Candida tropicalis Antifungal Cross-Resistance Is Related to Different Azole Target

13 Pages·2013·1.4 MB·English

Candida tropicalis Antifungal Cross-Resistance Is Related to Different
Azole Target (Erg11p) Modifications
A.Forastiero,a*A.C.Mesa-Arango,a,bA.Alastruey-Izquierdo,aL.Alcazar-Fuoli,aL.Bernal-Martinez,aT.Pelaez,cJ.F.Lopez,d
J.O.Grimalt,dA.Gomez-Lopez,aI.Cuesta,eO.Zaragoza,aE.Melladoa
MycologyReferenceLaboratory,CentroNacionaldeMicrobiologia,InstitutodeSaludCarlosIII,Majadahonda,Madrid,Spaina;GroupofInvestigativeDermatology,
UniversidaddeAntioquia,Medellín,Colombiab;MicrobiologyandInfectiousDiseasesDepartment,HospitalGeneralUniversitarioGregorioMarañón,andDepartmentof
Medicine,FacultyofMedicine,UniversidadComplutense,Madrid,Spainc;DepartamentodeQuímicaAmbiental,InstituteofEnvironmentalAssessmentandWater
Research(IDÆA),ConsejoSuperiordeInvestigacionesCientíficas,Barcelona,Spaind;BioinformaticUnit,CentroNacionaldeMicrobiologia,InstitutodeSaludCarlosIII,
Majadahonda,Madrid,Spaine
D
CandidatropicalisranksbetweenthirdandfourthamongCandidaspeciesmostcommonlyisolatedfromclinicalspecimens. o
w
InvasivecandidiasisandcandidemiaaretreatedwithamphotericinBorechinocandinsasfirst-linetherapy,withextended-spec-
n
trumtriazolesasacceptablealternatives.Candidatropicalisisusuallysusceptibletoallantifungalagents,althoughseveralazole lo
drug-resistantclinicalisolatesarebeingreported.However,C.tropicalisresistanttoamphotericinBisuncommon,andonlya a
d
fewstrainshavereliablydemonstratedahighlevelofresistancetothisagent.TheresistancemechanismsoperatinginC.tropi- e
d
calisstrainsisolatedfromclinicalsamplesshowingresistancetoazoledrugsaloneorwithamphotericinBcross-resistancewere
f
elucidated.Antifungaldrugresistancewasrelatedtomutationsoftheazoletarget(Erg11p)withorwithoutalterationsofthe ro
m
ergosterolbiosynthesispathway.Theantifungaldrugresistanceshowninvitrocorrelatedverywellwiththeresultsobtainedin

vivousingthemodelhostGalleriamellonella.Usingthispanelofstrains,theG.mellonellamodelsystemwasvalidatedasasim- h
t
ple,nonmammalianminihostmodelthatcanbeusedtostudyinvitro-invivocorrelationofantifungalsinC.tropicalis.The tp
:
developmentinC.tropicalisofantifungaldrugresistancewithdifferentmechanismsduringantifungaltreatmenthaspotential //
a
clinicalimpactanddeservesspecificprospectivestudies. a
c
.
a
s
m
Inrecentyears,anepidemiologicalchangeamongfungalinfec- expressionseemstoplayaroleinresistance.(ii)Theupregulation .
o
tionshasbeenobserved.AlthoughCandidaalbicansisstillthe of multidrug efflux transporters (ABC [ATP-binding cassette]/ r
g
mostcommonspeciesisolatedfrompatientsinfectedwithpatho- MFS[majorfacilitatorsuperfamily])leadstodecreaseddrugcon- /

