The unusual resistance of avian defensin AvBD7 to proteolytic enzymes preserves its antibacterial

21 Pages·2017·3.54 MB·English

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
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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