Macrocyclic Lactones Differ in Interaction with Recombinant P-Glycoprotein 9 of the Parasitic

23 Pages·2015·2.03 MB·English

RESEARCHARTICLE
Macrocyclic Lactones Differ in Interaction
with Recombinant P-Glycoprotein 9 of the
Parasitic Nematode
Cylicocylus elongatus
and Ketoconazole in a Yeast Growth Assay
MaximilianeKaschny1,JaninaDemeler1,I.JanaI.Janssen1,TetianaA.Kuzmina2,
BrunoBesognet3,TheoKanellos4,DominiqueKerboeuf5,GeorgvonSamson-
Himmelstjerna1,JürgenKrücken1*
1 InstituteforParasitologyandTropicalVeterinaryMedicine,FreieUniversitätBerlin,Berlin,Germany,
2 SchmalhausenInstituteofZoology,NationalAcademyofSciencesofUkraine,Kyiv,Ukraine,3 Zoetis,
Paris,France,4 ZoetisInternationalServices,Paris,France,5 INRA,ISP,AnimalInfectionandPublic
Health,Nouzilly,France
* [email protected]
OPENACCESS
Abstract
Citation:KaschnyM,DemelerJ,JanssenIJI,
KuzminaTA,BesognetB,KanellosT,etal.(2015)
MacrocyclicLactonesDifferinInteractionwith Macrocycliclactones(MLs)arewidelyusedparasiticidesagainstnematodesandarthro-
RecombinantP-Glycoprotein9oftheParasitic pods,butresistanceisfrequentlyobservedinparasiticnematodesofhorsesandlivestock.
NematodeCylicocyluselongatusandKetoconazole
Reportsclaimingresistanceordecreasedsusceptibilityinhumannematodesareincreas-
inaYeastGrowthAssay.PLoSPathog11(4):
ing.SincenotargetsitedirectedMLresistancemechanismshavebeenidentified,non-spe-
e1004781.doi:10.1371/journal.ppat.1004781
cificmechanismswerefrequentlyimplicatedinMLresistance,includingP-glycoproteins
Editor:DavidL.Williams,RushUniversityMedical
(Pgps,designatedABCB1invertebrates).Nematodegenomesencodemanydifferent
Center,UNITEDSTATES
Pgps(e.g.10inthesheepparasiteHaemonchuscontortus).MLtransportwasshownfor
Received:May14,2014
mammalianPgps,Pgpsonnematodeeggshells,andveryrecentlyforPgp-2ofH.contor-
Accepted:March3,2015
tus.Here,Pgp-9fromtheequineparasiteCylicocycluselongatus(Cyathostominae)was
Published:April7,2015 expressedinaSaccharomycescerevisiaestrainlackingsevenendogenouseffluxtrans-
Copyright:©2015Kaschnyetal.Thisisanopen porters.Pgpwasdetectedontheseyeastsbyflowcytometryandchemiluminescence
accessarticledistributedunderthetermsofthe usingthemonoclonalantibodyUIC2,whichisspecificfortheactivePgpconformation.Ina
CreativeCommonsAttributionLicense,whichpermits
growthassay,Pgp-9increasedresistancetothefungicidesketoconazole,actinomycinD,
unrestricteduse,distribution,andreproductioninany
valinomycinanddaunorubicin,butnottotheanthelminticfungicidethiabendazole.Sinceno
medium,providedtheoriginalauthorandsourceare
credited. fungicidalactivityhasbeendescribedforMLs,theirinteractionwithPgp-9wasinvestigated
inanassayinvolvingtwodrugs:Yeastswereincubatedwiththehighestketoconazolecon-
DataAvailabilityStatement:Allrelevantdataare
withinthepaperanditsSupportingInformationfiles centrationnotaffectinggrowthplusincreasingconcentrationsofMLstodeterminecompeti-
exceptforthesequenceofCylicocycluselongatus tionbetweenormodulationoftransportofbothdrugs.Alreadyequimolarconcentrationsof
Pgp-9whichisavailablefromGenbankunderthe
ivermectinandeprinomectininhibitedgrowth,andatfourfoldhigherMLconcentrations
accessionnumberKJ701410.
growthwasvirtuallyabolished.Selamectinanddoramectindidnotincreasesusceptibilityto
Funding:Thepresentstudywasperformedasa
ketoconazoleatall,althoughdoramectinhasbeenshownpreviouslytostronglyinteract
collaborativeresearchprojectbetweenZoetisandthe
InstituteofParasitologyandTropicalVeterinary withhumanandcaninePgp.Anintermediateinteractionwasobservedformoxidectin.This
Medicine,FreieUniversitätBerlin.Accordingly,the wassubstantiatedbyincreasedbindingofUIC2antibodiesinthepresenceofivermectin,
InstituteofParasitologyandTropicalVeterinary
Medicine,FreieUniversitätBerlinreceivedaProject
PLOSPathogens|DOI:10.1371/journal.ppat.1004781 April7,2015 1/23
MacrocyclicLactonesandPgp-9inNematodes
specificresearchgrantfromZoetis.Thefunders, moxidectin,daunorubicinandketoconazolebutnotselamectin.Theseresultsdemonstrate
exceptforBrunoBesognetandTheoKanellos,had directeffectsofMLsonarecombinantnematodePgpinanML-specificmanner.
noroleinstudydesign,datacollectionandanalysis,
decisiontopublish,orpreparationofthemanuscript.