o
genicyeasts,thefrequencyofisolationofnon-albicansCandida centrationswithinthefungalcell.(iii)Abypassintheergosterol n
species has increased (1, 2). The frequency of isolates of non- biosynthesispathwaymaydevelopasaresultofmutationsinste- J
albicansCandidaspeciesdifferswithgeographicallocation.Can- rol(cid:2)5,6-desaturase(ERG3)(18–20).InC.albicans,themostfre- an
didatropicalisranksbetweenthirdandfourthamongthespecies quentazoleresistancemechanismsreportedareincreasedefflux u
a
mostcommonlyisolated(3,4),althoughitisconsideredthemost ofthedrug(85%)andmutationsintheERG11sequence(65%) r
y
frequentlyisolatedspeciesintheAsia-Pacificregion(5–7)andthe (21).Infact,numerousErg11pmutationshavebeendescribed, 3
secondmostfrequentlyisolatedspeciesinBrazilandLatinAmer- butonlyafewhavebeendirectlyassociatedwithazoleresistance 1
,
ica(20.9%and13.2%,respectively)(1,8).Moreover,C.tropicalis (22).Also,theroleofaminoacidsubstitutionshasbeenelucidated 2
isalmostalwaysassociatedwiththedevelopmentoffungalinfec- 0
byheterologousexpressionoftheERG11gene(23,24).Inaddi- 1
tions,anditisoftenrelatedtohighermortalitythanothernon- 9
tion,increasingevidencepointstoarelationshipbetweenamino
albicansCandidaspeciesandC.albicans,particularlyinneutro- b
acidsubstitutionsandchangesinproteinconformationinvolved y
penicandoncologypatients(2,9,10).
inazoleresistance(25,26).ForC.tropicalis,overexpressionofC. g
Invasivecandidiasisandcandidemiaaretreatedwithampho- u
tropicalisERG11(CtERG11)associatedwithmissensemutations e
tericinBoranechinocandinasfirst-linetherapy,withextended- s
(Y132ForS154F)hasbeendescribedastheazoleresistancemech- t
spectrum triazoles as acceptable alternatives (11, 12). Candida
anisminclinicalisolates(14,27).Additionally,invitrodevelop-
tropicalisisusuallysusceptibletoallantifungalagents,although
severalcasesoffluconazoleandazolecross-resistanceinclinical
isolateshavebeenreported(5,13,14),especiallyintheAsia-Pa-
Received8March2013 Returnedformodification16April2013
cificregion(1,15).Ontheotherhand,C.tropicalisresistanceto
Accepted12July2013
amphotericinBisrare,andveryfewstrainshavereliablydemon-
Publishedaheadofprint22July2013
stratedhigh-levelresistancetothisagent(16,17).
AddresscorrespondencetoEmiliaMellado,[email protected].
Limitedspecificinformationisavailableaboutthemolecular
*Presentaddress:A.Forastiero,LaboratoriodeMicología,HospitalBritánico,
mechanismsofresistancetoazolesinC.tropicalis,althoughthey CiudadAutonomadeBuenosAires,Argentina.
arelikelytobesimilartothoseidentifiedinotherCandidaspecies. Supplementalmaterialforthisarticlemaybefoundathttp://dx.doi.org/10.1128
Todate,Candidasp.azoleresistancehasbeenlinkedtodifferent /AAC.00477-13.
molecular mechanisms. (i) Mutations in the azole target, 14(cid:1)- Copyright©2013,AmericanSocietyforMicrobiology.AllRightsReserved.
steroldemethylase(Erg11p),resultinareductionorlossofaffin- doi:10.1128/AAC.00477-13
itywithazolesoranincapacitytobindazoles.Also,ERG11over-
October2013 Volume57 Number10 AntimicrobialAgentsandChemotherapy p.4769–4781 aac.asm.org 4769
Forastieroetal.
ment of fluconazole resistance in C. tropicalis linked to the up- Testing(AFST-EUCAST)(39).ForamphotericinB,thebreakpointswere
regulation of two multidrug efflux transporter genes, CtMDR1 definedbasedonthedistributionofMICsforwild-typestrains,deter-
andCtCDR1,hasbeenreported(28). mined by a EUCAST method (susceptibility breakpoint, (cid:1)1.0 (cid:3)g/ml)
Ingeneral,adequatemeasurementofinvitroantifungalactiv- (40).Forfluconazole,voriconazole,andanidulafungin,breakpointspro-
ityintermsofMICsisconsideredofclinicalrelevanceandoften posedbytheAFST-EUCASTwereusedtointerpretsusceptibilityresults
(41).
givesusefultherapeuticinformationforpatientmanagement.The
Metabolicinhibitorsusceptibilityassays.Thesusceptibilitiesofthe
correlationbetweeninvitroandinvivodatacanbeexploredusing
strainstodifferentclassesofmetabolicinhibitorsweredeterminedbya
differentexperimentalmodels.Amousemodelofsystemiccandi-
visualspottingassayonYNBmediumwithdifferentdrugconcentrations
diasis has been used to evaluate the effects of azole resistance
(42).StrainsweregrowninSabouraudliquidmediumasdescribedabove,
mechanismsontheefficacyofazoletreatment(29,30).Murine andcellconcentrationswereadjustedforastartingsolutioncontaining
modelsarestillconsideredthegoldstandardforthemeasurement 105cellsperml.Then10-foldserialdilutionswerecarriedout,and5(cid:3)lof
ofinvivodrugefficacybuthaveasamaindisadvantagethatalarge eachdilutionwasspottedontoagarplatescontainingeither2(cid:3)g/mlam-
number of animals are needed to determine the dose-response photericinB,10(cid:3)g/mlbrefeldinA(Sigma-AldrichQuimicaSA,Madrid,
relationshipsoftheantifungal.Increasingconcernabouttheuse Spain), 200 (cid:3)g/ml hygromycin B (Sigma-Aldrich Quimica SA), or 2 D
of mammals, i.e., the cost, the number used, and ethical issues (cid:3)g/mlterbinafine(Novartis,Basel,Switzerland).Drug-freeYNBmedium o
relatedtothepainoftheanimalsduringtheprocedures,haspro- wasincludedasacontrol.Theplateswereincubatedfor48hat37°C w
n
beforereading.
motedtheuseofalternative,nonmammalianinvertebratemodels lo
for testing microbial virulence and for screening the efficacy of Amplificationandsequencingofergosterolbiosynthesisgenes.The a
ERG11andERG3geneswerePCRamplifiedandsequencedusingapanel d
antimicrobial agents. Among these, Galleria mellonella presents e
of oligonucleotide primers (synthesized by Sigma Genosys, Madrid, d
certainbenefitsincomparisonwithothernonmammalianmodels
Spain)(seeTableS1inthesupplementalmaterial).GenomicDNAwas f
ofinfection(31).PreviousstudieshavedocumentedthatG.mel- extracted using the DNeasy Plant minikit (Qiagen Inc., Valencia, CA, ro
lonellaisavalidmodelfortheevaluationofmicrobialvirulence USA)andwasusedasthetemplateforPCRamplification.Amplification m
and/ortheefficaciesofdifferentantimicrobialagentsagainstin- conditionsconsistedof5minofdenaturationat94°C,followedby30 h
t
fectionscausedbydifferentbacteriaandfungi(32–38). cyclesof30sat94°Cfordenaturation,40sat50°Cforannealing,and50 tp
Inthiswork,weinvestigatedtheresistancemechanismsoper- sat72°Cforelongation,andbyafinalelongationstepof10minat72°C. :/
/
atinginC.tropicalisstrainsshowingazolecross-resistancealone PCRproductswerepurifiedwiththeHighPurePCRproductpurification a
a
orcombinedwithamphotericinBresistance.Wealsotestedthein kit(RocheDiagnostics,Madrid,Spain)accordingtothemanufacturer’s c
vivoantifungalresponsesofC.tropicalisstrains,bothsusceptible recommendations. PCR products were semiquantified by agarose gel .a
electrophoresisandwereusedasthetemplatesforsequencing,whichwas s
andresistanttoamphotericinB,fluconazole,andvoriconazole,in m
performed with the Dye Terminator cycle sequencing quick-start kit
themodelhostG.mellonellainordertocorrelatethesusceptibility .o
(BeckmanCoulterInc.,Fullerton,CA).ThesequencingPCRproducts
phenotypesofthosestrainswiththoseshowninvitro. werethenpurifiedthroughG50Sepharosecolumns(AmershamBiosci- rg
/
ences)and,finally,weresequencedonaCEQ8000DNAanalysissystem o
MATERIALSANDMETHODS (BeckmanCoulter)withtheforwardandreverseprimersusedforPCR n

