INFEcrIONANDIMMUNITY,June1992,p.2153-21590019-9567/92/062153-07$02.00/ - PDF document

INFEcrIONANDIMMUNITY,June1992,p.2153-215
INFEcrIONANDIMMUNITY,June1992,p.2153-21590019-9567/92/062153-07$02.00/0Copyright©1992,AmericanSocietyforMicrobiologyPlasmid-EncodedOuterMembraneProteinYadAMediatesSpecificBindingofEnteropathogenicYersiniaetoVariousTypesofCollagenHENDRIKSCHULZE-KOOPS,lHARALDBURKHARDT,'JURGENHEESEMANN,2KLAUSVONDERMARK,1ANDFRANKEMMRICHl*Max-Planck-Society,ClinicalResearchUnitforRheumatology/ImmunologyattheInstituteforClinicalImmunologyoftheUniversityErlangen-Nurnberg,D-8520Erlangen,'andInstituteofHygieneandMicrobiology,UniversityHospital,D-8700Wurzburg,2GermanyReceived9December1991/Accepted6March1992Theplasmid-encodedoutermembraneproteinYadAofenteropathogenicyersiniaeisassociatedwithpathogenicity.Recently,collagenbindingofYadA-positiveyersiniaewasreportedwithoutdetailedcharacter-ization(L.Emody,J.Heesemann,H.Wolf-Watz,M.Skurnik,G.Kapperud,P.O'Toole,andT.Wadstrom,J.Bacteriol.171:6674-6679,1989).ToelucidatethenatureofcollagenbindingtoYadA,weusedarecombinantYersiniastrainexpressingtheclonedYadAgene.DirectbindingofYadA-positiveyersiniaetocollagenswasdemonstratedinaffinityblotexperimentsonnitrocellulosefilters.Aspectrumofcollagentypesinawideconcentrationrangeweretestedfortheirabilitytoblockbindingof'251-labeledcollagentypeIItoYadA-positiveyersiniae.Theresultsindicateaspecificbindingsite(s)forYadAincollagentypesI,II,III,IV,V,andXI.Incontrast,collagentypeVIdidnotbindtoYadA.Tocharacterizethebindingsite(s)moreprecisely,isolatedcollagenchainsandcyanogenbromidefragmentswereinvestigated.ThesestudiesrevealedthatbindingofYadAtocollagentypeIisconfinedtothecd(I)chain,whereasthebindingsitewithincollagentypeXIislocalizedintheo3(XI)chain.a2(I),cl(XI),anda2(XI)didnotbindtoYadA.Mostinterestingly,intheal(II)chainthespecificbindingsiteforYadAresidesinthecyanogenbromidefragmentCB10.Thelattermightindicateabindingsitethatdoesnotdependonconformation.Basedonthesefindings,furtherfragmentationandthesynthesisofpeptidesmayallowdefinitionofthepeptidesequence(s)relevantforYadAbinding.YersiniaenterocoliticaandYersiniapseudotuberculosisareenteropathogenicforhumans(4,7).Thediseasespec-trumcomprisesbothintestinalandextraintestinalmanifes-tationsinvolvingtheconnectivetissue,suchasreactivearthritis,erythemanodosum,anddifferenttypesofvasculi-tis(4,7,38).Recently,itwasdemonstratedthataplasmidofabout70kbisnecessaryforfullvirulenceexpressioninyersiniae(forreviews,seereferences6,15,29,and41).Differentplasmid-encodedproteins,includingoutermem-braneproteinswithpathogenicfunctions,havebeende-scribed(2,3,6,13,14).Oneofthemostprominentplasmid-encodedoutermembraneproteinsistheYersiniaadhesinYadA.Itformsmultimericfibrillaeonthesurfaceofentero-pathogenicyersiniae(25).ThereisplentyofevidencethatYadAisinvolvedinresistancetocomplementlysis,phago-cytosis,andadherencetomammaliancells(14,16).BindingofcollagentypesI,II,andIVtoYadA-positiveyersiniaehasbeenreported(8).Thispropertymightberelevantforthepathogenesisofdiseasesinducedbyenteropathogenicyer-siniae.However,thespecificityofthecollagen-YadAinter-actionstillremainsobscure,sincethereportedcompetitionexperimentswereperformedata400-to1,000-foldmolarexcessoftheunlabeledinhibitor.ThispromptedustoinvestigatethenatureofcollagenbindingbyYadAinmoredetail.InafirstseriesofexperimentswetesteddirectbindingofYadA-positiveyersiniaetoabroadspectrumofcollagensimmobilizedonnitrocellulosefilters.Wedemon-strateherethatYadAexpressionmediatesbindingofyers-iniaetoallcollagensinvestigatedexceptcollagentypeVI.Inblockingexperiments,differentcollagensinawideconcen-*Correspondingauthor.trationrangeweretestedfortheirabilitytoinhibitbindingofthemaincartilagecollagen,collagentypeII,toYadA-positiveyersiniae;blockingoccurredatpicomolarconcen-trations.Theexperimentsindicateaspecifichigh-affinitybindingsite(s)forYadAonmanycollagentypes.Conse-quently,theanalysiswasextendedtoassigntheputativebindingsitetoeitherisolatedcollagenchainsorcyanogenbromidefragments.MATERIALSANDMETHODSBacterialstrains.ThreeYenterocoliticastrainsofsero-type0:5wereusedinthisstudy.StrainNFOisofenviron-mentalorigin,plasmidfree,andYadAnegative.StrainNF-pCB9::Tn5harborsthevirulenceplasmidofYentero-coliticaserotype0:9withaninactivatedYadAgene.StrainNF-pRK29OB9-4harborsahybridplasmidconsistingofthemobilizablevectorpRK29OB(16)andaBamHIfragmentofthevirulenceplasmidofYenterocolitica0:9encodingYadAandisotherwisehomologoustoNFO;ithasbeendemonstratedthatthevectorpRK29OBdoesnotencodecollagenbindingfactors(8).Allstrainsweredescribedprev

iously(8,14,16,17).BacteriaweregrowninLu
iously(8,14,16,17).BacteriaweregrowninLuria-Bertanibouillonat37°Cfor24h,heatinactivatedat60°Cfor45min,andstoredinphosphate-bufferedsaline(PBS)-0.01%NaN3(pH7.3)at4°C.Collagenpreparation.HumantypeIandVcollagensfromplacentaltissuewerepreparedasdescribedbyBentzetal.(1)andkindlyprovidedbyK.Kuhn,Martinsried,Germany.TypeII,IX,andXIcollagenswerepreparedfromthechickenxyphoidprocessbyextractionwithguanidiniumhydrochlorideforremovalofproteoglycans,digestionwithpepsin,andfractionalsaltprecipitationasdescribedbyvon2153Vol.60,No.62154SCHULZE-KOOPSETAL.derMarketal.(40).Thefour-stepprocedureofcollagentypeIVpreparationfromhumanplacentawasperformedasdescribedbyGlanvilleandRauter(12)andincludedpepsinsolubilizationoftissue,fractionalsaltprecipitationofsolu-bilizedcollagens,nativereductionandcarboxymethylation,asecondpepsintreatment,andprecipitationofhigh-molec-ular-weightmaterialsoutofthedenaturedsamplewith3MNaCl.TypeIIIcollagenwaspreparedfromfetalbovineskin.Extractionofcollagenfromtheskin,precipitationbyam-moniumsulfateandNaCl,dialysisagainst2Mureacontain-ingNaCl-Trisbuffer,andseparationofcollagentypeIIIwithDEAE-cellulosecolumnswereperformedasdescribedbyTimpletal.(37).Pepsin-solubilizedcollagentypeVIfromhumanplacentawaspurifiedasdescribedbyvonderMarketal.(39).Proteoglycans.Proteoglycanswerepreparedbyextractionwith1MNaClofhumanfetalcartilagefollowedbyDEAEchromatography(23).TheywerekindlyprovidedbyT.Kirsch,MPG-Arbeitsgruppen,Erlangen,Germany.CNBrcleavageandCBfragmentpurification.Samplesof50mgofcollagentypeIIwerecleavedwithCNBr,andthecyanogenbromidefragments(CBfragments)werepurifiedasrecentlydescribedbyBurkhardtetal.(5).Briefly,colla-gentypeIIwasdissolvedin70%HCOOH(10ml),warmedto50°Ctoensuredenaturation,andflushedwithnitrogen.ThenCNBrwasaddedina100-foldmolarexcessovermethionineresidues(1.5mg/mgofcollagen)andallowedtoreactfor4hat37°C.ForfurtherpurificationtheCBfragmentsweresubjectedtofastproteinliquidchromatog-raphyin0.1Mammoniumacetate(pH5)onaSuperose12column.Homogeneityofthepeptideswascontrolledbysodiumdodecylsulfate(SDS)-gelelectrophoresisin18%acrylamideminigelswiththebuffersystemofLaemmli(26).Isolationofcl(I)anda2(I).(xl(I)and(2(I)collagenchainswereseparatedbychromatographyoncarboxymethylcellu-loseat42°Cin0.04Msodiumacetate(pH4.8)containing4Mureaasdescribedpreviously(40).ThepurityofthechainswascontrolledbySDS-polyacrylamidegelelectrophoresis(PAGE)in7%acrylamideminigelswiththebuffersystemofLaemmli(26).RadiolabelingofcollagentypeII.CollagentypeIIwasradiolabeledbytheiodogenmethod(10),andaspecificactivityof2x106to3x106cpm/,ugwasdetermined.BindingofradiolabeledcollagentypeIItoyersiniaeandblockingexperiments.Increasingamountsof1251I-labeledcollagentype11(2.7x106cpm/pRg)wereincubatedwith100Rlofparticulatebacterialsuspension(2x109bacteria)for75minatroomtemperatureundergentleagitation.Then1mlofPBS-0.01%Tween20wasadded,andthesuspensionwascentrifuged(30min,10°C,7,500xg).Thesupernatantwasaspirated,andtheradioactivityassociatedwiththepelletwasdeterminedinagammacounter(COBRA5005;CanberraandPackardInstruments).YadA-negativeyersin-iaewereusedasacontrolforunspecificbindingofcollagentypeIItoyersiniae.Intheblockingassays,100ngofradiolabeledcollagentypeIIwasused;withoutblockingproteins,thisgavesignalsofabout2.2x104cpmwithYadA-positiveyersiniaeandabout3.5x103cpmwithYadA-negativeyersiniaeafterthewashingprocedure.Forblockingexperiments,thedesiredamountofproteinin10pIofPBSwasincubatedwith100,uloftheparticulatebacterialsuspensionfor75minatroomtemperatureunlessotherwisestated.Samplesof100ngof1251-collagentypeIIin10,u1ofPBSwereaddedandallowedtoreactfor75minatroomtemperatureunlessotherwisestated.Washinganddetermi-nationofradioactivitywereperformedasdescribedabove.Allsampleswereanalyzedinatleasttwoindependentexperimentsinduplicateassays.Theplastictubesusedfortheassayswereprecoatedwithbovineserumalbumin(BSA)tominimizenonspecificbindingofcollagenandbacteriatothewallsofthetubes.Affinityblots.SDS-PAGEwasperformedoncollagenandcollagenfragmentswith18and10%acrylamideminigels,respectively.Theproteinswereelectrophoreticallytrans-ferredtonitrocellulosefilters.Afternonspecificproteinbindingsiteswereblockedwith1%BSAinPBSfor2hatroomtemperature,thefilterwasincubatedwithaparticulateYersiniasuspension(3x109bacteriapermlinPBS)for2hatroomtemperature.Arabbitanti-Yenterocolitica0:5antibodywasusedtodetectfilter-boundyersiniae,andperoxidase-conjugatedgoatanti-rabbitimmunoglobulinGwasusedasthedetectionsy

stem.ELISA.Fortheenzyme-linkedimmunosorb
stem.ELISA.Fortheenzyme-linkedimmunosorbentassay(ELISA),samples(10,ul)ofacollagensolution(variousconcentrationsinPBS)wereincubatedwith100,ulofthebacterialsuspension(2x109bacteria)inVmicrotiterplatesfor90minatroomtemperature.Theplateswerecentrifuged(20min,15°C,550xg),andthepelletswerewashedwith100p.1ofPBS-0.1%Tweenbyresuspension.Centrifugationandwashingwererepeated.Thebacteriawereincubatedwithananti-collagentypeII-specificantibody(in100p.lofPBS-1%BSA,4°C,overnight)andthenwashedasdescribedabove.Thepelletswereincubatedwithperoxidase-conju-gatedrabbitanti-mouseimmunoglobulinG(Bio-RadLabo-ratories)diluted1:5,000inPBS-1%BSAfor2hatroomtemperature.Bacteriawerewashedagain,andthesubstrate(3,3'-5,5'-tetramethylbenzidine[50ml],0.01%in100mMcitrate-phosphate(pH6)plus5Rlof30%H202)wasadded.TheA450wasdeterminedforthesupernatantaftercentrifu-gation.Themonoclonalanti-chickencollagentypeIIanti-bodiesusedinthisstudy(Bi,Cl,D3)werekindlyprovidedbyR.Holmdahl,Uppsala,Sweden.Theyweredescribedpreviously(5,19).TheepitopesoftheseantibodiesonchickencollagentypeIIarelocatedonspecificCBfrag-ments:CB8(B1),CB10(D3),andCB11(Cl).Thecurvesforbindingofallthreeantibodiestotheircorrespondingfrag-mentsweresimilar,andtheantibodyconcentrationswereadjustedtosimilarsignalintensities.RESULTSBindingofYadA-positiveyersiniaetoimmobilizedcollagen.Totestfordirectbindingofyersiniaetoaspectrumofcollagens,weperformedaffinityblotexperimentswithim-mobilizedcollagen.AfterSDS-PAGEandelectroblottingoftheproteinstoanitrocellulosefilter,yersiniaewereadded;thebacteriaboundtothefilterweredetectedbyusingananti-Y.enterocolitica0:5antibody.YadA-positiveyersiniaeboundtocollagentypesII,V,andXI,whereasnobindingwasdetectabletotypeVIcollagen(Fig.1).WithintheheterodimericcollagentypeV,YadA-positiveyersiniaeboundtobothchains,whereas,withintheheterotrimericcollagentypeXI,YadA-mediatedbindingwasconfinedtotheO3(XI)chain.CollagentypesIIIandIValsoboundYadA-positiveyersiniae(datanotshown).YadA-negativeyersiniaedidnotbindtoanyofthecollagens(Fig.1).ThemostabundantcollagenistypeIcollagen,ahetero-trimericcollagenconsistingoftwootl(I)chainsandonea2(I)chain.WewereinterestedindeterminingwhetherbindingofYadAtocollagentypeIisrestrictedtobothchains,whichshow65%homologyattheproteinlevel.ThechainswereINFECT.IMMUN.COLLAGENBINDINGOFYERSINIAENTEROCOLITICA2155abcdefghiFIG.1.BindingofYadA-positiveandYadA-negativeyersiniaetoimmobilizedcollagen.CollagentypesII(a,b),XI(c,d),V(e,f),andVI(g,h)wereelectroblottedtonitrocellulosefiltersafterSDS-PAGE.YadA-positive(a,c,e,g)orYadA-negative(strainNFpCB9::Tn5)(b,d,f,h)yersiniaewereadded,andfilter-boundbacteriawereassessedbyusingarabbitanti-Y.enterocolitica0:5antibody.Ananti-rabbitperoxidaseconjugatewasusedasthedetectionsystem.Thepositionsoftheproteinsnotboundbyyersiniae[axl(XI)and(x2(XI)inlanec,collagentypeVIinlanesgandh]areindicatedaccordingtotheirlocalizationbyPonceauredstaining.Coomassie-stainedSDS-PAGEofcollagentypeVIisalsoshown(i).isolatedbyion-exchangechromatographyandfurtherpuri-fiedbyreverse-phasehigh-pressureliquidchromatography.TheaffinityblotexperimentwiththeisolatedchainsofcollagentypeIshowedthatbindingtoYadAwaslimitedtotheotl(I)chain(Fig.2).Bindingof'251-labeledcollagentypeIItoYadA.Themaincartilagecollagen,collagentypeII,wasradiolabeledtofurthercharacterizebindingofYadA.Increasingamountsof1251I-labeledcollagentype11(2.7x106cpm/,ug)wereaddedto2x109YadA-positiveandYadA-negativeyersiniae.BindingofcollagentypeIItoYadA-negativeyersiniaewasusedtodetectnonspecificbinding.TheresultindicatessaturationofspecificbindingofcollagentypeIItoYadAathigherconcentrations(Fig.3).Basedonthesefindings,100ngof251I-labeledcollagentypeIIwaschosenasreferenceforfurtherbindingandblockingexperiments.Intheassay,afterincubationandwashing,100ngofradiolabeledcollagentype11(2x105to3x105cpm)gavesignalsofabout22,000cpmwithYadA-positiveyersiniaeandabout3,500cpmwithYadA-negativeyersiniae.BlockingofcollagentypeIIbindingtoYadAbyvariouscollagens.VariouscollagensandcollagenpreparationswereabcdFIG.2.BindingofYadA-positiveyersiniaetoimmobilizedcol-lagentypeIchains.Isolatedchainsoal(I)anda2(I)weresubjectedtoSDS-PAGE(aandb,respectively)andthenelectroblottedtonitrocellulosefilters(dandc,respectively).Thearrowheadmarksthepositionofthea2(I)chain,accordingtoitslocalizationbyPonceauredstaining.TheexperimentswereperformedasdescribedinthelegendtoFig.1.usedtoinhibitbindingofradiolabeledcollagentypeIItoYadA-positiveyersiniae.Heat-de

naturedandnativetypeIIcollagenswereequal
naturedandnativetypeIIcollagenswereequallyeffectiveinpreventingbindingofsubsequentlyaddednativeradioactivecollagentypeII(Fig.4).ForthisexperimentcollagentypeIIwasheatdenatured(42°C,60min)andtheblockingassaywasperformedat37°Ctopreventspontaneousrenaturation.CollagentypesII,III,IV,V,andXIshowedverysimilarinhibitioncurves(Fig.4and5).Thedatasuggesthigh-affinitybindingofallfivecollagenstoYadA.About60%inhibitionwasobservedalreadywithafivefoldmolarexcess(500ng)oftheinhibitor.ThecontrolproteinsBSAandcartilageproteoglycansdidnotshowanyinhibitionofcollagentypeIIbindingtoYadA(Fig.5A).However,theabilitytoblockcollagentype1l-YadAbindingisnotsimplyafeatureofalltriple-helicalcollagens.CollagentypeVIatuptoa20-foldmolarexcessoverradiolabeledcollagentypeIIdidnotblockcollagentypeII-YadAbinding.Onlyatahigherconcentration(50-foldmolarexcess)wasinhibitionobserved.ThecollagentypeIXinhibitioncurveshowedgreaterinhibitioncomparedwiththatofcollagentypeVIanddidnotshowthetypicalhighefficientblockingofcollagentypesII,III,IV,V,andXIinthelowconcentrationrange.BlockingofcollagentypeI1-YadAbindingbycollagentypeIandisolatedcollagentypeIchains.TypeIcollagendidnotblockcollagentypeII-YadAbindingasefficientlyastheotherfibril-formingcollagens,e.g.,typesII,III,V,andXI,did(Fig.5and6).SincetheaffinityblotexperimentswiththeisolatedcollagentypeIchainsrevealedthatthebindingsiteforYadAwasconfinedtothe(x1(I)chainonly(Fig.2)andbindingofcollagentypeIItoYadAwasindependentofthetriple-helicalconformation(Fig.4),weperformedblockingexperimentswiththeisolatedchainsofcollagentypeI.TheblockingcapacityofcollagentypeIatlowmolarconcentra-tionsresidedcompletelyintheal(I)chain(58%inhibitionat500ng)(Fig.6).Theblockingpotentialofot1(I)wascompa-rabletothatofcollagentypeIIinnativeanddenaturedforms(Fig.4).Thea2(I)chainfailedtoexhibitsignificantblockingcapacity(9%inhibitionat500ng).BlockingofcollagentypeII-YadAbindingbycyanogenbromidefragmentsofcollagentypeII.Tofurthercharacter-izethebindingsiteforYadAoncollagentypeII,CNBrcleavageatmethioninesitesofcollagentypeIIwasper-formed,andthenthefragmentswerepurifiedbyfastproteinliquidchromatography.Themodifiedsolid-phaseELISAwiththepurifiedfragmentsandfragment-specificmonoclo-nalantibodiesdemonstratedYadA-mediatedbindingforCB10only(datanotshown).Therefore,CB10(346aminoacids)andtheapproximatelyequalsizedCB11(279aminoacids)wereusedintheblockingexperimentsshowninFig.7.TheamountsofthethreeinhibitorproteinsshowninFig.7areindicatedinmolaritiesforbettercomparison[oxl(II),-95kDa;oxl(II)CB10,-31kDa;al(II)CB11,-25kDa].OnlyCB10wasefficientinblockingcollagentypeII-YadAbinding.Uptoaconcentrationof3pM,theblockingcapacitiesofCB10andthewholecollagentypeIImoleculeweresimilar.Atthisconcentrationthemaximuminhibition(55%)wasreachedfortheinhibitorCB10.Furtherriseofinhibitorconcentrationuptoa150-foldmolarexcess(corre-spondingto5,ug)didnotincreasetheblockingpotentialforthecollagentypeII-YadAinteraction.CB11didnotblockcollagentypeIIbindingtoYadA.InteractionsathighinhibitorconcentrationssuchasthoseobservedwiththecompletepolypeptidechainscouldnotbedetectedwiththeCBfragmentsusedforthebindingstudies.VOL.60,19922156SCHULZE-KOOPSETAL.C)0cv08Cu1*to'DC0.080-604020005101520251251-collagentype11added[106cpm]FIG.3.SpecificbindingofradiolabeledcollagentypeIItoYadA-positiveyersiniae.Increasingamountsof"WI-labeledcollagentype11(2.7x106cpm/I,g)wereincubatedwith2x109bacteria.UnspecificbindingtoYadA-negativeyersiniaewasdeterminedforeachconcentrationandsubtracted.TheinsertdemonstratestotalcountsobtainedwithYadA-positive(-)andYadA-negative(0)yersiniaeaswellastheresultingspecificbinding(A).Eachvaluerepresentstheaverageofduplicateassays.Thevariationofeachindividualvaluefromthemeanneverexceeded10%.DISCUSSION7100CD00~402001010025050010002000500025000inhibitor[nglFIG.4.InhibitionofthebindingofradiolabeledcollagentypeIItoYadAbynative(lightstippling)andheat-denatured(420C,60min)(darkstippling)collagentypeII.YadA-positiveyersiniae(2x10ibacteria)wereincubatedwithdifferentconcentrationsofthecolla-genpreparations,andthenradiolabelednativecollagentypeII(2x105to3x105cpm)wasadded.Theassayswereperformedat370Ctopreventspontaneousrenaturation.Radioactivityassociatedwiththepelletwasmeasuredinagammacounter.RadioactivityobtainedwithradiolabeledcollagentypeIIandYadA-positiveyersiniaewithoutpreincubationwithotherproteinswasdefinedas100%'25M-labeledcollagentypeIIbinding.Eachvaluerepresentsthemeanandstandarddeviationofatleastt

woindependentduplicateassays.Attachmento
woindependentduplicateassays.AttachmentofbacteriaorbacterialproductstohostcellsandhosttissueiscrucialforinfectiousdiseasescausedbyenteropathogenslikeYenterocolitica.Previousstudieshavereportedonbindingofdifferentbacteriatoextracellularmatrixproteinslikelaminin(9,27,32,34,35),fibronectin(24;forareview,seereference20),andcollagen(8,9,18,33)andhavemoreovershowntheimportanceoftheseinterac-tionsfortissueadhesion(21,22).TheinteractionofproteinsexpressedbyYenterocoliticawithcomponentsoftheextracellularmatrix,likecollagen,maycontributetodis-easesaffectingtheconnectivetissue.Therefore,weinvesti-gatedthebindingtocollagenofthevirulence-associatedoutermembraneproteinYadA,whichiscommontoallenteropathogenicyersiniae.WeidentifiedabroadspectrumofcollagentypesthatbindYadA-positiveyersiniaeinaffin-ityblotexperiments(collagentypesI,II,III,IV,V,andXI).Interestingly,YadA-positiveyersiniaedidnotbindtoallcollagenstested(e.g.,didnotbindtypeVIcollagen),andinsomecollagensthebindingpropertyforYadAwasrestrictedtoasinglechainonly[otl(I),a3(XI)].Collagenbindinghasbeenreportedforrathapatocytes(31)andStaphylococcusaureus(33),bothofwhichboundtoallcollagenstested(collagentypesI,II,III,IV,andVforrathepatocytesandcollagentypesI,II,III,IV,V,andVIforS.aureus).DifferentCNBr-generatedpeptidesoftheacl(I)chainandsyntheticpeptideswithcollagenlikestructures[e.g.,(Pro-Gly-Pro)n]werealsoboundindiscriminately.Incontrasttothesefindings,ourresultsargueinfavorofaYadAcollagenINFECT.IMMUN.COLLAGENBINDINGOFYERSINIAENTEROCOLITICA215710000.c8000)60to0cm40C.5C101102103104inhibitor[ng]:070100080CD060,600020101102103104inhibitor