The unusual resistance of avian defensin AvBD7 to proteolytic enzymes preserves its antibacterial activity Geoffrey Bailleul, Amanda Kravtzoff, Alix Joulin-Giet, Fabien Lecaille, Valérie Labas, Hervé Meudal, Karine Loth, Ana-Paula Teixeira-Mechin, Florence B. Gilbert, Laurent Coquet, et al. To cite this version: Geoffrey Bailleul, Amanda Kravtzoff, Alix Joulin-Giet, Fabien Lecaille, Valérie Labas, et al.. The unusual resistance of avian defensin AvBD7 to proteolytic enzymes preserves its antibacterial activity. PLoS ONE, 2016, 11 (8), pp.1-20. 10.1371/journal.pone.0161573. hal-01594888 HAL Id: hal-01594888 https://hal.science/hal-01594888 Submitted on 26 Sep 2017 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. 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Distributed under a Creative Commons Attribution| 4.0 International License RESEARCHARTICLE The Unusual Resistance of Avian Defensin AvBD7 to Proteolytic Enzymes Preserves Its Antibacterial Activity GeoffreyBailleul1,AmandaKravtzoff2,AlixJoulin-Giet1,FabienLecaille2,ValérieLabas3, HervéMeudal4,KarineLoth4,5,Ana-PaulaTeixeira-Gomes1,3,FlorenceB.Gilbert1, LaurentCoquet6,ThierryJouenne6,DieterBrömme7,CatherineSchouler1, CélineLandon4,GillesLalmanach2,Anne-ChristineLalmanach1* 1 ISP,INRA,UniversitéFrançoisRabelaisdeTours,UMR1282,Nouzilly,France,2 CEPR,INSERM, a11111 UniversitéFrançoisRabelais,UMR1100,Tours,France,3 PRC,INRA,UniversitéFrançoisRabelaisde Tours,UMR85,UMRCNRS7247,Plate-formed'AnalyseIntégrativedesBiomolécules,Laboratoirede SpectrométriedeMasse,Nouzilly,France,4 CBM,CNRS,UPR4301,Orléans,France,5 Collegium SciencesetTechniques,Universitéd’Orléans,Orléans,France,6 Plate-formedeProtéomique"PISSARO" del'IRIB,UniversitédeRouen,CNRS,UMR6270,Mont-SaintAignan,France,7 DepartmentofOral BiologicalandMedicalSciences,UniversityofBritishColumbia,Vancouver,BritishColumbia,Canada *[email protected] OPENACCESS Citation:BailleulG,KravtzoffA,Joulin-GietA, Abstract LecailleF,LabasV,MeudalH,etal.(2016)The UnusualResistanceofAvianDefensinAvBD7to ProteolyticEnzymesPreservesItsAntibacterial Defensinsarefrontlinepeptidesofmucosalimmunityintheanimalkingdom,including Activity.PLoSONE11(8):e0161573.doi:10.1371/ birds.Theirresistancetoproteolysisandtheirensuingabilitytomaintainantimicrobial journal.pone.0161573 potentialremainsquestionableandwasthereforeinvestigated.Wehaveshownbybottom- Editor:JérômeNigou,CentreNationaldela upmassspectrometryanalysisofproteinextractsthatbothavianbeta-defensinsAvBD2 RechercheScientifique,FRANCE andAvBD7wereubiquitouslydistributedalongthechickengut.CathepsinBwasfoundby Received:February24,2016 immunoblottinginjejunum,ileum,caecum,andcaecaltonsils,whilecathepsinsK,L,andS Accepted:August8,2016 weremerelyidentifiedincaecaltonsils.HydrolysisproductofAvBD2andAvBD7incubated Published:August25,2016 withapanelofproteaseswasanalysedbyRP-HPLC,massspectrometryandantimicrobial Copyright:©2016Bailleuletal.Thisisanopen assays.AvBD2andAvBD7wereresistanttoserineproteasesandtocathepsinsDandH. accessarticledistributedunderthetermsofthe ConverselycysteinecathepsinsB,K,L,andSdegradedAvBD2andabolisheditsantibac- CreativeCommonsAttributionLicense,whichpermits terialactivity.OnlycathepsinKcleavedAvBD7andreleasedIle4-AvBD7,aN-terminaltrun- unrestricteduse,distribution,andreproductioninany catednaturalpeptidoformofAvBD7thatdisplayedantibacterialactivity.Besidesthe3- medium,providedtheoriginalauthorandsourceare credited. strandedantiparallelbeta-sheettypicalofbeta-defensins,structuralanalysisofAvBD7by two-dimensionalNMRspectroscopyhighlightedtherestrictedaccessibilityoftheC-termi- DataAvailabilityStatement:Allrelevantdataare withinthepaperanditsSupportingInformationfiles. nusembeddedbytheN-terminalregionandgaveaformalevidenceofasaltbridge(Asp9- Arg12)thatcouldaccountforproteolysisresistance.Thedifferentialsusceptibilityofavian