ActaBiochimBiophysSin,2017,1–10doi:10.1093/abbs/gmx080ShortCommunication
ShortCommunication
QuantitativeproteomicanalysisofhostresponsestriggeredbyMycobacteriumtuberculosisinfectioninhumanmacrophagecells
HuaLi1,
2,ShaWei1,
2,YuanFang3,MinLi2,XiaLi2,ZheLi4,JibinZhang1,GuofengZhu5,ChuanyouLi6,LijunBi2,GuiminZhang3,DianbingWang2,*,andXian-EnZhang2,* 1StateKeyLaboratoryofAgromicrobiology,CollegeofLifeScienceandTechnology,HuazhongAgriculturalUniversity,Wuhan430070,China,2NationalLaboratoryofBiomacromolecules,CASCenterforExcellenceinBiomacromolecules,andKeyLaboratoryofRNABiology,InstituteofBiophysics,ChineseAcademyofSciences,Beijing100101,China,3CollegeofLifeScience,HubeiUniversity,Wuhan430062,China,4ChinaGoldenMarker(Beijing)BiotechCo.Ltd.,Beijing102206,China,5ShanghaiMunicipalCenterforDiseaseControl&Prevention,Shanghai200336,China,and6BeijingChestHospital,CapitalMedicalUniversity,Beijing101149,China *Correspondenceaddress.Tel:+86-10-64888148;E-mail:zhangxe@(
X.Z.)/Tel:+86-10-64888432;E-mail:wangdb@(
D.W.) Received17April2017;EditorialDecision27April2017 Abstract MacrophagesareprimaryhostofMycobacteriumtuberculosis(M.tb)andthecentraleffectorofinvivoinnateimmuneresponsesagainstbacteria.Thoughtheinteractionbetweenmacrophagesandmycobacteriahasbeenwidelyinvestigated,themolecularmechanismsofM.tbpathogenesisinmacrophagesarestillnotclear.Inthiswork,weinvestigatedthealteredproteinexpressionprofilesofmacrophagesaftervirulentH37RvstrainandavirulentH37Rastraininfectionbytandemmasstag-basedquantitativeproteomics.Among6762identifiedproteinsofmacrophages,theexpressionlevelsof235proteinsweresignificantlyaltered,whichissupposedtoberelatedtotheinfectionofdifferentstrains.Bybioinformaticsanalysisatsystemslevel,wefoundthattheseproteinsaremainlyinvolvedinthebiologicalprocessofapoptosis,bloodcoagulation,oxidativephosphorylation,andothers.TheenormousvariationinproteinprofilesbetweenmacrophagesinfectedwithH37RaandH37RvsuggeststheexistenceoffourdifferentimmunitymechanismsthatdecidethefatesofmacrophagesandM.tb.ThesedatamayprovideabetterunderstandingofM.tbpathogenesiswithinthehost,whichcontributestothepreventionandclinicaltreatmentoftuberculosis. Keywords:Mycobacteriumtuberculosis,virulentH37Rvstrain,avirulentH37Rastrain,macrophages,TMT-basedquantitativeproteomics Introduction Tuberculosis(TB)isaninfectiousdiseasethatgreatlyimpactshumanandanimalhealthworldwide.One-thirdoftheworld’s populationispersistentlyinfectedwiththecausativeagentMycobacteriumtuberculosis(M.tb)and~10%ofthemsufferfromactiveTB[1,2].AreportfromWorldHealthOrganization(WHO) ©TheAuthor2017.PublishedbyOxfordUniversityPressonbehalfoftheInstituteofBiochemistryandCellBiology,ShanghaiInstitutesforBiologicalSciences,Chinese AcademyofSciences.Allrightsreserved.Forpermissions,pleasee-mail:journals.permissions@
1 2 HostresponsestriggeredbyMycobacteriumtuberculosis indicatesthat1.8millionpeoplediedofTBin2015.OneofthemajorcausesofthishighincidenceisthedifficultyofdiagnosisandtreatmentthatcausedbythepoorunderstandingofplexM.tbpathogenesis. MacrophagesaretheprimaryhostcellsofM.tbinvivo.Theyareoneofthemostimportantimmunecellsparticipatingintheeliminationofinfectingmycobacteria,whichcannotonlyinducetheinnateimmuneresponseagainstpathogenicanismsbutalsoplaycentralrolesinTBcontrol[3].BactericidalresponsesofmacrophagesareactivatedbyintracellularM.tb,includinghagy,fusionofphagosomewithlysosome,apoptosis,necrosis,andproductionofantimicrobialagentssuchascytokines,hydrolases,freeradicals,reactiveoxygenspecies(ROS),andnitrogenintermediates[4–7].Inaddition,macrophagescanpresentmycobacterialantigenstoprimedlymphocytesandstarttheinflammatoryresponsebysecretingchemokine,cytokines,andchemicalmediatorsofinflammationtorecruitotherimmunecellstothesiteofinfection[8,9]. H37RvandH37RaaretwocloselyrelatedM.tbstrainswithdifferentvirulence.H37RvcauseshigherbacterialloadsofM.tbinlungsandansthanH37Ra[10–14].Furthermore,H37Rvcanevadehostimmunityanddisplaygreaterinvivoreplicationbyinhibitingthekillingprocessessuchasthefusionofphagosomewithlysosome,phagosomeacidification,hostcellsignaltransport,apoptosisofinfectedmacrophagesandinductionofsignificantnecrosis,granuloma,andlipiddroplet[15–23]. Therefore,prehensiveresearchondifferentmacrophageresponsesagainstvirulentH37RvstrainandavirulentH37RastraininfectionwillhelptounderstandplexmechanismsofM.tbinfectionanditssurvivalinhostcells[24].Recently,manyproteomicresearcheshavereportedtheinteractionbetweenhostandM.tb,whichprovedsignificantchangesintheexpressionofproteinsinmacrophagephagosomes,anellemembranes,exosomes,andendoplasmicreticulumafterM.tbinfection[25–31].Herein,weappliedtandemmasstag(TMT)-basedquantitativeproteomicstoassessthealteredproteinexpressionprofilesofmacrophagecell(THP-1)infectedwiththevirulentH37RvstrainandavirulentH37Rastrain,respectively.Among6762identifiedmacrophageproteins,235proteinsweredemonstratedtohavearelationshipwithvirulentinfection.Byanalysisofthesedataatsystemslevel,thesedifferentiallyexpressedproteins(DEPs)ofmacrophageswerefoundtobemainlyinvolvedinapoptosis,bloodcoagulation,oxidativephosphorylation,andothercellularprocesses.TheenormousvariationinproteinprofilesbetweenmacrophagesinfectedwithH37RaandH37RvsuggestedtheexistenceoffourdifferentimmunitymechanismsthatdecidethefatesofmacrophagesandM.tb.OurdatamayprovidenovelinsightsintothedifferentmolecularmechanismsofhostreactiontovirulentM.tbandavirulentM.tbinfection,therebygivenewthoughtsonthepreventionandclinicaltreatmentofTB. MaterialsandMethods M.tbstrainsandcultureconditions M.tbH37Rv(ATCC:AmericanTypeTissueCollection27294)andH37Ra(ATCC25177)weregrowninMiddlebrook7H9brothsupplementedwith10%albumindextrosecatalase(ADC)enrichment(BectonDickinson,SanJose,USA),0.5%glyceroland0.02%Tween80at37°Cfor6days.M.tbcellswerequantifiedbyseriallydilutingthebacterialstockandplating100μlaliquotsonMiddlebrook7H10agarwith10%ADC.Then,theCFUsofM.tbweredeterminedafter3weeksofincubationat37°
C. THP-1cellscultureanddifferentiation MonocyteleukemiacellTHP-1cellswereculturedinRPMI-1640medium(HyClone,Logan,USA)supplementedwith10%fetalbovineserum(FBS;Gibco,Carlsbad,USA)(V/V)at37°Cinahumidified,5%CO2atmosphere.Cellsatconcentrationof5×105cells/mlweredifferentiatedtomacrophagephenotypewith25ng/mlPMA(phorbol-12-myristate-13-acetate)inRPMl1640containing10%FBSfor48h.AfterremovalofthePMA-containingmedium,thecellswereincubatedforanother48hinfreshRPMI-1640mediumsupplementedwith10%FBS. Infectionexperiments TheM.tbwithOD600of0.3(CFU=2–3×107)andequivalentbeadswerewashedtwicewithRPMI-1640mediumandsuspendedinRPMl-1640mediumcontaining10%FBSasaprimaryinoculumformacrophageinfection.ThedifferentiatedTHP-1cellswereinfectedwithlatexbeads,H37RaorH37Rvatanmultiplicityofinfection(MOI)of35andthenincubatedfor4hat37°Cina5%CO2atmosphere.Toremovetheextracellularbacteria,theinfectedcellswerewashedtwicewithRPMI-1640mediumaftertheculturesupernatantwasdiscarded.Then,thecellswerefurtherculturedforanother12horasindicated.Everystimulationexperimentwasdonetwiceforbiologicalrepetition.TheblankcontrolgroupnamedTHP-1wastreatedsimultaneouslyastheothergroups. Quantitativereal-timepolymerasechainreaction TotalmRNAwasextractedusingTRIzolreagent(Invitrogen,Carlsbad,USA)andcDNAwassynthesizedusingthecDNAsynthesiskit(TransBionovo,Beijing,China),ordingtothemanufacturer’sinstructions.bfl-1(anti-apoptoticgene)andtnf-α(apoptoticgene)expressionlevelsweredeterminedbyquantitativereal-timepolymerasechainreaction(qPCR)atdifferenttimepointsafterinfection.TheqPCRmeasurementswereperformedonaBio-RadCFX96usingTransStartTipGreenqPCRSuperMix(TransBionovo)asfollows:pre-denaturation94°Cfor30s,and94°Cfor5s,52°Cfor15s,72°Cfor10sfor40cycles.Thespecificprimersforthegeneswere:tnf-α(F:5′-AGGCGGTGCTTGTTCCTC-3′;R:5′-GTTCGAGAAGATGATCTGACTGCC-3′),bfl-1(F:5′-TGCCAGAACACTATTCAACC-3′;R:5′-TTGCCTTATCCATTCTCCTG-3′)andgapdh(F:5′-GAAGGTCGGAGTCAACGGAT-3′;R:5′-CCTGGAAGATGGTGATGGG-3′).Two-tailedttests,type2,n=2wereusedforstatisticalevaluationofthemRNAlevels(mean±SD),whilegapdhmRNAwasusedasaninternalcontrolfornormalization. Samplepreparation Boththesupernatantandadherentcellswerewashedtwicewithphosphate-bufferedsaline(PBS).TheproteinswereextractedwithRIPAcelllysisbuffer(Amersco,Solon,USA)supplementedwith1×proteaseinhibitorCocktail(Sigma,StLouis,USA).Finally,theproteinconcentrationwasdeterminedusingthePierceBicinchoninicAcid(BCA;ThermoFisherScientific,Waltham,USA)ProteinAssayKit. Themacrophagesampleshavetwosetsofbiologicalrepetition,namedsample1andsample2.Bothofsamples(0.45–0.65mgeach)wereresuspendedin1×SDSloadingbufferattheconcentrationof5mg/ml.Thesampleswerereducedbyadditionof20mMdithiothreitol(DTT)andincubatedat90°Cfor10min.Aftercoolingtoroomtemperature,100μgofeachsamplewasresolvedbySDSPAGE(10%Bis-trisgel;Invitrogen)for1h.Then,thesamplesweresubjecttoanin-geltrypticdigestionafterreductionwith10mM HostresponsestriggeredbyMycobacteriumtuberculosis
3 DTTat60°Cfor30minandalkylationwith40mMiodoacetamideinthedarkfor1htoblockfreecysteine.Trypsinwasaddedat1:50(w:w;trypsin:sample)andthesampleswereincubatedat37°Covernight.Thedigestedpeptideswereextractedfromgelwith60%acetonitrile,5%formicacidandthendriedinaSpeed-Vac,afterwhichtheywerewashedwith50%acetonitriletoneutralpH.TheproteinsisolatedfromTHP-1macrophages,THP-1macrophagesstimulatedbyBeadsandTHP-1macrophagestreatedwithH37RaorH37RvwerelabeledwithTMT6-126,127,128,and129labelreagentordingtothemanufacturer’sinstructions(ThermoFisherScientific).Basically,thesamplesandthelabelingreagentswereincubatedfor60minatroomtemperature,then8μlofhydroxylamine(5%)wasaddedandincubatedforanother15mintoquenchthereactions.ThelabeledsampleswerenamedTMT-126,TMT-127,TMT-128,andTMT-129,respectively.Thefourlabeledsamplesbined,driedintheSpeed-Vacandsentforanalysis[32]. One-dimensionalseparationofTMT-labeledpeptides High-pHreversedphasehigh-performanceliquidchromatography(HPLC)wasusedforpeptidefractionationusingGilson300seriesequipment(Gilson,Worthington,USA).TheTMT-labeledbinedsamplesweresolubilizedin200μlof20mMammoniumformate(pH10)andtheninjectedintoanXbridgecolumn(C183.5μm2.1×150mm2;Waters,Milford,USA)andelutedwithalineargradient(2%–45%)ofbufferBin44min(bufferA:20mMammoniumformate,pH10;bufferB:20mMammoniumformatein90%acetonitrile,pH10).ThefractionswiththehighestUVabsorbanceat214nm(mostofpeptides)fromthehigh-pHHPLCwereselectedtobeanalyzedbyliquidchromatography/tandemmassspectrometry(LC–MS/MS).Thosefractions(1min)werecollectedanddriedinaSpeed-Vac. NanoLC–MS/MS PeptidesfromeachfractionwereanalyzedbynanoLC–MS/MSusinganRSLCsysteminterfacedwithaQExactive™hybridquadrupoleOrbitrapmassspectrometer(ThermoFisherScientific).Sampleswereloadedontoaself-packed100μm×2cmtrappackedwithMagicC18AQwith5μm200Å(MichromBioresources,Auburn,USA)andthenwashedwithBufferA(0.2%formicacid)for5minwithaflowrateof10μl/min.Thetrapwasbroughtin-linewithahomemadeanalyticalcolumn(MagicC18AQ,3μm200Å,75μm×50cm)andpeptideswerefractionatedat300nl/minwithamulti-steppedgradient[4%–15%BufferB(0.16%formicacid80%acetonitrile)in35minand15%–25%Bin65minand25%–50%Bin55min].Massspectrometrydatawereacquiredusingadata-dependentacquisitionprocedurewithacyclicseriesofafullscanacquiredinOrbitrapwitharesolutionof120,000.The20mostintenseionswithrepeatcountsofoneanddynamicexclusiondurationsof30swerescannedusingMS/MS(30%ofcollisionenergyinHCDcell,resolution30,000).Thesample1wasruntwiceandnamedAandB,andsample2wasrunonceandnamedC. Dataanalysis LC–MS/MSdataof14fractionsfromeachexperimentweresearchedinMUDPITstyleagainsttheEnsemblhumandatabaseusinganin-houseversionofX!
