonstratethattheUPC2transcriptionalnetworkisuniversallyessentialforazole h t resistanceinC.albicansandrepresentsanattractivetargetforenhancingazoleantifungalactivity. tp : / / e c Theincreasedfrequencyofinvasivefungalinfectionsinrecent tionsinERG11(18).Thesemechanismsarefrequentlycombined, .a decadesisdirectlyrelatedtoanexpansionintheimmunocom- resultinginastepwisedevelopmentofresistanceovertime. s m promised patient population, including those with HIV/AIDS, InC.albicans,UPC2encodesazincclustertranscriptionfactor . o cancerchemotherapypatients,neutropenicpatients,recipientsof thatregulatestheexpressionofgenesinvolvedinergosterolbio- r g transplantswithindwellingcatheters,andpatientsreceivinganti- synthesis, including ERG11 (19, 20). Disruption of ERG11 or / o biotics(1–3).Candidaspeciescollectivelyarethefourthleading pharmacologic inhibition of the enzyme it encodes leads to re- n causeofnosocomialinfectionsintheUnitedStates,andsuchin- duced ergosterol production and the accumulation of alternate J fectionsareassociatedwithunacceptablyhighratesofmortality sterols,includinglanosterol,eburicol,obtusifoliol,14(cid:2)-methylfe- an (2,4–6).Candidaalbicansisthemostprevalentopportunistichu- costerol, and 14(cid:2)-methylergosta-8,24(28)-dien-3(cid:3),6(cid:2)-diol (21). u a manfungalpathogenandcausesawidevarietyofinfections,from BiosynthesisofthesealternatesterolsdoesnotrequireERG11but ry superficial mucosal infections to invasive disseminated disease, doesrequireothergenesintheergosterolbiosynthesispathway, 9 despite the availability of effective antifungal treatment. More- someofwhichappeartoberegulatedbyUpc2.Wereasonedthat , 2 over,themostcommonopportunisticinfectionamongAIDSpa- sinceUpc2isrequiredforthetranscriptionalactivationofother 0 1 genesofthesterolbiosynthesispathway,thedisruptionofUPC2 tientsisoropharyngealcandidiasis(OPC),andchroniccasescon- 9 tinuedespitetheavailabilityofhighlyactiveantiretroviraltherapy mightresultinenhancedactivityoftheazoleantifungalsinazole- b resistantaswellasazole-susceptibleisolates.Inthepresentstudy, y (HAART),duetopoorcompliance,lackofaccess,andfailureof g HAART(7–11). wefurtherexaminedtheroleofUPC2inazoleantifungalactivity u againstbothazole-susceptibleandazole-resistantstrainsofC.al- e Theazoles,particularlyfluconazole(FLC),arethemostwidely s bicans,inparticularanazole-resistantclinicalisolateandmutant t usedclassofantifungalsforthetreatmentofCandidainfections strainsthatrepresentfourofthemajorazoleresistancemecha- (2, 12). Fluconazole acts by inhibiting the protein product of nisms.Takentogether,ourresultsindicatethattheUPC2tran- ERG11,lanosterol14(cid:2)-demethylase,whichleadstoergosterolde- scriptionalnetworkisuniversallyessentialforfluconazoleresis- pletionandtheaccumulationoftoxicmethylatedsterolprecur- tanceinC.albicans. sors,resultingintheinhibitionofgrowth(13).Inanimmuno- compromisedhost,thefungistaticnatureoffluconazolelimitsits efficacyagainstCandidaspecies(14,15).C.albicansexhibitsin- Received26August2013 Accepted18March2014 hibitedgrowthinthepresenceofthisfungistaticdrug,anditis Publishedaheadofprint21March2014 generallybelievedthatsuchfungistaticactivityfacilitatesthede- AddresscorrespondencetoP.DavidRogers,[email protected]. velopment of resistance, creating problems for treatment, espe- Supplementalmaterialforthisarticlemaybefoundathttp://dx.doi.org/10.1128 ciallyinimmunocompromisedpatientswithOPC(16,17).Cur- /EC.00221-13. rently known mechanisms of resistance to the azoles include Copyright©2014,AmericanSocietyforMicrobiology.AllRightsReserved. alterationsintheexpressionofdrugeffluxpump(CDR1,CDR2, doi:10.1128/EC.00221-13 andMDR1)andergosterolbiosynthesis(ERG)genesandmuta- July2014 Volume13 Number7 EukaryoticCell p.933–946 ec.asm.org 933 Vasiceketal. TABLE1C.albicansstrainsusedinthisstudy Sourceor Strain Designation Strainbackgrounda Relevantcharacteristic(s)orgenotypeb reference SC5314 SC5314 N/A UPC2-1/UPC2-2 ATCC Clinicalisolate2-79 2-79 N/A Susceptibleisolate 53 Clinicalisolate12-99 12-99 N/A Resistantisolate 53 UPC2M4A upc2(cid:7)/(cid:7)strain SC5314 upc2-1(cid:7)::FRT/upc2-2(cid:7)::FRT 25 UPC2M2K21A upc2(cid:7)/(cid:7)(cid:6)UPC2strain UPC2M2A upc2-1(cid:7)::FRT/UPC2S1-1-caSAT1 25 12-99UPC2A5C1A 12-99upc2(cid:7)/(cid:7) 12-99 upc2(cid:7)::FRT/upc2(cid:7)::FRT Thisstudy 12-99UPC2A5C1A4A 12-99upc2(cid:7)/(cid:7)(cid:6)UPC2 12-99UPC2A5C1A upc2(cid:7)::FRT/UPC212-99-caSAT1 Thisstudy 10C1B1M1 ERG11K143Rmutant SC5314 ERG11K143R::FRT/ERG11K143R::FRTUPC2-1/UPC2-2 Thisstudy 10C1B1M1UPC2C9H ERG11upc2(cid:7)/(cid:7)mutant 10C1B1M1 ERG11K143R::FRT/ERG11K143R::FRT Thisstudy upc2-1(cid:7)::FRT/upc2-2(cid:7)::FRT SCMRR1R34A MRR1P683Smutant SC5314 MRR1P683S::FRT/MRR1P683S::FRTUPC2-1/UPC2-2 20 (cid:7)upc2MRR1R34A MRR1upc2(cid:7)/(cid:7)mutant SCMRR1R34A MRR1P683S::FRT/MRR1P683S::FRT 20 D upc2-1(cid:7)::FRT/upc2-2(cid:7)::FRT o SCTAC1R34A TAC1G980Emutant SC5314 TAC1G980E::FRT/TAC1G980E::FRTUPC2-1/UPC2-2 32 w SCupc2TAC1R34A1A14A