Funding:GBwassupportedbyadoctoralfellowship (number00086685)fromtheInstitutNationaldela defensinstoproteolysisopensintriguingquestionsaboutadistinctiveroleinthemucosal RechercheAgronomique(http://www.inra.fr/)andthe immunityagainstpathogeninvasion. RégionCentreValdeLoire(http://www.regioncentre- valdeloire.fr/accueil.html).ThePlate-formed'Analyse IntégrativedesBiomolécules(VLAPTG)was financiallysupportedbytheRégionCentreValde Loire(http://www.regioncentre-valdeloire.fr/accueil. html),theCentreNationaldelaRecherche Scientifique(http://www.cnrs.fr/)andtheInstitut PLOSONE|DOI:10.1371/journal.pone.0161573 August25,2016 1/20 ResistanceofAvianDefensinstoProteolysis NationaldelaRechercheAgronomique(http://www. Introduction inra.fr/).TheNMRstudies(HMCL)werefinancially Theintestinaltractisconstantlyexposedtoacomplexcommunityofmicroorganismsthat supportedbytheTGIR-RMN-THCFr3050ofthe CentreNationaldelaRechercheScientifique(http:// includescommensalbacteriabutsometimesinvasivepathogensthattheepithelialinterfacehas www.cnrs.fr/).DBwasfinanciallysupportedbygrants tofight.Inthisbattle,defensinsplayanimportantroleinmucosalinnateimmunitybydisplay- (numberMOP89974andMOP201209)fromthe ingantimicrobialactivitytowardspathogens,inwoundrepaircapacityandininflammation CanadianInstitutesofHealthResearch(http://www. [1].Theyconstitutethelargestfamilyofcationicantimicrobialpeptidespresentthroughout cihr-irsc.gc.ca/e/193.html).Theworkwassupported byagrant(numberANR-10-EMID-001“HealthyGut”) theanimalkingdom,andmustbeconstantlyreadytoactintheirhost.Amongbirds,chicken fromtheEMIDAERA-NETprogramoftheEuropean possessarepertoireof14avianβ-defensins(AvBDs)butnoα-defensins,whicharerestricted Union(https://ec.europa.eu/research/fp7/index_en. tomammals,orθ-defensins,restrictedtoprimates[2].Thesechickendefensinsarecharacter- cfm?pg=eranet-projects-home). izedbyatypicalthree-strandedβ-sheetstructurestabilizedbythreedisulfidebridgesbetween CompetingInterests:Theauthorshavedeclared sixhighlyconservedcysteineresiduesasdeterminedforAvBD2[3],thatconstitutethehall- thatnocompetinginterestsexist. markofallβ-defensinsduringevolutionfrombirdstomammals.Twoofthem,AvBD1and AvBD2formerlyknownasgallinacin1andgallinacin2,havebeenisolatedfromgranulesof heterophils(avianpolynuclearneutrophils)[4,5].TheycanbepurifiedwithAvBD7fromthe bonemarrowanddisplayalargeantimicrobialspectrum[6].Heterophilscaninfiltratethe intestinaltissueofchickenduringinfectionsuchasSalmonellacolonizationofthecaecum,but arebarelypresentathomeostasis[7,8].However,geneexpressionofAvBD1andAvBD2has beenshowninchickenintestinalepithelialcells[9]andmoregenerallyinsmallandlargeintes- tineincludingcaecum[2].Thesedefensinshavebeenassociatedtothephenotypeofresistance toSalmonellacarriageinthecaecum[10].Bycontrast,littleisknownabouttheproteasesthat arepresentinthechickenintestinaltissueincomparisontothewelldescribedmammalian intestinalproteasesincludingserineproteases(neutrophilelastase,trypsin,chymotrypsin), aspartylcathepsinD(CatD),andcysteinecathepsins[11].Onestudyreportstheproteolytic activityinthechickenintestine,endorsedbycathepsins[12].Cathepsinshavebeenassociated inmammalswithinflammatoryprocessesand/orintissueremodeling.Theirfunctionsare determinedbysomestructuralmotifsconservedovermillionsofyearsaftertheevolutionary trailshavediverged,givingmultipleevolutionarygroupsofcysteinecathepsins[13].Ithas beenproposedthatcysteinecathepsinsincludingCatSmightbeinvolvedinpathological inflammatoryprocessessuchascolitis[14].Moreover,cysteinecathepsinscanimpairactivities ofantimicrobialpeptidesunderotherpathologicalconditionsinmucosaltissues[15–17]. Thisraisesthequestionofthesusceptibilityofaviandefensinstoproteolyticdegradationby