Gain of Function Mutations in CgPDR1 of Candida glabrata Not Only Mediate Antifungal ...

17 Pages·2008·0.94 MB·English

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
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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
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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
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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
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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,
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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
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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
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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.
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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
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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.
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