tandem[SLEDGEHAMMER(2013.09.01),]withcarbamidomethylationoncysteineandtheTMT6-plexlabelonlysine,withtheN-terminusofpeptidesasfixedmodificationsandoxidationofmethionineasavariablemodification.A±10ppmand±20ppmwereusedastoleranceforprecursorandproductions,respectively.Theintensitiesof TMT6-plexreporterionsineachspectrumwereextractedusinganin-houseperlscriptandcorrectedforecross-overusingvaluessuppliedbythemanufacturer.Thetreatment/blankcontrolratioofeachspectrumwascalculatedusingreporterionintensityandwasnormalizedtothemedianratioofallidentifiedspectrathatfitcertaincriteria:peptidebelongstoahomosapiensdatabase,peptideE-value<0.01(theexpectationthatanyparticularproteinassignment),totalreporterionintensity>60,000.Thefalsepositiverate(FPR)atthepeptidelevelwas<1.0%.Spectrawithratiosof‘Divide0’werereplacedwiththearbitrarynumber‘10’. Pairwiseratiosoffourgroups(groupBeads/THP-1:TMT-127/TMT-126,groupH37Ra/Beads:TMT-128/TMT-127,groupH37Rv/Beads:TMT-129/TMT-127,andgroupH37Rv/H37Ra:TMT-129/TMT-128)ofeachindividualproteinwerecalculatedusingthemedianratioofallpeptidesbelongingtotheproteinthathasatleasttwodatapointsthatfitthecriteriaofpeptideE-value<0.03andsumofreporterionsofbothchannel>20,000.ThemassspectrometryproteomicsdatahavebeendepositedtotheProteomeXchangeConsortium[33]viathePRIDEpartnerrepositorywiththedatasetidentifierPXD006331. ThemethodusedtoanalyzetheDEPsisdescribedinSupplementaryFig.S1.TheZ-test(P<0.05)wasusedtoscreentheDEPsfromeachgroupbyconvertinglog2ratiosofproteinsintoZscores[34].PANTHER(ProteinAnalysisThroughEvolutionaryRelationships;2February2017,datelastessed)wasusedtoclassifyproteinsbytheirfunctions[35].ForGenoOntology(GO)andpathwayenrichment,theuniqueproteinsidentifiedinthisstudywereanalyzedusingDatabaseforAnnotation,Visualization,andIntegratedDiscovery(DAVID)[36].Theprotein–proteininteraction(PPI)workswereanalyzedusingSTRINGsoftware,afterwhichtheparagraphswereeditedwithCytoscapev.3.2.1andBiNGOv.3.0.3[37,38]. Westernblotanalysis Approximately40μgofproteinsfromeachsamplewereresolvedby8%–15%SDS-PAGEat100Vfor2–3h.Theproteinbandswereelectro-blottedontopolyvinylidenefluoride(PVDF)microporousmembranes(Millipore,Billerica,USA),whichwereblockedwith5%skimmedmilkfor2hatroomtemperature.Forimmunoblotanalysis,themembraneswereincubatedwithantibodiesasfollows:anti-AHSG(1:1000;Boster,Wuhan,China),anti-IL-1β(1:100;Boster),andantiβ-Actin(1:2000;SangonBiotech,Shanghai,China)at4°Covernight.Afterbeingwashedfourtimeswith0.1%PBS-Tweenfor5min,themembraneswereincubatedwithhorseradishperoxidase(HRP)conjugatedgoatanti-rabbitIgGH&L(Abcam,Cambridge,UK)for60minatroomtemperature.AfterincubationwithchemiluminescentHRPsubstrate(Millipore),thechemiluminescencedetectionwasperformeddirectlyusinganAmershamImager600(GEHealthcare,Bethesda,USA).Forquantificationanalysis,ITQL(GEHealthcare)softwarewasusedtocalculatethefoldchanges,whileactinwasusedasaninternalcontrolfornormalization. Results InfectionwithH37RaorH37RvresultedindifferentM.tbgrowthinmacrophagesandtranscriptionlevelsofanti-apoptoticgenebfl-
1 InordertoconfirmthedifferentialgrowthrateofM.tbinmacrophagesafterinfection,THP-1differentiatedmacrophageswereinfectedwithavirulentH37RastrainorvirulentH37Rvstrainatan
4 HostresponsestriggeredbyMycobacteriumtuberculosis MOI(multiplicityofinfection)of10:
1.At0,1,3,and5daysafterinfection,M.tbcolony-formingunits(CFUs)ofmacrophagesweremeasuredafter0.1%SDStreatment(Fig.1A).H37RvshowedasignificantlyhigherintracellulargrowthrateinhumanmacrophagesthanH37Ra,althoughbothstrainshavesimilarcapacitiestoenterthemacrophages[23,39].ThedifferenceofthegrowthratesoftwoM.tbstrainsinhumanmacrophagesprobablyoriginatesfromthedifferentvirulencebetweenthem. TodeterminetheculturetimeofmacrophagesafterinfectionwithH37RaorH37Rv,theexpressionlevelsofbfl-1andtnf-αinTHP-1macrophagesweredetectedbyqPCR.Theanti-apoptotic-orapoptotic-relatedgenebfl-1andtnf-αwerethususedasthereferencetoindicatetheessfulinfections.Theexpressionlevelsofbfl-1andtnf-αinmacrophagesafterinfectionwithM.tbwerehigherat12hafterinfectionsthanthatatothertimepoints.Whatismore,expressionlevelsofbfl-1betweenH37RaandH37Rv-infectedmacrophagesweremostdifferentat12hafterinfections,asshowninSupplementaryFig.S2,whichisconsistentwithpreviousresults[40].Therefore,wefocusedontheproteinprofilesofTHP-1cellsat12hafterinfectionwithM.tb,fortheurrenceofapoptosisandvariantanti-apoptosisofmacrophagesafterinfectionwithtwostrains. TMT-basedquantitativeproteomicsrevealeddifferentexpressionprofilesbetweenH37Ra-andH37Rvinfectedmacrophages Tounderstandthemolecularmechanismsinvolvedincross-talkbetweenmacrophagesandM.tb,weappliedaTMT-basedmassspectrometryprofileofproteinsofTHP-1cellsinfectedwithH37RaorH37Rv(Fig.1B).Macrophageproteinswereextractedat12hafterinfectionwithH37RaorH37Rv,withbeadsstimulationgroupasanegativecontrolanduntreatedTHP-1macrophagesasablankcontrol.Asaresult,atotalof6762macrophageproteinswereidentifiedusingthecriteriadescribedinMaterialsandMethods(SupplementaryTablesS1andS2).ProteinsidentifiedbothinRunAorBandCwereselectedforfurtherstudy.Subsequently,thearithmeticmean,standarddeviation,andrelativestandarddeviation(RSD)ofindividualproteinswerecalculated.TheRSDof95%proteinsineachgroupwerelowerthan0.21,whichdisplayedlowvariabilitybetweentherepeats(SupplementaryFig.S3).Then,proteinswithRSD>thelargestRSDvalueof95%proteinsineachgroupwerefilteredoutfortheurateratios(SupplementaryFig.S1).TheproteinswithZ-score>1.96or<−1.96wereconsideredasDEPs(SupplementaryTableS3-1).Forprofilingtheproteinexpressionlevel,Hemlsoftwarewasusedtoexporttheheatmap[41] Figure1.TMT-labeledquantitativeproteomicsanalysisoftheproteinsofTHP-1cellsinfectedwithH37RaorH37Rv(A)H37RainfectionandH37RvinfectionresultedindifferentM.tbgrowthrateinmacrophages.**P<0.01,***P<0.001vs.H37RagrowthrateinTHP-1cells.Theseresultswerefromonerepresentativeexperiment.Similarresultswereobtainedintwootherexperiments.(B)ExperimentalworkflowofTMTlabelingquantitativeproteomics.Macrophageproteinswereextractedat12hafterbeadstreatment,H37RaorH37Rvinfection.Afterdenaturationanddigestion,sampleswerelabeledwithTMTlabelreagentsandanalyzedusingaQExactive™hybridquadrupole-Orbitrapmassspectrometer.Massspectrometrydatawerequantifiedusinganin-houseversionofX!
tandem.DEPsofmacrophagesbetweeninfectionswerediscussed.(C)HeatmapofDEPsinTHP-1cellsstimulatedbybeads,infectedwithH37RaorH37Rv.Thelog2ratiosofproteinsfromeachgroupwerevisualizedtotheheatmapusingHeml.Thecolorscaledemonstratestherelativeexpressionlevelsoftheproteins(log2ratio),eachrowrepresentsadifferentiallyexpressedproteinandeachcolumnrepresentsagroup.(D)PANTHERProteinClassontologyanalyzedDEPsbetweenTHP-1cellsinfectedwithH37RaandH37Rv(P<0.01)(groupH37Rv/H37Ra). HostresponsestriggeredbyMycobacteriumtuberculosis
5 whichdemonstratedclusteroffourgroupsordingtotheDEPsafterinfection(Fig.1C).NoconfidentproteinofM.tbwasidentifiedinthisstudy. Therewere140macrophageproteinswithincreasedexpressionand111proteinswithdecreasedexpressionafterbeadsstimulation(groupBeads/THP-1),asshowninSupplementaryTableS3-
2.Someofthemwereassociatedwithautolysosomesandimmunity,suchaslysosomemembraneprotein2(LIMPII),CD63antigen,andCathepsinG[42–45],indicatingthattheexpressionlevelsofsomemacrophageimmunityproteinschangedafterbeadsinfection.Therefore,thebeadsinfectiongroupservedasanegativecontroltoidentifymacrophageproteinsrelatedtolivebacterialinfection.ForH37Ra/Beadsgrouptheexpressionlevelsof130macrophageproteinssignificantlyincreasedand150macrophageproteinsdecreased,whichassociatedwithavirulentstraininfection(SupplementalTableS3-3).However,fortheH37Rv/Beadsgrouptheexpressionlevelsof115macrophageproteinsincreasedand79macrophageproteinsdecreased(SupplementalTableS3-4),whichwererelatedtovirulentM.tbinfection.AfterinfectionwithM.tbstrainH37RvorH37Ra,theexpressionlevelsoftypeIinterferon-inducibleproteinsinmacrophages,includingubiquitin-likeproteinISG15(ISG15),interferon-stimulatedgene20kDaprotein(ISG20),interferoninducedproteinwithtetratricopeptiderepeats1(IFIT1),IFIT2,IFIT3,interferon-inducedhelicaseCdomain-containingprotein1(IFIH1),bonemarrowstromalantigen2(BST2),interferonregulatoryfactor9(IRF9),andradicalS-adenosylmethioninedomaincontainingprotein2(RSAD2),werealmostequallyincreased.Theincreasehadbeendemonstratedbothattranscriptandproteinlevelsbypreviousresearchers[46–48]. Theexpressionlevelof142THP-1macrophagesproteinsincreasedand93proteinsdecreasedafterH37RvparedwithH37Rainfection(groupH37Rv/H37Ra)(SupplementalTableS3-5).TheseDEPsofmacrophagesweredirectlyassociatedwithM.tbvirulentinfectionandreflectedspecificresponsesofmacrophagestoH37RvinsteadofH37Ra. DEPsofmacrophageswereclassifiedbyGOanalysis First,weclassifiedproteinsinmacrophagesbyPANTHERProteinClassontologytoevaluatetheirbiologicalrelevance[35](Fig.1D).ForgroupH37Rv/H37Ra,theDEPsofmacrophageswereclassifiedto21terms.fivetermswereoxidoreductase(11),signalingmolecule(12),hydrolase(18),enzymemodulator(19),andnucleicacidbinding(44).Toobtainmorefunctioninformationandpathwaysformacrophageproteinsdifferentiallyexpressedbetweentwostrains’infections,theseproteinswerefunctionallyanalyzedusingDAVID.GOtermsweresortedintothe‘CellularComponent(CC)’,‘BiologicalProcesses(BP)’,and‘MolecularFunction(MF)’GOcategoriesandKyotoEncyclopediaofGenesandGenomes(KEGG)pathways(P<0.01)(SupplementaryFig.S4). FortheBeads/THP-1group,pathwaytermwaslysosomeGO-MFtermwascadherin-bindinginvolvedincell–celladhesion(SupplementaryFig.S4A),whichsuggestedthatbeadscouldinduceproteinexpressionchangesinthelysosomeandpathogenrecognitionofmacrophages[49,50].Becauseoftheactivationofmacrophagesbybeads,beadsgroupwasselectedasanegativecontroltoexcludeproteinexpressionchangesinducedbyphagocytosisofphysicalstimulation.FortheH37Ra/BeadsgrouporH37Rv/Beadsgroup,mostGOtermswereassociatedwithinfectionimmunityandpathogenicbacteriakilling(SupplementaryFig.S4B,C).However,when comparedwithH37Ra/Beadsgroup,thetermpositivelyregulatedTcellcytokineproductionandlysosomesdisappearedinH37Rv/Beadsgroup,possiblyowingtoinhibitionofH37Rv. Takentogether,thesefunctionalanalysesrevealedthatbothofH37RvandH37Rainfectioncouldleadtotheexpressionchangesofmacrophageproteinsassociatedwithapoptosisprocess,bloodcoagulation,nucleosomeassembly,focaladhesion,andexosome.Obviously,H37Rvinfectionresultedinexpressionchangesofmacrophageproteinsassociatedwithoxidativephosphorylationinmitochondrialinnermembrane,nucleosomeassembly,vesicle,apoptosisprocess,andbloodcoagulation,whichwasdifferentfromH37Rainfection(SupplementaryFig.S4D).TheseresultsprovidemoreevidencethatmacrophagesinfectedwithvirulentH37RvstrainoravirulentH37Rastraincouldtriggerdifferentimmuneresponses. workanalysisofDEPs TofurtherunderstandtheinteractionsbetweentheDEPsofmacrophage,proteinworkswereconstructedusingSTRING10.0andvisualizedwithCytoscapev.3.2.1.Nodeswerecoloredordingtothelog2ratioofproteinsfromeachgroupandworkswerecreatedmanuallyordingtotheGOtermsanalyzedbyBiNGOv3.0.3(Fig.2A,SupplementaryFig.S5).Undoubtedly,therelationshipsbetweentheseworksindicatedstrongassociationsamongthem,whichmightberelatedtoH37Rvinfection.FortheH37Rv/H37Ragroup,therewere23macrophageproteinsrelatedtobloodcoagulation(Fig.2B),26proteinscorrelatedwithapoptosisprocess(Fig.2C),19proteinsassociatedwiththeoxidativephosphorylationinmitochondrialinnermembrane(Fig.2D),andsomeotherproteinsassociatedwithvesicleornucleosomeassembly,indicatingthattheseDEPsofmacrophagesandtheirfunctionalclustersmaybeinvolvedinthevirulentM.tbinfection.Notably,amongtheapoptosisprocessandinflammatoryresponsecluster,themostobviousproteinswerealpha-2-hs-glycoprotein(AHSG)andinterleukin-1β(IL-1β),implyingthatthesetwoproteinsmayplayimportantrolesinH37RvimmunityofparedwithH37Ra.TheseresultsconfirmedthatmacrophagesinfectedwithH37RvorH37Rainduceddifferentwidespreadbiologicalprocessesandpowerfulcross-talkexistedamongthem. Westernblotanalysesvalidatethealterationsofproteinexpressionlevel ThemacrophageproteinsIL-1βandAHSGwhichareassociatedwiththeapoptosisprocessandinflammatoryresponseinmacrophageswereselectedforwesternblotanalysistoconfirmthedifferentialexpressionofproteinsidentifiedinaTMT-labeledLC−MS/MSsystem(Fig.3).EqualamountsoftotalproteinsfromM.tbinfectedTHP-1cellswereappliedandblottedwithantibodiestoIL1βandAHSG.Thedecreasedexpressionlevelofpro-IL-1β/IL-1βandtheincreasedexpressionlevelofAHSGinmacrophagesafterH37Rvinfectionwereconfirmedbywesternblotassay,whichisincontrasttotheirexpressionlevelsafterH37Rainfection.Theproteomicresultswerevalidatedusingthesedata. Discussion ThedifferentbiologicalprocessesofmacrophagesaftervirulentM.tbstrainH37RvoravirulentH37Rastraininfectioncontributeto