intestinalproteases.Whilethehighresistanceofdefensinstowardproteolysiscouldbe expected,thestructuralandfunctionaldatasupportingthishypothesisremainelusive.The mainobjectiveofthepresentstudywasthereforetoanalyzethesensitivityofmajorintestinal AvBDstowardproteolyticdegradationandtodeterminetheabilityoftruncatedAvBDsto retainantibacterialproperties,andthusmaintainhostdefensecapabilities. MaterialsandMethods Ethicsstatement WhiteLeghornchickens,histocompatiblefortheB13haplotype(GB1Athenschickenline), werehatchedandraisedfreeofspecificpathogensatINRAanimalfacility(PlatformforExper- imentalInfectiology,PFIE,INRAValdeLoire,Nouzilly,France)until10weeksofage,incom- pliancewithFrenchandEuropeanguidelinesfortheaccommodationandcareofanimalsused forscientificpurposes(EuropeanUnionDirective2010/63/EU)andunderauthorizationand supervisionofofficialveterinaryservices(agreementnumberC-37-175-3deliveredtothePFIE animalfacilitybytheveterinaryserviceoftheDepartementd’IndreetLoire,France).Inorder tocollecttissuespost-mortem,tenchickensweresacrificedattenweeksofagebyanaesthetic PLOSONE|DOI:10.1371/journal.pone.0161573 August25,2016 2/20 ResistanceofAvianDefensinstoProteolysis overdose(intravenousdoseof50mg/kgofbodyweight)ofsodiumpentobarbital(Merial, France),incompliancewithEuropeanUnionDirective2010/63/EUforanimalkilling.Chicken sacrificeswereperformedbyoneoftheauthorsandbyananimaltechnicianofthePFIEani- malfacility(INRAValdeLoire),bothlicensedpersonsaccordingtotheEuropeanUnion Directive2010/63/EU.Theprocedurewasperformedinstrictcompliancewithlegaldisposi- tionsapplicableinFrance,mentioninganimaleuthanasiawithonlypurposeoforganortissue useisnotconsideredasanexperimentalprocedureandthusnotunderobligationofsubmis- siontoethicscommitteeforapproval(Ordinance2013–118,articleR.214-89,publishedinthe JournalOfficieldelaRépubliqueFrançaise#0032ofthe7thofFebruary2013,pp2199). Proteinextractionfromchickentissues BonemarrowwascollectedfromfemurandtibiaofteneuthanatizedWhiteLeghornchickens (seeEthicsstatementabove)aspreviouslydescribed[6],forAvBDspreparation.Approxi- mately400mgofvariousintestinalsegments(jejunum,ileum,caecum,andcaecaltonsils) 1 werecollectedandhomogenizedwithUltra-Turrax (IKA-Werke)inaconical10mLtube (Sarstedt,Nümbrecht,Germany)containing4mLoflysisbuffer.Thelysisbuffercontained50 mMTris-Hydrochloricacid(Tris-HCl),1mMethylenediaminetetraaceticacid,0.1%Tween 20,and1×HaltEDTA-freeproteaseinhibitorcocktailtablets(RocheDiagnostics,Meylan, France)withafinalvolumeof50mLofPBSbuffer,pH7.4(Gibco,ThermoFisherScientific, SaintAubin,France).Tubeswereplacedat4°Cduring5minandthencentrifugedat 11,200×gfor10minat4°C.Thesupernatantswerecollectedandstoredat4°C.Proteincon- centrationsweremeasuredusingQuickStart™BradfordProteinAssay(Biorad,Marnes-la- Coquette,France)accordingtothemanufacturer’srecommendations. SDSPAGEandimmunoblottingofintestinalproteinextracts Totalproteins(10μg)weredilutedinLaemmlibuffer,heatedat90°Cfor5minandthensub- jectedtoelectrophoresisunderreducingconditions(12%SDSPAGE)[18].Prestainedmolecu- larmassmarker(KaleidoscopePrestainedStandards,Biorad)wasused.Proteinswerefurther transferredontoanitrocellulosemembrane(HybondECL,GEHealthcareLifeSciences, Velizy-Villacoublay,France).Themembranewassaturatedwith5%BSAinPBS,0.5%Tween 20(PBS-T)for1h.Afterthreewashesof5mininPBS-T,themembranewasincubatedover- nightat4°Cunderagitationwithagoatanti-humancathepsin(Cat)Bantibody(R&DSystems, Lille,France)diluted1:1,000inPBS-Tcontaining5%lowfatpowderedmilk.After3washes for5mininPBS-Tbuffer,themembranewasincubatedfor1hwitharabbitanti-goatanti- bodyconjugatedtohorseradishperoxidase(Nordic-MUbio,Susteren,Netherland)atthedilu- tionof1:1500inPBS-Tcontaining5%lowfatpowderedmilk.After3washesfor5minin PBS-T,proteinswerevisualizedbychemiluminescence(SuperSignalWestPico/SuperSignal