生物信息资源 杨军-系统生物学应用学习资料

系统生物学技术在生命科学研究中的应用杨军什么是系统生物学？SystemsBiology目的是通过对细胞内所有成分间的相互作用进行量化的描述，以达到在系统水平上理解这些相互作用这种研究的目标就是建立这些复杂系统的计算机模型，从而可以预测生物系统对任何干扰（如环境波动，遗传突变等）所发生的反应此类研究中最重要的部分就是确定系统中所有的成分，以及各成分如基因间的调控关系、蛋白质之间和生化通路之间的相互作用，并用这些知识建立一个原始模型（生化的或数学的）一旦这个系统结构建立了，系统行为就可以进一步通过遗传或环境因素的干扰进行分析分析得到的数据则可与原始模型整合，或用来改进模型，使得预期结果与实验观察结果一致因此，系统生物学是一个通过对比实验结果与预期结果而形成新模型并进行新实验验证的重复过程最终目的:通过这些详细的研究能够使研究者按照特定的设计原理进行模拟构建具有他们所需要的特性的生物系统技术手段对一个细胞反应进行系统化的定性需要能够在多水平上产生大量信息的定量的生物测量手段目前此类的方法中最为人知的就是对基因组（genome）进行研究的基因组学（genomics）和对蛋白组（proteome）进行研究的蛋白组学（proteomics）技术生物信息学（bioinformatics）基因组学：成千上万个基因的表达水平可以用DNA芯片的技术同时进行测量和分析，而且还可以用基因芯片技术进行测序，发现单核苷酸多态性，以及基因剪接的鉴定蛋白组学则侧重于决定尽可能多的蛋白的结构、表达、定位、生化活性及相互作用等目前常用的蛋白组学技术有双向电泳和质谱相结合的方法（如双向电泳与飞行质谱MALDI-TOF的结合）也有高效液相色谱与质谱相结合的方法（如LC-MS/MS）等应用这些技术已经得到了大量的生物学数据，并且已经被系统的组织成几个在线的数据库，如：TheNationalCenterforBiotechnologyInformation()ProteinDataBank(http://www.rcsb.rog/pdb)KyotoEncyclopediaofGenesandGenomes(http://www.genome.ad.jp/kegg)BiomolecularInteractionNetworkDatabase(/products/BIND)EncyclopediaofEscherichiacoliGenesandMetabolism()等包含了诸如核苷酸序列、氨基酸序列、分子特性、相互作用和通路等多种信息。这些数据库对于生物系统模型的建立是必不可少的在研究中的应用：举例：共调控基因的机理研究共调控基因可能参与一些紧密相关的生理功能可能由共同的转录因子激活环境致癌物诱导的细胞应激反应基因芯片分析GOanalysesofsomeoftheresultsTranscriptionfactorsInvolvedincellcycleregulationKinases蛋白组学分析为什么？许多信息仅从基因的研究是无法得到的蛋白质，而非基因，才决定了细胞的表型仅仅研究基因无法阐明疾病、衰老、环境影响等的机理部分被诱导的蛋白是否有共同的激活方式？基因表达的调控可在多环节上，但最重要的一个环节在转录的起始：转录因子（TF）结合到转录因子结合位点（TFBS）这些蛋白的基因上是否有共同的TFBS？传统方法：构建缺失体，DNaseI超敏感位点，DNA印迹，凝胶阻滞等缺点：长时间，人力，物力生物信息学：避免浪费大量人力、物力可行性：人类基因组全序列，TFBS预测软件缺点：假阳性如何解决假阳性？进化足迹法在DNA序列非功能区的突变率应高于功能区有功能的DNA序列一般比较保守如一段DNA序列在几个物种中都保守，则应有功能在非保守序列（即非功能序列）中的TFBS可去除软件BLASTN/BLAST/ClustalWversion1.81http://www.ebi.ac.uk/clustalw/MegaBLAST/BLAST/DNABlockAligner(DBA)http://www.ebi.ac.uk/Wise2/dbaform.htmlMatch1.0-public/pub/programs.html/matchPromoterPredition：FirstEF,PromoterInspector,PromoterScan,etc数据库GenBank/entrez/query.fcgi?db=NucleotideTRANSFAC5.0(transcriptionfactors)/cgi-bin/pub/databases/transfac/search.cgi?SWISS-PROT&tremble/sprot/