J
Strains,media,andgrowthconditions.FourCandidatropicalisstrains amplification(seeTableS1inthesupplementalmaterial). a
Theaminoacidsequencesofthegenesencodingputative14(cid:1)-sterol n
were used in this study: (i) two ATCC strains, ATCC 750 and ATCC
200956,and(ii)twoclinicalisolates(CL-6835andTP-13650)recovered demethylase(ERG11)andsterol(cid:2)5,6-desaturase(ERG3)werededuced ua
fromtwopatientswithcandidemiawhohadbeentreatedwithantifungal fromnucleotidesequencesandwereanalyzedusingtheMegAlignsoft- ry
drugspreviously(seeTable1).Theseisolateswereidentifiedbymorpho- warepackage(DNAStar,Inc.,Lasergene,Madison,WI,USA).Theamino 3
logicalfeaturesandwereconfirmedbyDNAinternaltranscribedspacer acidalignmentswerederivedbyCLUSTALanalysis(43). 1
,
(ITS)sequencing.YeastcellsweregrownovernightinSabouraudliquid Sterolanalysis.SterolswereextractedfromC.tropicalisafter18hof 2
medium(Becton,DickinsonandCompany,MD,USA)at30°Cwithcon- growthinSabouraudliquidmediumat37°Cwithshaking(150rpm), 0
1
stantshaking(150rpm).Cellswereharvestedbycentrifugationandwere usingtheprotocoldescribedpreviously(44,45).Briefly,cellswerehar- 9
washedtwicewithsteriledistilledwater.Inoculumsizeswereadjusted vested by centrifugation, washed with sterile water, lyophilized, and b
usingahemocytometerchamber.Yeastnitrogenbase(YNB)solidme- weighedtodeterminethedryweight(ingrams)ofthefungus.About150 y
diumwasusedinthemetabolicinhibitorassays. to200mgwasusedfortotal-sterolextraction.A3-mlvolumeofa25% gu
Antifungaldrugs.AmphotericinB(AMB)(Sigma-AldrichQuimica, alcoholic potassium hydroxide solution (methanol-ethanol, 3:2) was e
Madrid,Spain),fluconazole(FLC)(PfizerSA,Madrid,Spain),voricona- addedtothedriedpelletsandwasmixedbyvortexingfor1min.The st
zole(VRC)(PfizerSA),andanidulafungin(ANF)(Merck&Company, mixturewasincubatedat85°Cinawaterbathfor1h.Neutrallipidswere
Inc.,NJ,USA)wereusedforthedeterminationofMICsandforassess- extractedtwicebyadditionofamixtureof1mlofwaterand1.5mlof
mentoftheirefficaciesagainstC.tropicalisinfectionintheGalleriamel- hexane,followedbyvigorousvortexmixingfor3min.Theupperhexane
lonellamodel.Theantifungaldoseswerecalculatedbytakingintoaccount layer was transferred to a clean glass tube, evaporated, and stored at
thetherapeuticdoseusedforhumansandtheMICvaluesforthestrains (cid:4)20°Cuntilanalysis.Onehundredmicrogramsofandrostanol(Sigma)
studied(11).Stocksolutionsofvoriconazole,fluconazole,amphotericin wasaddedtothesamplesasaninternalstandardforsterolrecoveryand
B,andanidulafunginwerefirstpreparedindimethylsulfoxide(DMSO), quantification.Sterolswereconvertedintotheirtrimethylsilylethersby
andthendilutionsinwaterforinjectionweremadetoobtainthefollow- reactionwitha1:1solutionofN,O-bis-(trimethylsilyl)trifluoroacetamide
ingconcentrations:400and80(cid:3)g/mlofvoriconazole;800,360,and80 (85°C,60min)intoluene.NeutrallipidfractionswereanalyzedonaTrace
(cid:3)g/ml of fluconazole; 120 and 80 (cid:3)g/ml of amphotericin B; and 400 gas chromatograph coupled to a quadrupole mass analyzer (Thermo
(cid:3)g/mlofanidulafungin. FisherScientific,Manchester,UnitedKingdom).TheGCprogramand
Antifungalsusceptibilitytesting.Antifungalsusceptibilitywasdeter- MSoperationconditionswerethesameasthosedescribedpreviously(45,
minedbybrothmicrodilution(BMD).MICsweredeterminedbyfollow- 46).Sterolidentificationwasbasedoninterpretationandcomparisonof
ingtherecommendationsoftheAntifungalSusceptibilityTestingSub- massspectraandretentiontimedatawithpreviousdata(45,47).The
committee of the European Committee on Antibiotic Susceptibility amountofeachsterol(inmicrograms)relativetothedryweight(in
4770 aac.asm.org AntimicrobialAgentsandChemotherapy
ResistanceofCandidatropicalistoAntifungalDrugs
grams)ofthefunguswascalculatedfromthreebiologicalreplicates referencesequence(GenBankaccessionnumberM23673).Thefullnu-
analyzed in triplicate byGC-MS.Therelativesterolcompositionwas cleotidesequencesoftheERG11genesofATCC750,ATCC200956,and
expressed as the ratios of individual sterol concentrations to the total thetwoC.tropicalisclinicalisolates,CL-6835andTP-13650,andtheir
sterolcontent.Thesignificanceofdifferencesinsterolcomposition(P(cid:5) deducedaminoacidsequenceshavebeendepositedintheGenBankda-
0.0001)wasdeterminedbyanonparametricMann-Whitneytest. tabaseunderaccessionnumbersKC542323,KC542324,KC542325,and
HomologymodelingofErg11p.Homologymodelsofwild-typeC. KC542326,respectively.ThefullnucleotidesequenceoftheERG3gene
tropicalisErg11pandmutatedsequencescontainingaminoacidsubstitu- (encoding sterol (cid:2)5,6-desaturase) of C. tropicalis MYA-3404 was used
tions G464D, G464S, Y132H, and Y132F were constructed using the as the reference sequence for ERG3 (GenBank accession number
SWISS-MODEL platform (http://swissmodel.expasy.org ) 4(8–50). The XM002550136).ThefullnucleotidesequencesoftheERG3genesofthe
crystalstructureofhumanlanosterol14(cid:1)-demethylase(HS-CYP51)in fourstrainsusedinthisstudy(ATCC750,ATCC200956,CL-6835,and
complexwithketoconazole,depositedintheProteinDataBank(http: TP-13650)havebeendepositedintheGenBankdatabaseunderaccession
//www.rcsb.org) with code 3LD6 (51), was used as the template. The numbersKC542319,KC542320,KC542321,andKC542322,respectively.
amino acid sequence identity between this template and C. tropicalis
Erg11pwas39%.TheN-terminalregionofC.tropicalisErg11p(residues RESULTS
1 to 50), defined to comprise the membrane-spanning domains, and Antifungalsusceptibilitytesting.TheMICvaluesoffluconazole
D
thereforewithoutcounterpartsinthetemplateprotein,wasdeletedprior (FLC),voriconazole(VRC),amphotericinB(AMB),andanidu- o
toproteinmodeling. lafungin (ANF) are shown in Table 1. Candida tropicalis strain w
n
AnalysisofR6Guptakeandefflux.Theactivityofeffluxpumpsin ATCC 750 was susceptible to all the antifungals tested. Isolate lo
azole- and/or amphotericin B-susceptible and -resistant C. tropicalis TP-13650wasresistanttofluconazoleandvoriconazolebutsus- a
strains(ATCC200956,ATCC750,CL-6835,andTP-13650)wasevalu- d
ceptible to amphotericin B and anidulafungin. Resistance to all e
ated using rhodamine 6G (R6G) according to the protocol previously antifungal drugs except anidulafungin was observed for strains d
describedbyVandeputteetal.in2005(14)andbyMaesakietal.in1999 f
ATCC200956andCL-6835. r
(52).AccumulationofR6GwasmeasuredbyflowcytometryintheFL2 o
channel(collectingtheemissionsignalat585(cid:6)42nm).Atotalof10,000 Metabolicinhibitorsusceptibilityassays.Thesusceptibility m
cells were recorded using a FACSCalibur cytometer (FL2 channel; ofC.tropicalisstrainstodifferentmetabolicinhibitorswasusedas h
CellQuest;BDBiosciences),andthefluorescenceintensityofR6G-stained a screening method to identify those yeasts with alterations in ttp
cellswasdetermined.Unstainedcellswereincludedasanegativecontrol membrane fluidity. Candida tropicalis strains with cross-resis- :/
/
inallcases.DatawereprocessedusingFlowJosoftware(version7.1.6; tancetoazoledrugsandamphotericinBweremoresusceptibleto a
a
TreeStarInc.,Ashland,OR,USA).Experimentswereperformedatleast metabolicinhibitors(brefeldinAorhygromycinB)andtoterbi- c
twicewitheachstrainondifferentdays.Resultswereexpressedasmean nafinethanthewild-typestrainorthestrainwithazoledrugre- .a
fluorescenceintensities(MIF)andstandarddeviations(SD). sistanceonly.Changesinmembranefluiditywereclearlydetect- sm
Evaluation of antifungal efficacy in Galleria mellonella infected
ablewhenbrefeldinAwasused(seeFig.S2inthesupplemental .
o
withC.tropicalis.Galleriamellonellacaterpillarsinthefinalinstar(stage
oflarvaldevelopment)(R.J.MousLiveBait,theNetherlands)wereused. material).TheseresultssuggestthatamphotericinB-resistantiso- rg
Larvaewerestoredinthedarkat25°Cpriortouse.Tencaterpillars(body lates may have an alteration of the membrane that increases its o/
weight, 400 (cid:6) 100 mg) were employed per group. Candida tropicalis permeability,andtherefore,thesecompoundscouldpassthrough n

