JournalofInvertebratePathologyjournalhomepage:www.elsevier.com/locate/jipExpressionofHSP60andHSP70inwhiteshrimp,Litopenaeusvannameiinresponsetobacterialchallenge
JunZhou1,Wei-NaWang*,Wen-YinHe,YinZheng,LeiWang,YuXin,YuanLiu,An-LiWang
KeyLaboratoryofEcologyandEnvironmentalScienceinGuangdongHigherEducation,CollegeofLifeScience,SouthChinaNormalUniversity,Guangzhou510631,PRChinaarticleinfoabstract
Inthepresentstudy,cDNAencodingaheatshockprotein60(LvHSP60)geneinLitopenaeusvannameiwasclonedusingacombinationofhomologyandrapidamplificationofcDNAend(RACE)methods.ThefulllengthoftheLvHSP60cDNAwasfoundtobe2379bp,witha1737bpopenreadingframe.Thetranslatedaminoacidsequenceconsistedof579residueswithacalculatedmolecularmassof60.8kDandanisoelectronicpoint(pI)of5.97.Comparisonofthededucedaminoacidsequenceshowedthatithashighidentity(85–89%)withHSP60/chaperoninsfrominsectsandmammals.Quantitativereal-timePCRandWesternblotanalysiswerecarriedouttoinvestigatetheexpressionpatternsanddistribu-tionprofilesofLvHSP60beforeandafterstimulationwiththeGram-positivebacteriumStaphylococcusaureusandtheGram-negativebacteriumVibrioalginolyticus.LvHSP60mRNAwasfoundtobebothconstitutiveandinducible,andwashighlyexpressedinhaemocytesandalmostalltissuesexamined,includingmuscle,stomach,heart,hepatopancreasandgilltissue,butitwaslessstronglyexpressedintheintestine.TheexpressionanalysisrevealedthatLvHSP60wassignificantlyup-regulatedinthegills,hepatopancreasandhaemocytesafterbacterialchallenge.TranscriptionofLvHSP70wasalsoinducedinhaemocytesandthehepatopancreasafterdifferentbacteriainjection.SubsequentflowcytometryanalysisshowedthattheconcentrationofCa2+ionsincreasedsignificantlywithinbacteria-challengedhaemocytesby1.5hafterinjection.TheresultsindicatethatLvHSP60andLvHSP70mayplayimportantrolesinmediatingtheimmuneresponsesofL.vannameitobacterialchallenge,andthattheCa2+signal-lingtransductionpathwaymaybeinvolvedintheinitiationoftheshrimp’simmuneresponsesinearlystagesofinfection.Ó2009ElsevierInc.Allrightsreserved.Articlehistory:Received3October2009Accepted15December2009Availableonline4January2010Keywords:LitopenaeusvannameiHeatshockproteinsQuantitativereal-timePCRWesternblotFlowcytometry1.IntroductionMolecularchaperonescompriseseveralhighlyconservedfami-liesofheatshockproteins(HSPs)thatbindproteinsinnon-func-tionalstatesandassistthemtoreachfunctionalconformation(BukauandHorwich,1998;EllisandvanderVies,1991).Heatshockproteinsareconstitutivelyexpressedundercertainphysio-logicalconditionsandareinvolvedinmanyessentialcellularfunc-tions,suchasmetabolism,growth,differentiationandprogrammedcelldeath(Ranfordetal.,2000;Multhoff,2006).Inadditiontoheatshock,awidevarietyofstressstimuli,includingoxygenradicals,heavymetals,anti-inflammatorydrugs,nutrientdeprivation,bacterialandviralinfections,andmalignanttransfor-mation,caninitiatethesynthesisofHSPs(Aseaetal.,2008;Fulleretal.,1994;Gehrmannetal.,2004;Multhoff,2006).*Correspondingauthor.Fax:+862085217322.E-mailaddresses:wangwn@scnu.edu.cn,weina63@yahoo.com.cn(W.-N.Wang).1Presentaddress:SignalingandFunctionalGenomics,GermanCancerResearchCenter,ImNeuenheimerFeld580,69120Heidelberg,Germany.
0022-2011/$-seefrontmatterÓ2009ElsevierInc.Allrightsreserved.doi:10.1016/j.jip.2009.12.006RecentreportssuggestthatmembersoftheHSP60andHSP70familiesarethemajorHSPsinvolvedinautoimmunediseasesandinnateimmunity(RanfordandHenderson,2002;TsanandGao,2004).TheHSP60orchaperoninfamilyisagroupofproteinswithdistinctring-shaped,ortoroid(doubledoughnut)quaternarystructures(QuintanaandCohen,2005).AnimportantactivityofHSP60sismediationofthenativefoldingofproteinsinanATP-dependentmanner(EllisandvanderVies,1991).Thechaperoninshaveattractedconsiderableinterestamongimmunologistsinthecontextofbacterialinfectionandautoimmuneconditions,becausemammalianHSP60shavealsobeenidentifiedasendogenousstresssignalmolecules,withimplicatedinvolvementinautoimmunedis-ease(LiangandMacRae,1997;TsanandGao,2004;Vabulasetal.,2001).Thus,thechaperoninsappeartoplaycentralrolesinmam-maliandefencesagainstpathogenicattackandresponsestodam-ageorstressinadditiontonormalcellfunctions(Vabulasetal.,2001).MembersoftheHSP70familyarestress-inducibleandcon-stitutiveproteinsthatplayessentialrolesinproteinmetabolismunderbothnormalandstressconditions,includingdenovoproteinfolding,membranetranslocation,degradationofmisfoldedpro-teinsandotherregulatoryprocesses(Multhoff,2007;RanfordJ.Zhouetal./JournalofInvertebratePathology103(2010)170–178171andHenderson,2002).However,recentstudieshavesuggestedthatHSP70proteinscanalsospecificallyactivatehumannaturalkiller(NK)cellsbybindingtoToll-likereceptors(TLR)2and4,andexertimmunoregulatoryeffects(Aseaetal.,2002;Aseaetal.,2008).ThePacificwhiteshrimp,Litopenaeusvannamei(hereafterwhiteshrimp),hasbeenwidelyculturedintheworldandisanimportantcommercialspecies.However,duringthelasttwodecades,out-breaksofdiseaseassociatedwithbacteriasuchasVibrioalginolyt-icusandV.harveyi,andviruseshavecausedmassshrimpmortality,resultinginreductionsinfarmedshrimpproduction,andconsider-ableeconomiclosses(LiuandChen,2004;Liuetal.,1996).InthestudypresentedhereweclonedcDNAencodingHSP60fromthewhiteshrimpL.vannamei,andanalysedtheHSP60geneexpressionpatternsandtissueprofilesusingquantitativereal-timePCRandRT-PCR,respectively.Inaddition,theHSP60expressionpatternattheproteinlevelwasstudiedbyWesternblotanalysisusingantiserumraisedagainstrecombinantLvHSP60.Meanwhile,wealsoinvestigatedthechangesofHSP60,HSP70andtheintracel-lularCa2+concentration([Ca2+]i)inwhiteshrimp,L.vannamei,inresponsetobacterialchallenge(StaphylococcusaureusandV.alginolyticus).2.Materialsandmethods2.1.AnimalandsampledtreatmentShrimp(L.vannamei),averaging8.76±0.39cminbodylengthand4.85±0.58ginbodyweight,wereobtainedfromalocalshrimpfarminNanhai(Guangdong,China)andacclimatisedinsaltwatertanks(10m3)for1weekbeforetheexperiment.Thesalinityofthewaterinthetankswas10‰(dilutedseawater),thetemperaturewasmaintainedat22±1°Cthroughouttheaccli-matisationandexperimentalperiods,andthewaterwasaeratedcontinuouslyusinganairstone.L.vannameiwerefedwithcom-mercialshrimpfeedtwiceperdayuntil24hbeforetheexperimentstarted.Thebacterialchallengeexperimentwasinitiatedbyinjectingeachof200shrimpwith10llofV.alginolyticus(1Â107cellspershrimp)andeachofanothersetof200withS.aureusbacteriasuspended(ineachcase)insalineintotheventralsinusofthecephalothorax(1Â107cellspershrimp)(Zhangetal.,2009).Asimilarcontrolgroupof200shrimpwereeachinjectedwith10llofsaline.Experimentalandcontrolshrimpwerekeptin80lplasticaquaria(25/aquarium)containing60lof10‰saltwa-ter.Theinjectedshrimpwerereturnedtosaltwaterandnineindividualswererandomlycollectedat1.5,3,6,12and24h,post-injection.Sixindividualswererandomlytakenoutfortissuespreparationateachtimepointduringthechallengeexperiment.Thehaemolymphfromthecontrolandstimulatedgroupswerecollectedusingasyringefromtheventralsinusofeachshrimpwithsamevolumeofanticoagulant(30mMtrisodiumcitrate,0.34Msodiumchloride,and10mMEDTAatpH7.55,withtheosmolalityadjustedto780mOsmkgÀ1with0.115Mglucose)andcentrifugedat800g,4°Cfor10mintoharvestthehaemocytes.RNAwasimmediatelyextractedfromthehaemocytepelletsusingTrizolreagent(Invitrogen).Inaddition,afterbacterialinjection,thehaemolymphfromthreeshrimpwerecollectedforflowcytometryanalyses(Changetal.,2009).2.2.RNAextractionandcDNAsynthesisTotalRNAwasextractedfromthehepatopancreaswithTrizolreagent(Invitrogen,USA)followingthemanufacturer’sinstruc-tions,andtreatedwithRQ1Rnase-freeDNase(Promega,USA)toremovecontaminatingDNA.First-strandcDNAsynthesiswasper-formedusingM-MLVreversetranscriptase(Promega,Madison,WI,USA)totranscribepoly(A)RNAwitholigo-d(T)18astheprimersusingtotalRNAfromthehepatopancreas.First-strandcDNAwasgeneratedin25llreactionvolumes(incubatedat42°Cfor90min)containing2lgtotalRNA,2ÂRTbuffer,10mMdNTP,20lMoligo-d(T)18,20UofRNaseinhibitor(TakaraDalianChina)and100UofMMLV-reversetranscriptase.2.3.CloningoftheLvHSP60cDNAfragmentandrapidamplificationofcDNAends(RACE)Twodegenerateprimers(HSP60-dFandHSP60-dR)werede-signed,basedontheconservedaminoacidsequenceofHSP60sintheNationalCentreforBiotechnologyInformation(NCBI)data-base,toclonethemiddlefragmentoftheLvHSP60cDNAbyPCR.HepatopancreascDNAwasusedasatemplateforamplification.PolymeraseChainReaction(PCR)wasperformedusingprimersHSP60-dFandHSP60-dR,withadenaturationstepat94°Cfor1min,30cyclesof94°Cfor30s,56°Cfor30sand72°Cfor90s,followedbya5minextensionstepat72°C.PCRproductswereclonedintopMD-18T(Takara,China)andsequencedbyInvit-rogenCorp.