[ng]FIG.5.InhibitionofthebindingofradiolabeledcollagentypeIItoYadAbydifferentcollagensandotherpeptides.CollagentypesIV(A),V(-),andXI(A)(A)andtypesIII(0),VI(A),andIX(U)(B)wereinvestigated.TheexperimentswereperformedasdescribedinthelegendtoFig.4,exceptthatthetemperaturewasroomtemperature.Cartilageproteoglycans(O)andbovineserumalbumin(0)wereusedascontrols(A).Eachvaluerepresentsthemeanandstandarddeviationofatleasttwoindependentduplicateassays.bindingsitethatisnotdeterminedbythesimplerepetitivesequenceofcollagen(Gly-X-Y)butratherbyaspecificaminoacidmotif.StudieswithYadA-positiveandYadA-negativeyersiniaedemonstratedthatbindingofcollagentypeIItoYadAissaturable,whichisfurtherevidenceforaspecificinteraction.PreincubationofYadA-positiveyersiniaewithunlabeledcollagentypeIIpreventedbindingofsubsequentlyadded'"I-collagentypeIIinaconcentration-dependentmanner.Moreover,nativeandheat-denaturedtypeIIcollagensin-hibitedthecollagentypeII-YadAinteractiontosimilardegrees.Furthermore,ourexperimentsrevealedthat,incontrasttothea2(I)chain,theal(I)chainanda346-amino-acidCBfragment(CB10)ofcollagentypeIIharboraspecificbindingsiteforYadA.Therefore,YadAseemstointeractwithalocallyrestrictedpeptidesequence,whereasbindingtoaconformationallydependentepitopeformedbyscat-teredaminoacidsanddependentonthetriplehelicalstruc-tureofcollagenseemsratherunlikely.ItremainstobeshownwhethertheblockingcapacitiesofcollagentypeVIandofthect2(I)chainatvastmolarexcesses(Fig.SBand6)weredueto(i)minutecontamina-tions(5%)ofthepreparationswithothercollagensnotvisibleinCoomassie-stainedSDS-PAGE,(ii)aspecificbind-ingsitewithloweraffinity,or(iii)simplyastickinessbecauseofweaknoncovalentforcesalongthewholemole-VOL.60,19922158SCHULZE-KOOPSETAL.10CL03)0)0)CDC-.510080604020-l101102103FIG.6.InhibitionofthebindingofradiolabeledcoltoYadAbycollagentypeI(U)andisolatedcollagenal(I)(l)anda2(I)(0).TheexperimentswerepdescribedinthelegendtoFig.4,exceptthatthetemroomtemperature.Eachvaluerepresentsthemean,deviationofatleasttwoindependentduplicateassayscule.Consideringoneofthelasttwooptions,tEtheCBfragmentsinvestigatedinthisstudytoftcollagentypeII-YadAbindingathighinhibitortionsmightbedueto(i)anadditionalbindingsitoncollagentypeII,localizedonaCBfragmentrtheblockingassays,or(ii)destructionoflow-affiitoYadAbytheCNBrcleavage.ThecapacitiesofthecollagenstestedinthisstYadAaredifferent.However,somecollagensreactionpatterns.CollagentypesII,III,IV,V,nottypesVIandIXboundtoYadAwithhighwereabletoinhibitcollagentypeII-YadAbindiiingexperimentsatlowinhibitorconcentrationsformingcollagens(28)excepttypeIcollagen(i.e71008056040020.5W10-1100101FIG.7.InhibitionofthebindingofradiolabeledcoltoYadAbycollagentypeII(-)andthecollagentypeCB10(O)andCB11(0).Theamountsoftheinhibitorindicatedinmolarities[oxl(II),-95kDa;al(II)CB1(al(II)CB11,-25kDa).Theexperimentswereperfoscr

ibedinthelegendtoFig.4,exceptthatthetemp
ibedinthelegendtoFig.4,exceptthatthetemperatitemperature.Eachvaluerepresentsthemeanandstationofatleasttwoindependentduplicateassays.typesII,III,V,XI)exhibitedhighrelativeaffinitiesforYadA.WithincollagentypeI,theoa(I)chainalsoboundtoYadAtoadegreecomparabletootherfibril-formingcolla-gens.Moreover,collagentypeIIisahomotrimerofthreeal(II)chains,whereasthetypeXImoleculeconsistsofthreedifferentchains(al,ox2,a3),ofwhichonlythecc3(XI)chainwasshowntobindYadAintheaffinityblotexperiments.Thereissomeevidencethattheao3(XI)chainandtheoal(II)chainareencodedbythesamegene,withminordifferencesduetoposttranslationalmodifications(11,36).Therefore,itcouldwellbethatcollagentypesIIandXIinteractwithYadAviathesamebindingsite,whichisconservedinbothevolutionarilycloselyrelatedmolecules.Itmightbespecu-latedthattheinteractionsiteremainedconservedduringtheevolutionofthecollagenfamilyfromanancestral54-bpunit(30)incollagensexhibitingYadAbinding.WhetherthebindingsiteforYadAoncollagentypeIVishomologoustoinhibitor[ng]theYadAbindingsiteoncollagentypeIIiscurrentlyunderinvestigation.CollagentypesVIandIX,whichboundcon-[lagentypeIsiderablylesswelltoYadA,belongtodistinctclassesofthetypeIchainscollagensupergenefamily(28).performedasFurtherexperimentsarerequiredtolocalizepreciselytheandstandardinteractionsitesoncollagensandonYadAtoanswerthequestionofwhethercollagenbindingbyYadAcontributestothepathogenicityofyersiniae.BindingofYadAimmuno-complexestocollagentypeI,amajorcomponentofthe,,iefailureofextracellularmatrixoftheskin,couldbeimplicatedintheirtherblockinductionoferythemanodosum,askinafflictionthatisrtconcentra-frequentlycausedbyYenterocolitica.InthisstudywehaveteforYadAdemonstratedthatthecartilagecollagentypesIIandXIlottestedinspecificallybindYadA.Whetherbindingtocartilage-re-nitybindingstrictedcollagensmaybelinkedtothearthritogenicpoten-tialofenteropathogenicyersiniaeremainstobeinvestigated.tudytobindWehaveprovidedstructuraldataoncollagen-YadAinter-hadsimilaractionsthatmaystimulatefurtherstudies,includinginvivoandXIbutstudieswithcollagenpeptidesformodulationofYersinia-affinityandinducedexperimentalanimaldiseases.nginblock-s.Allfibril-ACKNOWLEDGMENTScollagenWethankEvaBauerforexperttechnicalassistance.ThisworkwaspartlysupportedbytheDeutscheForschungsge-meinschaft,SFB263,projectC3.TheMax-Planck-ArbeitsgruppeninErlangenarefundedbytheGermanMinistryforResearchandTechnology(grant01VM8702/0).REFERENCES1.Bentz,H.,H.P.Bachinger,R.W.Glanville,andK.Kuhn.1978.Physicalevidencefortheassemblyofaandbchainsofhumanplacentalcollageninasingletriplehelix.Eur.J.Biochem.92:563-567.2.Bolin,I.,L.Norlander,andH.Wolf-Watz.1982.Temperature-inducibleouter-membraneproteinofYersiniapseudotuberculo-sisandYersiniaenterocoliticaisassociatedwiththevirulenceplasmid.Infect.Immun.37:506-512.3.Bolin,I.,D.A.Portnoy,andH.Wolf-Watz.1985.Expressionofthetemperature-inducibleoutermembraneproteinsofyersin-iae.Infect.Immun.48:234-240.1024.Bottone,E.J.1977.Yersiniaenterocolitica:apanoramicviewofacharismaticmicroorganism.Crit.Rev.Microbiol.5:211-241.inhibitor[pml~5.Burkhardt,H.,R.Holmdahl,R.Deutzmann,H.Wiedemann,llagentypeIIH.vonderMark,S.Goodmann,andK.vonderMark.1991.IIfragmentsIdentificationofamajorantigenicepitopeonCNBr-fragment11proteinsareoftypeIIcollagenrecognizedbymurineautoreactiveBcells.0,-31kDa;Eur.J.Immunol.21:49-54.rmedasde-6.Cornelis,G.R.,T.Biot,C.LambertdeRouvroit,T.Michiels,B.arewasroomMulder,C.Sluiters,M.P.Sory,M.vanBouchaute,andJ.C.ndarddevia-Vanooteghem.1989.TheYersiniayopregulon.Mol.Microbiol.3:1455-1459.INFECT.IMMUN.COLLAGENBINDINGOFYERSINIAENTEROCOLITICA21597.Cover,T.L.,andR.C.Aber.1989.Yersiniaenterocolitica.N.Engl.J.Med.321:16-24.8.Emody,L.,J.Heesemann,H.Wolf-Watz,M.Skurnik,G.Kapperud,P.O'Toole,andT.Wadstrom.1989.BindingtocollagenbyYersiniaenterocoliticaandYersiniapseudotuber-culosis:evidenceforYopA-mediatedandchromosomallyen-codedmechanisms.J.Bacteriol.171:6674-6679.9.Fitzgerald,T.J.,L.A.Repesh,D.R.Blanco,andJ.N.Miller.1984.AttachmentofTreponemapallidumtofibronectin,lami-nin,collagenIVandcollagenI,andblockageofattachmentbyimmunerabbitIgG.Br.J.Vener.Dis.60:357-363.10.Fraker,P.J.,andJ.C.Speck,Jr.1978.Proteinandcellmembraneiodinationwithasparinglysolublechloroamide1,3,4,6-tetrachloro-3a,6a-diphenylglycoluril.Biochem.Bio-phys.Res.Commun.80:849-857.11.Furuto,D.K.,andE.J.Miller.1983.Differentlevelsofglycolisationcontributetotheheterogeneityofal(II)collagenchainsderivedfromatransplantableratchondrosarcoma

.Arch.Biochem.Biophys.226:604-611.12.Gla