WestFemto,ThermoFisherScientific,SaintAubin,France),accordingtothemanufacturer’s instructions.Allincubationswereperformedat4°C.Thesameprotocolwasrepeatedwithgoat anti-humancathepsinsLandS(R&DSystems).Alternatively,westernblottingwasperformed usingamouseanti-humancathepsinKantibodyatthedilutionof1:1,000(Calbiochem,Merck Millipore,Molsheim,France)andarabbitanti-mouseantibody-horseradishperoxidaseconju- gateassecondaryantibody(Nordic-MUbio). Bottom-upproteomicapproachtoidentifyproteinsfromintestinaltissues Theproteinsfromeachgelsliceweredigestedusingtrypsinaspreviouslydescribed[19].The extractedpeptideswereanalyzedbyon-linenanoflowliquidchromatography-tandemmass spectrometry(nanoLC-MS/MS)usingaduallineariontrapFourierTransformMass PLOSONE|DOI:10.1371/journal.pone.0161573 August25,2016 3/20 ResistanceofAvianDefensinstoProteolysis Spectrometer(FTMS)LTQOrbitrapVelos(ThermoFisherScientific)coupledtoanUlti- 1 mate 3000RSLCUltraHighPressureLiquidChromatographer(Dionex,ThermoFisherSci- entific).SampleswereloadedonanLCPackingstrapcolumn(AcclaimPepMap100C18,100 mmi.d62cmlong,3μmparticles)anddesaltedfor10minat5mL/minwith4%solventB. MobilephasesconsistedofsolventA(0.1%formicacid,97.9%water,2%acetonitrile,v/v/v) andsolventB(0.1%formicacid,15.9%water,84%acetonitrile,v/v/v).Separationwascon- ductedusingaLCPackingsnano-column(AcclaimPepMapC18,75mmi.d650cmlong,3μm particles)at300nl/minbyapplyinggradientconsistedof4to55%ofsolventBfor90min.The massspectrometryanalyseswereperformedinpositiveionmodeandindata-dependentmode withhighresolution(R=60,000)fullscanMSspectra(profilemode)andlow-resolution CID-MS/MS(centroidmode).Inthescanningrangeofm/z300–1800,the20mostintense peptideionswithchargestatesof(cid:1)2weresequentiallyisolated(isolationwidth,2m/z;1 microscan)andfragmentedbyCIDwithnormalizedcollisionenergyof35%.Anactivation q=0.25andactivationtimeof10mswereused.Dynamicexclusionwasappliedduring30s witharepeatcountof1.Polydimethylcyclosiloxane(m/z,445.1200025)ionswereusedaslock massforinternalcalibration.Allrawdatafileswereconverted,processedandconfrontedto thechordatasectionofareferencecopyofnrNCBI(3326079sequences,download01/22/ 2014),usingsearchparametersaspreviouslydescribed[20]. PreparationofAvBDsandtop-downproteomicanalysis AvBDswerepurifiedfromchickenbonemarrowaccordingtotheprocedurepreviously described[6].Aftersize-exclusionchromatographyofthebonemarrowpeptideextract,frac- tionswerediluted(v/v)withamixtureofwater-formicacid-methanol(v:v:v;49:1:50)andwere directlyanalyzedbytop-downproteomicapproachforstructuralidentificationofnative AvBDsusingaduallineariontrapFourierTransformMassSpectrometer(FTMS)LTQOrbi- trapVelos(ThermoFisherScientific)aspreviouslydescribed[21].Allanalyseswereperformed usingahigh-highstrategy,meaningthataFTMSspectrausingprofilemodeinthemassrange m/z400–1500,wasfollowedbyanFTMS2spectraobtainedbyHCD(normalizedcollision energybetween40–60%).Targetresolutionwas100,000forFTMSandFTMS2analysis.The spectrumshowninthisstudycorrespondtotheaccumulationofscansoverapproximately1 min,yetgoodsignaltonoiseratioscouldbeobtainedwithinlesstime.Rawdatawereinte- gratedinProSightPCsoftware(ThermoFisherScientific)andprocessedbyTHRASH(signal/ noise:2–3).FTMSdatawereconfronteddirectlytoAvBDsequencesusing“singleprotein” searchoption.ProsightPCwasusedwithmonoisotopicprecursors,15ppmforfragmentions masstoleranceandthedeltamassfeaturedeactivated.Post-translationalmodificationssuchas disulfidebridges,N-terminalpyroglutamicacidandC-terminalamidationwereinterpreted usingthemanualSequenceGazermode.ProposedresultswithaPscoreof<0.05wereconsid- eredpositivelyidentifiedandstructurallycharacterizedwithaminimal5fragmentionsmatch- ing.Finally,RP-HPLCpurificationwasperformedaspreviouslydescribed[6],frompoolsof