6 HostresponsestriggeredbyMycobacteriumtuberculosis Figure2.workofDEPsbetweenTHP-1cellsinfectedwithH37RaandH37Rv(groupH37Rv/H37Ra)(A)DEPsofmacrophageswereanalyzedbySTRINGanddividedintosevenclustersordingtothecorrespondingbiologicalprocesscategories(P<0.05).GraphsweregeneratedusingCytoscapev.3.2.1andtheBiNGOv3.0.3plugin.Nodeswerecoloredordingtothelog2ratiosofproteinsfromeachgroup.Thecolorscaledemonstratestherelativeexpressionlevelsoftheproteins(log2ratio).Proteinsarrangedinacircleinthecenterofareindependentfromotherworks.(B)workofbloodcoagulation-relatedproteins.(C)Apoptosisprocessandinflammatoryresponseassociatedproteins.(D)Mitochondrialinnermembraneandoxidativephosphorylation-associatedproteins. theunderstandingplexM.tbpathogenesis.ResearchershaveevaluatedthedifferentresponsesofmacrophagesinfectedwithH37RvorH37Raattranscriptomiclevel[11,51].However,ordingtoourresults,only5proteinsoutofthe235DEPsfrommacrophagesareidenticaltoprevioustranscriptomicresults(SupplementalTableS3-6).Thislackofcorrelationbetweenproteomicandtranscriptomicresultscanbecontributedtothepost-transcriptionalregulatorymechanisms. AmongDEPsofmacrophagesbetweenH37RvandH37Rainfection,someofthemwererelatedtonucleosomeassemblyandbloodcoagulation(Fig.2B),includinghistonesandgranuleconstituentsassociatedwithproteins[collagen,typeI,alpha1(COL1A1),thrombospondin1(THBS1),quiescinQ6sulfhydryloxidase1(QSOX1),proteinSalpha(PROS1),andcoagulationfactorV(F5)].Histonesreleasedintotheextracellularspaceofmacrophagescan activateinflammasomepathwaythroughToll-likereceptors(TLRs)[52].Furthermore,histonesandgranulestendtoformmacrophageextracellulartrap(MET)withnuclearDNA,andtherebykillbacteriabyreleasingofMETinvitroofmacrophages[53–55].ItisknownthatmacrophagesreleaseMETtoconstrainandkillM.tbduringM.tbinfection,weassumethatH37RvinfectioncouldinducemoreMETthanH37Rainfectionfortheup-regulationofassociatedproteins.Thisneedstobeprovedbyfurtherexperiments.Furthermore,thereexistsacross-talkbetweenbloodcoagulationandmacrophageimmunity:fibrinregulatesthechemokine/cytokineproductionandmacrophageadhesioninvivo[56],whichcouldhelptoexplaintheformationofgranulomasinvivowiththeparticipationofmacrophagesandothercellsstimulatedbyH37Rv[57]. AnotherinterestingproteinoftheDEPsisHLAclassIpatibilityantigen,A-2alphachain(HLA-A),amajor HostresponsestriggeredbyMycobacteriumtuberculosis
7 Figure3.ConfirmationofDEPs(IL-1βandAHSG)inTHP-1cellsinfectedwithH37RaorH37RvbywesternblotanalysisTheresultswereconsistentwithMSdata.Thepicturewasfromonerepresentativeexperimentandsimilarresultswereobtainedintwootherexperiments.TheWBratioswerecalculatedordingtothesethreeexperiments.β-Actinwasusedasaninternalcontroltonormalizethequantitativedata. plex(MHC)antigenspecifictohumans.TheHLA-AexpressionlevelofmacrophagesdecreasedafterH37RvparedwithH37Rainfection,indicatingthatthevirulentstraininfectioncoulddisturbthepresentationofantigenicpeptidestotheTcellreceptors(TCRs)onTcells.Thus,virulentM.tbinfectioninhibitsTcell-mediatedcellularimmuneresponse,whichconstitutesanotherprotectionmechanismofthevirulentstrain[58,59]. Notably,theproteinsinmitochondrialinnermembraneassociatedwithoxidativephosphorylationincreasedafterH37RvparedwithH37Rainfection(Fig.2D).Thenicotinamideadeninedinucleotidedehydrogenase(NADH)(ubiquinone)1betaplexsubunit1(NDUFB1),NDUFB7,NDUFB11,andNDUFA13arethemitochondrialenergygeneratingplexIsubunitsthattransferelectronsfromNADHtotherespiratorychain;cytochromeb–plexsubunit(UQCRFS1)andUQCR10arethesubunitsofubiquinol–cytochromecplexplexIII)whichgeneratesanelectrochemicalpotentialcoupledtoadenosinetriphosphate(ATP)synthesis[60–62].MitochondriaarethemajorsourceandthefirstsuffererofROS.TheincreaseinmacrophageproteinsofComplexIIIandI,theprincipalsourceofsuperoxide,indicatesmoresuperoxideproduction.ItwasreportedthatoverexpressionplexIsubunitNDUFA6couldreduceapoptosisinHIV-infectedcells[63].ROSproductioncanalsocontributetotheactivationofNF-κBandcellsurvival.Therefore,plexIandIIIproteinsmayplayanimportantroleinoxidationinducedapoptosis.Inaddition,superoxidecouldbeconvertedtoH2O2bymanganese-dependentsuperoxidedismutase(SOD2)[64].SOD2whichcandestroysuperoxideanionradicalsandprotectagainstoxidation-inducedapoptosis,wasdown-regulatedafterH37Rvinfectioninthisstudy[65].Therefore,H37RvinfectioncaninducemoresuperoxideproductioninparedwithH37Raandthedetailedfunctionneedsfurtherinvestigation. MitochondriaareessentialinproducingROSaswellasgeneratingATP[66].TheATPaseinhibitor(ATPIF1),ATPsynthaseF(0)plexsubunitC1(ATP5G1),ATP5E,ATP5IandUp-regulatedduringskeletalmusclegrowthprotein5(USMG5)ofmacrophages,expressionlevelsofwhichareincreasedafterH37Rvinfection,constituteanATPplex(ComplexV).ThelackofATPafterapoptosiscouldinducenecroticcelldeath.[67].SotheupregulatedmitochondriaATPsynthasesmaydependontheactivationofapoptosis.Mitochondrialribosomalprotein(MRPL41) interactswiththeanti-apoptosisregulatorBcl-2andpromotesapoptosis[68].AfterH37Rvinfection,theanti-apoptoticproteinisreducedduetotheup-regulationofMRPL4.Ontheotherhand,heatshockcognate71kDaprotein(HSPA8)thatpreventsapoptosisbyparticipatinginchaperone-mediatedhagy,isincreasedafterH37RvinfectionmorethanthatafterH37Rainfection[69–71].Asaresult,H37Rvincreasesitssurvivalrateinmacrophagesbypreventingmacrophageapoptosis.ThesemacrophagemitochondriaresponsesuraftertheinfectionofH37RvotherthanaftertheinfectionofH37Ra,whichprovidesevidenceanddetailsforelucidatingtherelationshipbetweenvirulentM.tbinfectionandtheoxidativephosphorylationinmitochondrialinnermembrane. Theexpressionlevelsofmacrophageproteinsassociatedwiththeapoptosisprocessandinflammatoryresponsewerealteredwhencellswereinfectedwiththesetwostrainsrespectively(Fig.2C).ThedifferentialexpressionlevelofAHSGandIL-1βbetweenH37RvandH37Rainfectionwereconfirmedbywesternblotanalysis.AHSG,alsocalledfetuin-A,functionsasapositiveornegativeacutephaseproteinininfectionandisregulatedbydifferentproinflammatorymediators[72].AHSGexpressionlevelincreasedsignificantlyafterM.tbinfection.H37RvinducedhigherAHSGexpressionthanH37Rainmacrophages,butthisstrategyremainsconfusingandrequiresfurtherinvestigation.Thewesternblotanalysisresultsshoweddecreasedexpressionlevelofpro-IL-1βinthemacrophagesinfectedwithparedwithH37Ra.Sincetheexpressionlevelofpro-IL-1βistightlycontrolledbythefirstsignalmediatedbyTLRligands,itispossiblethatH37Rvinhibitspro-IL-1βexpressioninmacrophagesbyblockingTLR-mediatedsignalingpathwaysafterinfection,whichalsodeservesfurtherinvestigation.Pro-IL-1βproteinsareactivatedbycaspase-1,whichisunderthecontrolofthesecondsignalmediatedbynucleotide-bindingoligomerizationdomain-likereceptors(NLRs)[73].ButbothH37Rv-andH37RainfectedmacrophagesexpressedincreasedproinflammatorycytokineIL-1β,whichisconsistentwiththepreviousreport[56].Notably,paredwithH37Rainfection,H37RvinfectiondecreasedtheexpressionsofnuclearfactorNF-κBp105/p50subunit(NFκB1),nuclearfactorNF-κBp100/p52subunit(NFκB2),TNFalphainducedprotein6(TSG6,TNFAIP6)andtumornecrosisfactor(TNF)receptor-associatedfactor1(TRAF1).NFκB1andNFκB2arepartofaplexintheNF-κBpathway[74],thedown-regulationofwhichmightinducemorecellulardeath.TRAF1isanegativeregulatorofTNFsignalingandpreventsNF-κBactivationbyTNFandIL-1[75],whichindicatesthedecreaseofTRAF1mayup-regulateNF-κ
B.Takentogether,theremustbesomeothermechanismsthatregulateNF-κBpathway.TheotherproteinTSG6hasanti-inflammatoryeffectsandcanbeinducedbyTNFandIL-1[76].plexmayplayimportantrolesinthefatedeterminationofmacrophagesandM.tb.H37Rvmayescapetheimmunesystembyregulatingtheexpressionlevelofimmunityfactors,themechanismsofwhichremainunclearandareworthfurtherinvestigation. Takentogether,theseanalysessuggestthatH37Rvinfectionresultsintheexpressionsofmacrophageproteinsassociatedwithoxidativephosphorylation,apoptosisprocess,bloodcoagulation,andnucleosomeassembly,incontrasttoH37Rainfection.Furthermore,thefateofmacrophagesafterH37Rvinfectionmaydependontheinterplayoftheseimmunityprocesses. Inaddition,someDEPsofmacrophagesbetweenH37RvandH37Rainfectionarealwayslocatedinserum,suchasAHSG,RBP4(retinol-bindingprotein4),andIL-1β,whichcouldbevirulentinfectionbiomarkercandidates[30,77,78].RBP4bindswithand
8 HostresponsestriggeredbyMycobacteriumtuberculosis NFκB2,pro-IL-1β,TNFAIP6,andTRAF1,whichparticipateintheimmunityofM.tbandregulatetheapoptosisofmacrophages,areallparedwiththeH37Ra-infectedcells.ThefatesofmacrophagesandM.tbaredeterminedbyalltheseDEPs-associatedimmunitymechanismsandtheircross-talk.TheseresultsprovidenewcluestothehostresponseaftervirulentM.tbinfection,whichareimportantforfurtherTBstudiesandtherapies. Acknowledgments WewouldliketothankProteinWorldBiotechnologyCo.,Ltd(Beijing,China)forassistancewithquantitativeproteomics. SupplementaryData SupplementarydataareavailableatActaBiochimicaetBiophysicaSinicaonline. Figure4.ProposedmodelsdepictingthespecificimmuneresponsesofmacrophagesinducedbyvirulentM.tbH37Rv-infectedmacrophagesformedmoreMETsthustrappingM.tbtopreventdissemination.H37RvdecreasestheexpressionlevelofHLA-Ainmacrophages,whichmaydisturbthepresentationofantigenicpeptidesfrommacrophagestotheTCRsofTcellsandtherebyavoidTcell-mediatedcellularimmunereaction.H37RvinfectioninducesROSoverproductioninthemitochondrialwhileH37Radoesnot.InH37Rv-infectedmacrophages,theexpressionlevelsofNFκB1,NFκB2,proIL-1β,TNFAIP6,andTRAF1areallparedwiththeH37Rainfectedcells.‘↑↓’RepresentsthedecreasedorincreasedexpressionlevelofmacrophageproteinsafterH37RvparedwithH37Ra.Thegreendottedlineindicatesdown-regulation,whilethereddottedlineindicatesupregulation. transportsbloodretinolintothecellsandleadstoincreasedintracellularretinoicacidanddecreasedserumfibronectin,whichisrelatedtogranulomaformation[79].TheexpressionlevelsofbothAHSGandRBP4frompatientplasmawithactiveTBaresignificantlylowerthanthoseofpatientswithoutactivedisease,whichhasbeenconfirmedbyothers[80–82].However,theexpressionlevelofAHSGandRBP4weresignificantlyincreasedinthemacrophagelasmafterinfectionwithH37Rv,whichagreeswithpreviouswork[29].TheoppositeexpressiontendencyofthesetwoproteinsbetweenthemacrophagesandserumafterM.tbinfectionisworthfurtherinvestigation. Inconclusion,thisworkrevealeddifferentexpressionprofilesbetweenmacrophagesinfectedwithH37RaandH37RvusingTMT-basedquantitativeproteomics.TheseDEPsaremainlyinvolvedinapoptosisprocess,bloodcoagulation,andparticularlyoxidativephosphorylation.TheenormousvariationinproteinprofilesbetweenmacrophagesinfectedwithH37RaandH37Rvsuggeststheexistenceofdifferentimmunitymechanisms(Fig.4).InbothH37Rv-andH37Ra-infectedmacrophages,theexpressionlevelofhistonesandgranuleconstituents,whichformtheMETs,areallupgraded,buttheformerexhibitsmorenotableeffect,whichisinfavorofformationofMETsandthustrappingM.tbtopreventdissemination.H37RvdecreasestheexpressionlevelofHLA-Ainmacrophages,whichmaydisturbthepresentationofantigenicpeptidesfrommacrophagestotheTCRsofTcellsandtherebyavoidTcell-mediatedcellularimmuneresponse.H37RvinfectioninducesROSoverproductioninthemitochondrialwhileH37Radoesnot.InH37Rv-infectedmacrophages,theexpressionlevelsofNFκB1, Funding ThisworkwassupportedbythegrantfromtheChineseAcademyofSciences(No.KJZD-EW-TZ-L04). References
1.HardyA.Captainofdeath:thestoryoftuberculosis.AmJHumBiol1999,43:149.