(Shanghai,China).RapidamplificationofcDNAends(50,30RACE)ofLvHSP60wasaccomplishedusingaBDSMARTRACEcDNAamplificationkit(BDBioscienceClontech,CA,USA)followingthemanufacturer’sinstructions.Gene-specificprimers(HSP60-5R1,HSP600-5R2andHSP60-3R1,showninTable1)for50and30RACEweredesignedfortheRT-PCRampliconobtainedusingthedegenerateprimers,followingitssequenceanalysis.Afull-lengthcDNAcloneforthecodingregionofL.vannameiHSP60wasrecoveredbyRT-PCRusingRNAisolatedfromthehepa-topancreas.ComplementaryPCRprimerstotheendsofthecodingregionweredesigned(HSP60-FeandHSP60-Re;Table1)andusedtoamplifythecDNAsequence(Zhouetal.,2009b).2.4.PhylogeneticanalysisofLvHSP60Afterdeterminingitsfulllength,theLvHSP60cDNAsequencewascomparedwithentriesintheNCBIdatabaseusingtheNCBI’sBLASTalgorithm(http://www.ncbi.nlm.nih.gov/BLAST/).TheOpenReadingFrame(ORF)ofLvHSP60waspredictedusingtheEditSeqprogram(DNAstarInc.,US)andmotifswerepredictedusingExP-ASy(http://www.au.expasy.org).PairwiseandmultiplealignmentoftheLvHSP60deducedaminoacidsequenceswereperformedusingtheClustalWmultiplealignmentprogram,andMegAlign(DNAstarInc.,US)wasusedtoproduceaphylogenetictreebytheNeighbour-Joining(NJ)method.2.5.TissueexpressionanalysisTostudythetissue-specificityofLvHSP60expressioninshrimpthathadnotbeenchallengedbybacteria,tissues,includingthehaemocytes,intestine,stomach,muscle,heart,hepatopancreasandgillswerecollectedfromsixcontrolindividuals.ExpressionofLvHSP60ineachtissuewasthenassayedbyRT-PCR,byre-verse-transcribingsamplescontaining2lgofRNAfromsixshrimpwitholigo-d(T)18.Twospecificprimers,HSP60-FcandHSP60-Rcwereusedtoamplifya410bpHSP60fragment,andtheprimersActin-FcandActin-Rcwereusedtoamplifytheb-actinfragment(637bp),whichwasusedasapositivecontrol.PCRwasperformedbyaninitialdenaturationstepof94°Cfor3min,followedby28cyclesof94°Cfor30s,57°CforHSP60or58°Cforb-actinfor30s,72°Cfor1min,anda10minstepat72°C.PCRproductswereanalysedon1%agarosegels(Fig.4).172J.Zhouetal./JournalofInvertebratePathology103(2010)170–178Table1
Primersforgeneamplification,characterization,andrecombinantproteinexpression.NameHSP60-dFHSP60-dRHSP60-FcHSP60-RcHSP60-5R1HSP60-5R2HSP60-3R1HSP60-FeHSP60-ReHSP60-FrHSP60-RrHSP70-FrHSP70-RrActin-FcActin-RcActin-FrActin-RrTargetGenefragmentcloningGenefragmentcloningRT-PCRamplificationRT-PCRamplification50RACEPCR50RACEPCR30RACEPCRRecombinantproteinexpressionRecombinantproteinexpressionReal-timePCRReal-timePCRReal-timePCRReal-timePCRRT-PCRamplificationRT-PCRamplificationReal-timePCRReal-timePCROrientationForwardReverseForwardReverseSequence(50?30)GAMGGATCKTGTWAATRATGTCVTCMTAGCCTRTCSATGAAGAGCTGCGGTTGAAGAGCATCGGTTAGGGCTGTGCGGAGTGATAGTATCCAAGGCAGGAAGGCATTCATATCCAACATCTCCAGACGCTTCCATGCGGTTGAAGAGGGCATAGTTCCAGGTGGATCCATGCATCGCGCAGCCTCCTTGAATTCCCTCCCACATATTTCTTTTCATTGTCCGCAAGGCTATCATCTCCAGACGCTTCCATCCTCCAGGACTTCTTCAACGGGTCACGTCCAACAGCAACCCCAGAGCAAGCGAGGTATCCATGCCCAGGAATGAGGCCCATCTACGAGGGATAGGTGGTCGTGAAGGTGTAAForward(withBamHI)Reverse(withEcoRI)ForwardReverseForwardReverseForwardReverseForwardReverse2.6.ExpressionandpurificationoftherecombinantLvHSP60proteinTheORFsofLvHSP60wereamplifiedwithspecificprimers(HSP60-FeandHSP60-ReshowninTable1)andclonedintothepMD-18Tvector.TheplasmidDNA(pMD-18Tvector)containingtheshrimpHSP60ORFwasdigestedwithBamHIandEcoRI,thensub-clonedintotheexpressionvectorpProEXHT-a(Invitrogen),whichhadbeenpreviouslycutwiththesamerestrictionenzymes.TheligatedproductwastransformedintoEscherichiacoliDH5acells,andthesuccessoftheligationwasconfirmedbyplasmiddetection,restrictionenzymedigestionandsequencing.TheexpressionconstructwasdesignatedpProEXHT-a/LvHSP60.TherecombinantL.vannameiHSP60wasthenover-expressedinE.coliDH5acellsbyinductionwithisopropyl-b-thiogalactopy-ranoside(IPTG)asfollows:10mlofstarterculturewasinoculatedinto100mlofLuriabrothwith100llampicillin(100mg/ml);theculturewasincubatedat37°Cwithshakingat200rpmuntilthecelldensity(OD600)reached0.5.Expressionwastheninducedbyadding1mMIPTG.Afterincubationat37°Cforafurther4h,thebacterialcellswereharvestedbycentrifugationat8000gfor10minat4°C,re-suspendedin50