NCBILocusLinkhttp:///LocusLinkNCBIRefSeqhttp:///RefSeq/

程序确定人和鼠中的同源基因对确定5’端用软件确定启动子区比较人和鼠基因发现保守序列在人和鼠中分别寻找TFBS比较，选择那些只在保守区出现的TFBS最后选择8个基因共同的TFBSStep1.identifythehomologouspairsQuery=gi|4758613|ref|NM_004791.1|Homosapiensintegrinbeta-like1

Query=gi|18044899|gb|BC020152.1|MusmusculusRIKENcDNAB930011D01gene

ThepairsStep2.Determinethe5’oftranscript:ClustalWExample:F9,mouseTSS:transcriptionstartsite

Step3.PromoterpredictionStep4.Identify

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conservedregion

Example:ITGB2Step5.SearchforTFBS(usingMatchprogram)usingMATCHtosearchF9geneupstream1000bp,find687TFBSStep6.PhylogeneticcomparisonOnly60leftAfterphylogeneticfootprintingThelaststepThecommontranscriptionfactors

AP1C/EBPCMYBELK1GATANFY

USFAREB6CAATNKX25HAND1E47Experimentalvalidation:gelmobilityshiftassay

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M:MNNGtreatedsample;D:DMSOcontrol;C:competitionassay

MycoplasmapneumoniaeinfectioninducesreactiveoxygenspeciesandDNAdamageinhumanlungcarcinomaA549cellsAnotherexampleMycoplasma

pneumoniae(M.pneumoniae)isoneofthesmallestself-replicatingorganismscapableofacell-freeexistencemajorcauseofcommunity-acquiredrespiratoryinfectionsinchildrenandadults,whichcanleadtotracheobronchitisandprimaryatypicalpneumoniaAlsoimplicatedinseveralextrapulmonarycomplicationsarisingfrominfection,suchasbeingafactorinthedevelopmentofarthritis,cardiovasculardiseases,andneurotropicinfectionsPathogenesisactivationofthehostimmuneresponseanddirectinvasionofcellsattachmentofM.pneumoniaetotherespiratoryepitheliumisaprerequisitefordiseaseThecloseinteractionbetweenM.pneumoniaeandhostcellsprotectsthebacteriumfromremovalbyhost’smucociliaryclearancemechanismandallowsittoproliferateandproducemetabolites,which,inturn,cancausecytotoxiceffectsPathogenesisStillnotfullyclearSystemsbiologyapproaches,suchasproteomictechniques,hasrevolutionizedourabilitytostudyproteininteractionsandcellularchangesonaglobalscale,revealingpreviouslyunknownandunanticipatedassociationsTherefore,weinvestigatedtheeffectsofM.pneumoniaeinfectiononA549hostcellproteinprofilestoelucidatethepathogenicmechanism(s)byusing2-dimensionalgelelectrophoresis(2-DE)andmatrix-assistedlaserdesorptionionization-timeofflightmassspectrometry(MALDI-TOF-MS)ResultsEffectsofM.pneumoniaeinfectiononcellviability

atconcentrationsbelow10CFU/cell,M.pneumoniaeinfectionhadlittleeffectoncellviabilityhigherconcentrationsofM.pneumoniaedidaffectcellviability,particularlyafterlongerincubationtimesM.pneumoniaeinfectioninduceschangesinproteinexpressionprofileofA549cells

10CFU/cell,12h967±101proteinspotsincontrolA549cellsand978±83spotsinM.pneumoniae-infectedA549cellsnoproteinspotappearednordisappearedinM.pneumoniae-infectedA549cells,but26proteinspotswereup-regulatedand3proteinspotsweredown-regulatedInterestingly,amongthe15proteinsidentified,therearethreeproteinswhichareinvolvedinregulatingcellularoxidativestatus:glucose-6-phosphate1-dehydrogenase(G6PD)NADHdehydrogenase(ubiquinone)Fe-Sprotein2(NDUFS2)ubiquinol-cytochrome-creductasecomplexcoreproteinImitochondrialprecursor(UQCRC1)M.pneumoniaeinfectionmightinduceoxidativestressincells?M.pneumoniaeinfectioninducesROSinA549cells