TAC1upc2(cid:7)/(cid:7)mutant SCTAC1R34A TAC1G980E::FRT/TAC1G980E::FRT Thisstudy n upc2-1(cid:7)::FRT/upc2-2(cid:7)::FRT lo a aN/A,notapplicable. d bFRT,FLPrecombinationtarget. ed f r o m MATERIALSANDMETHODS (Table 2), resulting in full-length sequence from both strands of the h Strains and growth conditions. All C. albicans strains (Table 1) were CaERG11gene.Thesequencingwasperformedusingsixsetsofclones tt p storedasfrozenstocksin40%glycerolat(cid:4)80°C.YPD(1%yeastextract, derivedfromthreeindependentPCRsforeachstrain/isolatesequenced. : / 2%peptone,and1%dextrose)agarplatesandYPDliquidmediumwere SequencedplasmidsoftheERG11openreadingframe(ORF)whose /e used for routine growth of strains at 30°C. Iron-poor agar plates and predictedtranslationindicatedanaminoacidsubstitutionweredigested c . mediumwerepreparedbyadding200(cid:5)Mbathophenanthrolinedisulfo- withrestrictionenzymesApaIandXhoI,whichexcisedthefull-length a s nicacid(BPS)toYPD.Iron-richagarplatesandmediumwereprepared ORFfromtheplasmid,andtheERG11alleleswereclonedupstreamofthe m byadding100(cid:5)Mironchloride(FeCl3)toYPD.ForCFUcountsduring SAT1-flippercassetteintotheApaIandXhoIsitesofplasmidpSFS2(24). .o time-killanalysis,PDA(0.4%potatostarch,2%dextrose,and1.5%agar) TheERG11downstreamsegmentswereamplifiedwithExTaqpolymerase r g plateswereused,andculturesgrownonPDAwereincubatedat35°C. (TaKaRa)usingprimersERG11-CandERG11-Dandwerecloneddown- / Drugsusceptibilitytesting.TheMICsoffluconazole(FLC)werede- streamoftheSAT1-flippercassetteinpSFS2usingtheNotIandSacIIsites. o n termined by using broth microdilution as described by NCCLS (now ConstructionofUPC2deletionstrains.PlasmidpSFS2containsthe J CLSI)standardM27-A2(22),modifiedbyusingYPDmedium(ironre- entireSAT1-flippercassette(24).TheSAT1-flippercassetteconsistsofthe a plete),iron-poormedium,oriron-richmedium,andwerereadbothvi- SAT1selectablemarker,whichconfersresistancetonourseothricin,and nu suallyandspectrophotometricallyat24,48,and72h.Minimumfungi- theFLPflipperrecombinasegene,bothflankedbyFRTsites(flipperre- a cidalconcentrations(MFCs)weremeasuredbyremoving2(cid:5)lfromeach combinasetargetsequences).TheUPC2deletioncassette(pUPC2M2), ry welloftheMICplateandplatingontoYPDagar.Also,serialdilutions wherethe5=upstreamsequencefromposition(cid:4)373to(cid:6)15wascloned 9 dfriolumteads4u-sfpoeldn,saionndw2i(cid:5)thlaonfeoapcthicdailludteinosnitwyaast6p0la0tendmon(OtoDY6P00D)oafga0r.1pwlaetrees dsiotiwonns(cid:6)tr2ea0m97otoft(cid:6)he24S3A7Tw1-aflsicplponeredcauspsestttreeawmhioleftthheeS3A=Tse1q-ufleipnpceerfrcoamssepttoe-, , 20 oriron-pooragarplateswithorwithout10(cid:5)g/mlfluconazoleandwas wasdevelopedbyDunkeletal.(25)(Table1).Upontransformationofthe 19 then incubated at 30°C for 24 and 48 h. Fluconazole activity was also parentstrainwiththegel-purifiedSacI-ApaIfragmentfrompUPC2M2, b assessedbyEpsilometerteststrips(Eteststrips)(bioMérieux)according theSAT1-flippercassettewasinsertedintothecodingregionofoneallele y tothemanufacturer’sinstructionswiththefollowingmodifications.A fromposition(cid:6)16to(cid:6)2096,andsuchpositivetransformants(NouR) g standardizedcellsuspension(a0.5McFarlandstandard)wasusedtocre- wereselectedonYPD-nourseothricinagarplatescontaining200(cid:5)g/mlof ue ateaconfluentlawnacrossYPDagarplatesoriron-pooragarplatesprior nourseothricin.TheFLPgenewasinducedbygrowingthetransformants s t toEteststripplacement,andthecellswerethenincubatedat30°Cfor24 inYPDmediumfor24hwithoutselectivepressure.Positivecells(NouS) and48h.Time-killanalyseswereperformedwithacellsuspensionata0.5 were selected by replica plating onto YPD plates with or without 200 McFarlandstandard,whichwas10-folddilutedintoYPDmediumwithor (cid:5)g/mlofnourseothricin.UponinductionoftheFLPgene,thecassette without10(cid:5)g/mlfluconazoleandwasincubatedat35°C.Aliquotswere wasexcisedsuchthatonlyonecopyoftheFRTsiteremainedinthelocus. removedat0,6,12,and24h,10-foldseriallydiluted,andplatedonto Another round was required to disrupt the second allele. Appropriate PDA.CFUwerecountedinduplicateafter48hat35°Candwereplotted genedisruptionandcomplementationfortwoindependentstrainswere onalog-scalecurveversustime(23). confirmedbySouthernhybridization(24). ConstructionofstrainswithERG11mutantalleles.Candidaalbicans IsolationofgenomicDNAandSouthernhybridization.Genomic ERG11(CaERG11)codingsequenceswereamplifiedbyPCR(PfuDNA DNAwasisolatedasdescribedpreviously(26).FourmicrogramsofDNA polymerase;Stratagene)fromC.albicansgenomicDNAusingprimers wasdigestedwithanappropriaterestrictionendonuclease,separatedona ERG11-A and ERG11-E. Products were cloned into pCR-BLUNTII- 1%agarosegel,and,afterstainingwithethidiumbromide,wastrans- TOPOusingaZeroBluntTOPOPCRcloningkit(Invitrogen)andwere ferredbyvacuumblottingtoanylonmembraneandfixedbyUVcross- transferredintoEscherichiacoliTOP10cellswithselectiononLBagar linking. Southern hybridization with enhanced-chemiluminescence-la- plates containing 50 (cid:5)g/ml kanamycin. Plasmid DNA was purified beledprobeswasperformedwiththeAmershamECLDirectnucleicacid (QIAprep; Qiagen, Germantown, MD) and was sequenced on an ABI labelinganddetectionsystemaccordingtothemanufacturer’sinstruc- model 3130XL genetic analyzer using the ERG11 sequencing primers tions. 