positivefractionsforAvBD2orAvBD7asdetermined,andaboutonemilligramofbothfull- lengthAvBD2andAvBD7wasobtainedforeachpreparation. HydrolysisofAvBD2andAvBD7 HumanCatKwasproducedasdescribedpreviously[22].HumanCatB,CatD,CatH,CatL andCatSwerepurchasedfromCalbiochem(VWRInternational,Fontenay-sous-Bois, France).TrypsinandchymotrypsinwerepurchasedfromEuromedex(Souffelweyersheim, France).Humanneutrophilelastase(HNE)wassuppliedbyBioCentrum(Krakow,Poland). Activesiteconcentrationsofproteasesweredeterminedaspreviouslydescribed[23].AvBD2 PLOSONE|DOI:10.1371/journal.pone.0161573 August25,2016 4/20 ResistanceofAvianDefensinstoProteolysis andAvBD7(12.5μM)wereincubatedinthepresenceof125nMoftrypsin,chymotrypsin, HNE,CatB,CatD,CatH,CatK,CatLorCatS.Assaysforcysteinecathepsins(B,H,K,Land S)werecarriedin0.1Msodiumacetatebuffer,pH5.5containing2mMDTTand0.01%Brij35 for4hat30°C.Thereactionwasstoppedbyadding4μMofE-64(L-3-carboxy-trans-2, 3-epoxypropionyl-leucylamido-(4-guanidino)butane,Sigma-Aldrich,Saint-Quentin-Fallavier, France).Alternatively,activitybufferswere0.1MTris/HClbuffer,pH7.8,20mMCaCl for 2 chymotrypsin,0.1MTris/HClbuffer,pH8.0,50mMCaCl ,100mMNaClfortrypsin,0.05M 2 HEPESbuffer,pH7.4,NP400.05%150mMNaClforHNE,and0.1Msodiumcitratebuffer, pH3.5,200mMNaClforCatD,respectively.Following4-hourincubation,serineproteases wereinactivatedby4-(2-Aminoethyl-benzenesulfonylfluoridehydrochloride(10μM)(Pefa- bloc,Sigma-Aldrich)whileCatDwasinhibitedbypepstatinA(10μM)(Sigma-Aldrich), beforeremovalof4μLofthereactionmixture.Incubationproductswereanalyzedbyreverse- phasehigh-performanceliquidchromatography(RP-HLC). Analysisofcleavageproductsofdefensins EachreactionmixturewassubmittedtoRP-HPLC(C-18Lichrocart55–2PurospherStarcol- umn)usingalinear0–90%water/acetonitrilegradientinthepresenceof0.1%TFA,ataflow rateof0.5mL/min.ChromatogramswereanalyzedusingtheChromQuestsoftware(Thermo FisherScientific).Majorpeakswerecollected,lyophilized,andresuspendedinH O. 2 ThemajorpeakproducedbytheAvBD7/CatKreactionwascollectedandconcentrated. Thesamplewassolubilizedwith10μLacetonitrile/trifluoroaceticacid/water(30/1/69v/v)and 0.5μLwasdepositedonMALDIplatewithequalvolumeofα-Cyano-4-HydroxyCinnamic Acid(CHCA)matrixsolutionat5mg/mLinacetonitrile/trifluoroaceticacid/water(50/0.1/ 49.9v/v).Afterdryingofthedropletinambientair,thesamplewasinspectedbyMALDI-TOF massspectrometryusingaMALDI-TOF-TOFUltraflex(BrukerDaltonics,Wissembourg, France)inreflectormodewithapulsed-ionextraction(PIE)delayof180nsandanaccelerat- ingvoltageintheionsourceof25kV.Calibrationwasperformedwithpeptidesofknown molecularmass(1−2.5kDarange):AngiotensinII,AngiotensinI,Neurotensin,ACTHclip(1– 17)andACTHclip(18–39).Theaccuracyofmassdeterminationswas±0.02%.Theidentifica- tionofAvBD7cleavagesiteswasdeterminedbycomparingtheexperimentallymeasuredpep- tidemassestothetheoreticalpeptidesmassesfromAvBDsequenceswiththeFindPeptTool accessibletoSIBBioinformaticsResourcePortal(http://web.expasy.org/cgibin/%22ndpept/% 22ndpept_form.pl). ThemajorpeakproducedbytheAvBD7/CatKreactionwasalsoanalyzedbyN-terminal sequencing.TheproteinofinterestwasloadedontoaprecycledBiobrenPlus-coatedglassfilter. TheN-terminalsequencewasdeterminedbyintroducingthefilterintoanAppliedBiosystems 494automatedproteinsequencer(LifeTechnologiesSAS,VillebonsurYvette,France)and runsofEdmandegradationwerecarriedout.Theresiduesobtainedwerematchedtothe expectedsequenceofdefensin. Antibacterialpropertiesofdefensinsandcleavageproducts TheantibacterialactivityofpurifiedAvBD2andAvBD7andofeachenzyme-AvBDreaction mixturewasmeasuredbyradialdiffusionassay[24]asdescribedpreviously[6],allowingthe determinationoftheminimalinhibitoryconcentration(MIC)againstStaphylococcusaureus ATCC29740andagainstEscherichiacoliATCC25922asrepresentativeGram-positiveand Gram-negativestrains,respectively.Thereactionmixturewithoutdefensinaddedwasusedas