ROSmediatestheDNAdamageelicitedbymanygenotoxicagentsSincetheforegoingexperimentslinkM.pneumoniaeinfectionwithelevatedROS,wepursuedthelogicalquestion:doesM.pneumoniaealsoinduceDNAdamage?AmongthevarioustypesofDNAdamage,doublestrandbreaks(DSBs)isthemostseveregammaH2AX(thephosphorylatedformofH2AX)fociformationhasbeengraduallybecomeacceptedasasensitiveindicatorforDSBsToxicologyinvitro,2006,Yu,Y.etal

M.pneumoniaeinfectioninducesgammaH2AXfociformationinA549cells

InhibitionofROSgenerationdecreasesM.pneumoniae-inducedgammaH2AXfociformation

indicateasignificantroleofROSinM.pneumoniaeinducedDNAdamageDiscussionProteomics,apowerfulsystemsbiologytool,hasbeenappliedinthestudyofbacterialpathogensaswellasanti-bacterialdrugdesigndeterminingtheproteincompositionofpathogens,thestructureandproteininteractionsofpathogens’proteins,andtheeffectsofinfectionandindividualpathogenicproteinsonthehostcellularproteomeThisknowledgecouldleadtothediscoveryofnewmoleculartargetsandbiomarkersformedicineandbiopharmacyG6PDisthefirstandrate-limitingenzymeinthepentosephosphatepathwaycatalyzethesynthesisofribosesfornucleicacidproductionandmoreimportantly,astheprincipalintracellularsourceofNADPH,whichparticipatesincellularanti-oxidationreactionsagainstROSincludingsuperoxideanion,hydrogenperoxide,andhydroxylradicalstheinductionofG6PDproteinasaconsequenceofbacterialorviralinfectionisnotwidelydocumentedalthoughincreasedG6PDactivityhasbeendetectedinRickettsiaconorii-infectedmicetissueandvirus-infectedtobaccoleavesEventhoughduringthe1980sthereweresomereportssuggestingthatROScontributetothecytotoxiceffectsofM.pneumoniae,ithassincerarelybeenmentionednorstudiedwithregardtothepathogenesisofM.pneumoniaebasedonourproteomicwork,theimportanceofROSshouldbereconsideredforthepathogenesisofM.pneumoniae,asROScanelicitseveredeleteriouseffectsoncellsHereforthefirsttime,wehaveshownthatM.pneumoniaealsoinducedDNAdamage,andROSparticipatedinthisprocessassupportedbytheNACexperimentraisesanotherinterestingquestion,withthepresenceofsevereDNAdamage,whynosignificantdecreaseincellviabilitywasobserved?Ononehand,thepresenceofgH2AXfocimaynotbedirectlyrelatedtoadecreaseincellviabilityOntheotherhand,thedisappearanceofgH2AXfociisgenerallyregardedastherepairofDSBsat24hthenumberofcellswithover20foci/cellwaslessthanthatat12h,indicatingtherepairofDNAdamagetherefore,itisquitepossiblethatthecellularDNArepairsystemcouldefficientlyfixtheproblem,thuspreventingthecellsfromgoingapoptosisorotherdeleteriousconsequencesFuturedirectionM.