934 ec.asm.org EukaryoticCell Upc2IsEssentialforAzoleResistanceinC.albicans TABLE2Primersusedinthisstudy RNA isolation for quantitative reverse transcription-PCR (qRT- PCR).RNAwasisolatedusingasmall-scalehotphenolmethodofRNA Primer Sequencea isolationdescribedbySchmittetal.(27).Briefly,overnightcultureswere qRT-PCR dilutedtoanOD of0.2in20mlYPDandwerethenincubatedat30°C ACT1-F 5=-ACGGTGAAGAAGTTGCTGCTTTAGTT-3= 600 with shaking for 3 h. Cells were collected by centrifugation and were ACT1-R 5=-CGTCGTCACCGGCAAAA-3= storedat(cid:4)80°C.Cellpelletswereresuspendedin950(cid:5)lofAEbuffer(50 BMR1-F 5=-ACATAAATACTTTGCCCATCCAGAA-3= BMR1-R 5=-AAGAGTTGGTTTGTAATCGGCTAAA-3= mMsodiumacetate[pH5.3],10mMEDTA[pH8.0])andwerethen CDR1-F 5=-ATTCTAAGATGTCGTCGCAAGATG-3= transferredtoa2-mlRNase-freemicrocentrifugetubecontaining950(cid:5)l CDR1-R 5=-AGTTCTGGCTAAATTCGTAATGTTTTC-3= acidphenol(pH4.3)with1%SDS.Cellswereincubatedat65°Cfor10 CDR2-F 5=-TAGTCCATTCAACGGCAACATT-3= min;thenlysateswereclarifiedbycentrifugation.Thesupernatantwas CDR2-R 5=-CACCCAGTATTTGGCATTGAAA-3= thendividedintotwonew2-mlmicrocentrifugetubescontaining950(cid:5)l CFL4-F 5=-GCAATGGTTGACAGGTTGGAA-3= ofchloroform,andthecontentsofeachtubeweremixed.Thesamplewas CFL4-R 5=-GCAATGTGACGATGATAAGTGACAA-3= then subjected to centrifugation again, and the top aqueous layer was ERG11-F 5=-CCCCTATTAATTTTGTTTTCCCTAATTTAC-3= transferredtoanewtubecontaining1mlofisopropanoland100(cid:5)l2M ERG11-R 5=-CACGTTCTCTTCTCAGTTTAATTTCTTTC-3= sodiumacetate.TheRNApelletwassubsequentlywashedwith500(cid:5)lof FET3-F 5=-GCCGGTGTCTTAGGTTTAGCC-3= 70%ethanolandwascollectedbycentrifugation.TheRNApelletwas D FET3-R 5=-CTAGCAACTCTTTCTTCAACATCGG-3= resuspendedinDNase/RNase-freeH O.Quantityandpurityweredeter- o FRP1-F 5=-CTTCCAATACCATCCATTCACGAT-3= 2 w minedspectrophotometricallybyA andA . FRP1-R 5=-ATCTCCCCACTTTCAGCAAGAC-3= 260 280 n FTR1-F 5=-ATTGTTGTTTCAGTGCTTTTGGC-3= QuantitativeRT-PCR.First-strandcDNAsweresynthesizedfrom1 lo (cid:5)goftotalRNAusingtheSuperScriptfirst-strandsynthesissystemfor a FTR1-R 5=-GGTCGGAACTACCACCCATAGA-3= d RT-PCR(Invitrogen).Gene-specificprimers(Table2)weredesignedus- e ERG11mutant ingPrimerExpresssoftware(AppliedBiosystems)andweresynthesized d construction byIntegratedDNATechnologies(Coralville,IA).QuantitativePCRswere fr o ERG11-A 5=-GGGCCCGGGTTATTTGAGAACAGCC-3= performedintriplicateusingthe7000sequencedetectionsystem(Ap- m ERG11-B 5=-ATCCGTTCTCGAGCACTAAGGGACAA-3= pliedBiosystems),independentlyamplifyingACT1(normalizinggene) ERG11-C 5=-GTAATCAATTGAGCTCTTTTAACTTT-3= andthegenesofinterest(GOI)asdescribedpreviously(28). ht ERG11-D 5=-GATTATAGTTCCGCGGTGGTTTTACC-3= RNAisolationformicroarray.RNAwasisolatedusingalarge-scale tp ERG11-E 5=-TGATGGTTTTTGTCCACTGGCTCGAG-3= versionofthehotphenolmethodofRNAisolationdescribedbySchmitt :// e ERG11sequencing 1et0a0l.m(2l7Y)P.BDriaenfldy,woevreernthigehnticnuclutubraetsedwearte3d0i°lCutewditthosahnaOkiDng60f0oorfa0n.0a0d5diin- c.a ERG11seqB 5=-TATTTTCACTGCTTCAAGATCT-3= tional8htoanOD of1.0.CulturesweredilutedagaintoanOD of s ERG11seqC 5=-CCAAAAGGTCATTATGTTTTAG-3= 600 600 m 0.025in100mlfreshYPD,allowedtoincubateat30°Cwithshakingfor EERRGG1111sseeqqEF 55==--ACACTCGTTATGAGCTCTGTATTACAAACCCTTGAGAAAGTTGA--33== onedoubling,inoculatedwithorwithout10(cid:5)g/mlFLC,andthenincu- .or g T7 5=-TAATACGACTCACTATAGGG-3= batedat30°Cwithshakingfor6h.Cellswerecollectedbycentrifugation / M13R 5=-CAGGAAACAGCTATGACC-3= andwerestoredat(cid:4)80°C.Cellpelletswereresuspendedin12mlofAE o n buffer(50mMsodiumacetate[pH5.3],10mMEDTA[pH8.0])andwere J UPC2mutant thentransferredto50-mlOakRidgetubestreatedwithRNaseAway(Mo- a construction lecularBioProducts)containing12mlacidphenol(pH4.3)with1%SDS. n UPC2-A 5=-GGGCCCGAGATCTTGATGTCATTAG-3= Cellswereincubatedat65°Cfor10min;thenlysateswereclarifiedby ua UPC2-B 5=-CTCGAGCTATATCTTCAATGAACTG-3= centrifugation.Thesupernatantwasthentransferredtoanewtubecon- ry UPC2-C 5=-CCGCGGACAGGTCAATACCGCGTAG-3= taining15mlofchloroform,andthecontentsofthetubeweremixed.The 9 UPC2-D 5=-GAGCTCGTTCCTCTAGTATCACTCTT-3= samplewasthensubjectedtocentrifugationagain,andthetopaqueous , UPC2-E 5=-CTCGAGCACCACAGTAACGAATCAC-3= 2 layerwastransferredtoanewtubecontaining1volumeofisopropanol 0 aUnderliningreflectstheintroductionofarestrictionsitesequence. and0.1volumeof2Msodiumacetate.TheRNApelletwassubsequently 1 9 b y TABLE3MICsandMFCsinYPDintheSC5314background g u MIC((cid:5)g/ml) MFC((cid:5)g/ml) e s Mediumandstrain Relevantcharacteristicsorgenotype 24h 48h 72h 24h 48h 72h t YPD SC5314 UPC2-1/UPC2-2 0.5 0.5 (cid:8)64 (cid:8)64 (cid:8)64 (cid:8)64 upc2(cid:7)/(cid:7)mutant upc2-1(cid:7)::FRT/upc2-2(cid:7)::FRT (cid:2)0.125 0.25 0.25 0.25 0.25 0.25 