AntimicrobialactivityoftheAnseriformoutereggshella

AntimicrobialactivityoftheAnseriformoutereggshellandcuticleOlivierWellman-Labadie,JaroslavPicman,MaxwellT.Hincke⁎DepartmentofBiology,UniversityofOttawa,30MarieCurie,Ottawa,Ontario,CanadaK1N6N5Received28October2007;receivedinrevisedform4January2008;accepted4January2008Availableonline12January2008AbstractTheavianeggshellisacomplex,multifunctionalbiomineralcomposedofacalciumcarbonatemineralphaseandanorganicphaseoflipidsandproteins.Theoutermostlayeroftheeggshell,theeggshellcuticle,isanorganiclayerofvariablethicknesscomposedofpolysaccharides,hydroxyapatitecrystals,lipidsandglycoprotein.Inadditiontoregulatinggasexchanges,theeggshellcuticlemaycontainantimicrobialelements.Inthisstudy,weinvestigatedtheantimicrobialactivityofeggshellcuticleandoutereggshellproteinextractsfromfourAnseriformspecies:woodduck(Aixsponsa),hoodedmerganser(Lophodytescucullatus),Canadagoose(Brantacanadensis)andmuteswan(Cygnusolor).CuticleandoutereggshellproteinwasextractedbyureaorHCltreatmentofeggs.C-typelysozyme,ovotransferrinandanovocalyxin-32-likeproteinweredetectedinallextracts.CuticleandoutereggshellproteinextractsinhibitedthegrowthofStaphylococcusaureus,EscherichiacoliD31,PseudomonasaeruginosaandBacillussubtilis.Thepresenceofactiveantimicrobialproteinswithintheaviancuticleandoutereggshellsuggestsaroleinantimicrobialdefense.Proteinextractsfromthecavitynestinghoodedmerganserwereespeciallypotent.Theuniqueenvironmentalpressuresexertedoncavity-nestingspeciesmayhaveledtotheevolutionofpotentantimicrobialdefenses.©2008ElsevierInc.Allrightsreserved.Keywords:Antimicrobial;Cuticle;Eggshell;Protein;Waterfowl1.IntroductionTheavianeggshellisacomplexmultifunctionalcalcitebiomineral,composedofhighlystructuredcalciumcarbonate,containingameshworkofproteins,glycoproteinsandproteo-glycans.Theeggshellpermitstheexchangeofgasesandwaterwhileprotectingtheembryoagainstsmallpredatorsandthecrushingweightoftheincubatinghen.Duringtheformationoftheegg,withinspecializedregionsoftheoviduct,theeggshellisgraduallyacquiredasuterinefluidcomponentsaredepositedontotheeggshellmembranes.Uterinefluidofthedomestichenchangesincompositionduringthedifferentstagesofeggshellformationandinfluencescalcitecrystalgrowthindifferentzonesofthecalcifiedshell(Gautronetal.,1997a;Dominguez-Veraetal.,2000;Nysetal.,2004).Theeggshellcuticleistheoutermostlayerofthemineralizedeggshellandisindirectcontactwiththeenvironment.Thecuticleisathinlayerofvariablethickness(0.5to12.8μm)composedofhydroxyapatitecrystals,polysaccharides,lipidsandglycoprotein(Whittow2000;Fernandezetal.,2001).Thisorganiclayerisdepositedontheeggshellsurfaceandregulateswaterexchangesaswellastheentryofmicro-organismsthroughblockingoftheeggshellpores(Chavezetal.,2002).Theeggshellcuticleenhancestheantimicrobialdefensesoftheeggbyphysicallypreventingmicrobialcontamination(DeReuetal.,2005,2006).Nonetheless,thechemicalcomponentsofthecuticle,includingeggwhiteproteins,mayalsobeinvolvedintheantimicrobialdefenseoftheegg.Moststudiesofeggshellstructureandfunctionhavebeencarriedoutusingeggsofthedomesticchicken,Gallusgallus.Mannetal.(2006)recentlyidentified520proteinswithintheacid-solubleorganicmatrixofthechickeneggshellusingMS-basedtechnology.Multipleeggshellmatrixproteins,includingovocleidin-17(Hinckeetal.,1995),ovocleidin-116(Hinckeetal.,1999;Panheleuxetal.,1999),ovocalyxin-32(GautronAvailableonlineat(2008)640–649⁎Correspondingauthor.DepartmentofCellularandMolecularMedicine,UniversityofOttawa,451SmythRoad,Ottawa,Ontario,CanadaK1H8M5.Tel.:[phone]x8193;fax:[phone].E-mailaddress:[e-mail](M.T.Hincke).1096-4959/$-seefrontmatter©2008ElsevierInc.Allrightsreserved.doi:10.1016/j.cbpb.2008.01.001etal.,2001a;Hinckeetal.,2003)andovocalyxin-36(Gautronetal.,2007)havebeenidentifiedwiththeG.galluseggshellmatrix.Ansocalcin,ahomologofovocleidin-17,wasisolatedfromtheeggshellmatrixofthedomesticgoose,Anseranser,andhasbeenreportedtoinfluencecalciumcarbonatecrystal-lization(Lakshminarayananetal.,2002).Inadditiontotheirinvolvementineggshellformation,avianeggshellproteinsmayplayadditionalroles.Lysozymeandovotransferrin,twoeggwhiteproteinsknownfortheiranti-microbialproperties,havebeenidentifiedwithintheeggshellmatrixofthechicken(Hinckeetal.,2000;Panheleuxetal.,1999;Gautronetal.,1997b).Mannetal.(2006)identifiedlowlevelsofgallinacin-8andβ-defensin11,aswellashistones2and4,ascomponentsofthechickeneggshellmatrixusingMS-basedtechnology.MatrixproteinsextractedfromdemineralisedeggshelldemonstrateantimicrobialactivityagainstPseudomonasaeruginosa,Bacillussubtilis,Staphylococcusaureus,Escheri-chiacoliandSalmonellaenteritidis(Mineetal.,2003).HistonesH1andH2B,twoantimicrobialproteinsdemonstratingactivityagainstGram-positiveaswellasGram-negativebacteria,wereidentifiedwithinovaryandoviducttissueextractsofthedomestichenbySilphaduangetal.(2006)suggestingthattheseproteinsareincorporatedduringformationoftheegg.Eggshellmatrixproteinsandeggshellcuticleproteins,inparticular,arestra