pneumoniae-generatedROSvshost-generatedROSInhibitionofhostenzymes,suchascatalaseandsuperoxidedismutase?Differenttypeofcells?Andmore…MorenuclearproteomicstudiesBenzo[a]pyrene(BaP)Arepresentativememberofthepolycyclicaromatichydrocarbons(PAHs)familyEnvironmentallyprevalentcarcinogensfoundincombustionproducts,suchascigarettesmokeanddieselexhaustImplicatedintumorinitiation,promotionandprogression10mMBaPfor4,12and24hOver600spots/gel128spotswithP<0.0567upregulated53downregulatedLC-MS/MSidentificationBub380outofthe128weresubjectedtoMS45wereidentified21wereexcludedforfurtheranalysisduetoTheoreticalpIorMWdidnotmatchwithgelimageOrusuallyregardedascytoplasmicproteinVerificationofLaminAVerificationofBub3Alternativesplicing(AS)?10outofthe24identifiedproteinsareinvolvedinpre-mRNAprocessingAlternativesplicingplaysanimportantroleindevelopment,physiology,anddiseasemorethanhalfofthehumangenesarealternativelysplicedisregulatedbyafairlylargenumberofregulatoryfactors,includingtwofamiliesofproteinsthatgenerallyfunctioninanantagonisticmannerhnRNPSR(serine/arginine-rich)proteinsASfactorsandDNAdamage?IthasbeenreportedthathnRNPKisup-regulatedinresponsetoDNAdamageinanATM-andATRdependentmannerputativeA18hnRNPmRNAisregulatedinresponsetoUV;andmayplaysomeroleintherepairofUV-inducedDNAdamageFurthermore,A18hnRNPisinducedandtranslocatedfromthenucleitothecytoplasmafterexposuretoUVASinDNAdamage?IthasalsobeenreportedthatAScanbeinducedforsomegenesbyDNAdamagep53-induciblegene3(PIG3)TATA-bindingprotein-associatedfactorI(TAFI)MDM2andMDM4FasCD44InBaP-treatedcellsHowaboutunderothertypeofDNAdamage?CisplatinWidelyusedinthetreatmentofvariouscancersFormsavarietyofDNAadducts,includingIntrastrandcrosslinksInterstrandcrosslinksMonoadductsDNA-proteincrosslinksNuclearproteomeanalysis35differentiallyexpressed14upregulated21downregulated19identifiedbyMSVerificationBub3PSP1AS?6outofthe19identifiedproteinsareinvolvedinpre-mRNAprocessingIncisplatin-treatedcellsASoccurredforFasbutnotCD44Sofar,DNAdamagecaninfluencetheexpressionlevelsofASfactorsAScanhappentocertaingenesinDNA-damagedcellsTheremightexistadifferenceforalternativelysplicedgenesforspecifictypeofDNAdamageWhyASConsideringthecomplexityofthemechanismsunderlyingtheDNAdamageresponse,theeffectorproteinsmayindeedbeinvolvedinmultiplebiologicalactivitiesProductsofalternativesplicingoftenpossessdifferent,evenoppositefunctionsthechangeofsplicingpatternsobservedislikelytoplayasignificantroleinthecomplicatedDNAdamageresponseHowever,onlyfewselectedgeneswereexamined,howgeneralisASinDNA-damagedcells?Andhowtodoit?ThesolutionExonarray:genome-wideanalysisofalternativesplicingAglobalview,detailedSplicemicroarrayHigherresolutionStillunderdevelopmentGeneChip

HumanExon1.0STArrayEachexonhas4probesEachgenehasover40probesEvaluategeneexpressionandASsimultaneouslyforover1millionexonsHasbeenusedinseveralstudiesPreliminaryresults0.5mMBaPtreatmentfor5h1754genesshowedchangesinASpatternt-testp<0.05,withspliceindex(SI)<-0.5orSI>0.5Morestrictanalysisfound70geneshavingchangedsplicingpatternst-testp<0.01,withSI<-1orSI>1SeveralgeneswereselectedforverificationNextstepCloneSequencingIdentificationofsplicingformsFunction?……在疾病研究中的应用：发现前列腺癌的新标记比较正常前列腺组织和一个前列腺肿瘤细胞系的mRNA表达，发现554个表达的转录因子中，有112个改变前列腺组织与其他组织mRNA比较，发现有近300个仅在前列腺组织表达其中60个含有信号肽，可能为分泌蛋白针对其中一个蛋白在血液中进行检测，可识别5/10早期前列腺癌和5/10晚期前列腺癌，而经典的PSA不能识别早期，但识别大部分晚期前列腺癌患者CancerOutlierProfileAnalysis(COPA)假设：一个癌基因的过度表达可以在基因芯片的结果中体现出来对132组基因表达数据，共10486基因芯片实验结果()进行分析对已知的两个在肿瘤(myeloma)中高表达的癌基因的验证：鉴定得到两个前列腺癌中高表达的基因：ETV1和ERG不同数据组可得到相同的结果部分通过COPA分析得到的结果ERG与ETV1高表达的原因前列腺特异基因TMPRSS2与ERG或ETV1的融合FISH验证系统生物学的新方向、新技术脂类组学糖类组学微流控芯片纳米机械生物分子检测器。。。。。。Howlipidomicsgetinvolved?MNNGinducestheclusteringofmembranereceptorsTNFRandEGFR,whichoccursatlipidraftsBothMNNGandEGFinducethetranslocationofacidsphingomyelinase(ASM)Sphingomyelinase

