UPC2 Is Universally Essential for Azole Antifungal Resistance in Candida albicans

14 Pages·2014·3.88 MB·English

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

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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
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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
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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
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aNG,nogrowth. d

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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
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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.
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FIG4ExpressionlevelsofERG11,CDR1,CDR2,andMDR1.LevelsofERG11,CDR1,CDR2,andMDR1expressioninvariousstrainsweremeasuredintriplicate 9
byqRT-PCRandwerecomparedtoexpressionlevelsinSC5314(A)and2-79(B).Errorbarsrepresentthestandarderrorsofthemeans.
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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
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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