strainsweregrowninliquidSabouraudmediumasdescribedabove.Pre- themembranerelativelyeasily,resultinginthehypersusceptible Ja
liminary experiments were performed with all the strains to define an phenotypeobservedinvitro. n
appropriate range of inoculum concentrations that would cause 80 to ERG11 and ERG3 gene sequence analyses. (i) ERG11 gene ua
100%mortalityat72to96hpostinfection.Theinoculaweredetermined sequence. No changes were observed between the ERG11 se- r
y
bycountinginahemocytometerchamberandwereadjustedinphos- quenceofstrainATCC750determinedhereandthatdepositedin 3
phate-bufferedsaline(PBS)to2(cid:7)108cellspermlforATCC750,CL- GenBank(accessionnumberM23673).ComparisonoftheERG11 1
6855,andTP-13650andto4(cid:7)108cellspermlforATCC200956.Viable gene sequence of strain ATCC 200956 with the available corre- , 2
countswereperformedtoconfirmthecorrectsizeoftheinocula. 0
A10-(cid:3)lHamiltonsyringe(FisherScientific,Madrid,Spain)wasused sbppotnhdaitncgosrerqesupeonncdesfotorstthreaianbAseTnCceCo7f5404raemveianleodaaciddeslewtiitohninofth1e32I 19
toinfectthelarvaebyinjecting10(cid:3)loftheinoculumsuspensionintothe helix((cid:2)276–319)oftheprotein(Table1;seealsoFig.S1Binthe b
hemocoelofeachG.mellonellathroughthelastleftproleg.Within2hof y
the infection, 10 (cid:3)l of an antifungal solution (either 400 or 80 (cid:3)g/ml supplementalmaterial)andanaminoacidsubstitution(D275V) g
voriconazole,800,360,or80(cid:3)g/mloffluconazole,120or80(cid:3)g/mlof with restoration of the open reading frame. PCR amplification ue
amphotericinB,or400(cid:3)g/mlofanidulafungin)wasinjectedintoadif- usingtwoprimercombinationswithoneoftheoligonucleotides s
t
ferentprolegusingthesametechnique.Afterantifungaltreatment,cater- designedwithinthedeletedsequenceclearlyshowedthatthede-
pillarswereincubatedat37°Cfor7days.Larvasurvivalwasmonitored letionhadoccurredinthetwoalleles(seeFig.S4inthesupple-
daily, and larvae were considered dead when they did not respond to mentalmaterial).Therefore,strainATCC200956washomozy-
physical pressure. The following control groups were included: un- gous for the ERG11 gene deletion. Analysis of the ERG11 gene
touchedlarvae,piercedlarvae,larvaereceivingPBS,andlarvaegiventoxic sequencesofstrainsCL-6835andTP-13650revealeduniquemis-
dosesofantifungals(400(cid:3)g/mlofvoriconazole,800(cid:3)g/mlofflucona- sensemutationsforeachstraincorrespondingtoaminoacidsub-
zole,120(cid:3)g/mlofamphotericinB,or400(cid:3)g/mlofanidulafungin).Each
stitutionsG464DandY132F,respectively(seeFig.S1CandAin
experiment was performed at least three times. and the results are re-
thesupplementalmaterial).Silentmutationswerealsoobserved
portedasmeanvalues.Tosimplifysomeofthefigures,thePBScontrol
inthesestrains(Table1).
groupresultsarenotpresented.
(ii)ERG3genesequence.Thefullnucleotidesequenceofthe
Thesurvivalrateofcaterpillarswasplottedagainsttime,andPvalues
ERG3 gene (encoding sterol (cid:2)5,6-desaturase) of C. tropicalis
werecalculatedbythelogrank(Mantel-Cox)testusingGraphPadPrism,
version5.Pvaluesof(cid:5)0.01wereconsideredsignificant. MYA-3404wasusedasareferencesequenceforERG3(GenBank
Nucleotide sequence accession numbers. The full nucleotide se- accessionnumberXM002550136).SequencingoftheERG3gene
quence of the ERG11 gene of C. tropicalis ATCC 750 was used as the showed single missense mutations, C773T and A334G, in the
October2013 Volume57 Number10 aac.asm.org 4771
Forastieroetal.
TABLE1MutationsinERG11andERG3genes,correspondingaminoacidsubstitutions,andMICsofantifungaldrugsforCandidatropicalis
isolates
Genesequenceanalysisb
14(cid:1)-Steroldemethylase Sterol(cid:2)5,6-desaturase MIC((cid:3)g/ml)c
Isolate Previoustreatmenta ERG11(bp) Erg11p(aa) ERG3(bp) Erg3p(aa) AMB FLC VRC ANF
ATCC750 Unknown G366A R122R 0.25 0.5 0.03 0.03
ATCC200956 Unknown (cid:2)132bp (cid:1)44aa C773T S258F 2 (cid:9)64 (cid:9)8 0.03
—d D275V
G1362A K454K
C1534G P511A
T1554C I517I
CL-6835(clinical) FLC,AMB,andCAS T906C I302I A334G S113G 2 (cid:9)64 (cid:9)8 0.03
G1391A G464D G366A R122R
T637C L212L D
TP-13650(clinical) FLC,VRC,andTRB T225C C75C 0.25 (cid:9)64 (cid:9)8 0.03 o
w
G264A L88L n
A395T Y132F lo
T783C I261I a
d
G1362A K454K e
T1554C I517I d

f
aCAS,caspofungin;TRB,terbinafine. ro
bSubstitutionsinaminoacidsequencesareshowninboldface. m
cDeterminedbybrothmicrodilutionaccordingtotheestablishedmethodofAFST-EUCAST.AMB,amphotericinB;FLC,fluconazole;VRC,voriconazole;ANF,anidulafungin.
EUCASTantifungalclinicalbreakpointsforC.tropicalisareasfollows:forAmB,(cid:1)1(cid:3)g/mlforsusceptibilityand(cid:9)1(cid:3)g/mlforresistance;forFLC,(cid:1)2(cid:3)g/mlforsusceptibilityand ht
(cid:9)4(cid:3)g/mlforresistance;forVRC,(cid:1)0.12(cid:3)g/mlforsusceptibilityand(cid:9)0.12(cid:3)g/mlforresistance;andforANF,(cid:1)0.06(cid:3)g/mlforsusceptibilityand(cid:9)0.06(cid:3)g/mlforresistance. tp
d—,132-bpdeletion,betweennucleotides824and945,resultsina44-amino-aciddeletion,betweenaa276and319.Thechangeintheopenreadingframeisthecauseofthe :/
/
D275Vaminoacidsubstitution. a
a
c
.
a
s
ERG3sequencesofATCC200956andCL-6835,respectively,re- cellsbythesametypesoftransportersasthoseusedforazolesin m
sultingintheS258FandS113Gaminoacidsubstitutions.Inad- yeasts(52).TherewerenoremarkabledifferencesamongtheR6G .
o
dition,CL-6835presentedtwopointmutationsintheERG3gene uptake/efflux ratios of the different C. tropicalis strains, despite rg
(T637CandG366A)thatdidnotaffecttheaminoacidsequence. theirdifferentantifungaldrugsusceptibilityprofiles(seeFig.S5in /
o
NeitherATCC750norisolateTP-13650presentedanymissense the supplemental material). Levels of retention of R6G, after n

mutationsintheentireERG3genesequence(Table1). washingwithPBSandincubationinfreshYPDmedium(15min), J
a
Sterol analysis. To investigate the contribution of mem- were similar for all strains. The percentages of retention of the n
branesterolcompositiontoantifungaldrugresistance,theste- totalR6Gquantifiedinitiallyrangedfrom58to83%.Somediffer- u
a
rolprofilesofsusceptibleandresistantstrainswereanalyzedby encesintheinitialmeanfluorescenceintensities(MIF)wereob- r
y
gas chromatography (GC)–mass spectrometry (MS). Sterols served.ForstrainsATCC750andTP-13650,bothsusceptibleto 3
wereclassifiedaccordingtotheirGCretentiontimesandmass amphotericinB,theMIFwere239(cid:6)28and365(cid:6)196,respec- 1
,

spectra,andatotalof22sterolsweredetected(Table2).The tively.Incontrast,fortheamphotericinB-resistantstrainsATCC 2
0
relative sterol composition for each strain is given in Table 2 200956andCL-6835,theintensitiesoffluorescencewerehigher 1
based on the retention times of the sterols. Ergosterol was (1,881(cid:6)28and949(cid:6)4,respectively). 9