2.DucatiRG,RuffitoA,BassoLA,SantosDS.Theresumptionofconsumption—areviewontuberculosis.MemInstOswaldoCruz2006,101:697–714.
3.KumarD,NathL,KamalMA,VarshneyA,JainA,SinghS,RaoKV.Genome-wideanalysisofthehostworkthatregulatessurvivalofMycobacteriumtuberculosis.Cell2010,140:731–743. 4.vanderWelN,HavaD,HoubenD,FluitsmaD,vanZonM,PiersonJ,BrennerM,etal.M.tuberculosisandM.lepraetranslocatefromthephagolysosometothecytosolinmyeloidcells.Cell2007,129:1287–1298.
5.SlyLM,Hingley-WilsonSM,ReinerNE,McMasterWR.SurvivalofMycobacteriumtuberculosisinhostmacrophagesinvolvesresistancetoapoptosisdependentuponinductionofantiapoptoticBcl-2familymemberMcl-
1.JImmunol2003,170:430–437.
6.BeharSM,DivangahiM,RemoldHG.EvasionofinnateimmunitybyMycobacteriumtuberculosis:isdeathanexitstrategy?
NatRevMicrobiol2010,8:668–674.
7.XuG,WangJ,GaoGF,LiuCH.InsightsintobattlesbetweenMycobacteriumtuberculosisandmacrophages.ProteinCell2014,5:728–736.
8.SaundersBM,CooperAM.Restrainingmycobacteria:roleofgranulomasinmycobacterialinfections.ImmunolCellBiol2000,78:334–341.
9.BendinelliM,FriedmanH.Mycobacteriumtuberculosis:interactionswiththeimmunesystemcontinued.Immunology1989,67:431. 10.ZhengH,LuL,WangB,PuS,ZhangX,ZhuG,ShiW,etal.icbasisofvirulenceattenuationrevealedparativegenomicanalysisofMycobacteriumtuberculosisstrainH37RaversusH37Rv.PLoSOne2008,3:e2375. 11.SilverRF,WalrathJ,LeeH,JacobsonBA,HortonH,BowmanMR,NockaK,etal.HumanalveolarmacrophagegeneresponsestoMycobacteriumtuberculosisstrainsH37RaandH37Rv.AmJRespirCellMolBiol2009,40:491–504. 12.PierceCH,DubosRJ,SchaeferWB.Multiplicationandsurvivaloftuberclebacilliinansofmice.JExpMed1953,97:189–206. 13.CollinsFM,SmithMM.parativestudyofthevirulenceofMycobacteriumtuberculosismeasuredinmiceandguineapigs.AmRevRespirDis1969,100:631–639. HostresponsestriggeredbyMycobacteriumtuberculosis
9 14.McmurrayDN,CarlomagnoMA,CumberlandPA.RespiratoryinfectionwithattenuatedMycobacteriumtuberculosisH37Rainmalnourishedguineapigs.InfectImmun1983,39:793–799. 15.KeaneJ,RemoldHG,KornfeldH.VirulentMycobacteriumtuberculosisstrainsevadeapoptosisofinfectedalveolarmacrophages.JImmunol2000,164:2016–2020. 16.DanelishviliL,McgarveyJ,LiYJ,BermudezLE.Mycobacteriumtuberculosisinfectioncausesdifferentlevelsofapoptosisandnecrosisinhumanmacrophagesandalveolarepithelialcells.CellMicrobiol2003,5:649–660. 17.ChenM,GanH,RemoldHG.Amechanismofvirulence:virulentMycobacteriumtuberculosisStrainH37Rv,butnotattenuatedH37Ra,causessignificantmitochondrialinnermembranedisruptioninmacrophagesleadingtonecrosis.JImmunol2006,176:3707–3716. 18.DavisMJ,RamakrishnanL.Theroleofthegranulomainexpansionanddisseminationofearlytuberculousinfection.Cell2009,136:37–49. 19.DanielJ,MaamarH,DebC,SirakovaTD,KolattukudyPE.Mycobacteriumtuberculosisuseshosttriacylglyceroltoumulatelipiddropletsandacquiresadormancy-likephenotypeinlipid-loadedmacrophages.PLoSPathog2011,7:e1002093. 20.TiwariB,RamakrishnanUM,RaghunandTR.TheMycobacteriumtuberculosisproteinpairPE9(Rv1088)-PE10(Rv1089)formsheterodimersandinducesmacrophageapoptosisthroughToll-likereceptor4.CellMicrobiol2015,17:1653–1669. 21.MeenaLS,Rajni.SurvivalmechanismsofpathogenicMycobacteriumtuberculosisH37Rv.FebsJ2010,277:2416. 22.WeissG,SchaibleUE.Macrophagedefensemechanismsagainstintracellularbacteria.ImmunolRev2015,264:182–203. 23.ZhangM,GongJ,LinY,BarnesPF.GrowthofvirulentandavirulentMycobacteriumtuberculosisstrainsinhumanmacrophages.InfectImmun1998,66:794. 24.YangY,HuM,YuK,ZengX,LiuX.Massspectrometry-basedproteomicapproachestostudypathogenicbacteria-hostinteractions.ProteinCell2015,6:265–274. 25.RaoPK,SinghCR,JagannathC,LiQ.Asystemsbiologyapproachtostudythephagosomalproteomemodulatedbymycobacterialinfections.IntJClinExpMed2009,2:233–247. 26.ShuiW,PetzoldCJ,ReddingA,LiuJ,PitcherA,SheuL,HsiehTY,etal.Organellemembraneproteomicsrevealsdifferentialinfluenceofmycobacteriallipoglycansonmacrophagephagosomematurationandhagosomeumulation.JProteomeRes2010,10:339–348. 27.DiazG,WolfeLM,Kruh-GarciaNA,DobosKM.Changesinthemembrane-associatedproteinsofexosomesreleasedfromhumanmacrophagesafterMycobacteriumtuberculosisinfection.SciRep2016,6:37975. 28.SaquibNM,JamwalS,MidhaMK,VermaHN,ManivelV.QuantitativeproteomicsandlipidomicsanalysisofendoplasmicreticulumofmacrophageinfectedwithMycobacteriumtuberculosis.IntJProteomics2015,2015:270438. 29.HareNJ,LeeLY,LokeI,BrittonWJ,SaundersBM,Thaysen-AndersenM.MycobacteriumtuberculosisinfectionmanipulatestheglycosylationmachineryandtheN-glycoproteomeofhumanmacrophagesandtheirmicroparticles.JProteomeRes2017,16:247–263. 30.KaewseekhaoB,NaranbhaiV,RoytrakulS,NamwatW,PaemaneeA,LulitanondV,ChaiprasertA,etal.ComparativeproteomicsofactivatedTHP-1cellsinfectedwithMycobacteriumtuberculosisidentifiesputativeclearancebiomarkersfortuberculosistreatment.PLoSOne2015,10:e0134168. 31.LiP,WangR,DongW,HuL,ZongB,ZhangY,WangX,etal.ComparativeproteomicsanalysisofhumanmacrophagesinfectedwithvirulentMycobacteriumbovis.FrontCellInfectMicrobiol2017,7:65. 32.DayonL,HainardA,LickerV,TurckN,KuhnK,HochstrasserDF,BurkhardPR,etal.RelativequantificationofproteinsinhumancerebrospinalfluidsbyMS/MSusing6-plexisobarictags.AnalChem2008,80:2921–2931. 33.AntonioVJ,AttilaC,NoemiDT,DianesJA,JohannesG,IliasL,GerhardM,etal.updateofthePRIDEdatabaseanditsrelatedtools.NucleicAcidsRes2016,44:D447–D456. 34.CoombsKM,BerardA,XuW,KrokhinO,MengX,CortensJP,KobasaD,etal.Quantitativeproteomicanalysesofinfluenzavirus-infectedculturedhumanlungcells.JVirol2010,84:10888–10906. 35.MiH.PANTHERversion7:improvedictrees,orthologsandcollaborationwiththeGeneOntologyConsortium.NucleicAcidsRes2010,38:204–210. 36.HuangdaW,ShermanBT,LempickiRA.SystematicandintegrativeanalysisoflargegenelistsusingDAVIDbioinformaticsresources.NatProtoc2009,4:44–57. 37.ShannonP,MarkielA,OzierO,BaligaNS,WangJT,RamageD,AminN,etal.Cytoscape:asoftwareenvironmentforintegratedmodelsofbiomolecularworks.GenomeRes2003,13:2498–2504. 38.MaereS,HeymansK,KuiperM.BiNGO:aCytoscapeplugintoassessoverrepresentationofgeneontologycategoriesinworks.Bioinformatics2005,21:3448–3449. 39.SchlesingerL.MacrophagephagocytosisofvirulentbutnotattenuatedstrainsofMycobacteriumtuberculosisismediatedbymannosereceptorsinadditionplementreceptors.JImmunol1993,150:2920–2930. 40.DhimanR,RajeM,MajumdarS.DifferentialexpressionofNF-κBinmycobacteriainfectedTHP-1affectsapoptosis.BiochimBiophysActa2007,1770:649–658. 41.DengW,WangY,LiuZ,ChengH,XueY.HemI:atoolkitforillustratingheatmaps.PLoSOne2014,9:e111988. 42.LadelCH,SzalayG,RiedelD,KaufmannS.Interleukin-12secretionbyMycobacteriumtuberculosis-infectedmacrophages.InfectImmun1997,65:1936–1938. 43.ReczekD,SchwakeM,SchröderJ,HughesH,BlanzJ,JinX,BrondykW,etal.LIMP-2isareceptorforlysosomalmannose-6-phosphateindependenttargetingofβ-glucocerebrosidase.Cell2007,131:770–783. 44.DoyleEL,RidgerV,FerraroF,TurmaineM,SaftigP,CutlerDF.CD63isanessentialcofactortoleukocyterecruitmentbyendothelialP-selectin.Blood2011,118:4265–4273. 45.DanelishviliL,EvermanJ,McNamaraM,BermudezL.Inhibitionoftheplasma-membrane-associatedserineproteasecathepsinGbyMycobacteriumtuberculosisRv3364csuppressescaspase-1andpyroptosisinmacrophages.FrontMicrobiol2012,2:281. 46.BerryMP,GrahamCM,abFW,XuZ,BlochSA,OniT,WilkinsonKA,etal.Aninterferon-inducibleneutrophil-drivenbloodtranscriptionalsignatureinhumantuberculosis.Nature2010,466:973–977. 47.Otte
2,ShaWei1,
2,YuanFang3,MinLi2,XiaLi2,ZheLi4,JibinZhang1,GuofengZhu5,ChuanyouLi6,LijunBi2,GuiminZhang3,DianbingWang2,*,andXian-EnZhang2,* 1StateKeyLaboratoryofAgromicrobiology,CollegeofLifeScienceandTechnology,HuazhongAgriculturalUniversity,Wuhan430070,China,2NationalLaboratoryofBiomacromolecules,CASCenterforExcellenceinBiomacromolecules,andKeyLaboratoryofRNABiology,InstituteofBiophysics,ChineseAcademyofSciences,Beijing100101,China,3CollegeofLifeScience,HubeiUniversity,Wuhan430062,China,4ChinaGoldenMarker(Beijing)BiotechCo.Ltd.,Beijing102206,China,5ShanghaiMunicipalCenterforDiseaseControl&Prevention,Shanghai200336,China,and6BeijingChestHospital,CapitalMedicalUniversity,Beijing101149,China *Correspondenceaddress.Tel:+86-10-64888148;E-mail:zhangxe@(
X.Z.)/Tel:+86-10-64888432;E-mail:wangdb@(
D.W.) Received17April2017;EditorialDecision27April2017 Abstract MacrophagesareprimaryhostofMycobacteriumtuberculosis(M.tb)andthecentraleffectorofinvivoinnateimmuneresponsesagainstbacteria.Thoughtheinteractionbetweenmacrophagesandmycobacteriahasbeenwidelyinvestigated,themolecularmechanismsofM.tbpathogenesisinmacrophagesarestillnotclear.Inthiswork,weinvestigatedthealteredproteinexpressionprofilesofmacrophagesaftervirulentH37RvstrainandavirulentH37Rastraininfectionbytandemmasstag-basedquantitativeproteomics.Among6762identifiedproteinsofmacrophages,theexpressionlevelsof235proteinsweresignificantlyaltered,whichissupposedtoberelatedtotheinfectionofdifferentstrains.Bybioinformaticsanalysisatsystemslevel,wefoundthattheseproteinsaremainlyinvolvedinthebiologicalprocessofapoptosis,bloodcoagulation,oxidativephosphorylation,andothers.TheenormousvariationinproteinprofilesbetweenmacrophagesinfectedwithH37RaandH37RvsuggeststheexistenceoffourdifferentimmunitymechanismsthatdecidethefatesofmacrophagesandM.tb.ThesedatamayprovideabetterunderstandingofM.tbpathogenesiswithinthehost,whichcontributestothepreventionandclinicaltreatmentoftuberculosis. Keywords:Mycobacteriumtuberculosis,virulentH37Rvstrain,avirulentH37Rastrain,macrophages,TMT-basedquantitativeproteomics Introduction Tuberculosis(TB)isaninfectiousdiseasethatgreatlyimpactshumanandanimalhealthworldwide.One-thirdoftheworld’s populationispersistentlyinfectedwiththecausativeagentMycobacteriumtuberculosis(M.tb)and~10%ofthemsufferfromactiveTB[1,2].AreportfromWorldHealthOrganization(WHO) ©TheAuthor2017.PublishedbyOxfordUniversityPressonbehalfoftheInstituteofBiochemistryandCellBiology,ShanghaiInstitutesforBiologicalSciences,Chinese AcademyofSciences.Allrightsreserved.Forpermissions,pleasee-mail:journals.permissions@