negativecontrolandthedefensinalonewasusedaspositivecontrol.ForAvBD7anditsmajor degradationproduct,MICwasdeterminedtowardsthreeGram-negativeandthreeGram- PLOSONE|DOI:10.1371/journal.pone.0161573 August25,2016 5/20 ResistanceofAvianDefensinstoProteolysis positivebacterialstrains,respectively,includingPseudomonasaeruginosaATCC25010, EscherichiacoliATCC25922,SalmonellaentericaserovarTyphimuriumLT2ATCC700720, ListeriamonocytogenesstrainEGD,StaphylococcusaureusATCC29740,andStreptococcussal- ivariusJIM8780.ThestrainSalmonellaentericaserovarTyphimuriumLT2waskindlypro- videdbyDrBenoîtDoublet(INRA,UMR1282ISP,Nouzilly,France).ThestrainStreptococcus salivariuswasaclinicalstrainisolatedfrombloodculture,kindlyprovidedbyDrChristine Delorme(INRA,UMR1319MICALIS,JouyenJosas,France). Statisticalanalysis AMann-Whitneynon-parametricUtestwasusedtocomparetheminimalinhibitoryconcen- trationofeachdefensinbeforeandafterincubationwithcathepsins. AnalysisofAvBD7structure 2D1HTOCSY(80ms)andNOESY(160ms)andasofast-HMQC[25](15Nnaturalabun- dance,704scans)experimentswereperformedat298Kona0.23mMsolutionofAvBD7in H O:D O(9:1ratio)atpH4.5onanAvanceIIIHDBRUKER950MHzspectrometer 2 2 equippedwithacryoprobe.IdenticalsetsofTOCSY/NOESYexperimentswererecordedat 288Kand308KonanAvanceIIIHDBRUKER700MHzspectrometerequippedwithacryo- probe,toresolveassignmentambiguitiesduetospinoverlaps.Afterlyophilization,thesame samplewasdissolvedinD Otorecordthesamehomonuclearexperimentsanda13C-HSQC 2 (naturalabundance,1184scans,700MHzspectrometerwithacryoprobe).1Hchemicalshifts werereferencedtothewatersignal(4.87ppmat288K,4.77ppmat298Kand4.68ppmat 308K).NMRdatawereprocessedusingBruker'sTopspin3.2™andanalyzedwithCCPNMR (version2.2.2)[26]. Structurecalculation StructureswerecalculatedwithNOEderiveddistances,hydrogenbonds(inaccordancewith theobservationofaβ-sheettypicallongdistanceNOEcrosspeaksnetwork—HN/HN,HN/Hα, α α H /H )andbackbonedihedralanglerestraints(determinedwiththeDANGLEprogram[27]) usingCNS[28,29]throughtheautomaticassignmentsoftwareARIA2(version2.3)[30].The pyroglutamicacidresidue(pQ)locatedattheN-terminalpositionoftheAvBD7sequenceis consideredasanonstandardresidueinCNS.Topologylibraries(topalldg5.3.proand topalldg5.3.pep)weremodifiedasdescribedintheARIA2.3tutorials.Covalentbondswere addedbetweensulfuratomsinvolvedineachbridge(Cys11-Cys40,Cys18-Cys33and Cys23-Cys41)byhomologywithotherβ-defensinsandinaccordancewithintra-cysteineNOE connectivities[Cys11(HN)/Cys40(Hβ),Cys18(HN)/Cys33(Hβ),Cys18(Hα)/Cys33(Hβ),Cys23 (Hβ)/Cys41(Hβ),Cys41(HN)/Cys23(Hβ),Cys41(Hα)/Cys23(Hβ)].TheARIA2protocolused simulatedannealingwithtorsionangleandCartesianspacedynamicswiththedefaultparame- ters.TheiterativeprocesswasrepeateduntiltheassignmentoftheNOEcrosspeakswascom- plete.Thelastrunusedalistof904NOE-deriveddistancerestraintsandwasperformedwith 1000structuresateachiteration.Finally200structureswererefinedinwater,andthe10best structureswereselectedonthebasisoftotalenergiesandrestraintviolationstatisticstorepre- sentAvBD7insolution.ThequalityofthefinalsetofstructureswasevaluatedusingPRO- CHECK-NMR[31]andPROMOTIF[32]software.ThefigureswerepreparedwithPYMOL [33].ElectrostaticandhydrophobicareaswerecalculatedattheConnollysurface,byAPBS [34]andbyPlatinum[35]software,respectively.Theatomicsolventaccessibleareaswere determinedbytheNACCESSprogram[36]. PLOSONE|DOI:10.1371/journal.pone.0161573 August25,2016 6/20 ResistanceofAvianDefensinstoProteolysis Results Detectionofcathepsinsanddefensinsinchickenintestinaltissues Weinitiallystudiedthepresenceofcysteinecathepsinsinsequentialsegmentsofchicken intestinaltract(jejunum,ileum,caecumandcaecaltonsils)thatwerepreviouslyreportedto expressAvBDgenes[2].Itshouldbenotedthatnocommercialanti-chickencathepsinanti- bodiesarecurrentlyavailable.However,anti-humanantibodiesdirectedtowardsCatB,Cat