upc2(cid:7)/(cid:7)(cid:6)UPC2strain upc2-1(cid:7)::FRT/UPC2S1-1-caSAT1 0.5 0.5 (cid:8)64 (cid:8)64 (cid:8)64 (cid:8)64 YPD(cid:6)BPS SC5314 UPC2-1/UPC2-2 0.5 0.5 0.5 (cid:8)64 (cid:8)64 (cid:8)64 upc2(cid:7)/(cid:7)mutant upc2-1(cid:7)::FRT/upc2-2(cid:7)::FRT NGa NG (cid:9)0.125 0.125 0.125 (cid:9)0.125 upc2(cid:7)/(cid:7)(cid:6)UPC2strain upc2-1(cid:7)::FRT/UPC2S1-1-caSAT1 0.5 0.5 0.5 (cid:8)64 (cid:8)64 (cid:8)64 YPD(cid:6)FeCl 3 SC5314 UPC2-1/UPC2-2 1 1 (cid:8)64 (cid:8)64 (cid:8)64 (cid:8)64 upc2(cid:7)/(cid:7)mutant upc2-1(cid:7)::FRT/upc2-2(cid:7)::FRT (cid:9)0.125 0.25 0.5 0.25 0.25 1 upc2(cid:7)/(cid:7)(cid:6)UPC2strain upc2-1(cid:7)::FRT/UPC2S1-1-caSAT1 1 1 (cid:8)64 (cid:8)64 (cid:8)64 (cid:8)64 aNG,nogrowth. July2014 Volume13 Number7 ec.asm.org 935 Vasiceketal. D o w n lo a d e d f r o m h t t p : / / e c . a s m . o r g / o n J a n u a r y 9 , 2 0 1 FIG1(A)EffectsofUPC2onMICsandgrowthonYPDagarasdeterminedbyEtest.AconfluentlawnofC.albicanswasstreakedpriortotheadditionofEtest 9 stripsandwasthenincubatedfor48h.(B)MICheatmapofSC5314,theUPC2mutant,andacomplementedderivative.Susceptibilitywasdeterminedbybroth microdilutioninYPDat72h(MICsin(cid:5)g/mlaboveheatmap).Growthwasquantifiedspectrophotometricallyandwasassignedtoacolorimetricscale.(C)Effect by ofUPC2ontheabilitytogrowonasolidmediumcontainingfluconazole.From4-foldserialdilutionsofC.albicansstrains,2-(cid:5)laliquotswerespottedontoYPD g agarwith(right)orwithout(left)10(cid:5)g/mlFLCandwereincubatedfor48h.(D)EffectoffluconazoleonUPC2inatime-killassay.SC5314orupc2(cid:7)/(cid:7)cellswere u dilutedinYPDmediumcontainingfluconazole(10(cid:5)g/ml)orthesolventdimethylsulfoxide(DMSO).After0,6,12,and24h,samplesfromeachstrainand e s mediumweredilutedandwereplatedforCFU. t washedwith10mlof70%ethanolandwascollectedbycentrifugation. Microarraydataaccessionnumber.Allmicroarraydataareavailable TheRNApelletwasresuspendedinDNase/RNase-freeH O.Quantity fordownloadfromtheNCBIunderGEOaccessionnumberGSE57929. 2 andpurityweredeterminedspectrophotometricallybyA andA . 260 280 Transcriptionalprofiling.Geneexpressionprofileswereobtainedby RESULTS hybridizinglabeledcRNAsgeneratedfromC.albicanstotalRNAonto UPC2disruptionresultsinenhancedfluconazoleactivity.Inor- AffymetrixC.albicanscustomexpressionarrays(CAN07;49-5241array dertofurtherinvestigatetherequirementofUPC2forsuscepti- format)(25),whichhavebeendescribedpreviously(29).Microarrayhy- bility to fluconazole, we subjected the upc2(cid:7)/(cid:7) mutant derived bridization and analysis were performed as described previously (29). fromazole-susceptibleisolateSC5314tovariousazolesusceptibil- Geneswereconsideredtobedifferentiallyexpressedinresponsetothe drugiftheirexpressionchanged(cid:3)1.5-foldintwoindependentexperi- itytests,examiningbothMICsandMFCsbyusingnutrient-rich ments.GenesinducedbyFLCwereconsideredtobeUPC2dependentif YPDmediuminordertodetectstrongphenotypesinanenviron- theinductioninthedeletionmutantwas(cid:3)2.0-fold(50%)lessthanthatin mentthatpromotesgrowth.Inagreementwithpreviousobserva- thewildtype. tions,disruptionofUPC2resultedinmarkedreductionsinflu- 936 ec.asm.org EukaryoticCell Upc2IsEssentialforAzoleResistanceinC.albicans TABLE4MICsandMFCsinYPDinthebackgroundsofstrainsexpressingresistancemechanisms MIC((cid:5)g/ml) MFC((cid:5)g/ml) Strain Relevantcharacteristicsorgenotype 24h 48h 72h 24h 48h 72h SC5314 UPC2-1/UPC2-2 0.5 0.5 (cid:8)64 (cid:8)64 (cid:8)64 (cid:8)64 ERG11K143Rmutant ERG11K143R::FRT/ERG11K143R::FRTUPC2-1/UPC2-2 4 8 8 8 8 32 ERG11upc2(cid:7)/(cid:7)mutant ERG11K143R::FRT/ERG11K143R::FRT 0.5 1 1 1 1 1 upc2-1(cid:7)::FRT/upc2-2(cid:7)::FRT MRR1P683Smutant MRR1P683S::FRT/MRR1P683S::FRTUPC2-1/UPC2-2 16 (cid:8)64 (cid:8)64 (cid:8)64 (cid:8)64 (cid:8)64 MRR1upc2(cid:7)/(cid:7)mutant MRR1P683S::FRT/MRR1P683S::FRT 2 2 4 4 4 4 upc2-1(cid:7)::FRT/upc2-2(cid:7)::FRT TAC1G980Emutant TAC1G980E::FRT/TAC1G980E::FRTUPC2-1/UPC2-2 16 (cid:8)64 (cid:8)64 (cid:8)64 (cid:8)64 (cid:8)64 TAC1upc2(cid:7)/(cid:7)mutant TAC1G980E::FRT/TAC1G980E::FRT 2 2 2 2 2 2 upc2-1(cid:7)::FRT/upc2-2(cid:7)::FRT D conazoleMICsbybrothmicrodilution(Table3),Etest(Fig.1A), h,theMICforSC5314,asmeasuredbyEtest,was1.0(cid:5)g/ml,and o 72-hregrowth(Fig.1B),andspotassays(Fig.1C).Interestingly,at ahaloofreducedgrowth(butnotaclearzoneofinhibition)was w 24hinYPD,thefluconazoleMFCwas(cid:8)64(cid:5)g/mlforSC5314, observeduptotheEteststrip,aresultconsistentwiththefungi- nlo whereasfortheupc2(cid:7)/(cid:7)mutantitwas0.25(cid:5)g/ml(Table3).At48 static nature of fluconazole. The 48-h MIC by Etest for the a d e d f r o m h t t p : / / e c . a s m . o r g / o n J a n u a r y 9 , 2 0 1 9 b y g u e s t FIG2(A)EffectsofUPC2inresistantbackgroundsonMICsandgrowthonYPDagarasdeterminedbyEtest.AconfluentlawnofC.albicanswasstreakedpriorto theadditionofEteststripsandwasthenincubatedfor48h.