CompetingInterests:BBandTKareemployeesof
Zoetis.ExceptofBBandTK,Zoetiswasnotinvolved
AuthorSummary
instudydesign,datacollection,dataanalysisor
preparationofthemanuscript.Thedecisiontopublish
Macrocycliclactones(MLs)arewidelyuseddrugsagainstparasiticnematodes,butdrug
themanuscriptwasjointlytaken.Thisdoesnotalter
resistanceisrapidlyincreasinginprevalenceandspatialdistributioninparasitesofrumi-
ouradherencetoallPLOSpoliciesonsharingdata
andmaterials. nantsandhorses,andissuspectedinhumannematodesaftermassdrugapplications.
ChangesinexpressionlevelsortheaminoacidsequencesofP-glycoprotein(Pgp)trans-
portershavefrequentlybeenimplicatedinMLresistance,butdirectevidencefortransport
ofMLsbynematodePgpsisstillmissing.Here,cloningofpgp-9oftheequineparasite
CylicocycluselongatusanditsfunctionalrecombinantexpressioninaSaccharomycescere-
visiaeyeaststraindeficientinsevenendogenousABCtransportersisdescribed.Expression
decreasedsusceptibilitytoseveralfungicidalmammalianPgpsubstratesincludinge.g.ac-
tinomycinDandketoconazole,buthadnoinfluenceonsusceptibilitytothebenzimid-
azolethiabendazole,whichisactiveagainstboth,yeastsandnematodes.Additionofsome
MLsstronglyincreasedketoconazolesusceptibilityinyeastsexpressingC.elongatusPgp-
9,whileotherMLshadnoeffect.TheseinteractionsareastronghintthatsomeMLsactas
substratesoratleastasinhibitorsofPgp-9mediateddrugtransport.
Introduction
Duetotheirbroad-spectrumantiparasiticactivitywitheffectsagainstboth,nematodesandar-
thropods(endectocides),macrocycliclactones(MLs)areamongthemostimportantantiparasitic
drugsinveterinaryandhumanmedicine[1].However,resistancetoMLsiswidespreadinnema-
todesofsmallruminantsandcurrentlyincreasinginprevalenceandspatialdistributioninnema-
todesofcattle,horsesandhumans[2–9]althoughforthelatterthenumberofreportsdescribing
unresponsivenessofparasitestodrugsisstillonlylowandfutureinvestigationsarerequiredto
formallyproveresistance.Inequines,MLresistancewasinitiallyobservedinParascarisequorum
[10]butrecentlyreportsofMLresistantcyathostominaehavealsoemerged[11–15].
Innematodes,themostimportantMLtargetsareglutamate-gatedchloridechannels
(GluCl-Rs)whereasionotropicγ-amino-butyricacidreceptorsrespondonlyathigherdrug
concentrations[9].
Specificresistancemechanismsinvolvingsinglenucleotidepolymorphisms(SNPs)intheβ-
tubulinisotype1geneofnematodesfromtheorderStrongylidaarewellknowntoberesponsi-
blefororatleaststronglycorrelatewithresistancetobenzimidazoles(BZs)inruminantsand
equines[16–20].Forlevamisoleresistancedecreaseddensityandopenprobabilityofnicotinic
acetylcholinereceptors,whichareactivatedbylevamisole,andcertainsplicevariantsencoding
onlytruncatedsubunitsofthereceptors[21–23]havebeendescribedinresistantisolates.In
contrast,nogenotypeshavebeenclearlyinvolvedinMLresistanceexceptforasinglereport
describingaSNPinaCooperiaoncophoraGluCL-Rsubunit[24]butthisspecificchangehas
neverbeenobservedinMLresistantnematodesinthefield[25].Intherecentpast,ABC
(ATP-bindingcassette)transportersandinparticularP-glycoproteins(Pgps,i.e.orthologsof
themammalianABCB1)havefrequentlybeenimplicatedinMLresistancemechanisms[26].