.Arch.Biochem.Biophys.226:604-611.12.Glanville,R.W.,andA.Rauter.1981.Pepsinfragmentsofhumanplacentalbasement-membranecollagensshowinginter-ruptedtriple-helicalaminoacidsequences.Hoppe-Seyler'sZ.Physiol.Chem.362:943-951.13.Guan,K.,andJ.E.Dixon.1990.ProteintyrosinephosphataseactivityofanessentialvirulencedeterminantinYersinia.Sci-ence249:553-556.14.Heesemann,J.,B.Algermissen,andR.Laufs.1984.GeneticallymanipulatedvirulenceofYersiniaenterocolitica.Infect.Im-mun.46:105-110.15.Heesemann,J.,andK.Gaede.1989.MechanismsinvolvedinthepathogenesisofYersiniainfections.Rheumatol.Int.9:213-217.16.Heesemann,J.,andL.Gruter.1987.GeneticevidencethatoutermembraneproteinYOP1ofYersiniaenterocoliticamediatesadherenceandphagocytosisresistancetohumanepithelialcells.FEMSMicrobiol.Lett.40:37-41.17.Heesemann,J.,andR.Laufs.1983.Constructionofamobiliz-ableYersiniaenterocoliticavirulenceplasmid.J.Bacteriol.155:761-767.18.Holderbaum,D.,R.A.Spech,andL.A.Erhart.1985.SpecificbindingofcollagentoStaphylococcusaureus.CollagenRelat.Res.5:261-271.19.Holmdahl,R.,K.Rubin,L.Klareskog,E.Larssen,andH.Wigzell.1986.CharacterizationoftheantibodyresponseinmicewithtypeIIcollagen-inducedarthritis,usingmonoclonalanti-typeIIcollagenantibodies.ArthritisRheum.29:400-412.20.H66k,M.,L.M.Switalski,T.Wadstrom,andM.Lindberg.1989.Interactionsofpathogenicmicroorganismswithfibronec-tin,p.295-308.InD.F.Mosher(ed.),Fibronectin.AcademicPress,Inc.,SanDiego,Calif.21.Isberg,R.R.1991.Discriminationbetweenintracellularuptakeandsurfaceadhesionofbacterialpathogenes.Science252:934-938.22.Isberg,R.R.,andJ.M.Leong.1988.CulturedmammaliancellsattachtotheinvasinproteinofYersiniapseudotuberculosis.Proc.Natl.Acad.Sci.USA85:6682-6686.23.Kirsch,T.,andK.vonderMark.1990.IsolationofbovinetypeXcollagenandimmunolocalizationingrowth-platecartilage.Biochem.J.265:453-459.24.Kuusela,P.1978.FibronectinbindstoStaphylococcusaureus.Nature(London)276:718-720.25.Lachica,R.V.,D.L.Zink,andW.R.Ferries.1984.AssociationoffibrilstructureformationwithcellsurfacepropertiesofYersiniaenterocolitica.Infect.Immun.46:272-275.26.Laemmli,U.K.1970.CleavageofstructuralproteinsduringtheassemblyoftheheadofbacteriophageT4.Nature(London)227:680-685.27.Lopes,J.D.,M.DosReis,andR.R.Brentani.1985.PresenceoflamininreceptorsinStaphylococcusaureus.Science229:275-277.28.Miller,E.J.,andS.Gay.1987.Thecollagens:anoverviewandupdate.MethodsEnzymol.144:3-41.29.Portnoy,D.A.,andR.J.Martinez.1985.RoleofaplasmidinthepathogenicityofYersiniaspecies,p.29-51.InW.Goebel(ed.),Geneticapproachestomicrobialpathogenicity.Springer-Verlag,NewYork.30.Ramirez,F.1989.Organizationandevolutionofthefibrillarcollagengenes,p.21-30.InB.R.Olsen,andM.E.Nimni(ed.),Collagen,vol.4.Molecularbiology.CRCPress,Inc.,BocaRaton,Fla.31.Rubin,K.,M.Hook,B.Obrink,andR.Timpl.1981.Substrateadhesionofrathepatocyte:mechanismofattachmenttocolla-gensubstrates.Cell24:463-470.32.Speziale,P.,M.Hook,T.Wadstrom,andR.Timpl.1982.BindingofthebasementmembraneproteinlaminintoEsche-richiacoli.FEBSLett.146:55-58.33.Speziale,P.,G.Raucci,L.Visai,L.M.Switalski,R.Timpl,andM.Hook.1986.BindingofcollagentoStaphylococcusaureusCowan1.J.Bacteriol.167:77-81.34.Switalski,L.M.,H.Murchison,R.Timpl,R.CurtissIII,andM.Hook.1987.Bindingoflaminintooralandendocarditisstrainsofviridansstreptococci.J.Bacteriol.169:1095-1101.35.Switalski,L.M.,P.Speziale,M.Hook,T.Wadstrom,andR.Timpl.1984.BindingofStreptococcuspyogenestolaminin.J.Biol.Chem.259:3734-3738.36.Swoboda,B.,R.Holmdahl,H.Stod3,andK.vonderMarlk1989.Cellularheterogeneityinculturedhumanchondrocytesidenti-fiedbyantil--diesspecificfora2(XI)collagenchains.J.CellBiol.109:13t0o-1369.37.Timpl,R.,R.W.Glanville,H.Nowack,H.Wiedemann,P.P.Fietzek,andK.Kuhn.1975.Isolation,chemicalandelectronmicroscopicalcharacterizationofneutral-salt-solubletypeIIIcollagenandprocollagenfrom'fetalbovineskin.Hoppe-Seyler'sZ.Physiol.Chem.356.±.792.38.Toivanen,A.,K.Granfors,R.Lahesmaa-Rantala,R.Leino,T.Stahlberg,andR.Vuento.1985.PathogenesisofYersinia-triggeredreactivearthritis:immunological,microbiologicalandclinicalaspects.Immunol.Rev.86:47-70.39.vonderMark,H.,M.Aumailley,G.Wick,R.Fleischmajer,andR.Timpl.1984.Immunochemistry,genuinesizeandtissuelocalisationofcollagenVI.Eur.J.Biochem.142:493-502.40.vonderMark,K.,M.vanMenxel,andH.Wiedemann.1982.Isolationandcharacterisationofnewcollagenfromchickcar-tilage.Eur.J.Biochem.124:57-620.41.Wolf-Watz,H.1987.PlasmidmediatedvirulenceofYersiniae.Prog.Vet.Microbiol.Immunol.3:159-178.VOL.6