mMphosphatebuffersolution(PBS,pH8.0)containing0.3MNaCland10mMimidazole,andsonicatedonice.Thecelldebriswasremovedbycentrifugationat15,000gfor20min,andthesupernatantwascollectedforpro-teinpurification.Theself-ligatedpProEXHTwasusedasanegativecontrolinalltheexperiments.TherecombinantLvHSP60waspuri-fiedusinga6ÂHis-taggedaffinitycolumn(10mLNi–NTAagaroseresin,Novagen)followingthemanufacturer’sinstructions.Theelutedfractionwasdialysedovernightandthepooledproteinfrac-tionswereanalysedby12%SDS–PAGE.ProteinconcentrationsweredeterminedbythemethodofBradford(1976)usingbovineserumalbuminasastandard.2.7.Quantitativereal-timePCRassayfollowingbacterialinjectionTotalRNAwaspreparedfromthegillandhepatopancreastis-sues,andhaemocytes,ofshrimp.First-strandcDNAwasthensyn-thesised,asdescribedabove,andstoredatÀ20°Cuntilusedforreal-time-PCR.mRNAexpressionofthetargetgeneineachofthetissueswasmeasuredbyqPCR.ThespecificprimersweredesignedbasedonpublishedL.vannameimRNA[GenbankaccessionNos.FJ710169,AY645906andAF300705]usingPrimerPremier5(PRE-MIERBiosoftInternational,PaloAlto,CA).TheprimersequencesareshowninTable1.TheqRT-PCRamplificationswerecarriedoutintotalvolumesof20llcontaining10ll2ÂSYBRPremixExTaq(TakaraDalian,Chi-na),5ulcDNA(diluted2:5withPCR-gradeH2O),0.2lleachof20lMforwardandreverseprimers,0.4llROXand4.2llPCR-gradewater.Real-timePCRwascarriedoutwithanABI7300real-timePCRmachine(AppliedBiosystemFosterCityCA,USA).TheLvHSP60,LvHSP70andb-actinQPCRprimersarelistedinTable1.PCRamplificationwasperformedintriplicatewells,withthefol-lowingprogram:95°Cfor10s,followedby40cyclesof95°Cfor5sand60°Cfor31s.Meltingcurveanalysisofamplificationprod-uctswasperformedattheendofeachPCRreactiontoconfirmthatonlyonePCRproductwasamplifiedanddetected.Samplesfromcontrolshrimpandtheshrimpinjectedwiththetwospeciesofbacteriawereanalysed,intriplicate,theresultswereexpressedasthefold-changeinexpression,relativetotheb-actingene,bythe2ÀDDCTmethod(LivakandDavey,2001),thendifferencesintherelativeexpressionratiosoftheLvHSP60(orLvHSP70)genetotheb-actingenebetweenthecontrolandinducedgroupswereevaluatedtoassesschangesintheexpressionofthegenesfollow-inginfection.Quantitativedatawereexpressedasmeans±SD(standarddeviation)representingtherelativeexpressionratio.Thesignificanceofdifferencesinexpressionwasdeterminedusingtwo-wayANOVAfollowedbyDuncan’smultiplerangetests;withathresholdsignificancelevelofP<0.05.AllstatisticalanalyseswereperformedusingSPSS13.0(SPSS,Chicago,IL,USA).2.8.PolyclonalantibodypreparationandWesternblottingThepurifiedrecombinantL.vannameiHSP60wasusedtoraiseantibodiesinaNewZealandwhiterabbitbyemulsifyingapproxi-mately1mgofthepurifiedrm-HSP60withFreund’scompleteadjuvantandinjectingitsubcutaneouslyatmultiplesitesoftherabbit.Threeboosterinjectionsof1.5mgantigen(0.5mgeachtime)mixedwithFreund’sincompleteadjuvantweresubsequentlyadministeredsubcutaneouslyatintervalsof3weeks.Eightdaysafterthefinalbooster,bloodwascollectedandserumwaspre-pared.Serumfromthesamerabbitcollectedpriortoimmunisationwasusedasacontrol.Theanti-serawerealiquotedandstoredatÀ80°C.Shrimpgillsthathadbeensampledafterbacterialtreatmentwereeachhomogenisedin5mlof50mMTris–HCl(pH7.2)with50mMNaClonice,andcentrifugedat10,000gfor20minat4°C(5mineachtime).Theresultingsupernatantswerecollected,andportionscontainingca.30lgofprotein(measuredasdescribedabove)wereloadedinseparatelanesofa12%SDS–PAGEgel,andelectrophoreticallyseparated.Thegelswerethenwashedfor15minin20mMPBScontaining0.1%Tween-20,andtheproteinsinthegelswereblottedontoanitrocellulosemembrane(Hybond,J.Zhouetal./JournalofInvertebratePathology103(2010)170–178173AmershamPharmacia).Blottedmembraneswereincubatedin20mMPBScontaining3%bovineserumalbumin(BSA)at4°Covernight,andthenintherabbitanti-seradiluted1:800with20mMPBScontaining0.1%Tween-20for1h.Afterwashingin20mMPBS,themembraneswereincubatedwithperoxidase-con-jugatedanti-rabbitantibodydiluted1:2000at25°Cfor1h,afterwhichthebandswerevisualizedusingDABand0.03%H2O2(Fengetal.,1999;Zhouetal.,2009a).2.9.Intracellularfree-Ca2+detectionbyflowcytometryIntracellularcalciumconcentrationsweremonitoredusingthecalcium-sensitivedyeFluo-3,whichwasdissolvedinDMSOtogiveastocksolutionof1mg/mlandstoredatÀ20°Cinthedark.Fluo-3isawidely-used,longwavelengthfluorescentcalciumindicatordevel-opedbyRogerTsienandcolleagues(Kaoetal.,1989).Theindicatorabsorbsat526nmandcanbeefficientlyexcitedbya488argon-ionlaser.Fluo-3isnon-fluorescentintheabsenceofCa2+,butthefluo-rescentsignal(at526nm)increasesbyafactorofatleast40uponCa2+binding(Kresgeetal.,2006).CellsuspensionsfromeachofthethreereplicateswerestainedwithFluo-3(finalconcentration,10lm)andincubatedina37°Cwaterbathfor30mininthedark.Thecellswerethencentrifugedfor5minat700g,washedandre-suspendedinPBS,filteredthrougha200lmmesh,andtrans-ferredtoflowcytometrytubes(1–2Â106cells/ml).ThecontrolcellswereincubatedunderidenticalconditionsexceptthatFluo-3wasabsent(BaileyandMacardle,2006).3.Results3.1.CloningandsequenceanalysisofcDNAencodingLvHSP60Thefull-lengthLvHSP60cDNAwas2305bplongwithanORFof1737bpandapolyadenylationsignalwithapoly(A)tail(Fig.1).TheORFputativelyencodesapolypeptideof579aminoacidswithanestimatedmolecularmassof60.8kDaandapredictedisoelectricpoint(pI)of5.97.ThededucedaminoacidsequenceofLvHSP60wascomparedwiththesequencesofseveralheatshockprotein60sfromotherspeciesthatwereavailableinGenBank,andsubmittedtotheNCBIGenBankunderaccessionnumberFJ710169.TheBLAST(blastp)searchoftheNCBIdatabasewiththede-ducedaminoacidsequencefortheheatshockprotein60fromL.vannameirevealedthatithassimilar,conservedsubstitutions(po-sitive)withHSP60s/chaperoninsfromthreeanimalphylums;Arthropoda:BlackleggedTick(Ixodesscapularis,EEC05122)(88%positive,e0.0),FruitFly(Drosophilamelanogaster,NP_511115)(88%positive,e0.0),Australiansheepblowfly(Luciliacuprina,ABO09590)(88%positive,e0.0),Pealeafminer(Liriomyzahuidobr-ensis,AAW30392)(88%positive,e0.0),Bodylouse(Pediculushum-anuscorporis,EEB15946)(87%positive,e0.0),Southernhousemosquito(Culexquinquefasciatus,XP_001850501)(87%positive,e0.0),Yellowfevermosquito(Aedesaegypti,XP_001661764)(87%positive,e0.0);Mollusca:Bloodflukeplanorb(Biomphalariaglabrata,ACL00842)(87%positive,e0.0);Chordata:Human(Homosapiens,AAA36022)(86%positive,e0.0),Norwayrat(Rattusnorvegicus,AAC53362)(86%positive,e0.0).AconserveddomainsearchoftheNCBIdatabaseandapplicationoftheprogrampat-matmotifsinEMBOSSidentifiedaconservedATP-binding/Mg2+bindingsite,hingeregionsandstackinginteractionsites(March-ler-Baueretal.,2007).3.2.PhylogeneticanalysisofLvHSP60Aphylogenetictree,basedonthededucedaminoacidsequencesfromtheHSP60s/chaperoninsof10species(includinghumanandrat),wascreatedusingtheNeighbour-Joining(NJ)methodtodeterminetheevolutionarypositionofLvHSP60.TheconstructedphylogenetictreeindicatedthatknowninsectHSP60sequencesareclusteredtogetherandmammaliansequencesformanotherseparategroup.OurwhiteshrimpHSP60sequencewasslightlydifferentfrombothinsectandmammalHSP60sequenceswithinaseparatesubgroup(Fig.2).3.3.ExpressionofrecombinantLvHSP60andantibodyspecificityAnexpressionvectorpProEXHT-a,includingtheentireORFofL.vannameiHSP60,wasconstructedandtransformedintoE.coli,fol-lowingwhichtherecombinantLvHSP60waspurifiedbyaffinitychromatography.ThepurifiedrecombinantLvHSP60withtheHis6tagyieldedasinglebandof63kDaonSDS–PAGEgelsafterCoomassiebluestaining(Fig.3A).Rabbitantiserumwasobtainedagainstthepurifiedrecombi-nantLvHSP60withatitreof1:600,whichreactedinWesternblotanalysiswithaconstituent(apparentmolecularweight,63kDa)ofthesupernatantofthecelllysateofIPTG-inducedE.coliDH5acar-ryingtheexpressionvector,butnotwithanyconstituentsofacor-respondingpreparationofE.coliDH5acontainingtheexpressionvectorpriortoinductionbyIPTG(Fig.3B).Theantiserumwasalsoreactivewithaconstituentthathadanapparentmolecularweightca.63kDa,correspondingtothemolecularmasspredictedforLvHSP60cDNAoftheshrimpandgillhomogenates(Fig.5B1).Thesefindingsshowthattherabbitantiserumhadmarkedanti-gen-specificreactivity.3.4.TissueexpressionofshrimpHSP60ExpressionanalysisofLvHSP60mRNAbyreversetranscriptionRT-PCR,withb-actinasaninternalcontrol,showedthattheshrimpHSP60genewasexpressedinallexaminedtissues.However,LvHSP60transcriptlevelsweresignificantlyhigher(Fig.4)inthestomachthaninalloftheothertissues,andonlyverylowlevelsoftheLvHSP60transcriptweredetectedintheintestine(Fig.4).3.5.LvHSP60expressionafterinjectionwithbacteriaRNAandproteinlevelexpressionofHSP60inL.vannameihepa-topancreas,gillsandhaemocyteswasdetectedbyquantitativeRT-PCR(QPCR)andWesternblotanalysisafterinjectionwithtwotypesofbacteria(Gram-positive,S.aureus;andGram-negative,V.alginolyticus).Thetime-courseQPCRanalysisofgeneexpressionshowedthatHSP60mRNAlevelsincreasedinthegillsapproximately1.9-fold1.5hafterstimulationbyS.aureus,thenrecoveredtocontrol(orlower)levelsafter24h.FollowinginjectionwithV.alginolyticus,theHSP60transcriptlevelwassignificantlyup-regulatedafter3h,butthenreturnedtoitsoriginallevelby12h(Fig.5A1).TheQPCRresultsforhepatopancreastissueshowedthatwheninjectedwiththeGram-positivebacteriatherewasalsosignificantHSP60expressionby1.5h,butexpressionthendecreasedtobelownor-mallevelsby24h.Gram-negativebacterialinjectionfirstreduceHSP60mRNAexpressioninthehepatopancreasby1.5h,andexpressionfelltoitslowestlevelby3h;itwasthenup-regulatedsignificantlyby6h,andreacheditshighestlevelby12hpost-injection(Fig.5A2).Inthebacteria-challengedhaemocytes,LvHSP60mRNAwasinducedbetween1.5and3h,butexpressionthendecreasedgraduallyfrom3to24h(Fig.5A3).TheproteinexpressionlevelofLvHSP60wasalsoinducedatanearlystageinthegillsfollowingbacterialinjection(Fig.5B1),inaccordancewiththeQPCRresultsforthistissue.174J.Zhouetal./JournalofInvertebratePathology103(2010)170–178Fig.1.NucleotideanddeducedaminoacidsequencesofLvHSP60(GenbankaccessionNos.FJ710169).Thenucleotidesequenceisnumberedfromthe50end,andthesingleletteraacodeisshownbelowthecorrespondingcodon.ThestartcodeATGandtheterminationcodeareshowninbold.Theclassicalmt-HSP60signature(underlined)motifsandtheATP-bindingmotif(boxed)areindicated.AtypicalGGMrepeatmotifattheCterminusisshowninlightgrey.J.Zhouetal./JournalofInvertebratePathology103(2010)170–178175Fig.2.AphylogenetictreeofLvHSP60with10otherHSP60/chaperoninspeciesreconstructedusingtheNeighbour-Joiningmethod.Thetreeisbasedonanalignmentcorrespondingtofull-lengthaasequences,usingClustalWandmegAlign.Thenumbersshownatthebranchesdenotebootstrapmajorityconsensusvaluesof1000replicates.TheGenBankaccessionnumbersforthesequencedesignationsareasfollows:HSP60/chaperoninfromFruitfly(D.melanogaster,NP_511115);Australiansheepblowfly(L.cuprina,ABO09590);Pealeafminer(L.huidobrensis,AAW30392);Yellowfevermosquito(A.aegypti,XP_001661764);Southernhousemosquito(C.quinquefasciatus,XP_001850501);Bodylouse(P.humanuscorporis,EEB15946);BlackleggedTick(I.scapularis,EEC05122);Norwayrat(R.norvegicus,AAC53362);Human(H.sapiens,AAA36022)andBloodflukeplanorb(B.glabrata,ACL00842).Fig.3.ExpressionandpurificationoftherecombinantLvHSP60fusionprotein.(A)ProteinsamplesofpurifiedrecombinantLvHSP60separatedby12%SDS–PAGEandstainedwithCoomassiebrilliantblue.(B)Westernblottingwithanti-LvHSP60polyclonalantibody.M,Prestainedproteinsizemarkers(Fermentas);1,vectorcontrolfromDH5a;2,crudeextractfromDH5ainducedwith1mMIPTGandgrownat37Cfor4h;3,purifiedrecombinantLvHSP60fusionprotein.3.6.LvHSP70expressionafterinjectionwithbacteriaHSP70mRNAexpressioninwhiteshrimpgillssignificantlyde-creased3,6,and24haftertheywereinjectedwithS.aureus.Fur-thermore,expressionwasdown-regulatedbetween3and12hinthesametissuewhenstimulatedwithV.alginolyticus(Fig.6A1).WiththestimulationbyGram-positivebacteria,theLvHSP70Fig.4.TissuespecificityofHSP60geneexpressioninthewhiteshrimp(Litopenaeusvannamei).TotalRNAfromvarioustissuesofsixshrimp(n=6)wasanalysedbyRT-PCRusingsequence-specificprimers(Table1)forHSP60asdescribedinSection2,usingtheb-Actingeneasaninternalcontrol.He,haemocytes;In,intestine;St,stomach;Mu,muscle;Ht,heart;Hp,hepatopancreas;Gi,gill.genetranscriptsincreasedandreachedamaximum(4.5-foldhigherthanincontrols)after1.5hinthehepatopancreas,butthenreturnedtonormallevels.SignificantincreasesintheexpressionlevelofLvHSP70werealsoobservedbetween6and24hinthehepatopancreasoftheV.alginolyticus-challengedgroup(Fig.6A2).Similarly,theHSP70mRNAexpressioninthehaemocytesofthewhiteshrimpwassignificantlyup-regulatedbetween1.5and6hafterinjectionwiththebacteria,butthenre-turnedtooriginallevelsbetween12and24h(Fig.6A3).3.7.IntracellularcalciuminhaemocytesIntheS.aureus-challengedgroup,theintracellularcalciumlevel(determinedbyFluo-3-AMfluorescencemeasurements)showedanincreasebetween1.5and3h.Thereafter,asignificantdecreaseincalciumlevelswasobservedbetween6and24hpost-injection(Fig.7).Wealsonoticedasignificantinductionof[Ca2+]iinV.alg-inolyticus-challengedshrimphaemocytes1.5hpost-injection,fol-lowedbydown-regulationtothesamelevelasinthecontrolsbetween3and6h(Fig.7).4.DiscussionGlobally,shrimpaquaculturehasbeenseriouslyaffectedbydis-easeoutbreaks,mainlycausedbyVibriospeciesofbacteriaandviruses.Currentlythereisnoeffectivemethodtocontrolthesedis-easeoutbreaks,butvariousaspectsofshrimp’defenceresponseshavebeencharacterizedtosomedegree.Incrustaceans,gillsarerespiratoryorgansthatareindirectcontactwiththeexternalenvi-ronmentandareknowntoplaykeyrolesinosmoregulation,detoxificationanddefencemechanisms(Clavero-Salasetal.,2007).Further,Burgents(2005a)reportedthatgillsarethesiteofaccumulationofviablebacteriaduringinfection,andunderhyp-oxicconditionsmoreVibrioaccumulateingillsofwhiteshrimp,indicatingthattheybecomemoresusceptibleunderenvironmen-talstress(Burgentsetal.,2005b).Thehepatopancreasisthemajormetaboliccentreresponsiblefortheproductionofreactiveoxygenspeciesandareimportanttissuesforimmunedefencesincrusta-ceans(SoderhallandCerenius,1998).Circulatinghaemocytesalsoplayanimportantroleintheimmuneresponseagainstpathogens.Thehumoraldefencesystemincludesefficient,sensitivenon-selfrecognitionmolecules,phenoloxidase-mediatedmelanisationactivity,antimicrobialpeptides,andahaemolymphclotting–coag-ulationreaction(CereniusandSoderhall,2004).Haemocytesalsoparticipateincellularencapsulationandphagocytosis(CereniusandSoderhall,2004;SoderhallandCerenius,1998).However,de-spitetheseobservations,betterunderstandingofthemolecularmechanismofshrimpimmuneresponsesisrequiredtofacilitatethedevelopmentofnewmethodsofdiseasecontrol.InthestudyreportedherewedeterminedthecompletecDNAsequenceofHSP60fromL.vannamei.Conservedsequencesandcharacteristicmotifs,suchasHSP60familysignatures,ATP-bind-ingsites(from194to212aminoacidresidues)(BrocchieriandKarlin,2000),andconservedGGMrepeatsattheC-terminalend176J.Zhouetal./JournalofInvertebratePathology103(2010)170–178A1The relative expression of LvHSP603S.aureusSalineV.alginolyticusa*2a*bbba**1bbc*ab*b00h1.5h3h6h12h24hTime after bacterial challengeA2The relative expression of LvHSP602.01.5b1.00.50.0The relative expression of LvHSP60a*S.aureusSalineV.alginolyticusA3a*a*bbc*2.52.01.51.00.50.00ha*a*aa**bS.aureusSalineV.alginolyticusac*b*c*0h1.5h3hc*ba*baab*b*ab**6h12h24h1.5h3h6h12h24hTime after bacterial challengeTime after bacterial challengeFig.5.Quantitativereal-timePCRandWesternblotexpressionanalysisofHSP60inwhiteshrimpgills(A1,B1)at0,1.5,3,6,12,24hafterstimulationwithGram-positivebacteria(G+:Staphylococcusaureus)andGram-negativebacteria(GÀ:Vibrioalginolyticus).AnalysisofLvHSP60expressioninthehepatopancreas(A2)andhaemocytes(A3)ofthebacteria-challengedshrimpbyreal-timePCR.ThestatisticalanalysiswasbasedoncomparisonsoftherelativeexpressionratiooftheLvHSP60genetotheb-actingeneineachofthetissuesbycalculating2ÀDDCTvalues.Significantdifferences(P<0.05)ofLvHSP60expressionbetweenthebacteria-challengedandcontrol(saline)groupsareindicatedbydifferencesinletters(a,b,c).Significantdifferences(P<0.05)inLvHSP60expressionbetweenthechallengedanduntreatedgroups(0h)areindicatedwithasterisks.Valuesaremeans±SD(standarddeviation)ofdataobtainedfromsamplesofsixanimals(n=6).ForWesternblotexpressionanalysis,30lgofproteinwereloadedintoeachlane.(thestructureandfunctionofwhichareunknown(Sanchezetal.,1999)),aswellasthemajorstructuralandfunctionaldomainstyp-icallyfoundinHSP60proteins(Choreshetal.,2004),werefoundinthededucedLvHSP60aminoacidsequence.SequencesimilaritysearchesrevealedthatthededucedaminoacidsequenceofLvHSP60shareshighsimilaritywithpreviouslydescribedHSP60sequencesfrominsectsandmammals(morethan83%similarityinallmatches).OurtranscriptionexpressionrevealedthatLvHSP60wasup-regulatedinthegillsandhepatopancreaspost-injection.WesternblotanalysesconfirmedthatLvHSP60wasin-ducedinthegillfollowinginjection.Clavero-Salasetal.