b
found to be the major sterol in strains ATCC 750 and TP- HomologymodelingofErg11p.Thecrystalstructureofhu-
y
13650.Incontrast,noergosterolwasdetectedineitherATCC manlanosterol14(cid:1)-demethylase(HS-CYP51)wasusedtogener- g
200956orCL-6835.Thisabsenceofergosterolsuggestedpos- atea3-dimensional(3D)homologymodeloftheaminoacidse- u
e
siblealterationsinthesterolcontentsofthesetwostrains.We quenceofwild-typeC.tropicalisErg11p.Thehemecofactorwas s
t
then grouped these metabolites according to the presence of extractedfromtheHS-CYP51structureandwasmergedintothe
themethylgrouponcarbonatom14.FigureS3inthesupple- modelsobtained.Alignmentofthetwosequencesrevealedthat
mentalmaterialshowsstatisticallysignificantaccumulationsof there were no insertions or deletions in any of the secondary-
C-14-methylated sterols in ATCC 200956 and CL-6835 that structureregionsoftheprotein.Thefungus-specificinsertionin
werenotobservedinstrainsATCC750andTP-13650.C-14- theC-terminalpartofthesequencewasmodeledasaloopandwas
methylated sterols in ATCC 200956 and CL-6835 comprised supportedbytheANOLEAprediction(53),whichreportedneg-
14-methyl fecosterol, 14-methylcholesta-8,24-dien-3(cid:8)-ol, ativeenergyvaluesformostoftheresiduesofthissection.More-
4,14-dimethylcholesta-8,24-dien-3(cid:8)-ol,lanosterol,andeburi- over,noneoftheresiduesstudiedinthispaperwerelocatedwithin
col. thisregionofthemodel;rather,theywerelocatedinconserved
AnalysisofR6Guptakeandefflux.Rhodamine6G(R6G)isa regions that have been intensively studied, as discussed below.
fluorescent substrate of the multidrug transporter superfamily Therefore,modelsforErg11pwiththeG464SorY132Fsubstitu-
(conferring multidrug resistance [MDR]) that has already been tion were generated using the same approach. Modeling of the
usedtodemonstratetheactivityoftheseproteinsinazole-resis- partiallydeletedErg11pfromstrainATCC200956wasnotpossi-
tantisolatesofCandidaalbicans.Infact,thisdyeisexportedfrom ble because of the lack of sufficient identity with the available
4772 aac.asm.org AntimicrobialAgentsandChemotherapy
ResistanceofCandidatropicalistoAntifungalDrugs
TABLE2SterolcompositionsofCandidatropicalisstrains
Sterol %oftotalsterolsainstrain:
Compound
no. Systematicnameb Commonname ATCC750 ATCC200956 CL-6835 TP-13650
1 Squalene 8.54 0.17 1.45 ND
2 24-Methylcholesta-5,7,9(11),22-tetraen-3(cid:8)-ol 0.15 ND ND ND
3 S28:3 0.08 ND ND ND
4 Cholesta-8,24-dien-3(cid:8)-ol Zymosterol 8.97 ND ND 20.60
5 14-Methylcholesta-8,24-dien-3(cid:8)-ol ND 0.11 0.07 ND
6 24-Methylcholesta-5,7,22-trien-3(cid:2)-ol Ergosterol 64.96 ND ND 60.84
7 24-Methylcholesta-7,22-dien-3(cid:8)-ol 0.20 ND ND 0.51
8 24-Methylcholesta-5,7,22,24(28)-tetraen-3(cid:8)-ol 0.81 ND ND 5.20
9 14,24-Dimethylcholesta-8,24(28)-dien-3(cid:8)-ol 14-Methylfecosterol ND 61.08 52.67 ND
10 24-Methylcholesta-8,24(28)-dien-3(cid:8)-ol Fecosterol 1.91 ND ND 2.31
11 4,14-Dimethylcholesta-8,24-dien-3(cid:8)-ol 0.12 1.64 0.63 ND D
12 4(cid:1)-S28:2 ND ND ND 0.21 o
13 24-Methylcholesta-5,7,24(28)-trien-3(cid:8)-ol 2.56 ND ND 3.43 w
14 24-Methylcholesta-5,7-dien-3(cid:8)-ol 4.11 ND ND 2.23 n
15 24-Methylcholesta-7,24(28)-dien-3(cid:8)-ol Episterol 4.45 ND ND 2.06 lo
a
16 24-Methylcholest-7-en-3(cid:8)-ol ND ND ND 0.03 d
17 4(cid:1),4(cid:8),14-Trimethylcholesta-8,24-dien-3(cid:8)-ol Lanosterol 2.32 25.80 27.75 1.83 e
d
18 4(cid:1),24-Dimethylcholesta-8,24(28)-dien-3(cid:8)-ol 0.11 ND ND ND
f
19 4(cid:1),4(cid:8)-Dimethylcholesta-8,24(28)-dien-3(cid:8)-ol 0.53 ND ND 0.65 ro
20 4(cid:1),4(cid:8),14,24-Tetramethylcholesta-8,24(28)-dien-3(cid:8)-ol Eburicol 0.15 8.68 17.42 0.10 m
21 4(cid:1),4(cid:8),24-Trimethylcholesta-8,24(28)-dien-3(cid:8)-ol 0.03 ND ND ND h
22 Unidentified ND 2.51 ND ND tt
p
aND,notdetected. :/
/
bS28:3and4(cid:1)-S28:2refertosterolswith28carbonatomsand3or2doublebonds,respectively. a
a
c
.
a
crystal structures of proteins that could be useful as templates. acid substitutions affected the heme environment in different s
m
Therefore,thismodelisnotshownbutisdiscussedbelow.Figure ways.Thewild-typestrainshowsatyrosineatposition132(Fig.
.
1Ashowsthepositionsofthemutationsanalyzed,bothcloseto 1B),whichinteractsdirectlywiththehemethroughthehydroxyl o
r
the heme group. The different models revealed that the amino group. Two mutations were analyzed for this site, Y132F and g
/