(B)EffectsofUPC2inresistantbackgroundsontheabilitytogrowonasolidmediumcontaining fluconazole.From4-foldserialdilutionsofC.albicansstrains,2-(cid:5)laliquotswerespottedontoYPDagarwith(right)orwithout(left)10(cid:5)g/mlFLCandwere incubatedfor48h. July2014 Volume13 Number7 ec.asm.org 937 Vasiceketal. TABLE5MICandMFCsinYPDforstrainsinthe12-99background MIC((cid:5)g/ml) MFC((cid:5)g/ml) Mediumandstrain Relevantcharacteristicsorgenotype 24h 48h 72h 24h 48h 72h YPD 2-79 Susceptibleisolate 1 1 (cid:8)64 (cid:8)64 (cid:8)64 (cid:8)64 12-99 Resistantisolate (cid:8)64 (cid:8)64 (cid:8)64 (cid:8)64 (cid:8)64 (cid:8)64 12-99upc2(cid:7)/(cid:7) upc2(cid:7)::FRT/upc2(cid:7)::FRT 4 4 4 4 4 4 12-99upc2(cid:7)/(cid:7)(cid:6)UPC2 upc2(cid:7)::FRT/UPC2-caSAT1 64 (cid:8)64 (cid:8)64 (cid:8)64 (cid:8)64 (cid:8)64 YPD(cid:6)BPS 2-79 Susceptibleisolate 2 2 4 (cid:8)64 (cid:8)64 (cid:8)64 12-99 Resistantisolate 32 32 32 (cid:8)64 (cid:8)64 (cid:8)64 12-99upc2(cid:7)/(cid:7) upc2(cid:7)::FRT/upc2(cid:7)::FRT NGa NG NG NG NG NG 12-99upc2(cid:7)/(cid:7)(cid:6)UPC2 upc2(cid:7)::FRT/UPC2-caSAT1 32 32 32 (cid:8)64 (cid:8)64 (cid:8)64 D YPD(cid:6)FeCl o 2-79 3 Susceptibleisolate 2 (cid:8)64 (cid:8)64 (cid:8)64 (cid:8)64 (cid:8)64 w 12-99 Resistantisolate (cid:8)64 (cid:8)64 (cid:8)64 (cid:8)64 (cid:8)64 (cid:8)64 nlo 12-99upc2(cid:7)/(cid:7) upc2(cid:7)::FRT/upc2(cid:7)::FRT 4 4 8 8 8 16 a 12-99upc2(cid:7)/(cid:7)(cid:6)UPC2 upc2(cid:7)::FRT/UPC2-caSAT1 64 (cid:8)64 (cid:8)64 (cid:8)64 (cid:8)64 (cid:8)64 d e aNG,nogrowth. d f r o m upc2(cid:7)/(cid:7)mutantwas0.032(cid:5)g/ml,andaclearzoneofinhibition ducedfrom1.5(cid:5)g/ml,4(cid:5)g/ml,and8(cid:5)g/mlintheirbackground h t aroundtheEteststripwasobserved(Fig.1A).Wealsouseda72-h strainsto0.19(cid:5)g/ml,0.5(cid:5)g/ml,and0.5(cid:5)g/ml,respectively(Fig. tp : endpointforabrothmicrodilutionassayinYPDasawaytoassess 2A).ThegrowthoftheMRR1upc2(cid:7)/(cid:7)andTAC1upc2(cid:7)/(cid:7)strains // e theabilityoftheorganismtoresumegrowthinthepresenceof whenplatedonYPDagarplatescontaining10(cid:5)g/mlfluconazole c . fluconazole.SC5314wasabletoresumegrowthinfluconazoleat wasalsoreducedfromthatoftheirbackgroundstrains(Fig.2B). a all concentrations tested, whereas the upc2(cid:7)/(cid:7) mutant was not FortheERG11K143Rstrainanditsupc2(cid:7)/(cid:7)derivative,theresults sm (Table 3; Fig. 1B). When cells were plated on YPD agar plates ofgrowthexperimentswereconsistentwiththefluconazoleMICs: .o containing10(cid:5)g/mlfluconazole,thelevelofgrowthinthepres- bothwereunabletogrowinthepresenceoffluconazoleatthis rg enceoffluconazolewaslowerfortheupc2(cid:7)/(cid:7)mutantthanfor concentration. o/ SC5314 (Fig. 1C). Time-kill analysis showed higher fungistatic DisruptionofUpc2overridesclinicaldrugresistance.Since n activityof10(cid:5)g/mlfluconazoleagainsttheupc2(cid:7)/(cid:7)mutantthan J UPC2disruptioninstrainscontainingoneresistancemechanism a againstitsparentstrain(Fig.1D).Allphenotypesrevertedwith resultedinenhancedfluconazoleactivity,wewantedtoexamine n reintegrationofonealleleofthedisruptedgene. u furthertheextenttowhichdisruptionofUPC2influenceshigh- a UPC2disruptioninstrainscontainingresistancemutations r levelazoleresistanceinthepresenceofmultipleresistancemech- y inMRR1,TAC1,orERG11alsoenhancesfluconazoleactivity.In anisms. We constructed upc2(cid:7)/(cid:7) mutant strains in the back- 9 order to investigate the requirement for UPC2 in the setting of , groundofanazole-resistantclinicalisolate(isolate12-99)known 2 specific mechanisms of azole resistance, independent mutants 0 tocarryfourofthemostcommonmechanismsofazoleresistance: 1 wereconstructedinstrainscontainingtwocopiesofagenecon- 9 overexpression of CDR1 and CDR2, overexpression of MDR1, ferring reduced susceptibility to fluconazole: the MRR1P683S, b TAC1G980E,orERG11K143Rgene.TheMRR1P683SandTAC1G980E overexpressionofERG11,andamutationinERG11(35).Aswas y observedforisolateSC5314andtheisogenicresistantstrains,the g allelescontaingain-of-functionmutationsthatrenderthetran- u scriptionfactorstheyencodeconstitutivelyactive,resultinginthe disruptionofUPC2resultedinmarkedreductionsinMICsand e upregulation of either MDR1 (MRR1P683S) orCDR1 and CDR2 MFCsasdeterminedbyallmethods.TheMFCat24hinYPDwas st (TAC1G980E),respectively,anddecreasedfluconazolesusceptibil- (cid:8)64(cid:5)g/mlfor12-99,whereasitwas4(cid:5)g/mlfor12-99upc2(cid:7)/(cid:7) ity(30–33).TheERG11K143Rallelecontainsapointmutationpos- (Table5).ThefluconazoleMICbyEtestat48hwas(cid:8)256(cid:5)g/ml for12-99,andconfluentgrowthwasobserved,whereasaMICof3 tulatedtobelocatedneartheazoleaccesschannel,interferingwith (cid:5)g/ml and a clear zone of inhibition were observed for 12- theentryoffluconazoleandthusresultingindecreasedflucona- zole susceptibility (34). Again, the disruption of UPC2 in each 99upc2(cid:7)/(cid:7)(Fig.3A).Likewise,inbrothmicrodilutionassaysafter backgroundresultedinmarkedreductionsinMICsandMFCsas 72h,theparentstrainwasabletogrowinthepresenceofflucona- determinedbyallmethods,andthetrendinsusceptibilityseenfor zoleatallconcentrationstested,whereas12-99upc2(cid:7)/(cid:7)grewless