1 2 HostresponsestriggeredbyMycobacteriumtuberculosis indicatesthat1.8millionpeoplediedofTBin2015.OneofthemajorcausesofthishighincidenceisthedifficultyofdiagnosisandtreatmentthatcausedbythepoorunderstandingofplexM.tbpathogenesis. MacrophagesaretheprimaryhostcellsofM.tbinvivo.Theyareoneofthemostimportantimmunecellsparticipatingintheeliminationofinfectingmycobacteria,whichcannotonlyinducetheinnateimmuneresponseagainstpathogenicanismsbutalsoplaycentralrolesinTBcontrol[3].BactericidalresponsesofmacrophagesareactivatedbyintracellularM.tb,includinghagy,fusionofphagosomewithlysosome,apoptosis,necrosis,andproductionofantimicrobialagentssuchascytokines,hydrolases,freeradicals,reactiveoxygenspecies(ROS),andnitrogenintermediates[4–7].Inaddition,macrophagescanpresentmycobacterialantigenstoprimedlymphocytesandstarttheinflammatoryresponsebysecretingchemokine,cytokines,andchemicalmediatorsofinflammationtorecruitotherimmunecellstothesiteofinfection[8,9]. H37RvandH37RaaretwocloselyrelatedM.tbstrainswithdifferentvirulence.H37RvcauseshigherbacterialloadsofM.tbinlungsandansthanH37Ra[10–14].Furthermore,H37Rvcanevadehostimmunityanddisplaygreaterinvivoreplicationbyinhibitingthekillingprocessessuchasthefusionofphagosomewithlysosome,phagosomeacidification,hostcellsignaltransport,apoptosisofinfectedmacrophagesandinductionofsignificantnecrosis,granuloma,andlipiddroplet[15–23]. Therefore,prehensiveresearchondifferentmacrophageresponsesagainstvirulentH37RvstrainandavirulentH37RastraininfectionwillhelptounderstandplexmechanismsofM.tbinfectionanditssurvivalinhostcells[24].Recently,manyproteomicresearcheshavereportedtheinteractionbetweenhostandM.tb,whichprovedsignificantchangesintheexpressionofproteinsinmacrophagephagosomes,anellemembranes,exosomes,andendoplasmicreticulumafterM.tbinfection[25–31].Herein,weappliedtandemmasstag(TMT)-basedquantitativeproteomicstoassessthealteredproteinexpressionprofilesofmacrophagecell(THP-1)infectedwiththevirulentH37RvstrainandavirulentH37Rastrain,respectively.Among6762identifiedmacrophageproteins,235proteinsweredemonstratedtohavearelationshipwithvirulentinfection.Byanalysisofthesedataatsystemslevel,thesedifferentiallyexpressedproteins(DEPs)ofmacrophageswerefoundtobemainlyinvolvedinapoptosis,bloodcoagulation,oxidativephosphorylation,andothercellularprocesses.TheenormousvariationinproteinprofilesbetweenmacrophagesinfectedwithH37RaandH37RvsuggestedtheexistenceoffourdifferentimmunitymechanismsthatdecidethefatesofmacrophagesandM.tb.OurdatamayprovidenovelinsightsintothedifferentmolecularmechanismsofhostreactiontovirulentM.tbandavirulentM.tbinfection,therebygivenewthoughtsonthepreventionandclinicaltreatmentofTB. MaterialsandMethods M.tbstrainsandcultureconditions M.tbH37Rv(ATCC:AmericanTypeTissueCollection27294)andH37Ra(ATCC25177)weregrowninMiddlebrook7H9brothsupplementedwith10%albumindextrosecatalase(ADC)enrichment(BectonDickinson,SanJose,USA),0.5%glyceroland0.02%Tween80at37°Cfor6days.M.tbcellswerequantifiedbyseriallydilutingthebacterialstockandplating100μlaliquotsonMiddlebrook7H10agarwith10%ADC.Then,theCFUsofM.tbweredeterminedafter3weeksofincubationat37°
C. THP-1cellscultureanddifferentiation MonocyteleukemiacellTHP-1cellswereculturedinRPMI-1640medium(HyClone,Logan,USA)supplementedwith10%fetalbovineserum(FBS;Gibco,Carlsbad,USA)(V/V)at37°Cinahumidified,5%CO2atmosphere.Cellsatconcentrationof5×105cells/mlweredifferentiatedtomacrophagephenotypewith25ng/mlPMA(phorbol-12-myristate-13-acetate)inRPMl1640containing10%FBSfor48h.AfterremovalofthePMA-containingmedium,thecellswereincubatedforanother48hinfreshRPMI-1640mediumsupplementedwith10%FBS. Infectionexperiments TheM.tbwithOD600of0.3(CFU=2–3×107)andequivalentbeadswerewashedtwicewithRPMI-1640mediumandsuspendedinRPMl-1640mediumcontaining10%FBSasaprimaryinoculumformacrophageinfection.ThedifferentiatedTHP-1cellswereinfectedwithlatexbeads,H37RaorH37Rvatanmultiplicityofinfection(MOI)of35andthenincubatedfor4hat37°Cina5%CO2atmosphere.Toremovetheextracellularbacteria,theinfectedcellswerewashedtwicewithRPMI-1640mediumaftertheculturesupernatantwasdiscarded.Then,thecellswerefurtherculturedforanother12horasindicated.Everystimulationexperimentwasdonetwiceforbiologicalrepetition.TheblankcontrolgroupnamedTHP-1wastreatedsimultaneouslyastheothergroups. Quantitativereal-timepolymerasechainreaction TotalmRNAwasextractedusingTRIzolreagent(Invitrogen,Carlsbad,USA)andcDNAwassynthesizedusingthecDNAsynthesiskit(TransBionovo,Beijing,China),ordingtothemanufacturer’sinstructions.bfl-1(anti-apoptoticgene)andtnf-α(apoptoticgene)expressionlevelsweredeterminedbyquantitativereal-timepolymerasechainreaction(qPCR)atdifferenttimepointsafterinfection.TheqPCRmeasurementswereperformedonaBio-RadCFX96usingTransStartTipGreenqPCRSuperMix(TransBionovo)asfollows:pre-denaturation94°Cfor30s,and94°Cfor5s,52°Cfor15s,72°Cfor10sfor40cycles.Thespecificprimersforthegeneswere:tnf-α(F:5′-AGGCGGTGCTTGTTCCTC-3′;R:5′-GTTCGAGAAGATGATCTGACTGCC-3′),bfl-1(F:5′-TGCCAGAACACTATTCAACC-3′;R:5′-TTGCCTTATCCATTCTCCTG-3′)andgapdh(F:5′-GAAGGTCGGAGTCAACGGAT-3′;R:5′-CCTGGAAGATGGTGATGGG-3′).Two-tailedttests,type2,n=2wereusedforstatisticalevaluationofthemRNAlevels(mean±SD),whilegapdhmRNAwasusedasaninternalcontrolfornormalization. Samplepreparation Boththesupernatantandadherentcellswerewashedtwicewithphosphate-bufferedsaline(PBS).TheproteinswereextractedwithRIPAcelllysisbuffer(Amersco,Solon,USA)supplementedwith1×proteaseinhibitorCocktail(Sigma,StLouis,USA).Finally,theproteinconcentrationwasdeterminedusingthePierceBicinchoninicAcid(BCA;ThermoFisherScientific,Waltham,USA)ProteinAssayKit. Themacrophagesampleshavetwosetsofbiologicalrepetition,namedsample1andsample2.Bothofsamples(0.45–0.65mgeach)wereresuspendedin1×SDSloadingbufferattheconcentrationof5mg/ml.Thesampleswerereducedbyadditionof20mMdithiothreitol(DTT)andincubatedat90°Cfor10min.Aftercoolingtoroomtemperature,100μgofeachsamplewasresolvedbySDSPAGE(10%Bis-trisgel;Invitrogen)for1h.Then,thesamplesweresubjecttoanin-geltrypticdigestionafterreductionwith10mM HostresponsestriggeredbyMycobacteriumtuberculosis
3 DTTat60°Cfor30minandalkylationwith40mMiodoacetamideinthedarkfor1htoblockfreecysteine.Trypsinwasaddedat1:50(w:w;trypsin:sample)andthesampleswereincubatedat37°Covernight.Thedigestedpeptideswereextractedfromgelwith60%acetonitrile,5%formicacidandthendriedinaSpeed-Vac,afterwhichtheywerewashedwith50%acetonitriletoneutralpH.TheproteinsisolatedfromTHP-1macrophages,THP-1macrophagesstimulatedbyBeadsandTHP-1macrophagestreatedwithH37RaorH37RvwerelabeledwithTMT6-126,127,128,and129labelreagentordingtothemanufacturer’sinstructions(ThermoFisherScientific).Basically,thesamplesandthelabelingreagentswereincubatedfor60minatroomtemperature,then8μlofhydroxylamine(5%)wasaddedandincubatedforanother15mintoquenchthereactions.ThelabeledsampleswerenamedTMT-126,TMT-127,TMT-128,andTMT-129,respectively.Thefourlabeledsamplesbined,driedintheSpeed-Vacandsentforanalysis[32]. One-dimensionalseparationofTMT-labeledpeptides High-pHreversedphasehigh-performanceliquidchromatography(HPLC)wasusedforpeptidefractionationusingGilson300seriesequipment(Gilson,Worthington,USA).TheTMT-labeledbinedsamplesweresolubilizedin200μlof20mMammoniumformate(pH10)andtheninjectedintoanXbridgecolumn(C183.5μm2.1×150mm2;Waters,Milford,USA)andelutedwithalineargradient(2%–45%)ofbufferBin44min(bufferA:20mMammoniumformate,pH10;bufferB:20mMammoniumformatein90%acetonitrile,pH10).ThefractionswiththehighestUVabsorbanceat214nm(mostofpeptides)fromthehigh-pHHPLCwereselectedtobeanalyzedbyliquidchromatography/tandemmassspectrometry(LC–MS/MS).Thosefractions(1min)werecollectedanddriedinaSpeed-Vac. NanoLC–MS/MS PeptidesfromeachfractionwereanalyzedbynanoLC–MS/MSusinganRSLCsysteminterfacedwithaQExactive™hybridquadrupoleOrbitrapmassspectrometer(ThermoFisherScientific).Sampleswereloadedontoaself-packed100μm×2cmtrappackedwithMagicC18AQwith5μm200Å(MichromBioresources,Auburn,USA)andthenwashedwithBufferA(0.2%formicacid)for5minwithaflowrateof10μl/min.Thetrapwasbroughtin-linewithahomemadeanalyticalcolumn(MagicC18AQ,3μm200Å,75μm×50cm)andpeptideswerefractionatedat300nl/minwithamulti-steppedgradient[4%–15%BufferB(0.16%formicacid80%acetonitrile)in35minand15%–25%Bin65minand25%–50%Bin55min].Massspectrometrydatawereacquiredusingadata-dependentacquisitionprocedurewithacyclicseriesofafullscanacquiredinOrbitrapwitharesolutionof120,000.The20mostintenseionswithrepeatcountsofoneanddynamicexclusiondurationsof30swerescannedusingMS/MS(30%ofcollisionenergyinHCDcell,resolution30,000).Thesample1wasruntwiceandnamedAandB,andsample2wasrunonceandnamedC. Dataanalysis LC–MS/MSdataof14fractionsfromeachexperimentweresearchedinMUDPITstyleagainsttheEnsemblhumandatabaseusinganin-houseversionofX!