K,CatL,andCatSdisplayedasubstantialcrossreactivitywiththeavianenzymeswhentested bywesternblotting(Fig1).Thiscrossreactivityissupportedbythepercentageofidentityof 77%forCatB,80%forCatK,69%forCatLand67%forCatSbetweenhumanandchicken proteases(determinedbyNCBIBLASTtool,http://blast.ncbi.nlm.nih.gov/Blast.cgi).CatB wasidentifiedinproteinextractsfromjejunum,ileum,caecum,andcaecaltonsilsofbirds (Fig1).ConverselyCatK,CatLandCatSwereonlyrevealedincaecaltonsils.Inparallel, aspartylCatDwasdetectedbyhigh-resolutionmassspectrometryinileum,caecumandton- sils,butnotinjejunum,whileCatHwasuncoveredonlyincaecaltonsils(Table1).AvBD1 wasalsodetectedinintestinalsections,exceptileum.Theexhaustivelistofalltheproteins identifiedineachintestinalsegmentisprovidedassupplementarydata(seeS1Tableinthe supplementalmaterial).BothAvBD2(withahigherrelativeabundance)andAvBD7were Fig1.Immunodetectionofcathepsinsinchickenintestinalsegments.Proteinextractsfromchicken jejunum(J),ileum(I),caecaltonsil(To),andcaecum(C),aswellashumancathepsinsusedaspositive control(C+),weresubjectedtoSDS-PAGE(12%)beforeimmunoblottinganalysis.Eachhumancathepsin (hCat)isindicatedontherightside,withitsmolecularweight(MW)indicatedontheleftside. doi:10.1371/journal.pone.0161573.g001 PLOSONE|DOI:10.1371/journal.pone.0161573 August25,2016 7/20 ResistanceofAvianDefensinstoProteolysis Table1. IdentificationofAvBDsandcathepsinsinproteinextractsfromintestinalsegmentsanalysedbybottom-upmassspectrometry (nanoLC-MS/MS). IdentifiedProteins AccessionNumbera Numberof ProteinIdentification EmPAIc UniquePeptides Probability Tob C I J To C I J To C I J AvBD2 gi|385251609 4 5 3 4 100% 100% 100% 100% 197.44 259.43 30.79 67.44 AvBD7 gi|304282216 3 3 2 2 100% 100% 100% 100% 4.48 2.62 2.77 1.58 CathepsinDprecursor[Gallusgallus] gi|45384002 3 3 3 0 100% 100% 100% 42% 0.60 0.15 0.48 0 CathepsinB[Homosapiens] gi|16307393 1 0 1 0 98% 99% 100% 99% 0.15 0 0.09 0 CathepsinBprecursor[Gallusgallus] gi|46195455 0 0 1 1 0 98% 100% 98% 0 0 0.09 0.22 AvBD1Precursor gi|73915343 1 1 0 1 86% 84% 99% 99% 0.85 0.29 0 0.57 AvBD1Precursor gi|114053822 1 0 0 1 99% 0 92% 81% 0.88 0 0 0.59 CathepsinH[Gallusgallus] gi|330376140 1 0 0 0 99% 32% 38% 33% 0.16 0 0 0 CathepsinL1[Homosapiens] gi|148745204 1 0 0 0 100% 27% 0 0 0.15 0 0 0 aNCBInraccessionnumber. bTo,caecaltonsils;C,caecum;I,ileum;J,jejunum. cExponentiallymodifiedproteinabundanceindex. doi:10.1371/journal.pone.0161573.t001 foundinthefourintestinalsegments,i.e.jejunum,ileum,caecumandtonsils(Table1). Finally,caecaltonsilisarestrictedintestinalsitewheredefensinsandcathepsinsareexpressed andthusmaybothbeencountered. ResistanceofAvBDstoproteolysis AllformsofAvBDsseparatedbysize-exclusionchromatographyfromthechickenbonemar- rowproteinextractwereanalyzedbyhigh-resolutiontop-downmassspectrometry(seeS1Fig inthesupplementalmaterial).ResultsrevealedthepresenceofpeptidoformsofAvBD1, AvBD2,andAvBD7withvariabletruncationsoftheirN-and/orC-termini(upto3amino acids)assummarizedinTable2,accordingtothemassofthepeptidoformsidentified(seeS1 Figinthesupplementalmaterial).Thefull-lengthformsofAvBD2andAvBD7werefurther purifiedbyRP-HPLC.AvBD2andAvBD7wereincubatedwithtrypsin,chymotrypsin,and HNE,aspartylCatDandcysteineCatB,CatH,CatK,CatL,CatS(4hours;substrate: enzymemolarratioof100)respectively.Resultinghydrolysisproductswerefurtheranalyzed byRP-HPLC.Trypsin,chymotrypsin,neutrophilelastaseandCatDdidnotcleaveAvBD2 andAvBD7(seeS2Figinthesupplementalmaterial).Moreoverbothdefensinswereresistant tohydrolysisbyCatH(Figs2Band3B).AvBD2waspartiallydegradedbyCatB,CatL,and