isresponsibleforhydrolysissphingomyelintogenerateceramideHowever,thedownstreamsignalingpathwayisdifferentMNNGandEGFinducedifferentchangesinsphingolipidsmetabolismBlankDMSOEGFMNNGEffectsofMycoplasmapneumoniaeinfectiononsphingolipidmetabolisminhumanlungcarcinomaA549cellsYuanyuanYu,GongpingSun,GuangyiLiu,YingshuoWang,ZhengpingShao,ZhiminChen,JunYangMicrobialPathogenesis,2009,46:63-72IntroductionMycoplasmapneumoniae(M.pneumoniae)isamajorcauseofcommunity-acquiredpneumoniaOneofthesmallestandsimplestfree-livingandself-replicatingmicroorganismsMayalsoexperienceextrapulmonarycomplicationssuchasencephalitisorpolyradiculitisCloserelationshipwithasthmaMechanismofpathogenesisAttachmentpre-requisiteDirectcytotoxiceffectsToxicsubstances:ROS(?),othermetabolitesInflammationInductionofpro-inflammatorycytokinesetc.MolecularmimicryM.pneumoniaeinfectioncaninduceacutemotoraxonalneuropathyGuillain–Barre′(GBS)orFisher(FS)syndromeAntibodiestargetedtogangliosides,asubgroupofglycosphingolipids,areresponsiblefortheseautoimmunediseasesSphingolipidsAmajorclassoflipidsintheeukaryoticcellmembraneTogetherwithcholesterolformlipidraftsthatfunctionasplatformsforproteininteractionCeramideisatthecenterstageinthesphingolipidbiosynthesispathwayItcanbeconvertedtoSphingomyelinorglucosylceramideandgalactosylceramideThelattertwocanbefurtherglycosylatedtoformmorecomplexglycosphingolipidsThefunctionofsphingolipidsStructuralcomponentsmanysphingolipidspecies,suchasceramide,sphingosine,sphingosine-1-phosphate,andglucosylceramide,arealsoimportantsignalingmoleculesthatareinvolvedinmanycellularfunctions:cellproliferation,differentiation,apoptosis,stressresponse,inflammation,angiogenesis,geneticdiseases,andresistancetochemotherapyInfectiousdisease?ManypathogensusehostmembranesphingolipidsasreceptorsLipidraftscanbeusedasplatformsforinfectionsignalingandentryofintracellularparasitesOntheotherhand,hostdefenseagainstpathogensalsorequiresceramide-enrichedmembraneplatformsSphingolipidsinteractionMostbacteriaandvirusesdonotproducesphingolipidsHowever,theyhavetheabilitytoincorporatetheselipidsfromthesurroundingsintotheirmembranes,andsometimesmodifytheselipidsCanleadtoquitedifferentoutcomesSphingolipidinteractionApathogencanobtainmammaliansphingolipidstoevadethehostimmuneresponseorconvertthemtonewsphingolipidsbymicrobialenzymestoelicitnovelordifferentfunctionsIncontrast,amicrobialsphingolipidmayinterferewithhostintracellularsignalingorinduceanautoimmuneresponsethroughmolecularmimicry(likeinthecasesofGBSandFS)SignificanceTherefore,itisimportanttohaveaclearpictureofthesphingolipidprofilesofboththepathogenandthehostaswellasthesphingolipidinteractiontounderstandthedelicatebalancebetweenhostandpathogenLipidomicsafastgrowingresearchareawhichfocusesonthesystems-levelanalysisoflipidsandfactorsthatinteractwithlipidsUtilizationofmassspectrometry(MS)techniques,includingmatrix-assistedlaserdesorptionionization-timeofflight(MALDITOF)-MSliquidchromatography-electrosprayionization(LC-ESI)-MS/MSPurposeofthisstudyAsoneofthesmallestself-replicatingorganismscapableofacell-freeexistence,M.pneumoniaehasbeenwidelyusedasamodelsystemtoevaluatesuchsystemsbiologyapproachesasgenomicsandproteomicsTherefore,inthepresentstudy,M.pneumoniaeinfectionwasalsousedasamodeltoexaminetheapplicabilityoflipidomicsinthestudyofbacterialpathogenesisResults