theMRR1upc2(cid:7)/(cid:7)mutantwasconsistentwithwhatwehaveob- well (Fig. 3B). The growth of 12-99upc2(cid:7)/(cid:7) was also reduced served previously (20). The MFCs at 24 h in YPD for the fromthatofitsparentstrainwhentheywereplatedonYPDagar ERG11upc2(cid:7)/(cid:7), MRR1upc2(cid:7)/(cid:7), and TAC1upc2(cid:7)/(cid:7) mutants platescontaining10(cid:5)g/mlfluconazole(Fig.3C).Aswasobserved werereducedfrom8(cid:5)g/ml,(cid:8)64(cid:5)g/ml,and(cid:8)64(cid:5)g/mlintheir intheSC5314background,time-killanalysisalsorevealedanin- backgroundstrainsto1(cid:5)g/ml,4(cid:5)g/ml,and2(cid:5)g/ml,respectively creasedfungistaticeffectforfluconazoleat10(cid:5)g/mlagainst12- (Table 4). At 48 h, the MICs for the ERG11upc2(cid:7)/(cid:7), 99upc2(cid:7)/(cid:7)(Fig.3D).Allphenotypesrevertedwiththereintegra- MRR1upc2(cid:7)/(cid:7), and TAC1upc2(cid:7)/(cid:7) mutants by Etest were re- tionofonealleleofthedisruptedgene. 938 ec.asm.org EukaryoticCell Upc2IsEssentialforAzoleResistanceinC.albicans D o w n lo a d e d f r o m h t t p : / / e c . a s m . o r g / o n J a n u a r y 9 , FIG3(A)EffectsofUPC2in12-99onMICsandgrowthonYPDagarasdeterminedbyEtest.AconfluentlawnofC.albicanswasstreakedpriortotheaddition 2 ofEteststripsandwasthenincubatedfor48h.(B)MICheatmapof2-79,12-99,theUPC2mutant,andacomplementedderivative.Susceptibilitywas 0 determinedbybrothmicrodilutioninYPDat72h(MICsin(cid:5)g/mlaboveheatmap).Growthwasquantifiedspectrophotometricallyandwasassignedtoa 1 9 colorimetricscale.(C)EffectofUPC2in12-99ontheabilitytogrowonasolidmediumcontainingfluconazole.From4-foldserialdilutionsofC.albicansstrains, 2-(cid:5)laliquotswerespottedontoYPDagarwith(right)orwithout(left)10(cid:5)g/mlFLCandwereincubatedfor48h.(D)EffectoffluconazoleonUPC2in12-99 by byatime-killassay.12-99or12-99upc2(cid:7)/(cid:7)cellsweredilutedinYPDmediumcontainingfluconazole(10(cid:5)g/ml)orthesolventdimethylsulfoxide(DMSO). g After0,6,12,and24h,samplesfromeachstrainandmediumweredilutedandwereplatedforCFU. u e s t ExpressionofERG11,CDR1,CDR2,andMDR1whenUPC2 disrupted.DisruptionofUPC2didnotresultindecreasedexpres- isdisruptedinresistantbackgrounds.Inordertodetermineif sionofCDR1,CDR2,orMDR1inanybackground.Interestingly, theenhancedfluconazoleactivitywasduetodecreasedexpression disruptionofUPC2intheERG11upc2(cid:7)/(cid:7)mutantresultedinin- ofERG11orgenesencodingeffluxpumps,wemeasuredtheabun- creases in the expression of these transporter genes, the signifi- dancesofERG11,CDR1,CDR2,andMDR1mRNAsbyqRT-PCR canceofwhichisunclear.Thesedatasuggestthattheenhanced in the strains containing a single resistance mechanism, clinical activityoffluconazoleobservedinresistantstrainslackingUPC2 isolate12-99,andtheirrespectiveupc2(cid:7)/(cid:7)mutants(Fig.4).As isnotduetochangesintransportergeneexpressionlevelsbutmay expected,theupc2(cid:7)/(cid:7)mutantconstructedintheSC5314back- beassociatedwithareductioninthelevelofexpressionofERG ground showed a reduction in baseline ERG11 expression from genes,particularlyERG11. that of its parent strain. This was also the case for the upc2(cid:7)/(cid:7) Comparison of the gene expression profiles of wild-type mutantsconstructedintheERG11K143Rand12-99backgrounds. strainSC5314andtheupc2(cid:2)/(cid:2)mutantexposedtofluconazole. However, both the TAC1G980E and MRR1P683S strains exhibited In order to identify genes whose expression in response to flu- levels of ERG11 expression lower than that of SC5314, with no conazoleisinfluencedbyUpc2,wecomparedthetranscriptional appreciable additional reduction in expression when UPC2 was profilesofSC5314anditsupc2(cid:7)/(cid:7)derivativeaftertreatmentwith July2014 Volume13 Number7 ec.asm.org 939 Vasiceketal. D o w n lo a d e d f r o m h t t p : / / e c . a s m . o r g / o n J a n u a r y 9 , 2 0 1 FIG4ExpressionlevelsofERG11,CDR1,CDR2,andMDR1.LevelsofERG11,CDR1,CDR2,andMDR1expressioninvariousstrainsweremeasuredintriplicate 9 byqRT-PCRandwerecomparedtoexpressionlevelsinSC5314(A)and2-79(B).Errorbarsrepresentthestandarderrorsofthemeans. b y g u orwithout10(cid:5)g/mlfluconazolefor6h.Geneswereconsideredto interestbyusingthesameRNAisolatedforthemicroarrayexper- e s bedifferentiallyexpressedinresponsetofluconazoleiftheirex- iments.InadditiontoERG11,fourothergeneswerechosenbased t pressionchangedby(cid:3)1.5-foldintwoindependentexperiments. ontheirinvolvementinirontransportandhomeostasis.Thecor- Fluconazole-induciblegeneswerealsoconsideredtobeUPC2de- relationbetweenthemicroarraydataandthoseobtainedbyreal- pendentiftheirinductionwasreduced(i.e.,thelevelofexpression timeRT-PCRwasgood(Fig.5).TheexpressionofCFL4,FET3, was(cid:3)2.0-fold[50%]lowerthanthatinSC5314)inthedeletion FRP1,andFTR1wasupregulatedinthewild-typestrainSC5314 mutant.Byuseofthesecriteria,therewere127genesupregulated whentreatedwithfluconazolebutcouldnotrespondtothesame byfluconazolewhoseinductionwasabrogatedintheabsenceof extentwhenUPC2wasdisrupted.Asexpected,ERG11wasalso