CatS(Fig2A,2Dand2E),leadingtothepartiallossofantimicrobialactivitytowardsEscheri- chiacoliandStaphylococcusaureusasattestedbythesignificantincreaseofitsMIC(Fig2F). Undertheseexperimentalconditions,AvBD2wastotallydigestedbyCatK(Fig2C),ascon- firmedbycompletelossofantimicrobialactivity(Fig2F).Bycontrast,AvBD7wasfullyresis- tanttohydrolysisbyCatB,CatLandCatS(Fig3A,3Dand3E)andretainedantimicrobial activity(Fig3F).CatKwastheonlyproteasethathydrolyzedextensivelyAvBD7(Fig3C), withoutincreasingitsMICtowardsE.colibutabolishingitsantibacterialeffecttowardsS. aureus(Fig3F).CatKwasabletogenerateamajordegradationproduct(peak4,Fig3C)for whichantimicrobialactivitywasfurtherdeterminedtowardsadditionalGram-positiveand Gram-negativestrains.Ofmajorinterest,theAvBD7-derivedpeptide(i.e.peak4)displayed similarantibacterialpropertiestothatofuncleavednativeAvBD7,exceptagainstStaphylococ- cusaureus(Table3). PLOSONE|DOI:10.1371/journal.pone.0161573 August25,2016 8/20 ResistanceofAvianDefensinstoProteolysis Table2. CharacterizationofAvBDspeptidoformsfrombonemarrowpeptideextractbytop-downmassspectrometry. Peptidename Sequence Modifications Observed Theoretical Monoisotopic[M+H]+ Monoisotopic[M+H]+ AvBD1full GRKSDCFRKSGFCAFLKCPSLTLISGKCSRFYLCCKRIW 3DBa+C-ter 4501.267 4501.223 length amidation AvBD1 —KSDCFRKSGFCAFLKCPSLTLISGKCSRFYLCCKRIW 3DB+C-ter 4288.140 4288.101 peptidoform1 amidation AvBD2full LFCKGGSCHFGGCPSHLIKVGSCFGFRSCCKWPWNA 3DB 3913.738 3913.717 length AvBD2 LFCKGGSCHFGGCPSHLIKVGSCFGFRSCCKWPWN- 3DB 3842.709 3842.680 peptidoform1 AvBD2 LFCKGGSCHFGGCPSHLIKVGSCFGFRSCCKWPW— 3DB 3728.664 3728.637 peptidoform2 AvBD2 -FCKGGSCHFGGCPSHLIKVGSCFGFRSCCKWPWN- 3DB 3729.667 3729.596 peptidoform3 AvBD2 -FCKGGSCHFGGCPSHLIKVGSCFGFRSCCKWPWNA 3DB 3800.667 3800.633 peptidoform4 AvBD7full QPFIPRPIDTCRLRNGICFPGICRRPYYWIGTCNNGIGSCCARGWRS 3DB+N-ter 5350.556 5350.513 length pyroglutamicacid AvBD7 QPFIPRPIDTCRLRNGICFPGICRRPYYWIGTCNNGIGSCCARGWR- 3DB+N-ter 5263.527 5263.481 peptidoform1 pyroglutamicacid AvBD7 ---IPRPIDTCRLRNGICFPGICRRPYYWIGTCNNGIGSCCARGWRS 3DB 4995.393 4995.360 peptidoform2 AvBD7 ---IPRPIDTCRLRNGICFPGICRRPYYWIGTCNNGIGSCCARGWR- 3DB 4908.362 4908.328 peptidoform3 aDB:disulfidebridges doi:10.1371/journal.pone.0161573.t002 StructuralinvestigationsonAvBD7 ThemajorandactivehydrolysisproductofAvBD7byCatKwasfurthercharacterizedbyN- terminalpeptidesequencingandbymassspectrometry(Fig4).Itssequencecorrespondedto the3aminoacidtruncatedformofAvBD7startingfromIle4(full-lengthAvBD7sequence numbering)anditsmassmatchedtothenaturalpeptidoform2ofAvBD7(Table2).Moreover, takentogethertheseresultsindicatethatthethreeN-terminalresiduesarenotessentialamino acidsforAvBD7antibacterialactivity. The3DstructureofAvBD7wasdeterminedbyNMRspectroscopy.The1Hhomonuclear andthenatural-abundance15Nheteronuclearspectrarevealedagooddispersionoftheamide chemicalshifts,indicativeofahighlystructuredpeptide.Severalminorformsco-existedin solution.However,theanalysisofthesetsof2D-TOCSYandNOESYspectraallowedacom- pleteassignmentof1Hchemicalshifts(BRMBentry34014)ofthemainform.Natural-abun- danceheteronuclearNMRspectraallowed39oftheNH,35oftheCαand33oftheCβshiftsto beassignedalongwithmanysidechaincarbonchemicalshifts(BRMBentry34014).This helpedustounambiguouslyassign1Hchemicalshifts,particularlyincrowdedregionsofthe 1HTOCSYandNOESYspectracorrespondingtosidechains.TheknowledgeofHN,Hα,Cα β andC chemicalshiftsallowedustousetheDANGLEprogramandobtain70dihedralangle restraintswhichsupplementthe904distancerestraintsderivedfromNOEs(Table4).Eight hydrogenbondsandthethreedisulfidebridgeswerealsointroducedinthecalculation.More- over,theArg25-Pro26amidebondwassettoacisconformationasattestedbythetypical NOEs:Arg25(Hα)/Pro26(Hα)andArg25(HN)/Pro26(Hα).Theuseofalltheserestraintswas necessarytoreachagoodconvergenceinthecalculation.Amongthe200refinedstructures,10 PLOSONE|DOI:10.1371/journal.pone.0161573 August25,2016 9/20
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