I.SphingolipidprofilesofM.pneumoniaeandA549cellssphingolipidswereextractedfromM.pneumoniaeandA549cellsthensubjectedtoMALDI-TOF-MSanalysismolecularions[M+Na]+,[M+H]+,[M+K]+,or[M+H2O+H]+fortwomajorclassesofsphingolipids,e.g.,ceramide(Cer)orsphingomyelin(SM)speciescorrespondingtospecificpeakswereassignedRepresentativemassspectraofsphingolipidsinM.pneumoniaeandA549cells.Characteristicions(m/z)M.pneumoniaeUninfectedA549cells502.9Cer[d18:1C12:1+Na]+++506.9Cer[d18:1C11:0+K]+++507.8Cer[d16:1C16:1+H]++-508.9Cer[d18:1C14:1+H]+-+511.1Cer[d18:1C14:0+H]++-518.8Cer[d18:1C12:1+K]+-+528.8Cer[d18:1C14:0+H2O+H]+++539.0Cer[d18:1C16:0+H]+++549.7Cer[d18:1C17:1+H]++-574.5Cer[d18:1C16:1+K]++-701.6SM[d18:1C16:1+H]+++723.5SM[d18:1C16:1+Na]+++729.6SM[d18:1C18:1+H]++-751.5SM[d18:1C18:1+Na]++-811.4SM[d18:1C22:0+Na]+++833.3SM[d18:0C24h:0+H]++-SphingolipidsspeciesinM.pneumoniaeanduninfectedA549cellsM.pneumoniaeandA549cellssharemanycommonsphingolipidmoleculesyet7peakswereonlypresentinM.pneumoniaetheyweredefinedasamarkerofM.pneumoniaesphingolipidsOntheotherhand,severalpeakswereonlyfoundinA549cellsII.M.pneumoniaeinfectioninducessignificantchangesincellularsphingolipidcompositionM.pneumoniaeatconcentrationsof1,10,and100CFU/cellSphingolipidswerethenextractedfromcellsat2,12,and24hpostinfection,andanalyzedbyMALDI-TOF-MSRepresentativemassspectraofsphingolipidsininfectedanduninfectedA549cellsafter2hinfectionTimeanddoseeffects1CFU/cellinfectionAt2h,therelativeamountsofsomeceramideandsphingomyelinspecieswerechangedAt12and24h,however,therewasthegenerationofnewceramidespecies10CFU/cell:strongerthanthoseof1CFU/cellat2hpostinfectiontherewasalreadytheinductionofnewceramidespecies100CFU/cell:weremostdramaticonlyat24h,withthegenerationofmanynewceramideandsphingomyelinspeciesReferencemassspectraofsphingolipidsinA549cellsaftervariousdosesofM.pneumoniaeinfection.(A)2hpostinfection;(B)12hpostinfection;(C)24hpostinfection.III.M.pneumoniaeinfectionaffectsserinepalmitoyltransferaseexpressioninA549cellsfurtherexaminedtheeffectsofM.pneumoniaeontheexpressionoftwokeyenzymesinthesphingolipidmetabolismpathwaybyRT-PCRserinepalmitoyltransferase(SPT)acidsphingomyelinase(ASM)EffectsofM.pneumoniaeinfectiononSPTandASMexpression.(A)RepresentativeimagesofRT-PCRresults.(B)DensitometryanalysisofSPTmRNAexpressionat24hpostinfection.(C)WesternblotofSPTexpressionIV.M.pneumoniaeinfectioninducestheclusteringofASMTheactivationofASMwasusuallyaccompaniedbyitsclusteringonlipidraftsTherefore,immunofluorescentmicroscopywasperformedtoobservetheclusteringofASMat2,12,and24hpostinfectionwith1,10,and100CFU/cellM.pneumoniaeCholeratoxinB,whichspecificallybindstoGM1(atypeofglycosylsphingolipids)inlipidrafts,wasusedtolabellipidraftsM.pneumoniaeinfectioninducesASMclustering.(A)12hpostinfectionA549cellswerefixedandstainedwithanti-ASMantibody.Theformationof‘cap’structureindicatesASMclustering(red).Choleratoxin-Bwasusedtolabelmembrane(green).(B)PercentageofA549cellswithASMclusteringNoASMclusteringwasobservedat2h24hpostinfectionHowever,at12h,ASMclusteringonlipidraftscanbeinducedby1and10butnot100CFU/cellM.pneumoniae10