FirsthintsthatPgpsareinvolvedinresistancewereobtainedbycomparisonofpgp-2allelesbe-
tweenMLsusceptibleandresistantisolatesofHaemonchuscontortus[27].Usingantibodies
PLOSPathogens|DOI:10.1371/journal.ppat.1004781 April7,2015 2/23
MacrocyclicLactonesandPgp-9inNematodes
againstahighlyconservedepitope,largeramountsofactivePgpsweredetectedontheegg
shellofMLresistantH.contortus[26]andC.oncophora[28].Moreover,thecompetitivePgp
inhibitorverapamilwasshowntostronglysensitizenon-parasiticstagesofC.oncophora,
whereastheverapamileffectsoninhibitionofdevelopmentofthemodelnematodeCaenorhab-
ditiselegansinthepresenceofivermectin(IVM)wereonlysmall[29].Moderatelyincreasedef-
ficacyofMLsinC.elegansstrainswhicharedeficientinindividualPgpswasdescribedin
severalassays[29–31].UsingH.contortuseggsitwasalsodemonstratedthatPgpsareactivated
byMLs[32].Despitethislargeamountofwork,directevidencethatanindividualnematode
PgpisabletointeractwithMLswasmissingforalongtime.InthepresentstudyaSaccharo-
mycescerevisiaeyeaststraindeficientinsevenendogenousABCtransporters[33]wasusedto
expressarecombinantPgp-9clonedfromtheequineparasiticnematodeCylicocycluselongatus
(Cyathostominae)andtocompareinteractionofseveralfungicidalPgpsubstrateswiththis
CegPgp-9inasimpleandcheapyeastgrowthassay.EffectsofdifferentMLswerecomparedin
thepresenceorabsenceofketoconazole(Ket)toidentifyanypotentialcompetitiveorenhanc-
ingeffectsofthedrugcombinations.BindingassaysusingthemonoclonalantibodyUIC2,
whichbindstoaPgpepitopeonlypresentduringactivetransport,demonstratedactivationof
PgpbyMLsandfungicidalsubstancesintheabsenceofaseconddrug.
MaterialsandMethods
Ethicsstatement
AdultC.elongatuswerecollectedfromeuthanizednaturallyinfectedhorses,whichwere
boughtfromtheirowners.Theseanimalexperimentswereinaccordancewiththe“Tierschutz-
gesetz”inGermanyandwiththeEuropeanUniondirective2010/63/EU.Experimentswereap-
provedbytheLandesamtfürVerbraucherschutzundLebensmittelsicherheit(LAVES)in
Hannover(Germany)underthereferencenumber06A435andbytheLandesamtfürGesund-
heitundSoziales(LaGeSo)inBerlin(Germany)underthereferencenumberL0088/10.
Cloningoffull-lengthPgps
Thefull-lengthcDNAofC.elongatuspgp-9wasobtainedusingastrategydescribedpreviously
[34].Amplificationofsmallfragmentswithdegeneratedprimerswasfollowedbyrapidampli-
ficationofcDNAends(RACE)PCRandamplificationoffull-lengthcDNAs.Initially,degener-
atedprimersweredesignedbasedonsequencealignmentsoforthologousPgpsequencesofC.
elegans,Caenorhabditisbriggsae,C.oncophoraandH.contortus.Oligonucleotidesequencesare
listedinS1Table.RNAwasextractedfromadultnematodesandapproximately100ngwere
reversetranscribedtocDNAaccordingtothemanufacturer’sprotocolusingrandomhexamer
primers(RevertAidFirstStrandcDNASynthesisKit,ThermofisherScientific).ThePCRcon-
tained16μlH O,10μMeachforwardandreverseprimer,2.5μlAccuPrimebuffer1(with
2
dNTPs),0.5μlAccuPrimeTaqDNApolymerase(LifeTechnologies)and1μlcDNA.PCRpro-
tocolswerecarriedoutasfollows:After2minat94°Cforinitialdenaturation,10cyclesof
94°Cfor15s,50°Cfor30sand68°Cfor1minwerefollowedby30cycleswithequalsettings
butanincreasedannealingtemperatureat60°C.PCRfragmentsweregel-purifiedandcloned
intopCR4-TOPOvectorandsequencedbyGATCBiotech(Konstanz).
RACE-PCRwascarriedoutaccordingtomanufacturer’sinstructions(3'/5'-RACE2ndgen-
erationKit,Roche)asdescribedrecently[34]withprimersandtemperatureprofilesaslistedin
S1Table.For5'-RACE,cDNAsynthesiswasstartedwithagene-specificprimer(S1Table)and
purifiedcDNAsweretailedwithdATPtoallowannealingoftheoligo-dTanchorprimer.PCR
mixturesconsistedof18.75μlH O,2.5μlAccuPrimeBuffer1,0.5μMofeachprimer,0.5μl
2
AccuPrimeTaqDNApolymeraseand1.0μlcDNA.PCRprotocolsforamplificationwereset
PLOSPathogens|DOI:10.1371/journal.ppat.1004781 April7,2015 3/23
MacrocyclicLactonesandPgp-9inNematodes
asfollows:Initialdenaturationat94°Cfor2min,followedby40cyclesof94°Cfor15s,55°C
for30sand72°Cfor1min,andaterminalelongationat72°Cfor10min.