o
n

J
a
n
u
a
r
y

3
1
,

2
0
1
9

b
y

g
u
e
s
t
FIG1PositionsofthepointmutationsofCandidatropicalisErg11panalyzedaccordingtothehomologymodelsgeneratedinthisstudy.(A,B,andD)Wild-type
CandidatropicalisErg11p(Y132andG464).(C,EandF)Positionsofthemutationsanalyzedintheprotein:Y132F,G464D,andG464S,respectively.Theresidues
studiedareshowninstickrepresentation(green,carbonatoms;red,oxygenatoms;blue,nitrogenatoms).Hemeisrepresentedinred.Thedistancebetweenthe
hemeandtheclosestatomoftheaminoacidstudiedisindicatedbyadashedgreenlineandanumbergivingthedistanceindaltons.Thecrystalstructureof
human lanosterol 14(cid:1)-demethylase (HS-CYP51) in complex with ketoconazole (Protein Data Bank code 3LD6) was used as a template. Y, tyrosine; F,
phenylalanine;G,glycine;D,asparticacid;S,serine.
October2013 Volume57 Number10 aac.asm.org 4773
Forastieroetal.
Y132H,butonlytheformer(theonefoundinthiswork)isshown. nisms responsible for azole drug resistance in Candida species
Interactionoftheproteinwiththehemegroupwouldbeavoided havebeenextensivelystudiedandreviewed(18–20,55).InCan-
because of the absence in the F residue of the hydroxyl group didaalbicans,numerousERG11mutationshavebeendescribed,
presentintheYresidue,positionedinthe3-Åenvironmentofthe but only a few of them have been associated with amino acid
hemegroup(Fig.1C).Anothertwomutationswereanalyzedfor substitutionsandazoleresistance(22).Themostfrequentlyre-
theaminoacidatposition464.IncontrastwithY132,glycineat portedmodificationsarethesubstitutionsY132H,D278E,S405F,
position464(Fig.1D)isclosetothehemegroupbutnotwithin G464S,andR467K(14,22).However,littleinformationisavail-
the3-Åenvironmentandprobablydoesnotinteractwithit.In ableconcerningthemolecularmechanismsleadingtoazoleresis-
contrast,thesubstitutionG464SorG464Dmightaffecttheenvi- tanceinthepathogenicyeastC.tropicalis.Acquiredresistanceto
ronment of the heme group, changing the interaction, due not azoledrugsinclinicalisolatesofC.tropicalisduetooverexpres-
onlytothenucleophiliccharacterofserine(Fig.1F)ortheacidic sion of a CtERG11 gene associated with missense mutations
characterofasparticacid(Fig.1E)butalsotothefactthattheside (Y132FandS145F)hasbeenreported(13,26).
chainsofthesetwoaminoacidsarelargerthanthatofglycine. Inthisstudy,weintensivelyinvestigatedthemolecularbases
Galleria mellonella in vivo infection model and antifungal forC.tropicalisstrainspresentingapatternofresistancetoazole
D
response.Galleriamellonellalarvaewereinfectedwithaninocu- drugs,aloneorincombinationwithresistancetoamphotericinB. o
lumof2(cid:7)106CFU/larvawhenstrainATCC750,CL-6835,or Cross-resistancetoazolesandamphotericinBdrugswasobserved w
n
TP-13650wasused.Aninoculumof4(cid:7)106CFU/larvawasused in two of the C. tropicalis strains analyzed (ATCC 200956 and lo
forATCC200956,sincethisstrainshowsreducedvirulenceinthis CL-6835),whilestrainTP-13650wasresistantonlytoazoledrugs. a
d
model(37).Inallcases,80%ofinfectedanduntreatedlarvaedied We also confirmed this in vitro phenotype in vivo by using an e
betweenthe3rdand4thdaypostinfection.Foreachstraininocu- alternativeminihostmodelofcandidiasis. d

f
lated,thesurvivalofthelarvaewasdependentonboththeanti- Enhanced drug efflux activation is one of the major mecha- r
o
fungaldoseadministeredandtheinvitroMIC(Fig.2to4). nismscausingazoleresistanceinyeast,althoughitcouldbeac- m
Theresultsshowedthatattherapeuticdosesofazoledrugs(10 companiedbyotherresistancemechanisms.Thismechanismisa h
mg of voriconazole/kg of body weight/day, equivalent to 4 (cid:3)g/ consequenceoftranscriptionalactivationofgenesencodingdrug tt
p
larva,and9mgoffluconazole/kgofbodyweight/day,equivalent effluxpumpproteinsbelongingtotheABC(encodedbyCandida :/
to 3.6 (cid:3)g/larva), larvae infected with azole-susceptible strain drugresistance[CDR]genes)andmajorfacilitator(encodedby /a
a
ATCC750showedsignificantlyimprovedsurvivalrelativetothat MDRgenes)superfamiliesoftransporters(20).Inordertosearch c
.
of their untreated control groups (P, 0.0033 for both VRC and forconcomitantresistancemechanismsrelatedtoincreasedefflux a
s
FLC)(Fig.2).Incontrast,whenG.mellonellalarvaewereinfected pump activity, we tested this possibility using rhodamine 6G m
withanazole-resistantstrain(ATCC200956,TP-13650,orCL- (R6G).Thiscompoundisafluorescentsubstrateofthemultidrug .
o
6835)andtreatedwithatherapeutic(10mgofvoriconazole/kg/ transporter superfamily and has been used to demonstrate the rg
day, equivalent to 4 (cid:3)g/larva, or 9 mg of fluconazole/kg/day, activityoftheseproteinsinazole-resistantisolatesofC.albicans, /
o
equivalent to 3.6 (cid:3)g/larva) or subtherapeutic (2 mg/kg/day, becausethisdyeusesthesametransportersasazolesinyeasts(52). n
equivalent to 0.8 (cid:3)g/larva) dose of an azole, the percentages of OurresultsshowedthatallstrainscapturedR6G,althougham- J
a
survivalofthesegroupsdidnotdiffersignificantlyfromthoseof photericinB-resistantstrainsshowedhigheruptake/effluxratios n
theirrespectiveuntreatedcontrolgroups(Fig.2).Inaddition,an than amphotericin B-susceptible strains at time zero. This phe- u
a
increaseinthefluconazoledosageto20mg/kg/day(equivalentto nomenoncouldbeexplainedbyhighermembranepermeability r
y
8(cid:3)g/larva)failedtoimprovesurvivalwhenlarvaewereinfected duetochangesinthesterolcompositionofthemembrane.How- 3
withthesestrains(Fig.2). ever,analysisoftheuptakeandeffluxofR6Gforallazole-resistant 1
,
Amphotericin B treatment (1.2 (cid:3)g/larva, equivalent to a orwild-typeC.tropicalisstrainsshowsthattheeffluxpumpsdo 2
0
therapeuticdoseof3mg/kg/day)improvedthesurvivaloflar- notseemtoplayafundamentalroleinazoleresistanceinC.tropi- 1
vaeinfectedwithstrainATCC750(P,0.0015)orTP-13650(P, calis,inagreementwithpreviousresults(14,27). 9

b
0.0002) but not that of those infected with a strain (ATCC The antifungal susceptibility phenotype of strains ATCC
y
200956orCL-6835)cross-resistanttoazoleandamphotericin 200956andCL-6835resembledthatofC.albicansERG11/ERG11 g
B(Fig.3). doublemutantstrainsshowingresistancetoazolesaswellasto u
e
In contrast, anidulafungin was the most active antifungal amphotericinBoneitherthewild-typeortheERG3/ERG3mutant s
t
drugagainstallstrainsregardlessoftheirazole/amphotericinB background(42).Also,ourdatawereconsistentwiththoseforthe
susceptibilityprofiles,withalarvalsurvivalrangebetween30 C. albicans ERG11-null mutation when the strains were tested
and 50% at the end of the experiment (Fig. 4). None of the againstdifferentmetabolicinhibitors(seeFig.S2inthesupple-
drugsweretoxictothelarvaeatanyoftheconcentrationsused mental material). These data suggest that the susceptibilities of
(datanotshown). bothantifungalcross-resistantC.tropicalisstrainstothesemeta-
boliccompoundsmightberelatedtochangesinthecomposition
DISCUSSION andpermeabilityofthefungalmembraneandarelikelyassociated
Candida tropicalis is usually susceptible to all antifungal agents. withthefunctionalityofErg11pandalterationsinergosterolbio-
However,antifungalshavebeenusedextensivelyfortheprophy- synthesis(42,56).Sterolanalysisconfirmedtheabsenceofergos-
laxisortreatmentofcandidiasis,andthereareincreasingreports terol(Table2)andtheaccumulationof14-methylsterolsinC.
of azole resistance for this species (13, 14, 27, 54). In fact, the tropicalisstrainsATCC200956andCL-6835(Fig.5;seealsoFig.
azole-resistantclinicalisolatesofC.tropicalisstudiedinthiswork S3 in the supplemental material). In fact, this sterol profile was
wererecoveredfromtwopatientssufferingfromcandidemiaand similar to those of different Candida species with ERG11 gene
treatedwithdifferentantifungals(Table1).Theprecisemecha- deletions(42,56).Sequencingandstructuralanalysisofthepro-
4774 aac.asm.org AntimicrobialAgentsandChemotherapy
ResistanceofCandidatropicalistoAntifungalDrugs
D
o
w
n
lo
a
d
e
d