(2007)alsoidentifiedapartialsequenceofHSP60fromaWSSV-infectedwhiteshrimpgillcDNAlibrary.InshrimpchallengedbybothbacteriaexpressionofLvHSP70mRNAwasalsoinducedinthehepatopan-creas.Inaddition,levelsofHSP60andHSP70transcriptsinhaemo-cytesweresignificantlyincreasedaftershrimpwerechallengedbyS.aureusandV.alginolyticus.ThesefindingssuggestthatHSP60andHSP70playkeyrolesinshrimpimmuneresponsesfollowingbac-terialinfection.Inhumans,thepotentialfunctionofeukaryoticHSPsasendog-enousdangersignallingmoleculeshasbeendiscussed(Matzinger,2001;Osterlohetal.,2004),andrecentworkhasprovidedevi-dencethatendogenousHSPs,whicharethoughttobereleasedfromdyinginfectedcells,canincreasetheactivationofinnateim-muneresponsesviainteractionswithToll-likereceptors(Asea,2008;Aseaetal.,2000;Aseaetal.,2002;Akiraetal.,2006;Matz-inger,2001;).However,themechanismsunderlyingHSPinductionandtheroleofHSPsduringbacterialinfectionhavenotbeenfullyelucidated.Indecapodcrustaceans,animportantimmunedefencereactionofhaemocytesisphagocytosis(LeeandSoderhall,2002).Duringthecourseofphagocytosis,NADPH-oxidaseisproduced,therebyinducingtheproductionofreactiveoxygenspecies(ROS),whichcanefficientlykillforeigninvaders(Bogdanetal.,2000;Schwarz,1996).However,highlevelsofROSmaydamagethehost’sDNAandinactivateproteins,resultinginseriouscelldamage(Yu,1994).Asaresultofbacterialinfectionandsubse-quentROSproduction,theaccumulationofdenaturedproteinsinthehostcellmighttriggertheexpressionofHSPs(Gaoetal.,2008),andLeeetal.(2008)reportedthatHSP60isakeyplayerinresistanceagainstoxidativestress.Hence,theinductionofHSP60andHSP70post-injectionmaybeaprotectivemechanism.InaccordancewiththishypothesisHSPsarecytoprotectiveandactaschaperoneproteins,assistinginthefoldingofnewly-pro-ducedproteinsandsolubilisingdenaturedproteinaggregates,restoringproteinfunctionandassistingintheclearanceofdam-agedproteinsfollowingincreasesinlevelsofROSortoxicsub-stancesproducedbypathogensinbacteria-challengedanimals(Deanetal.,1991;KiangandTsokos,1998;Ramagliaetal.,2004).Inconclusion,weclonedfull-lengthHSP60fromacrustacean,anddescribedtheexpressionpatternsofLvHSP60andLvHSP70afterbacterialinfectionforthefirsttimeinshrimp.OurresultsindicatethatLvHSP60andLvHSP70arepotentiallyinvolvedinshrimpimmuneresponsestobacterialinfection.TheCa2+signal-lingtransductionpathwaymaybeinvolvedintheinitiationoftheshrimpimmuneresponseinearlypost-infectionstages.ThesefindingswillbehelpfulinunderstandingthesignificanceofHSPsJ.Zhouetal./JournalofInvertebratePathology103(2010)170–178177A1The relative expression of LvHSP701.61.20.80.40.00hb*The relative expression of LvHSP70abS.aureusSalineV.alginolyticusA25.0abaaaab*b*a*S.aureusSalineV.alginolyticus4.5a*1.51.00.50.00hbb**a*bbbba*aabb*ba*1.5h3h6h12h24hTime after bacterial challenge1.5h3h6h12h24hTime after bacterial challengeA3The relative expression of LvHSP704.03.22.41.60.80.00ha*a*a*b*b*S.aureusSalineV.alginolyticusa*a*b*ba*aab1.5h3h6h12h24hTime after bacterial challengeFig.6.(A)Quantitativereal-timePCRexpressionanalysisofHSP70inwhiteshrimpgills(A1),hepatopancreas(A2)andhaemocytes(A3)at0,1.5,3,6,12and24hafterstimulationwithGram-positivebacteria(G+:Staphylococcusaureus)andGram-negativebacteria(GÀ:Vibrioalginolyticus).ThestatisticalanalysiswasbasedoncomparisonsoftherelativeexpressionratiooftheLvHSP70genetotheb-actingeneineachofthetissuesbycalculating2ÀDDCTvalues.Significantdifferences(P<0.05)inLvHSP70expressionbetweenthebacteria-challengedandcontrol(saline)groupsareindicatedbydifferencesinletters(a,b,c).Significantdifferences(P<0.05)inLvHSP70expressionbetweenthechallengedanduntreatedgroup(0h)areindicatedwithasterisks.Valuesaremeans±SD(standarddeviation)ofdataobtainedfromsamplesofsixanimals(n=6).(The mean intensity of fluo-3 fluorescence)2+]i levels in the haemocytes1008060402000h***S.aureusSalineV.alginolyticustheNaturalScienceFoundationofGuangdongProvince,PRChina(Grantnos.06025052and8151063101000035),andGuangdongProvincialOceanicFisheriesScienceandTechnologyProject(Grantno.A200899D01),andtheProjectoftheDepartmentofEducationofGuangdongProvincefortheYoungResearcherofHigherSchool.WearegratefultoProfs.QiliFengandSichunZhengfromthelab-oratoryofMolecularEntomology,SouthChinaNormalUniversity.References*1.5h3h6h12h24hTime after bacterial 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