UPC2(Table6;seealsoDataSetS1inthesupplementalmaterial). showntorespondtofluconazoleinaUPC2-dependentfashion. The most common biological processes represented by these Thesedatasuggestthattheenhancedactivityoffluconazoleob- genesincludedthelipidmetabolicprocess,ironiontransportand servedinbothsusceptibleandresistantstrainslackingUPC2may iron homeostasis, transport, responses to stress and chemical be due to dysregulation of iron homeostasis, in addition to the stimuli,andtheoxidation-reductionprocess. inabilitytoupregulategenesinvolvedintheergosterolbiosynthe- Validationofmicroarraydatabyreal-timeRT-PCR.Inorder sispathway. to validate the differential expression of genes identified by the Upc2isrequiredforgrowthunderiron-poorconditions.In microarray,weexaminedthemRNAabundancesforfivegenesof ordertoinvestigatetherelationshipbetweenUpc2andirontrans- 940 ec.asm.org EukaryoticCell Upc2IsEssentialforAzoleResistanceinC.albicans TABLE6Genesupregulatedatleast1.5-foldbyfluconazolethataredependentonUpc2 Foldchangeinexpressionbin: Ratio(foldchangein theupc2(cid:7)/(cid:7)strain/ SC5314 upc2(cid:7)/(cid:7)strain foldchangeinSC5314) Processa orf19designation CGDname Expt1 Expt2 Expt1 Expt2 Expt1 Expt2 Lipidmetabolicprocess orf19.1598 ERG24 2 1.6 0.9 0.7 0.5 0.4 orf19.1631 ERG6 1.8 1.7 0.4 0.5 0.2 0.3 orf19.2670 1.6 1.8 0.4 0.7 0.2 0.4 orf19.3240 ERG27 2.8 2.4 1.4 1.1 0.5 0.4 orf19.4982 2.1 2.3 0.7 0.8 0.3 0.4 orf19.7585 INO1 9.2 20.5 0.9 0.8 0.1 0 orf19.922 ERG11 1.6 1.5 0.8 0.7 0.5 0.4 Ironiontransport orf19.1415 FRE10 2 4.2 0.2 0.1 0.1 0 orf19.1932 CFL4 35.6 359 8 22 0.2 0.1 orf19.4211 FET3 2.8 6.2 0.7 0.2 0.3 0 orf19.4215 FET34 2.1 5.2 0 0.1 0 0 D orf19.5634 FRP1 8.9 8.2 0.1 0.2 0 0 o w orf19.7219 FTR1 3.2 7.8 0.1 0 0 0 n Ironionhomeostasis orf19.1264 CFL2 2 6 0.2 0.8 0.1 0.1 lo a orf19.1715 IRO1 4.2 8.1 2.2 1.4 0.5 0.2 d orf19.5636 RBT5 1.7 1.6 0.2 0.2 0.1 0.2 e d orf19.7114 CSA1 1.7 3.2 0.7 0.6 0.4 0.2 f r Transport orf19.1352 TIM22 3.6 4.4 1.3 2 0.4 0.4 o m orf19.2023 HGT7 20.8 21.6 9.4 7.8 0.5 0.4 orf19.2292 OPT4 5.2 3.7 0 0 0 0 h t orf19.2350 2.5 4.1 0.9 0.6 0.4 0.1 t p orf19.2785 ATP7 3.1 3.1 1.3 1.5 0.4 0.5 : / orf19.3026 MAS1 1.7 1.6 0.9 0.8 0.5 0.5 /e orf19.3195 HIP1 1.5 2.6 0.7 0.8 0.5 0.3 c . orf19.3232 24.6 5.3 1.6 1.6 0.1 0.3 a orf19.3668 HGT2 48.3 35.5 20.1 12.3 0.4 0.3 s m orf19.3746 IFC1 2.3 2.7 0.2 0 0.1 0 . orf19.4335 TNA1 194.9 29.8 0.4 0.2 0 0 o r orf19.4384 HXT5 70.6 75.5 16.9 9.7 0.2 0.1 g orf19.4682 HGT17 45 24.7 4.2 4.5 0.1 0.2 / o orf19.4690 16.3 20.2 1 1.4 0.1 0.1 n orf19.5307 JEN2 10.3 2.7 0.4 1.3 0 0.5 J orf19.5753 HGT10 20.6 1.6 2.6 0.8 0.1 0.5 a orf19.6148 4.4 47.3 2 13.8 0.4 0.3 n u orf19.6249 HAK1 5.4 5.8 1.4 1.7 0.3 0.3 a orf19.6993 GAP2 30.6 10.6 6 3 0.2 0.3 r y orf19.7093 HGT13 40.9 18.8 9.8 2.6 0.2 0.1 9 , Responsetostress orf19.1434 1.7 2.7 0.8 0.9 0.5 0.3 2 orf19.3239 CTF18 2.7 2.4 1.4 0.8 0.5 0.4 0 1 orf19.3672 GAL10 6 7.3 2.7 3.8 0.5 0.5 9 orf19.4082 DDR48 5.8 5.7 1.9 1.3 0.3 0.2 b orf19.4093 PES1 2.7 3.5 0.6 1.3 0.2 0.4 y orf19.4317 GRE3 1.5 1.5 0.3 0.7 0.2 0.5 g orf19.496 2.6 2.3 1.1 0.6 0.4 0.3 u orf19.5902 RAS2 7.6 6.2 2.3 1.5 0.3 0.2 e s orf19.7221 SET3 4.6 2.9 2.1 1.4 0.5 0.5 t orf19.921 HMS1 2.6 2.8 0.8 1.3 0.3 0.5 Responsetochemicalstimulus orf19.4645 BEM1 1.5 1.9 0.4 0.6 0.3 0.3 orf19.5591 ADO1 2.4 2.9 0.7 0.8 0.3 0.3 orf19.6102 RCA1 2.5 3.4 1.1 1.4 0.5 0.4 orf19.7374 CTA4 1.7 2.2 0.8 1 0.5 0.5 Oxidation-reductionprocess orf19.1411 2.7 4.8 1.4 1.1 0.5 0.2 orf19.1710 ALI1 2 1.9 0.7 0.8 0.4 0.4 orf19.1940 1.7 1.5 0.4 0.7 0.2 0.5 orf19.2091 2.2 2.3 0.8 0.9 0.4 0.4 orf19.2108 SOD6 7.9 10.6 1.1 1.6 0.1 0.1 orf19.4274 PUT1 6.8 8.8 2.5 0.6 0.4 0.1 orf19.4747 HEM14 1.7 2 0.1 0.2 0 0.1 aDescriptionsarefromtheCandidaGenomeDatabase(CGD)(http://www.candidagenome.org). bGivenastheratioofexpressioninthepresenceofFLCtoexpressionintheabsenceofFLC. July2014 Volume13 Number7 ec.asm.org 941 Vasiceketal. DISCUSSION Identifying novel drug targets that improve the efficacy of flu- conazoleisimportantinordertodevelopnewtherapeuticstrate- giestopreservetheazoleclassofantifungalsandovercomeazole resistance. UPC2 has been well characterized with regard to its impact on fluconazole susceptibility and its role in regulating genesoftheergosterolbiosynthesispathway(19,29,36,37).Silver etal.andMacPhersonetal.identifiedUpc2pasthekeyregulator ofergosterolmetabolisminC.albicans,showingthatazole-induc- ibleexpressionofERG2,ERG7,ERG11,andERG25isdiminished intheabsenceofUPC2(19,36).Weandothershaveestablished that in some azole-resistant isolates, specific mutations render UPC2constitutivelyactive,resultinginincreasesintheexpression of ERG genes (including ERG11), cellular ergosterol levels, and D fluconazoleresistance(25,28,29,38–41).Moreover,UPC2dis- o ruptionresultsinareductionincellularergosterolcontent(28). w n FIG5Validationoffluconazole-inducibleandUpc2-dependentirongene This suggests that UPC2 influences azole susceptibility through lo expression.LevelsofCFL4,FET3,FRP1,FTR1,andERG11expressionwere