Foramplificationofafull-lengthproduct,cDNAsynthesiswascarriedoutusingoligo-(dT)
primers.PCRmixtureforamplificationofCegpgp-9contained10μlQ-solution(Qiagen),
0.5μMeachforwardandreverseprimer,10nMdNTPs,0.5μlPhusionIIHotStartPolymerase
(ThermofisherScientific)and4μlcDNAin50μl1×HFbuffer.Afterinitialdenaturationat
98°Cfor30s,40cyclesconsistingof98°Cfor10s,68°Cfor30sand72°Cfor2minwerecar-
riedoutfollowedbyaterminalelongationat72°Cfor10min.
Phylogeneticanalysis
Pgpproteinsequencesfromnematodesaswellasrepresentativesequencesfromvertebrates,
insects,mollusksandplatyhelmintheswerealignedusingClustalX2[35].Theoptimalamino
acidsubstitutionmodelwasidentifiedusingProttest3.0[36]withthenumberofevolutionrate
categoriessetto8.PhylogenetictreeswerecalculatedwithPhyML3.01[37,38]assumingthe
samenumberofratecategoriesandtheLG+I+F+Gmodel[39]usingboth,nearestneighborin-
terchange(NNI)andsubtreepruningandregraftment(SPR)moves.Toavoidtrappingofthe
iterativeoptimizationprocessinalocalmaximumofthelikelihoodfunction,calculations
startedwithoneneighborjoiningandfiverandomtrees.Branchsupportwasobtainedbycon-
ductingtheShimodaira-Hasegawa[SH]approximatelikelihoodratiotestandtheBayesian
transformationoftheapproximatelikelihoodratiotest.Finally,thebesttreewasvisualized
usingMEGA5[40].
ExpressionofC.elongatusPgp-9inyeast. Tore-amplifytheopenreadingframe(ORF)
ofC.elongatuspgp-9withandwithoutstopcodon,twosetsofprimerpairswereused.There-
actioncontained41.8μlH O,5μlAccuPrimeBuffer,0.4μMofeachforwardandreverse
2
primer,0.2μlAccuPrimeTaqDNAPolymeraseHighFidelity(Lifetechnologies)andapproxi-
mately80ngplasmidDNA.After2minofinitialdenaturationat94°C,35cyclesofdenatur-
ationat94°Cfor15s,annealingat55°Cfor30sandelongationat68°Cfor4minwere
performed.Terminalelongationwascarriedoutat68°Cfor10min.PCRproductswereligated
intothepYes2.1TOPOvectorandtransformedintoE.coliTop10cells(Lifetechnologies).
Afterverificationoftheinsertbysequencing,AD1234567(AD1-7)yeastcells,whicharedefi-
cientinsevenendogenouseffluxtransporters[33],weretransformedwithpurifiedplasmid
DNAusingthelithiumacetatemethodaccordingtothepYes2.1TOPOmanual.Transformed
yeastswereidentifiedafter48hofincubationat30°Conagarplateslackinguracil.
AnalysisoftranscriptionbyRT-PCR. YeastRNAwasisolatedfromtransformantsselect-
edforcomplementationofuracilauxotrophyusingtheMaxwellSimplyRNAkit.AfterDNase
digestion,cDNAsynthesiswithanoligod(T)anchorprimerwasperformed.Transcription
wasconfirmedbyRT-PCRusingprimerstargetingfragmentsatthe3'endofthesequence,i.e.
Ceg-Pgp-9-3R2combinedwitheitherpYes-Pgp-9-loorpYes-Pgp9-his(S1Table).Thereac-
tionmixturecontained0.4μMforwardprimerand0.4μMreverseprimereitherwithorwith-
outstopcodon,2.5μlAccuPrimerbuffer1,0.5μlAccuPrimeTaqDNApolymerase
(Invitrogen),18μlbidest.H Oand2μlcDNA.Aninitialdenaturationwasperformedat94°C
2
for2minfollowedby35cyclesofdenaturationat94°Cfor15s,annealingat55°Cfor30sand
elongationat72°Cfor1min.CorrectPCRproductswereidentifiedbyfragmentlengthafter
gelelectrophoresisandsequenceanalysis.
Westernblotting. SDSPageandtransfertonitrocellulosemembraneswereperformedes-
sentiallyasdescribedpreviously[41].Theprimaryanti-V5antibody(LifeTechnologies)was
diluted1:500andthesecondarygoatanti-mouseantibody(Dianova)1:5000.Detectionwas
carriedoutusingSuperSignalWestPicoreagents(ThermoScientific).