f
r
o
m

h
t
t
p
:
/
/
a
a
c
.
a
s
m
.
o
r
g
/

o
n

J
a
n
u
a
r
y

3
1
,

2
0
1
9

b
y

FIG2Efficaciesoffluconazole(FLC)andvoriconazole(VRC)forGalleriamellonellainfectedwithdifferentstrainsofCandidatropicalis.Tenlarvaepergroup g
wereinfectedwith2(cid:7)106cellsofC.tropicalisATCC750,CL-6835,orTP-13650.ForC.tropicalisATCC200956,aninoculumof4(cid:7)106cellswasused.After u
e
inoculation,larvaeweretreatedwith0.8or4(cid:3)gofVRC/larva(subtherapeuticandtherapeuticdoses,respectively)orwith0.8,3.6,or8(cid:3)gofFLC/larva s
(subtherapeuticandtherapeuticdosesandtwicethetherapeuticdose,respectively).Anuntreatedcontrolgroupwasincluded.Larvasurvivalwasmonitored t
daily;thelarvaeareconsidereddeadwhentheydonotrespondtotouch.
teinsshowedthatstrainATCC200956hasadeletionof44amino proteinthatislacking(helixI)islikelytobethecauseofthelackof
acidsinErg11pthatcorrespondstotheexcisionofthe“I”helixin interaction either with the substrate (lanosterol) or with azole
theazoletarget.Thatdeletionwouldpresumablyrenderthepro- drugs (26, 57). In fact, in Candida glabrata and Saccharomyces
teinnonfunctional,whichwouldexplainboththelackofergos- cerevisiae,asinglemutationatthatlevelisenoughtonullifythe
terolandtheaccumulationof14(cid:1)-methylatedsterols,andthere- functionofnativeErg11p(56,58).
foretheresistancetoamphotericinB.Infact,duringtherevision The effects of Erg11p mutations at G464 and Y132 on azole
ofthiswork,anidenticalazole/amphotericinBresistancemecha- susceptibilityhavebeenreportedpreviouslyindifferentyeastsand
nismwasdescribedinaclinicalisolateofC.tropicalisisolatedin filamentousfungi(59–63).TheY132HandG464Smodifications
Tunisia(54). conferredazoleresistanceonC.albicans(59).Also,theeffectsof
ExaminationoftheC.albicansErg11p3Dproteinmodelsin differentERG11mutationshavebeenexploredbyheterologous
combination with azole drugs suggests that the portion of the expression in S. cerevisiae (24, 64). In addition, several studies
October2013 Volume57 Number10 aac.asm.org 4775
Forastieroetal.
D
o
w
n
lo
a
d
e
d
FIG3EfficacyofamphotericinB(AMB)inGalleriamellonellainfectedwithdifferentstrainsofCandidatropicalis.Tenlarvaepergroupwereinfectedwith2(cid:7) fr
106cellsofC.tropicalisATCC750,CL-6835,orTP-13650.ForC.tropicalisATCC200956,aninoculumof4(cid:7)106cellswasused.Afterinoculation,larvaewere o
treatedwith0.8or1.2(cid:3)gofAMB/larva(subtherapeuticandtherapeuticdoses,respectively).Anuntreatedcontrolgroupwasincluded.Larvasurvivalwas m

monitoreddaily;thelarvaeareconsidereddeadwhentheydonotrespondtotouch. h
t
t
p
:
/
performedwithmicrosomalfractionsandpurifiedenzymeprep- ThatisthecaseinstrainTP-13650;thepresenceofphenylala- /a
arations have been used to analyze the relationship between nineatposition132producesthelossofabridgeofhydrogenthat a
c
Erg11p amino acid substitutions and reductions in the level of isnormallyformedbetweenhemeandtyrosine.Thismodification .
a
azole binding (22, 65). Those authors concluded that the azole in the environment of heme would alter the binding to azoles s
m
nitrogencouldnotinteractwiththeironofthehemeofC.albicans withoutmodifyingtheactivityoftheenzyme.Thisdifferenceis
.
o
Erg11p(CaErg11p)withY132HorG464S,asnormallyoccursto markedbyawild-typesterolprofile,resistancetometabolicin- r
g
produceinhibition.Incontrast,thealteredproteinretainedthe hibitors(duetoanormalcytoplasmicmembrane),andsuscepti- /

abilitytometabolizethesubstrate,comparablytotheERG11wild- bilitytoamphotericinB.Themodelingofwild-typeproteinanda o
n
type strain, allowing resistant mutants to produce ergosterol variantwithequivalentresiduesmutatedinfungalplantpatho-
J
(23,64). gens,suchasY137FinMycosphaerellagraminicola,hasprovided a
n
u
a
r
y

3
1
,

2
0
1
9

b
y

g
u
e
s
t
FIG4 Efficacyofanidulafungin(ANF)inGalleriamellonellainfectedwithdifferentstrainsofCandidatropicalis.Tenlarvaepergroupwereinfectedwith2(cid:7)106
cellsofC.tropicalisATCC750,CL-6835,orTP-13650.ForC.tropicalisATCC200956,aninoculumof4(cid:7)106cellswasused.Afterinoculation,larvaewere
treatedwith4(cid:3)gofANF/larva.Anuntreatedcontrolgroupwasincluded.Larvasurvivalwasmonitoreddaily;thelarvaeareconsidereddeadwhentheydonot
respondtotouch.
4776 aac.asm.org AntimicrobialAgentsandChemotherapy
ResistanceofCandidatropicalistoAntifungalDrugs
D
o
w
n
lo
a
d
e
d