tandem[SLEDGEHAMMER(2013.09.01),]withcarbamidomethylationoncysteineandtheTMT6-plexlabelonlysine,withtheN-terminusofpeptidesasfixedmodificationsandoxidationofmethionineasavariablemodification.A±10ppmand±20ppmwereusedastoleranceforprecursorandproductions,respectively.Theintensitiesof TMT6-plexreporterionsineachspectrumwereextractedusinganin-houseperlscriptandcorrectedforecross-overusingvaluessuppliedbythemanufacturer.Thetreatment/blankcontrolratioofeachspectrumwascalculatedusingreporterionintensityandwasnormalizedtothemedianratioofallidentifiedspectrathatfitcertaincriteria:peptidebelongstoahomosapiensdatabase,peptideE-value<0.01(theexpectationthatanyparticularproteinassignment),totalreporterionintensity>60,000.Thefalsepositiverate(FPR)atthepeptidelevelwas<1.0%.Spectrawithratiosof‘Divide0’werereplacedwiththearbitrarynumber‘10’. Pairwiseratiosoffourgroups(groupBeads/THP-1:TMT-127/TMT-126,groupH37Ra/Beads:TMT-128/TMT-127,groupH37Rv/Beads:TMT-129/TMT-127,andgroupH37Rv/H37Ra:TMT-129/TMT-128)ofeachindividualproteinwerecalculatedusingthemedianratioofallpeptidesbelongingtotheproteinthathasatleasttwodatapointsthatfitthecriteriaofpeptideE-value<0.03andsumofreporterionsofbothchannel>20,000.ThemassspectrometryproteomicsdatahavebeendepositedtotheProteomeXchangeConsortium[33]viathePRIDEpartnerrepositorywiththedatasetidentifierPXD006331. ThemethodusedtoanalyzetheDEPsisdescribedinSupplementaryFig.S1.TheZ-test(P<0.05)wasusedtoscreentheDEPsfromeachgroupbyconvertinglog2ratiosofproteinsintoZscores[34].PANTHER(ProteinAnalysisThroughEvolutionaryRelationships;2February2017,datelastessed)wasusedtoclassifyproteinsbytheirfunctions[35].ForGenoOntology(GO)andpathwayenrichment,theuniqueproteinsidentifiedinthisstudywereanalyzedusingDatabaseforAnnotation,Visualization,andIntegratedDiscovery(DAVID)[36].Theprotein–proteininteraction(PPI)workswereanalyzedusingSTRINGsoftware,afterwhichtheparagraphswereeditedwithCytoscapev.3.2.1andBiNGOv.3.0.3[37,38]. Westernblotanalysis Approximately40μgofproteinsfromeachsamplewereresolvedby8%–15%SDS-PAGEat100Vfor2–3h.Theproteinbandswereelectro-blottedontopolyvinylidenefluoride(PVDF)microporousmembranes(Millipore,Billerica,USA),whichwereblockedwith5%skimmedmilkfor2hatroomtemperature.Forimmunoblotanalysis,themembraneswereincubatedwithantibodiesasfollows:anti-AHSG(1:1000;Boster,Wuhan,China),anti-IL-1β(1:100;Boster),andantiβ-Actin(1:2000;SangonBiotech,Shanghai,China)at4°Covernight.Afterbeingwashedfourtimeswith0.1%PBS-Tweenfor5min,themembraneswereincubatedwithhorseradishperoxidase(HRP)conjugatedgoatanti-rabbitIgGH&L(Abcam,Cambridge,UK)for60minatroomtemperature.AfterincubationwithchemiluminescentHRPsubstrate(Millipore),thechemiluminescencedetectionwasperformeddirectlyusinganAmershamImager600(GEHealthcare,Bethesda,USA).Forquantificationanalysis,ITQL(GEHealthcare)softwarewasusedtocalculatethefoldchanges,whileactinwasusedasaninternalcontrolfornormalization. Results InfectionwithH37RaorH37RvresultedindifferentM.tbgrowthinmacrophagesandtranscriptionlevelsofanti-apoptoticgenebfl-
1 InordertoconfirmthedifferentialgrowthrateofM.tbinmacrophagesafterinfection,THP-1differentiatedmacrophageswereinfectedwithavirulentH37RastrainorvirulentH37Rvstrainatan
4 HostresponsestriggeredbyMycobacteriumtuberculosis MOI(multiplicityofinfection)of10:
1.At0,1,3,and5daysafterinfection,M.tbcolony-formingunits(CFUs)ofmacrophagesweremeasuredafter0.1%SDStreatment(Fig.1A).H37RvshowedasignificantlyhigherintracellulargrowthrateinhumanmacrophagesthanH37Ra,althoughbothstrainshavesimilarcapacitiestoenterthemacrophages[23,39].ThedifferenceofthegrowthratesoftwoM.tbstrainsinhumanmacrophagesprobablyoriginatesfromthedifferentvirulencebetweenthem. TodeterminetheculturetimeofmacrophagesafterinfectionwithH37RaorH37Rv,theexpressionlevelsofbfl-1andtnf-αinTHP-1macrophagesweredetectedbyqPCR.Theanti-apoptotic-orapoptotic-relatedgenebfl-1andtnf-αwerethususedasthereferencetoindicatetheessfulinfections.Theexpressionlevelsofbfl-1andtnf-αinmacrophagesafterinfectionwithM.tbwerehigherat12hafterinfectionsthanthatatothertimepoints.Whatismore,expressionlevelsofbfl-1betweenH37RaandH37Rv-infectedmacrophagesweremostdifferentat12hafterinfections,asshowninSupplementaryFig.S2,whichisconsistentwithpreviousresults[40].Therefore,wefocusedontheproteinprofilesofTHP-1cellsat12hafterinfectionwithM.tb,fortheurrenceofapoptosisandvariantanti-apoptosisofmacrophagesafterinfectionwithtwostrains. TMT-basedquantitativeproteomicsrevealeddifferentexpressionprofilesbetweenH37Ra-andH37Rvinfectedmacrophages Tounderstandthemolecularmechanismsinvolvedincross-talkbetweenmacrophagesandM.tb,weappliedaTMT-basedmassspectrometryprofileofproteinsofTHP-1cellsinfectedwithH37RaorH37Rv(Fig.1B).Macrophageproteinswereextractedat12hafterinfectionwithH37RaorH37Rv,withbeadsstimulationgroupasanegativecontrolanduntreatedTHP-1macrophagesasablankcontrol.Asaresult,atotalof6762macrophageproteinswereidentifiedusingthecriteriadescribedinMaterialsandMethods(SupplementaryTablesS1andS2).ProteinsidentifiedbothinRunAorBandCwereselectedforfurtherstudy.Subsequently,thearithmeticmean,standarddeviation,andrelativestandarddeviation(RSD)ofindividualproteinswerecalculated.TheRSDof95%proteinsineachgroupwerelowerthan0.21,whichdisplayedlowvariabilitybetweentherepeats(SupplementaryFig.S3).Then,proteinswithRSD>thelargestRSDvalueof95%proteinsineachgroupwerefilteredoutfortheurateratios(SupplementaryFig.S1).TheproteinswithZ-score>1.96or<−1.96wereconsideredasDEPs(SupplementaryTableS3-1).Forprofilingtheproteinexpressionlevel,Hemlsoftwarewasusedtoexporttheheatmap[41] Figure1.TMT-labeledquantitativeproteomicsanalysisoftheproteinsofTHP-1cellsinfectedwithH37RaorH37Rv(A)H37RainfectionandH37RvinfectionresultedindifferentM.tbgrowthrateinmacrophages.**P<0.01,***P<0.001vs.H37RagrowthrateinTHP-1cells.Theseresultswerefromonerepresentativeexperiment.Similarresultswereobtainedintwootherexperiments.(B)ExperimentalworkflowofTMTlabelingquantitativeproteomics.Macrophageproteinswereextractedat12hafterbeadstreatment,H37RaorH37Rvinfection.Afterdenaturationanddigestion,sampleswerelabeledwithTMTlabelreagentsandanalyzedusingaQExactive™hybridquadrupole-Orbitrapmassspectrometer.Massspectrometrydatawerequantifiedusinganin-houseversionofX!
tandem.DEPsofmacrophagesbetweeninfectionswerediscussed.(C)HeatmapofDEPsinTHP-1cellsstimulatedbybeads,infectedwithH37RaorH37Rv.Thelog2ratiosofproteinsfromeachgroupwerevisualizedtotheheatmapusingHeml.Thecolorscaledemonstratestherelativeexpressionlevelsoftheproteins(log2ratio),eachrowrepresentsadifferentiallyexpressedproteinandeachcolumnrepresentsagroup.(D)PANTHERProteinClassontologyanalyzedDEPsbetweenTHP-1cellsinfectedwithH37RaandH37Rv(P<0.01)(groupH37Rv/H37Ra). HostresponsestriggeredbyMycobacteriumtuberculosis
5 whichdemonstratedclusteroffourgroupsordingtotheDEPsafterinfection(Fig.1C).NoconfidentproteinofM.tbwasidentifiedinthisstudy. Therewere140macrophageproteinswithincreasedexpressionand111proteinswithdecreasedexpressionafterbeadsstimulation(groupBeads/THP-1),asshowninSupplementaryTableS3-
2.Someofthemwereassociatedwithautolysosomesandimmunity,suchaslysosomemembraneprotein2(LIMPII),CD63antigen,andCathepsinG[42–45],indicatingthattheexpressionlevelsofsomemacrophageimmunityproteinschangedafterbeadsinfection.Therefore,thebeadsinfectiongroupservedasanegativecontroltoidentifymacrophageproteinsrelatedtolivebacterialinfection.ForH37Ra/Beadsgrouptheexpressionlevelsof130macrophageproteinssignificantlyincreasedand150macrophageproteinsdecreased,whichassociatedwithavirulentstraininfection(SupplementalTableS3-3).However,fortheH37Rv/Beadsgrouptheexpressionlevelsof115macrophageproteinsincreasedand79macrophageproteinsdecreased(SupplementalTableS3-4),whichwererelatedtovirulentM.tbinfection.AfterinfectionwithM.tbstrainH37RvorH37Ra,theexpressionlevelsoftypeIinterferon-inducibleproteinsinmacrophages,includingubiquitin-likeproteinISG15(ISG15),interferon-stimulatedgene20kDaprotein(ISG20),interferoninducedproteinwithtetratricopeptiderepeats1(IFIT1),IFIT2,IFIT3,interferon-inducedhelicaseCdomain-containingprotein1(IFIH1),bonemarrowstromalantigen2(BST2),interferonregulatoryfactor9(IRF9),andradicalS-adenosylmethioninedomaincontainingprotein2(RSAD2),werealmostequallyincreased.Theincreasehadbeendemonstratedbothattranscriptandproteinlevelsbypreviousresearchers[46–48]. Theexpressionlevelof142THP-1macrophagesproteinsincreasedand93proteinsdecreasedafterH37RvparedwithH37Rainfection(groupH37Rv/H37Ra)(SupplementalTableS3-5).TheseDEPsofmacrophagesweredirectlyassociatedwithM.tbvirulentinfectionandreflectedspecificresponsesofmacrophagestoH37RvinsteadofH37Ra. DEPsofmacrophageswereclassifiedbyGOanalysis First,weclassifiedproteinsinmacrophagesbyPANTHERProteinClassontologytoevaluatetheirbiologicalrelevance[35](Fig.1D).ForgroupH37Rv/H37Ra,theDEPsofmacrophageswereclassifiedto21terms.fivetermswereoxidoreductase(11),signalingmolecule(12),hydrolase(18),enzymemodulator(19),andnucleicacidbinding(44).Toobtainmorefunctioninformationandpathwaysformacrophageproteinsdifferentiallyexpressedbetweentwostrains’infections,theseproteinswerefunctionallyanalyzedusingDAVID.GOtermsweresortedintothe‘CellularComponent(CC)’,‘BiologicalProcesses(BP)’,and‘MolecularFunction(MF)’GOcategoriesandKyotoEncyclopediaofGenesandGenomes(KEGG)pathways(P<0.01)(SupplementaryFig.S4). FortheBeads/THP-1group,pathwaytermwaslysosomeGO-MFtermwascadherin-bindinginvolvedincell–celladhesion(SupplementaryFig.S4A),whichsuggestedthatbeadscouldinduceproteinexpressionchangesinthelysosomeandpathogenrecognitionofmacrophages[49,50].Becauseoftheactivationofmacrophagesbybeads,beadsgroupwasselectedasanegativecontroltoexcludeproteinexpressionchangesinducedbyphagocytosisofphysicalstimulation.FortheH37Ra/BeadsgrouporH37Rv/Beadsgroup,mostGOtermswereassociatedwithinfectionimmunityandpathogenicbacteriakilling(SupplementaryFig.S4B,C).However,when comparedwithH37Ra/Beadsgroup,thetermpositivelyregulatedTcellcytokineproductionandlysosomesdisappearedinH37Rv/Beadsgroup,possiblyowingtoinhibitionofH37Rv. Takentogether,thesefunctionalanalysesrevealedthatbothofH37RvandH37Rainfectioncouldleadtotheexpressionchangesofmacrophageproteinsassociatedwithapoptosisprocess,bloodcoagulation,nucleosomeassembly,focaladhesion,andexosome.Obviously,H37Rvinfectionresultedinexpressionchangesofmacrophageproteinsassociatedwithoxidativephosphorylationinmitochondrialinnermembrane,nucleosomeassembly,vesicle,apoptosisprocess,andbloodcoagulation,whichwasdifferentfromH37Rainfection(SupplementaryFig.S4D).TheseresultsprovidemoreevidencethatmacrophagesinfectedwithvirulentH37RvstrainoravirulentH37Rastraincouldtriggerdifferentimmuneresponses. workanalysisofDEPs TofurtherunderstandtheinteractionsbetweentheDEPsofmacrophage,proteinworkswereconstructedusingSTRING10.0andvisualizedwithCytoscapev.3.2.1.Nodeswerecoloredordingtothelog2ratioofproteinsfromeachgroupandworkswerecreatedmanuallyordingtotheGOtermsanalyzedbyBiNGOv3.0.3(Fig.2A,SupplementaryFig.S5).Undoubtedly,therelationshipsbetweentheseworksindicatedstrongassociationsamongthem,whichmightberelatedtoH37Rvinfection.FortheH37Rv/H37Ragroup,therewere23macrophageproteinsrelatedtobloodcoagulation(Fig.2B),26proteinscorrelatedwithapoptosisprocess(Fig.2C),19proteinsassociatedwiththeoxidativephosphorylationinmitochondrialinnermembrane(Fig.2D),andsomeotherproteinsassociatedwithvesicleornucleosomeassembly,indicatingthattheseDEPsofmacrophagesandtheirfunctionalclustersmaybeinvolvedinthevirulentM.tbinfection.Notably,amongtheapoptosisprocessandinflammatoryresponsecluster,themostobviousproteinswerealpha-2-hs-glycoprotein(AHSG)andinterleukin-1β(IL-1β),implyingthatthesetwoproteinsmayplayimportantrolesinH37RvimmunityofparedwithH37Ra.TheseresultsconfirmedthatmacrophagesinfectedwithH37RvorH37Rainduceddifferentwidespreadbiologicalprocessesandpowerfulcross-talkexistedamongthem. Westernblotanalysesvalidatethealterationsofproteinexpressionlevel ThemacrophageproteinsIL-1βandAHSGwhichareassociatedwiththeapoptosisprocessandinflammatoryresponseinmacrophageswereselectedforwesternblotanalysistoconfirmthedifferentialexpressionofproteinsidentifiedinaTMT-labeledLC−MS/MSsystem(Fig.3).EqualamountsoftotalproteinsfromM.tbinfectedTHP-1cellswereappliedandblottedwithantibodiestoIL1βandAHSG.Thedecreasedexpressionlevelofpro-IL-1β/IL-1βandtheincreasedexpressionlevelofAHSGinmacrophagesafterH37Rvinfectionwereconfirmedbywesternblotassay,whichisincontrasttotheirexpressionlevelsafterH37Rainfection.Theproteomicresultswerevalidatedusingthesedata. Discussion ThedifferentbiologicalprocessesofmacrophagesaftervirulentM.tbstrainH37RvoravirulentH37Rastraininfectioncontributeto