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MacrocyclicLactonesandPgp-9inNematodes
Fluorescenceactivatedcellscanning
BasedonthemonoclonalantibodyUIC2,whichisspecificforaconservedepitopepresentin
activePgps,expressionlevelsofCegPgp-9weredeterminedbyfluorescenceactivatedcellscan-
ning.Briefly,yeastcellswerecollectedfromovernightculturesbycentrifugationandwashed
twicewithPBS.Afterre-suspensionin3mlPBS,threealiquotsof10μlweretransferredto
1.5mltubesandincubatedwith1mlblockingbuffer(50mgbovineserumalbuminin25ml
PBS)forsaturationofnon-specificbindingsites.Sampleswerecentrifugedandwashedbefore
incubationwiththemonoclonalantibodyspecificforactivePgps(UIC2,Drako),PBSorthe
isotypicantibody(mouseIgG2aλ,cloneHOPC-1,BeckmanCoulter).Beforeflowcytometry
analysiscellswereagainwashedandfilteredtoremovecellagglomerates(30μMmeshsize).
ThecellularfluorescenceintensitiesweremeasuredwithaMoFlocytometer(BeckmanCoul-
ter).Instrumentsettingscorrespondedtotheprotocolasdescribedelsewhere[42].
Growthassayswithdirectlyfungicidaldrugs. Growthassayswerecarriedoutessentially
asdescribedelsewhere[43]withsmallmodifications.Inbrief,S.cerevisiaecells(AD1-7lacZ,
AD1-7CegPgp-9orAD1-7CegPgp-9V5His)weregrownovernightat30°Cand250rpmin
5mlsyntheticminimalmedium(6.7g/lyeastnitrogenbasewithaminoacids,1.92g/lyeast
drop-outmediumsupplementwithouturacil)supplementedwith2%galactoseand1%raffi-
nose(SD+GR)toinduceexpressionoftransgenes.AftercountinginaNeubauerchamber,
therequiredcellnumberwasharvestedbycentrifugationandsubsequentlydilutedin1ml
SD+GR.Growthassayswereperformedin96-wellnon-tissueculturetreated,flatbottom
plates(BectonDickinson).Eachwellwasinoculatedwith4×104cellsin5μlSD+GRand95μl
SD+GRwith1%DMSOcontainingdifferentdrugconcentrations.ForKet,finalconcentrations
of0.18μM,0.37μM,1.09μM,1.46μM,2.2μMand2.94μMwereused.Concentrationsforac-
tinomycinD,valinomycinanddaunorubicinwere6.25μM,12.5μM,25μM,50μM,100μM
and200μM.Thiabendazol(TBZ)wasusedatfinalconcentrationsof155μM,310μM,
620μM,1240μMand2490μM.TheplatewasclosedandsealedwithParafilmMtoavoid
evaporation.Duringincubationover48hinaplatereader(Synergy4,Biotech)at30°C,plates
werecontinuouslyshakenwithashortdelaybeforedeterminationofabsorptionat600nm
every10mintorecordgrowthcurves.Cellsincubatedwithmediumcontaining1%DMSO
servedaspositivecontrols.
Growthassayswithmacrocycliclactones. AD1-7lacZandAD1-7CegPgp-9V5Hiscells
wereculturedandprocessedasdescribedabove.Controlswerepreparedcontainingeitherno
fungicide(bothyeaststrains)or0.18μMKet(AD1-7lacZ)or0.72μMKet(AD1-7CegPgp-
9V5His)inSD-GRwith1%DMSO.Foridentificationofpossibledirecteffectsonyeastgrowth,
allMLsweretestedat0.18,0.36,0.72,1.44,2.88μM(AD1-7lacZ)and0.72,1.44,2.88,4.32,
5.76μM(AD1-7CegPgp-9V5His)withoutadditionofKet.ToanalyzeanyinteractionofMLs
withKetinthepresenceofheterologousCegPgp-9,cellswereincubatedwithacombinationof
KetandMLsatconcentrationsmentionedabove.Plateswerepreparedandanalyzedas
describedabove.
DetectionofchangesinUIC2bindinginresponsetodrugs. Yeastcells(AD1-7lacZand
AD1-7CegPgp-9V5His)wereobtainedfromfreshovernightculturesinSD+GRmedium,
washedonceandthenre-suspendedinfreshSD+GRwith2%BSAatanOD of0.5.Aliquots
600
(200μl)wereshakenat30°Cand250rpmfor60minbeforeadditionofdrugsin2μlDMSO.
VehiclecontrolsreceivedonlyDMSO(1%finalconcentrationasinallsampleswithdrugs).