f
r
o
m

h
t
t
p
:
/
/
a
a
c
.
a
s
m
.
o
r
g
/

o
n

J
a
n
u
a
r
y

3
1
,

2
0
1
9

b
y

g
u
e
s
t
FIG5ErgosterolbiosyntheticpathwayinCandidatropicalis.MajorsterolintermediatesthataccumulateinC.tropicalisstrainswithnonfunctionalErg11pare
boxed.Brokenarrowsindicatemultipleenzymaticsteps;solidarrowsindicatesingleenzymaticsteps.Thenumbersinparenthesesrefertothesterolsdescribed
inTable2.
similarresults(65),whichareconsistentwiththeresistancere- interdomainconformationalchangesuponinhibitororsubstrate
cordedforotherplantpathogens(Y136FinMycosphaerellafijien- binding(57).InstrainCL-6835,theabsenceofergosterolanda
sis, Y136F in Erysiphe graminis, and Y136F in Uncinula necator patternofsterolmetabolitessimilartothatofstrainATCC200956
[66–68]). would suggest that the Erg11p amino acid substitution G464D
On the other hand, although G464 cannot interact with flu- (Fig.1E),locatedatthecatalytichemebindingdomain,willaffect
conazoledirectly,thispositionmightbeimportantforthecon- equallytheaffinityoftheenzymefortheazoleanditsaffinityfor
formationofthehemeenvironment.Changingglycinetoother thesubstrate.Thiscouldbeduetothenegativechargeorthelarger
residueswouldbeexpectedtodecreasetheflexibilityrequiredfor sizeofasparticacid,producingagreatdisturbanceintheenviron-
October2013 Volume57 Number10 aac.asm.org 4777
Forastieroetal.
ment of the heme and preventing the binding of the substrate/ gillusfumigatus(36).Recently,Mesa-Arangoetal.(37)demon-
inhibitor.Infact,inC.albicansErg11pwiththeG464Ssubstitu- stratedthatC.tropicalisisabletoinfectandkillG.mellonella.The
tion, although no difference in the affinity of the substrate was lethaleffectofC.tropicalisdependsonboththeyeastdoseandthe
observedbetweenthemutantandthewild-typeprotein,reduced incubation temperature. In larvae infected with antifungal-sus-
steroldemethylasecatalyticactivitywasobserved(63).Thesterol ceptibleC.tropicalis,thefungalburdensincreasedinatime-de-
profileconfirmedthatpossibility,sincethisstrainhasacomplete pendent manner, and treatment with either amphotericin B or
lackofergosterolandanaccumulationof14(cid:1)-methylatedsterols, fluconazolereducedthenumberofCFUintheworm.Also,anti-
thusshowingcross-resistancetoazoleandamphotericinB,aswas fungaltreatmentwasassociatedwiththeformationofcellaggre-
thecasewithstrainATCC200956.Interestingly,strainCL-6835is gatesaroundinfectedareas.Takingthisintoaccount,alongwith
thefirstreportedisolatethatcarriesanasparticacidsubstitutionat otherpublishedworks(34,35),weusedtheG.mellonellamodelto
positionG464. evaluate the efficacies of different antifungal drugs against C.
Ithasbeenreportedthatdisruptionofergosterolbiosynthesis tropicalisinfection.Theinvivoresponsecorrelatesverywellwith
attheErg11plevelresultsintoxicmetabolites.Cellgrowtharrest theantifungalsusceptibilityphenotypeshowninvitro.Flucona-
correlates with the accumulation of 14(cid:1)-methyl-ergosta- zoleorvoriconazoletreatmentiscapableofimprovingthesur-
D
8,24(28)-dien-3(cid:8),6(cid:1)-diolinyeastswithasterol14(cid:1)-demethylase vivaloflarvaeinfectedwithazole-susceptibleC.tropicalis(ATCC o
genedisruption.Inthissituation,14(cid:1)-methylatedsterolsarecon- 750).Thesedrugsimprovesurvivalinadose-dependentmanner. w
n
verted to toxic 3,6-diol derivatives by (cid:2)5,6 desaturation per- Infact,voriconazoleandfluconazoleareeffectivewhenthedoses lo
formed by the enzyme Erg3p (Fig. 5). However, cells can over- recommendedforhumanstotreatinvasivecandidiasisorcandi- a
d
cometheeffectofsuchablockbyasuppressormutationinsterol demiaareused(11).Ontheotherhand,theseantifungaldrugsare e
C5,6desaturationandcanthusacquireazoleresistance(69).In ineffectiveforlarvaeinfectedwithazole-resistantstrains(ATCC d

f
agreement with this observation, azole-resistant fungal isolates 200956, CL-6835, and TP-13650). However, amphotericin B r
o
withnonfunctionalERG3alleleshavebeendescribedforC.albi- treatmentattherapeuticdoses(1.2(cid:3)g/larva)improveslarvalsur- m
cansandCandidadubliniensis(70–72).Bothstrainswithaputa- vivalwhenstrainsATCC750andTP-13650(amphotericinBsus- h
tiveblockattheErg11plevel(CL-6835andATCC200956)carried ceptible)areusedforinfectionbutfailtoimprovethesurvivalof tt
p
ERG3 mutations (Table 1) and showed an accumulation of 24- larvaeinfectedwithstrainCL-6835orATCC200956(amphoter- :/
/
methylcholesta-5,7,24(28)-trien-3-ol (Table 2), suggesting that icin B resistant). The response to anidulafungin was used as a a
a
thesetwostrainsmighthaveevolvedtoavoidtheproductionof controlfordrugefficacyandtoevaluateifthemodelwasableto c
toxic sterols like 14,24-dimethylcholesta-8,24(28)-3(cid:8),6(cid:1)-diol reproducetheinvitrophenotypeofsusceptibilitytoechinocandin .a
s
(Fig.5).Ontheotherhand,itispossiblethatchangesinoverall drugsforallthestrains.Anidulafungintreatmentshowedade- m
membrane composition could compensate for perturbations in creaseinthemortalityoflarvae,independentlyofthestrain.We .
o
sterolcontent,ascenarioconsistentwithreportsofotherergos- confirmedthattheG.mellonellahostisveryusefulforevaluation rg
terol-deficientyeastswithdysfunctionalErg11pthatremainaer- of the in vivo efficacies of antifungal agents in an experimental /
o
obicallyviable(56).Althoughourresultsagreewiththoseofother modelofcandidiasis. n

studies regarding activity alterations of Erg11p and Erg3p (54), Insummary,wereportthestudyofdifferentmechanismsre- J
a
future work would include functional characterization of these sponsibleforsingleandcombinedazoleandamphotericinBre- n
enzymes by gene deletion or other genetic approaches, such as sistanceinC.tropicalisclinicalisolates.Althoughincreasedefflux u
a
heterologousexpressionandsite-directedmutagenesis,inorder or missense mutations in ERG11 are the most frequent mecha- r
y
to fully understand the enzymatic activities of these proteins in nismsinvolvedinazoleresistance,thedecreasedsusceptibilityof 3
theseparticularclinicalisolates. theseisolatestoazolesseemedtobeduetoanERG11genedele- 1
,

Sincetwoofthestrains,CL-6835andTP-13652,wereisolated tion/aminoacidsubstitutioninanareaoftheproteinaffectingthe 2
0
frompatientswithcandidiasisrefractorytoantifungaltreatment, siteforbothsubstratebindingandazolebinding.Theinterrup- 1
weattemptedtodeterminethetherapeuticeffectsofazolesinanin tion of the ergosterol biosynthesis pathway would, in turn, be 9

b
vivomodel.Duetothestrongstructuralandfunctionalsimilari- responsibleforamphotericinBresistanceinstrainslackingfunc-
y
tiesbetweentheinnateimmuneresponsesofinsectsandmam- tionalErg11p.Theresultsofourstudyelucidateimportantchar- g
mals (73), several minihosts are now being employed to study acteristicsandpotentialmechanismsoffluconazoleandampho- u
e
alterationsinmicrobialvirulenceandtheefficacyofantimicrobial tericin B resistance in C. tropicalis. In addition, we validated G. s
t
agents.Amongthem,Galleriamellonellapresentscertainbenefits mellonellaasasimplenonmammalianmodelsystemthatcanbe
in comparison with other nonmammalian models of infection usedtostudyinvitro-invivocorrelationofantifungals.Galleria
(31).Forexample,thelarvaecanbemaintainedatatemperature mellonellalarvaeinfectedwithCandidatropicalisstrainsshowing
rangeof25°Cto37°C,thusreproducingbothatthetemperature invitroresistancetoamphotericinB,fluconazole,andvoricona-
atwhichfungiexistintheenvironmentandatthetemperatureat zolefailedtreatmentwiththeseantifungalsbutrespondedtoechi-
which they are capable of producing invasive infections in hu- nocandins.
mans.Inaddition,anaccurateinoculumofthepathogencanbe
delivereddirectlyintothehost’sbody(34).Finally,thenumberof ACKNOWLEDGMENTS
larvae used and the ease of handling also represent important
E.M.wassupportedbytheEuropeanScienceFoundation(Fuminomics
advantages.PreviousstudieshavedocumentedthatG.mellonella
06-RNP-132),theResearchProjectsfromtheSpanishMinistryofScience
isavalidmodelfortheevaluationofmicrobialvirulenceand/or
andInnovation(ERA-NETPathogenomics[7thFP],BFU2008-04709-E/
theefficaciesofdifferentantimicrobialagentsininfectionscaused BMC),andtheFondodeInvestigacionSanitaria(PI12_02376,MPY1003/
bydifferentbacteria(32–38)andfungi,suchasCryptococcusneo- 13).L.A.-F.isfundedbytheSpanishFondodeInvestigaciónSanitaria
formans(34),Candidaspp.(38),Fusariumspp.(35),andAsper- withaMiguelServetfellowship(FISref.CP11/00026).A.F.isfundedbya
4778 aac.asm.org AntimicrobialAgentsandChemotherapy