theregulationofthispathway. a d measured in triplicate by qRT-PCR and were compared to the expression In the present study, we observed that UPC2 disruption re- e levelsinSC5314.Shownaretherelativen-foldchangesingeneexpressionin sulted not only in enhanced fluconazole susceptibility as mea- d SC5314andupc2(cid:7)/(cid:7)cellstreatedwithfluconazole(FLC).Errorbarsrepresent suredbyMICsbutalsoinasubstantialreductioninfluconazole fr thestandarderrorsofthemeans. o MFCsat24,48,and72h.Indeed,UPC2disruptioninanazole- m portandhomeostasis,andtoexaminetheimpactofirononsus- susceptiblestrainpreventeditsregrowthinYPDmediuminthe h presenceofhighfluconazoleconcentrationsafter72h,resultedin tt ceptibilitytofluconazole,weexaminedthegrowthandflucona- p a clear zone of inhibition around a fluconazole Etest strip, and : zole susceptibilities of SC5314, 12-99, and their respective // upc2(cid:7)/(cid:7)derivativesinmediawithvaryingconcentrationsofiron. prerleevveannttecdognrcoewntthraotinonasooflflidumcoendaizuomlec(o1n0t(cid:5)aign/imngl)a.Tthimeraep-keiulltiacnaallly- ec. Inbrothmicrodilutionassaysusingiron-repletemedium(YPD a ysis also demonstrated a greater effect of 10 (cid:5)g/ml fluconazole s only),isolatesSC5314and12-99wereabletoresumegrowthin m againsttheupc2(cid:7)/(cid:7)mutantthanagainstitsparentstrain.Taken thepresenceofallconcentrationsoffluconazoletestedafter72h, . o together,thesedataunderscorethecontributionoftheUpc2tran- whereasiniron-poormedium(YPDplusBPS),thesestrainswere rg unabletogrowatconcentrationsexceedingtheir24-hMICs(Ta- scriptionalactivationpathwaytoazolesusceptibility. / bles 3 and 5). The MICs at all time points were 0.5 (cid:5)g/ml for We then wanted to determine if disruption of UPC2 might on SC5314and32(cid:5)g/mlfor12-99.NochangeinMFCwasobserved havesimilareffectsonfluconazole-resistantisolates.Forthispur- J forthesestrainsatanytimepoint.Meanwhile,bothupc2(cid:7)/(cid:7)mu- pose,wechoseisogenicstrainscontainingresistancemutationsin an tantsshowedlittletonogrowthatalltimepointsbasedonboth ERG11,MRR1,orTAC1.ForthestraincontainingtheERG11K143R u mutation,thefluconazoleMICwas8(cid:5)g/mlat48and72hinYPD. a MICsandMFCs.Inaniron-poormedium,thefluconazoleMIC r y forSC5314at48hbyEtestwas0.38(cid:5)g/ml,comparedto1(cid:5)g/ml Accordingly,thisstrainwasunabletogrowinthepresenceof10 9 in an iron-replete medium (Fig. 1A); however, a clear zone of (cid:5)g/mlfluconazole.Althoughthisbackgroundwasnotashighly , 2 inhibitionwasobserved(Fig.6A).AMICof(cid:9)0.016(cid:5)g/mlanda resistantasothers,itsrespectiveUPC2deletionmutantexhibited 0 clearzoneofinhibitionwereobservedfortheupc2(cid:7)/(cid:7)mutantin amarkeddropinboththeMICandtheMFCoffluconazole.The 19 aniron-poormedium(Fig.6A),comparedto0.032(cid:5)g/mlanda MICs and MFCs for the resistant MRR1P683S and TAC1G980E b clearzoneofinhibitioninaniron-repletemedium(Fig.1A).A strainswere(cid:8)64(cid:5)g/mlat48and72hinYPDmedium,andthose y MICof64(cid:5)g/mlandasmallclearzonewereobservedfor12-99in fortherespectiveupc2(cid:7)/(cid:7)mutantswerereducedmarkedlyatall gu aniron-poormedium(Fig.6A),comparedto(cid:8)256(cid:5)g/mland timepoints.Thistrendinsusceptibilityisconsistentwithwhathas e confluentgrowthinaniron-repletemedium(Fig.3A),andaMIC been observed previously for this MRR1P683Supc2(cid:7)/(cid:7) mutant st of(cid:9)0.016(cid:5)g/mlandaclearzoneofinhibitionwereobservedfor (20). However, in contrast to the halo of reduced confluent 12-99upc2(cid:7)/(cid:7)inaniron-poormedium(Fig.6A),comparedto growth observed around the Etest strip with clinical isolate 1.5(cid:5)g/mlandaclearzoneinaniron-repletemedium(Fig.3A). SC5314(andgenerallyobservedwithotherC.albicansisolates), GrowthwasalsoreducedforallstrainswhenplatedonYPDagar suchareductioningrowthwasnotobservedwiththeseresistant plates containing BPS compared to YPD alone and was further strains,despitehigherMICsandMFCsof(cid:8)64(cid:5)g/ml.Ithasbeen reducedby10(cid:5)g/mloffluconazole(Fig.6B).Only12-99andits shown recently that the constructed MRR1P683S and TAC1G980E complementedderivativewereabletogrowonYPDagarcontain- mutantsexhibitafitnessdefectassociatedwiththeintroductionof ingbothBPSand10(cid:5)g/mlfluconazole.Importantly,disruption thesespecificresistancemutations,whereasclinicalisolatescarry- ofUPC2inbothSC5314and12-99precludedthegrowthofeither ing such mutations appear to have regained fitness (42). Such strainunderiron-poorconditions.Conversely,high-ironcondi- clinicalisolateshavelikelyevolvedcompensatorymutationsthat tions(YPDplusFeCl )onlyverymodestlyenhancedtheabilities mitigate these fitness defects. This may explain the unusual 3 oftheseisolatestogrowinthepresenceoffluconazole(Tables3 growth pattern of these mutants when their susceptibilities are and5).ThesedataindicatethatUPC2isrequiredforgrowthun- testedbyEtest.Despitetheabsenceofreducedconfluentgrowth, deriron-poorconditions. theupc2(cid:7)/(cid:7)mutantsineachbackgroundshowedincreasedsus- 942 ec.asm.org EukaryoticCell