FinalconcentrationsforIVM,MOXandSLMwere4μMand1μMwhile2and0.5μMwere
usedforKetand200μMand6.25μMfordaunorubicin.Aftershakingat30°Cand250rpm
for10min,themonoclonalantibodiesUIC2(recognizingactivePgp,LEAFpurifiedanti-
humanCD243,Biolegend)andMg2a-53(IgG2aLEAFpurifiedisotypecontrol,Biolegend)
PLOSPathogens|DOI:10.1371/journal.ppat.1004781 April7,2015 5/23
MacrocyclicLactonesandPgp-9inNematodes
wereaddedin2μltoachieveafinalconcentrationof2μg/ml.Afterfurtherincubationwith
shakingat30°Cfor20min,cellswererapidlycooledonice,washedoncewithicecoldPBSand
fixedat4°Cin2.5%paraformaldehydeinPBSovernight.AftertwowashesinPBS/2%BSAat
roomtemperature,cellsweresuspendedinsecondaryantibodysolution(goat-antimousecou-
pledtohorseradish-peroxidasediluted1:2000inPBS/2%BSA)andshakenfor1hatroom
temperature.AftertwowashstepswithPBS,cellswerere-suspendedin25μlofsolutionI(con-
tainingstabilizedH O )fromtheSuperSignalPicoWestPicochemiluminescencesubstrate
2 2
(ThermoScientific)andtransferredtowhite,flatbottom96wellplates.Then25μlofsolution
II(containingluminol)wereaddedtoeachwell.Plateswereimmediatelytransferredintoa
Synergy4(Biotek)platereaderandrapidlyshakenatroomtemperaturefor1min.After10s
delay,chemiluminescencesignalswereintegratedover1sandrecordedasrelativelightunits.
Statisticalanalysis. GrowthcurveswerefittedandstatisticallyanalyzedusingtheRsoft-
ware[44]withthegrofitpackage[45]andGraphPadPrism5.04(GraphPadSoftware,San
Diego,California).Forcomparingdifferentyeaststrains,relativegrowthrateswerecalculated
bydividingtheareaunderthecurve(AUC)undercertainexperimentalconditionstothemean
AUCofthevehicle(negativecontrol)withandwithoutKet,respectively.Forcalculationof
concentrationresponsecurves,concentrationswerelog transformed.Inordertoincludethe
10
negativecontrolsdespitelog transformationofthedrugconcentrations,negativecontrols
10
weresetto0.001μM(Ket),0.01μM(actinomycinD,valinomycinanddaunorubicin)or10μM
(TBZ)beforetransformation.EC -valuesofactinomycinD,valinomycin,daunorubicin,TBZ
50
andKetwerecalculatedwithafourparametriclogisticregressionanalysisinGraphPadand
duetopreviousnormalization,thedatawereconstraintto1asmaximumvalue.Statisticaldif-
ferencesinEC valueswerecalculatedwiththeextrasumofsquareFtestimplementedin
50
GraphPadPrism.DirecteffectsofMLsonyeastgrowthwereanalyzedusingaoneway
ANOVAwithBonferroni’sposthoctesttocompareMLexposedsamplestothenodrugcon-
trol.DifferencesandputativeinteractionsbetweenvehiclesandMLconcentrationswithand
withoutKetweredeterminedwithatwowayANOVAandBonferroni’sposthoctest.
Duetothelownumberofrepeats(7–8)andunequalvariances,non-parametricanalysis
waschosentoanalyzedatafromUIC2bindingassaysusingchemiluminescencedetection
sincenormaldistributionandequalvariancesasrequiredforANOVAanalysiswerenotsup-
ported.Therefore,Kruskal-WallisanalysesfollowedbyBonferroni’sposthoctestswerecon-
ducted.First,AD1-7expressingPgp-9incubatedwithUIC2werecomparedwithAD1-7Pgp-
9V5HisincubatedwiththeisotypecontrolandAD1-7lacZwitheitherUIC2ortheisotypecon-
trol.Inaddition,allAD1-7yeastsampleswithPgp-9expressionincubatedwithdrugsand
UIC2werecomparedtotheDMSOcontrolinthepresenceofthesameantibody.
Results
FulllengthPgps
AfteramplificationofasmallRT-PCRfragmentusingdegeneratedprimersfollowedby5'and
3'RACEPCR,ampliconscontainingtheentireORFswereobtainedforC.elongatus.Thefrag-
mentsshowedhighsimilaritytoC.elegansPgp-9asdeterminedbyBlastanalyses.ThecDNA
sequencewasdepositedinGenBankundertheaccessionno.KJ701410.Thededucedamino
acidsequencerevealsthetypicaldomainarrangementofPgpswithtwosimilarhalves,each
consistingofanABCtransportertransmembranedomain(CDDaccessionnumbercd03249)
containingsixtransmembranehelicesfollowedbyanucleotide-bindingdomain(CDDacces-
sionnumbercl005249)containingthetypical,highlyconservedWalkerAandBmotifsaswell
asQ,DandHloops.ResultsofphylogeneticanalysisusingallPgpproteinsencodedinthege-
nomesofC.elegansandC.briggsaeaswellasmanypreviouslypublishedPgpsfromother
PLOSPathogens|DOI:10.1371/journal.ppat.1004781 April7,2015 6/23
MacrocyclicLactonesandPgp-9inNematodes
parasiticnematodesconfirmedthattheproteinwasaclearorthologofC.elegansPgp-9and
wasthereforedesignatedC.elongatusPgp-9(Fig1).