6 HostresponsestriggeredbyMycobacteriumtuberculosis Figure2.workofDEPsbetweenTHP-1cellsinfectedwithH37RaandH37Rv(groupH37Rv/H37Ra)(A)DEPsofmacrophageswereanalyzedbySTRINGanddividedintosevenclustersordingtothecorrespondingbiologicalprocesscategories(P<0.05).GraphsweregeneratedusingCytoscapev.3.2.1andtheBiNGOv3.0.3plugin.Nodeswerecoloredordingtothelog2ratiosofproteinsfromeachgroup.Thecolorscaledemonstratestherelativeexpressionlevelsoftheproteins(log2ratio).Proteinsarrangedinacircleinthecenterofareindependentfromotherworks.(B)workofbloodcoagulation-relatedproteins.(C)Apoptosisprocessandinflammatoryresponseassociatedproteins.(D)Mitochondrialinnermembraneandoxidativephosphorylation-associatedproteins. theunderstandingplexM.tbpathogenesis.ResearchershaveevaluatedthedifferentresponsesofmacrophagesinfectedwithH37RvorH37Raattranscriptomiclevel[11,51].However,ordingtoourresults,only5proteinsoutofthe235DEPsfrommacrophagesareidenticaltoprevioustranscriptomicresults(SupplementalTableS3-6).Thislackofcorrelationbetweenproteomicandtranscriptomicresultscanbecontributedtothepost-transcriptionalregulatorymechanisms. AmongDEPsofmacrophagesbetweenH37RvandH37Rainfection,someofthemwererelatedtonucleosomeassemblyandbloodcoagulation(Fig.2B),includinghistonesandgranuleconstituentsassociatedwithproteins[collagen,typeI,alpha1(COL1A1),thrombospondin1(THBS1),quiescinQ6sulfhydryloxidase1(QSOX1),proteinSalpha(PROS1),andcoagulationfactorV(F5)].Histonesreleasedintotheextracellularspaceofmacrophagescan activateinflammasomepathwaythroughToll-likereceptors(TLRs)[52].Furthermore,histonesandgranulestendtoformmacrophageextracellulartrap(MET)withnuclearDNA,andtherebykillbacteriabyreleasingofMETinvitroofmacrophages[53–55].ItisknownthatmacrophagesreleaseMETtoconstrainandkillM.tbduringM.tbinfection,weassumethatH37RvinfectioncouldinducemoreMETthanH37Rainfectionfortheup-regulationofassociatedproteins.Thisneedstobeprovedbyfurtherexperiments.Furthermore,thereexistsacross-talkbetweenbloodcoagulationandmacrophageimmunity:fibrinregulatesthechemokine/cytokineproductionandmacrophageadhesioninvivo[56],whichcouldhelptoexplaintheformationofgranulomasinvivowiththeparticipationofmacrophagesandothercellsstimulatedbyH37Rv[57]. AnotherinterestingproteinoftheDEPsisHLAclassIpatibilityantigen,A-2alphachain(HLA-A),amajor HostresponsestriggeredbyMycobacteriumtuberculosis
7 Figure3.ConfirmationofDEPs(IL-1βandAHSG)inTHP-1cellsinfectedwithH37RaorH37RvbywesternblotanalysisTheresultswereconsistentwithMSdata.Thepicturewasfromonerepresentativeexperimentandsimilarresultswereobtainedintwootherexperiments.TheWBratioswerecalculatedordingtothesethreeexperiments.β-Actinwasusedasaninternalcontroltonormalizethequantitativedata. plex(MHC)antigenspecifictohumans.TheHLA-AexpressionlevelofmacrophagesdecreasedafterH37RvparedwithH37Rainfection,indicatingthatthevirulentstraininfectioncoulddisturbthepresentationofantigenicpeptidestotheTcellreceptors(TCRs)onTcells.Thus,virulentM.tbinfectioninhibitsTcell-mediatedcellularimmuneresponse,whichconstitutesanotherprotectionmechanismofthevirulentstrain[58,59]. Notably,theproteinsinmitochondrialinnermembraneassociatedwithoxidativephosphorylationincreasedafterH37RvparedwithH37Rainfection(Fig.2D).Thenicotinamideadeninedinucleotidedehydrogenase(NADH)(ubiquinone)1betaplexsubunit1(NDUFB1),NDUFB7,NDUFB11,andNDUFA13arethemitochondrialenergygeneratingplexIsubunitsthattransferelectronsfromNADHtotherespiratorychain;cytochromeb–plexsubunit(UQCRFS1)andUQCR10arethesubunitsofubiquinol–cytochromecplexplexIII)whichgeneratesanelectrochemicalpotentialcoupledtoadenosinetriphosphate(ATP)synthesis[60–62].MitochondriaarethemajorsourceandthefirstsuffererofROS.TheincreaseinmacrophageproteinsofComplexIIIandI,theprincipalsourceofsuperoxide,indicatesmoresuperoxideproduction.ItwasreportedthatoverexpressionplexIsubunitNDUFA6couldreduceapoptosisinHIV-infectedcells[63].ROSproductioncanalsocontributetotheactivationofNF-κBandcellsurvival.Therefore,plexIandIIIproteinsmayplayanimportantroleinoxidationinducedapoptosis.Inaddition,superoxidecouldbeconvertedtoH2O2bymanganese-dependentsuperoxidedismutase(SOD2)[64].SOD2whichcandestroysuperoxideanionradicalsandprotectagainstoxidation-inducedapoptosis,wasdown-regulatedafterH37Rvinfectioninthisstudy[65].Therefore,H37RvinfectioncaninducemoresuperoxideproductioninparedwithH37Raandthedetailedfunctionneedsfurtherinvestigation. MitochondriaareessentialinproducingROSaswellasgeneratingATP[66].TheATPaseinhibitor(ATPIF1),ATPsynthaseF(0)plexsubunitC1(ATP5G1),ATP5E,ATP5IandUp-regulatedduringskeletalmusclegrowthprotein5(USMG5)ofmacrophages,expressionlevelsofwhichareincreasedafterH37Rvinfection,constituteanATPplex(ComplexV).ThelackofATPafterapoptosiscouldinducenecroticcelldeath.[67].SotheupregulatedmitochondriaATPsynthasesmaydependontheactivationofapoptosis.Mitochondrialribosomalprotein(MRPL41) interactswiththeanti-apoptosisregulatorBcl-2andpromotesapoptosis[68].AfterH37Rvinfection,theanti-apoptoticproteinisreducedduetotheup-regulationofMRPL4.Ontheotherhand,heatshockcognate71kDaprotein(HSPA8)thatpreventsapoptosisbyparticipatinginchaperone-mediatedhagy,isincreasedafterH37RvinfectionmorethanthatafterH37Rainfection[69–71].Asaresult,H37Rvincreasesitssurvivalrateinmacrophagesbypreventingmacrophageapoptosis.ThesemacrophagemitochondriaresponsesuraftertheinfectionofH37RvotherthanaftertheinfectionofH37Ra,whichprovidesevidenceanddetailsforelucidatingtherelationshipbetweenvirulentM.tbinfectionandtheoxidativephosphorylationinmitochondrialinnermembrane. Theexpressionlevelsofmacrophageproteinsassociatedwiththeapoptosisprocessandinflammatoryresponsewerealteredwhencellswereinfectedwiththesetwostrainsrespectively(Fig.2C).ThedifferentialexpressionlevelofAHSGandIL-1βbetweenH37RvandH37Rainfectionwereconfirmedbywesternblotanalysis.AHSG,alsocalledfetuin-A,functionsasapositiveornegativeacutephaseproteinininfectionandisregulatedbydifferentproinflammatorymediators[72].AHSGexpressionlevelincreasedsignificantlyafterM.tbinfection.H37RvinducedhigherAHSGexpressionthanH37Rainmacrophages,butthisstrategyremainsconfusingandrequiresfurtherinvestigation.Thewesternblotanalysisresultsshoweddecreasedexpressionlevelofpro-IL-1βinthemacrophagesinfectedwithparedwithH37Ra.Sincetheexpressionlevelofpro-IL-1βistightlycontrolledbythefirstsignalmediatedbyTLRligands,itispossiblethatH37Rvinhibitspro-IL-1βexpressioninmacrophagesbyblockingTLR-mediatedsignalingpathwaysafterinfection,whichalsodeservesfurtherinvestigation.Pro-IL-1βproteinsareactivatedbycaspase-1,whichisunderthecontrolofthesecondsignalmediatedbynucleotide-bindingoligomerizationdomain-likereceptors(NLRs)[73].ButbothH37Rv-andH37RainfectedmacrophagesexpressedincreasedproinflammatorycytokineIL-1β,whichisconsistentwiththepreviousreport[56].Notably,paredwithH37Rainfection,H37RvinfectiondecreasedtheexpressionsofnuclearfactorNF-κBp105/p50subunit(NFκB1),nuclearfactorNF-κBp100/p52subunit(NFκB2),TNFalphainducedprotein6(TSG6,TNFAIP6)andtumornecrosisfactor(TNF)receptor-associatedfactor1(TRAF1).NFκB1andNFκB2arepartofaplexintheNF-κBpathway[74],thedown-regulationofwhichmightinducemorecellulardeath.TRAF1isanegativeregulatorofTNFsignalingandpreventsNF-κBactivationbyTNFandIL-1[75],whichindicatesthedecreaseofTRAF1mayup-regulateNF-κ
B.Takentogether,theremustbesomeothermechanismsthatregulateNF-κBpathway.TheotherproteinTSG6hasanti-inflammatoryeffectsandcanbeinducedbyTNFandIL-1[76].plexmayplayimportantrolesinthefatedeterminationofmacrophagesandM.tb.H37Rvmayescapetheimmunesystembyregulatingtheexpressionlevelofimmunityfactors,themechanismsofwhichremainunclearandareworthfurtherinvestigation. Takentogether,theseanalysessuggestthatH37Rvinfectionresultsintheexpressionsofmacrophageproteinsassociatedwithoxidativephosphorylation,apoptosisprocess,bloodcoagulation,andnucleosomeassembly,incontrasttoH37Rainfection.Furthermore,thefateofmacrophagesafterH37Rvinfectionmaydependontheinterplayoftheseimmunityprocesses. Inaddition,someDEPsofmacrophagesbetweenH37RvandH37Rainfectionarealwayslocatedinserum,suchasAHSG,RBP4(retinol-bindingprotein4),andIL-1β,whichcouldbevirulentinfectionbiomarkercandidates[30,77,78].RBP4bindswithand
8 HostresponsestriggeredbyMycobacteriumtuberculosis NFκB2,pro-IL-1β,TNFAIP6,andTRAF1,whichparticipateintheimmunityofM.tbandregulatetheapoptosisofmacrophages,areallparedwiththeH37Ra-infectedcells.ThefatesofmacrophagesandM.tbaredeterminedbyalltheseDEPs-associatedimmunitymechanismsandtheircross-talk.TheseresultsprovidenewcluestothehostresponseaftervirulentM.tbinfection,whichareimportantforfurtherTBstudiesandtherapies. Acknowledgments WewouldliketothankProteinWorldBiotechnologyCo.,Ltd(Beijing,China)forassistancewithquantitativeproteomics. SupplementaryData SupplementarydataareavailableatActaBiochimicaetBiophysicaSinicaonline. Figure4.ProposedmodelsdepictingthespecificimmuneresponsesofmacrophagesinducedbyvirulentM.tbH37Rv-infectedmacrophagesformedmoreMETsthustrappingM.tbtopreventdissemination.H37RvdecreasestheexpressionlevelofHLA-Ainmacrophages,whichmaydisturbthepresentationofantigenicpeptidesfrommacrophagestotheTCRsofTcellsandtherebyavoidTcell-mediatedcellularimmunereaction.H37RvinfectioninducesROSoverproductioninthemitochondrialwhileH37Radoesnot.InH37Rv-infectedmacrophages,theexpressionlevelsofNFκB1,NFκB2,proIL-1β,TNFAIP6,andTRAF1areallparedwiththeH37Rainfectedcells.‘↑↓’RepresentsthedecreasedorincreasedexpressionlevelofmacrophageproteinsafterH37RvparedwithH37Ra.Thegreendottedlineindicatesdown-regulation,whilethereddottedlineindicatesupregulation. transportsbloodretinolintothecellsandleadstoincreasedintracellularretinoicacidanddecreasedserumfibronectin,whichisrelatedtogranulomaformation[79].TheexpressionlevelsofbothAHSGandRBP4frompatientplasmawithactiveTBaresignificantlylowerthanthoseofpatientswithoutactivedisease,whichhasbeenconfirmedbyothers[80–82].However,theexpressionlevelofAHSGandRBP4weresignificantlyincreasedinthemacrophagelasmafterinfectionwithH37Rv,whichagreeswithpreviouswork[29].TheoppositeexpressiontendencyofthesetwoproteinsbetweenthemacrophagesandserumafterM.tbinfectionisworthfurtherinvestigation. Inconclusion,thisworkrevealeddifferentexpressionprofilesbetweenmacrophagesinfectedwithH37RaandH37RvusingTMT-basedquantitativeproteomics.TheseDEPsaremainlyinvolvedinapoptosisprocess,bloodcoagulation,andparticularlyoxidativephosphorylation.TheenormousvariationinproteinprofilesbetweenmacrophagesinfectedwithH37RaandH37Rvsuggeststheexistenceofdifferentimmunitymechanisms(Fig.4).InbothH37Rv-andH37Ra-infectedmacrophages,theexpressionlevelofhistonesandgranuleconstituents,whichformtheMETs,areallupgraded,buttheformerexhibitsmorenotableeffect,whichisinfavorofformationofMETsandthustrappingM.tbtopreventdissemination.H37RvdecreasestheexpressionlevelofHLA-Ainmacrophages,whichmaydisturbthepresentationofantigenicpeptidesfrommacrophagestotheTCRsofTcellsandtherebyavoidTcell-mediatedcellularimmuneresponse.H37RvinfectioninducesROSoverproductioninthemitochondrialwhileH37Radoesnot.InH37Rv-infectedmacrophages,theexpressionlevelsofNFκB1, Funding ThisworkwassupportedbythegrantfromtheChineseAcademyofSciences(No.KJZD-EW-TZ-L04). References
1.HardyA.Captainofdeath:thestoryoftuberculosis.AmJHumBiol1999,43:149.
2.DucatiRG,RuffitoA,BassoLA,SantosDS.Theresumptionofconsumption—areviewontuberculosis.MemInstOswaldoCruz2006,101:697–714.
3.KumarD,NathL,KamalMA,VarshneyA,JainA,SinghS,RaoKV.Genome-wideanalysisofthehostworkthatregulatessurvivalofMycobacteriumtuberculosis.Cell2010,140:731–743. 4.vanderWelN,HavaD,HoubenD,FluitsmaD,vanZonM,PiersonJ,BrennerM,etal.M.tuberculosisandM.lepraetranslocatefromthephagolysosometothecytosolinmyeloidcells.Cell2007,129:1287–1298.
5.SlyLM,Hingley-WilsonSM,ReinerNE,McMasterWR.SurvivalofMycobacteriumtuberculosisinhostmacrophagesinvolvesresistancetoapoptosisdependentuponinductionofantiapoptoticBcl-2familymemberMcl-
1.JImmunol2003,170:430–437.
6.BeharSM,DivangahiM,RemoldHG.EvasionofinnateimmunitybyMycobacteriumtuberculosis:isdeathanexitstrategy?
NatRevMicrobiol2010,8:668–674.
7.XuG,WangJ,GaoGF,LiuCH.InsightsintobattlesbetweenMycobacteriumtuberculosisandmacrophages.ProteinCell2014,5:728–736.
8.SaundersBM,CooperAM.Restrainingmycobacteria:roleofgranulomasinmycobacterialinfections.ImmunolCellBiol2000,78:334–341.
9.BendinelliM,FriedmanH.Mycobacteriumtuberculosis:interactionswiththeimmunesystemcontinued.Immunology1989,67:431. 10.ZhengH,LuL,WangB,PuS,ZhangX,ZhuG,ShiW,etal.icbasisofvirulenceattenuationrevealedparativegenomicanalysisofMycobacteriumtuberculosisstrainH37RaversusH37Rv.PLoSOne2008,3:e2375. 11.SilverRF,WalrathJ,LeeH,JacobsonBA,HortonH,BowmanMR,NockaK,etal.HumanalveolarmacrophagegeneresponsestoMycobacteriumtuberculosisstrainsH37RaandH37Rv.AmJRespirCellMolBiol2009,40:491–504. 12.PierceCH,DubosRJ,SchaeferWB.Multiplicationandsurvivaloftuberclebacilliinansofmice.JExpMed1953,97:189–206. 13.CollinsFM,SmithMM.parativestudyofthevirulenceofMycobacteriumtuberculosismeasuredinmiceandguineapigs.AmRevRespirDis1969,100:631–639. HostresponsestriggeredbyMycobacteriumtuberculosis
9 14.McmurrayDN,CarlomagnoMA,CumberlandPA.RespiratoryinfectionwithattenuatedMycobacteriumtuberculosisH37Rainmalnourishedguineapigs.InfectImmun1983,39:793–799. 15.KeaneJ,RemoldHG,KornfeldH.VirulentMycobacteriumtuberculosisstrainsevadeapoptosisofinfectedalveolarmacrophages.JImmunol2000,164:2016–2020. 16.DanelishviliL,McgarveyJ,LiYJ,BermudezLE.Mycobacteriumtuberculosisinfectioncausesdifferentlevelsofapoptosisandnecrosisinhumanmacrophagesandalveolarepithelialcells.CellMicrobiol2003,5:649–660. 17.ChenM,GanH,RemoldHG.Amechanismofvirulence:virulentMycobacteriumtuberculosisStrainH37Rv,butnotattenuatedH37Ra,causessignificantmitochondrialinnermembranedisruptioninmacrophagesleadingtonecrosis.JImmunol2006,176:3707–3716. 18.DavisMJ,RamakrishnanL.Theroleofthegranulomainexpansionanddisseminationofearlytuberculousinfection.Cell2009,136:37–49. 19.DanielJ,MaamarH,DebC,SirakovaTD,KolattukudyPE.Mycobacteriumtuberculosisuseshosttriacylglyceroltoumulatelipiddropletsandacquiresadormancy-likephenotypeinlipid-loadedmacrophages.PLoSPathog2011,7:e1002093. 20.TiwariB,RamakrishnanUM,RaghunandTR.TheMycobacteriumtuberculosisproteinpairPE9(Rv1088)-PE10(Rv1089)formsheterodimersandinducesmacrophageapoptosisthroughToll-likereceptor4.CellMicrobiol2015,17:1653–1669. 21.MeenaLS,Rajni.SurvivalmechanismsofpathogenicMycobacteriumtuberculosisH37Rv.FebsJ2010,277:2416. 22.WeissG,SchaibleUE.Macrophagedefensemechanismsagainstintracellularbacteria.ImmunolRev2015,264:182–203. 23.ZhangM,GongJ,LinY,BarnesPF.GrowthofvirulentandavirulentMycobacteriumtuberculosisstrainsinhumanmacrophages.InfectImmun1998,66:794. 24.YangY,HuM,YuK,ZengX,LiuX.Massspectrometry-basedproteomicapproachestostudypathogenicbacteria-hostinteractions.ProteinCell2015,6:265–274. 25.RaoPK,SinghCR,JagannathC,LiQ.Asystemsbiologyapproachtostudythephagosomalproteomemodulatedbymycobacterialinfections.IntJClinExpMed2009,2:233–247. 26.ShuiW,PetzoldCJ,ReddingA,LiuJ,PitcherA,SheuL,HsiehTY,etal.Organellemembraneproteomicsrevealsdifferentialinfluenceofmycobacteriallipoglycansonmacrophagephagosomematurationandhagosomeumulation.JProteomeRes2010,10:339–348. 27.DiazG,WolfeLM,Kruh-GarciaNA,DobosKM.Changesinthemembrane-associatedproteinsofexosomesreleasedfromhumanmacrophagesafterMycobacteriumtuberculosisinfection.SciRep2016,6:37975. 28.SaquibNM,JamwalS,MidhaMK,VermaHN,ManivelV.QuantitativeproteomicsandlipidomicsanalysisofendoplasmicreticulumofmacrophageinfectedwithMycobacteriumtuberculosis.IntJProteomics2015,2015:270438. 29.HareNJ,LeeLY,LokeI,BrittonWJ,SaundersBM,Thaysen-AndersenM.MycobacteriumtuberculosisinfectionmanipulatestheglycosylationmachineryandtheN-glycoproteomeofhumanmacrophagesandtheirmicroparticles.JProteomeRes2017,16:247–263. 30.KaewseekhaoB,NaranbhaiV,RoytrakulS,NamwatW,PaemaneeA,LulitanondV,ChaiprasertA,etal.ComparativeproteomicsofactivatedTHP-1cellsinfectedwithMycobacteriumtuberculosisidentifiesputativeclearancebiomarkersfortuberculosistreatment.PLoSOne2015,10:e0134168. 31.LiP,WangR,DongW,HuL,ZongB,ZhangY,WangX,etal.ComparativeproteomicsanalysisofhumanmacrophagesinfectedwithvirulentMycobacteriumbovis.FrontCellInfectMicrobiol2017,7:65. 32.DayonL,HainardA,LickerV,TurckN,KuhnK,HochstrasserDF,BurkhardPR,etal.RelativequantificationofproteinsinhumancerebrospinalfluidsbyMS/MSusing6-plexisobarictags.AnalChem2008,80:2921–2931. 33.AntonioVJ,AttilaC,NoemiDT,DianesJA,JohannesG,IliasL,GerhardM,etal.updateofthePRIDEdatabaseanditsrelatedtools.NucleicAcidsRes2016,44:D447–D456. 34.CoombsKM,BerardA,XuW,KrokhinO,MengX,CortensJP,KobasaD,etal.Quantitativeproteomicanalysesofinfluenzavirus-infectedculturedhumanlungcells.JVirol2010,84:10888–10906. 35.MiH.PANTHERversion7:improvedictrees,orthologsandcollaborationwiththeGeneOntologyConsortium.NucleicAcidsRes2010,38:204–210. 36.HuangdaW,ShermanBT,LempickiRA.SystematicandintegrativeanalysisoflargegenelistsusingDAVIDbioinformaticsresources.NatProtoc2009,4:44–57. 37.ShannonP,MarkielA,OzierO,BaligaNS,WangJT,RamageD,AminN,etal.Cytoscape:asoftwareenvironmentforintegratedmodelsofbiomolecularworks.GenomeRes2003,13:2498–2504. 38.MaereS,HeymansK,KuiperM.BiNGO:aCytoscapeplugintoassessoverrepresentationofgeneontologycategoriesinworks.Bioinformatics2005,21:3448–3449. 39.SchlesingerL.MacrophagephagocytosisofvirulentbutnotattenuatedstrainsofMycobacteriumtuberculosisismediatedbymannosereceptorsinadditionplementreceptors.JImmunol1993,150:2920–2930. 40.DhimanR,RajeM,MajumdarS.DifferentialexpressionofNF-κBinmycobacteriainfectedTHP-1affectsapoptosis.BiochimBiophysActa2007,1770:649–658. 41.DengW,WangY,LiuZ,ChengH,XueY.HemI:atoolkitforillustratingheatmaps.PLoSOne2014,9:e111988. 42.LadelCH,SzalayG,RiedelD,KaufmannS.Interleukin-12secretionbyMycobacteriumtuberculosis-infectedmacrophages.InfectImmun1997,65:1936–1938. 43.ReczekD,SchwakeM,SchröderJ,HughesH,BlanzJ,JinX,BrondykW,etal.LIMP-2isareceptorforlysosomalmannose-6-phosphateindependenttargetingofβ-glucocerebrosidase.Cell2007,131:770–783. 44.DoyleEL,RidgerV,FerraroF,TurmaineM,SaftigP,CutlerDF.CD63isanessentialcofactortoleukocyterecruitmentbyendothelialP-selectin.Blood2011,118:4265–4273. 45.DanelishviliL,EvermanJ,McNamaraM,BermudezL.Inhibitionoftheplasma-membrane-associatedserineproteasecathepsinGbyMycobacteriumtuberculosisRv3364csuppressescaspase-1andpyroptosisinmacrophages.FrontMicrobiol2012,2:281. 46.BerryMP,GrahamCM,abFW,XuZ,BlochSA,OniT,WilkinsonKA,etal.Aninterferon-inducibleneutrophil-drivenbloodtranscriptionalsignatureinhumantuberculosis.Nature2010,466:973–977. 47.Otte
声明：
该资讯来自于互联网网友发布，如有侵犯您的权益请联系我们。