RecombinantexpressionofPgp-9inyeasts
TheORFofC.elongatuspgp-9wasamplifiedwithandwithoutstopcodontoallowexpression
withoutandwithaCOOH-terminaltag(V5/6×His).PCRproductswereclonedintothe
pYes2.1TOPOvectorallowinggalactose-inducedexpression.Plasmidsweretransformedinto
theS.cerevisiaestrainAD1-7whichisdeficientinsevenmajorendogenousABCtransporters
[33].Initially,expressionofPgpsinyeastwasanalyzedbyRT-PCRandtranscriptionofpgp-9
mRNAscouldbeconfirmed(S1Fig).Disappointingly,expressionofC.elongatusPgpsinin-
ducedyeastculturesusingWesternblottingwasnotsuccessfulneitherusingthemonoclonal
antibodyC219(knowntodetectinWesternblottingahighlyconservedPgpepitopewhichis
presentinCegPgp-9)norwiththeanti-V5antibodyalthoughdetectionofβ-galactosidasein
thecontrolstrainwassuccessfulusingtheanti-V5antibody(S2Fig).FACSanalysisusingthe
monoclonalantibodyUIC2,whichisspecificforahighlyconservedepitopepresentonactive
Pgptransporters,revealedspecificbindingoftheUIC2antibodycomparedtotheisotypecon-
trol.ResultsofarepresentativeexperimentforcellsexpressingCegPgp-9areshowninFig2.
Accordingtoforward(FS)andside(SS)scatter,twomajorpopulationsofyeastcellwerede-
fined,i.e.small,non-granular(regionR1)vs.large,granular(regionR2)cells(Fig2A).Noob-
viousdifferenceswerefoundbetweencellsexpressingCegPgp-9withorwithoutV5/6×Histag
andthefollowingdatasummarizeexperimentsirrespectiveofthepresenceofatag:Inregion
R1,onlyasmallfractionofthecells(4.4–8.9%)waspositiveintermsofincreasedbindingof
UIC2incomparisontotheisotypecontrol(Fig2B–2D).Despitemuchhigherbackground
fluorescenceinregionR2,amuchhighernumberoftheyeastcellsinthisregion,i.e.16.3–
20.4%,haddetectableamountsofactivePgpontheirsurface(Fig2E–2G).Nospecificbinding
ofUIC2wasdetectedforAD1-7lacZyeasts,i.e.percentageofpositivecellswasbetween0.23
and0.6%(n=3)inregionR1and0.31and1.3%inregionR2(n=3).
EfficacyoffungicidalPgpsubstratesandthefungicidalanthelmintic
thiabendazoleinadirectyeastgrowthassay
InordertodemonstratethattheexpressionofrecombinantPgp-9influencesdrugsusceptibili-
tyoftheyeastcells,thesubstratesofmammalianPgpsactinomycinD,daunorubicin,valino-
mycinandKetwereused[46,47].ForselectedconcentrationsofTBZaswellasforavehicle
controltypicalgrowthcurvesareshowninS3Figasexamples.Initialexperimentswerecarried
outusingKetandcellsexpressingPgp-9withouttag(AD1-7CegPgp-9)orwithV5/6×Histag
(AD1-7CegPgp-9V5His),whichwerecomparedwithcellsexpressinglacZfromthesamevec-
torbackbone(AD1-7lacZ).CegPgp-9expressiondecreasedsusceptibilitytoKetirrespectively
ofthepresenceofaV5/6×Histag(Fig3A).Theconcentrationresponsecurveswereextremely
steepandevenlessthantwofolddifferencesinconcentrationsledtochangesfrom(cid:1)95%to
(cid:3)10%growth,inparticularinthestrainexpressingCegPgp-9withoutatag.Nevertheless,due
toverysmalldilutionsteps,theEC valuesandconfidenceintervalsofallthreestrainscould
50
bedetermined(Table1).TheEC valuesofthestrainsexpressingCegPgp-9wereapproxi-
50
mately2.2(withouttag)and3.0(withtag)foldincreased.Althoughthedifferencebetween
EC valuesofbothstrainswasstatisticallynotsignificant(p=0.19),allfurtherexperiments
50
wereconductedusingtheAD1-7CegPgp-9V5Hisstrain.ForactinomycinD,valinomycinand
daunorubicinsignificantlyhigherEC valueswereobservedwhencomparingAD1-7CegPgp-
50
9V5HisstrainwiththecontrolstrainAD1-7lacZ(Fig3B–3D,Table1)withincreasesbetween
2.1and2.3fold.
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Fig1.PhylogeneticanalysisofnematodePgps.ThetreewascalculatedusingtheLG+I+F+Gsubstitution
modelandPhyML3.01.Thenumberofaminoacidsubstitutionratecategorieswassettoeight.Theresultsof
theapproximatelikelihoodratiotestmodifiedaccordingtoShimodairaandHasegawaandofaBayesian-like
transformationoftheapproximatelikelihoodratiotestareprovidedbeforeandaftertheslash,respectively.If
onlyonevalueisgiven,theresultsofbothtestswereidentical.Asoutgroup,Pgpsequencesfrommouse
(Mmu),theinsectsDrosophilamelanogaster(Dme)andPediculushumanuscorporis(Phu),thetrematode
Schistosomamansoni(Sma)andthemusselsMytiluscalifornianus(Mca)andMytilusgalloprovincialis(Mga)
wereused.ThecompletePgpproteinfamiliesofCaenorhabditiselegans(Cel)andCaenorhabditisbriggsae
(Cbr)werealsoincluded.Moreover,availableannotatedPgpsequencesofPristionchuspacificus(Ppa)and
oftheparasitesAscarissuum(Asu),Brugiamalayi(Bma),Haemonchuscontortus(Hco),Cooperia
oncophora(Con)andOnchocercavolvolus(Ovo)wereincludedintheanalysis.ThenewC.elongatusPgp-9
sequenceishighlightedinblue.Pgp,P-glycoprotein;MDR,multi-drugresistanceprotein.Thescalebar
representstheindicatednumberofsubstitutionspersite.Accessionnumbersforproteinsequencesinthe
treeareprovidedinS2Table.
doi:10.1371/journal.ppat.1004781.g001
SincetheanthelminticTBZisalsoaneffectivefungicide—althoughatrelativelyhighcon-
centrations—theabilityofCegPgp-9toreducesusceptibilityofAD1-7yeastcellstoTBZcould
alsobeanalyzeddirectly.Concentrationresponsecurves(Fig3E)revealedonlyminimaldiffer-
encesbetweenthecontrolstrainandthestrainexpressingCegPgp-9andEC valueswerevir-
50
tuallyidentical(Table1).
EffectsofMLsintheabsenceandpresenceofketoconazoleinan
indirectyeastgrowthassay
SinceMLswerenotknowntoexertanysignificantfungicidaleffects,anindirectmethodwas
usedtoquantifyMLinteractionswithCegPgp-9.Forthispurpose,acriticalconcentration(i.e.
thehighestconcentrationshowingnosignificanteffectonyeastgrowth)ofthefungicidalPgp
substrateKetwasused.ForAD1-7lacZthiswas0.18μMwhileAD1-7CegPgp-9V5Hisstill
grewnormallyat0.72μM.MLswerethenusedinaconcentrationrangestartingwithequimo-
larconcentrationsofKetandMLandendingwithupto16foldhigherMLconcentrations(Fig
4).TheseassayswereconductedwiththeaimtotestwhetherMLscaninhibittransportofKet
byCegPgp-9,whichwouldleadtoincreasedsusceptibilitytoKet.
IntheabsencesofKet,highconcentrationsofIVMandeprinomectin(EPM)surprisingly
showedadirecteffectoftheMLongrowthofAD1-7CegPgp-9V5His(Fig4Aand4B).Inthe
samestrain,nosignificantdirectconcentrationdependenteffectsofmoxidectin(MOX),sela-
mectin(SLM)anddoramectin(DRM)wereobserved(Fig4Dand4EandS4AFig).Withthe
exceptionofEPM,whichsignificantlyreducedgrowthatthehighestconcentrationof2.94μM,
therewerenodirectseffectsongrowthofAD1-7lacZ(Fig4E–4HandS4BFig),whichcanbe
explainedbythefactthatMLconcentrationsusedinthisstrainweregenerallylower.
WhenusedincombinationwithKet,evenequalconcentrationsofEPM,IVMandMOX
(butnotofSLMandDRM)withKetwereabletosignificantlyenhancethefungicidaleffectsof
KetinCegPgp-9expressingyeastcells(Fig4A–4C,S4AFig).Effectsrangedbetween31and
79%inhibitionofrelativegrowthwiththestrongesteffectsexertedbyEPMandtheleastpro-
nouncedeffectsbyMOX.TwofoldhigherMLconcentrationsvirtuallyabolishedrelative
growthinthepresenceofEPM,allowingonlyminimalgrowthinthepresenceofIVMandre-
ducinggrowthinthepresenceofMOXbyapproximately59%.Remarkably,evenathigher
concentrationsMOXonlyinhibitedgrowthofAD1-7CegPgp-9V5Hisbyabout92%whereas
maximalinhibitionofgrowthinthepresenceofIVMandEPMwasapproximately95%and
98%.IncontrasttoEPM,IVMandMOX,noeffectswereobservedforSLMandDRMevenat
thehighestconcentrationsused(Fig4D,S4AFig).
Incontrast,effectsofMLsongrowthofAD1-7lacZcellsweremuchsmallerandateightfold
higherconcentrationsofIVM,EPMorDRMthanthoseofKet,inhibitionratesintherageof
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