生物信息資源 楊軍-系統(tǒng)生物學(xué)應(yīng)用學(xué)習(xí)資料

系統(tǒng)生物學(xué)技術(shù)在生命科學(xué)研究中的應(yīng)用楊軍什么是系統(tǒng)生物學(xué)？SystemsBiology目的是通過(guò)對(duì)細(xì)胞內(nèi)所有成分間的相互作用進(jìn)行量化的描述，以達(dá)到在系統(tǒng)水平上理解這些相互作用這種研究的目標(biāo)就是建立這些復(fù)雜系統(tǒng)的計(jì)算機(jī)模型，從而可以預(yù)測(cè)生物系統(tǒng)對(duì)任何干擾（如環(huán)境波動(dòng)，遺傳突變等）所發(fā)生的反應(yīng)此類(lèi)研究中最重要的部分就是確定系統(tǒng)中所有的成分，以及各成分如基因間的調(diào)控關(guān)系、蛋白質(zhì)之間和生化通路之間的相互作用，并用這些知識(shí)建立一個(gè)原始模型（生化的或數(shù)學(xué)的）一旦這個(gè)系統(tǒng)結(jié)構(gòu)建立了，系統(tǒng)行為就可以進(jìn)一步通過(guò)遺傳或環(huán)境因素的干擾進(jìn)行分析分析得到的數(shù)據(jù)則可與原始模型整合，或用來(lái)改進(jìn)模型，使得預(yù)期結(jié)果與實(shí)驗(yàn)觀(guān)察結(jié)果一致因此，系統(tǒng)生物學(xué)是一個(gè)通過(guò)對(duì)比實(shí)驗(yàn)結(jié)果與預(yù)期結(jié)果而形成新模型并進(jìn)行新實(shí)驗(yàn)驗(yàn)證的重復(fù)過(guò)程最終目的:通過(guò)這些詳細(xì)的研究能夠使研究者按照特定的設(shè)計(jì)原理進(jìn)行模擬構(gòu)建具有他們所需要的特性的生物系統(tǒng)技術(shù)手段對(duì)一個(gè)細(xì)胞反應(yīng)進(jìn)行系統(tǒng)化的定性需要能夠在多水平上產(chǎn)生大量信息的定量的生物測(cè)量手段目前此類(lèi)的方法中最為人知的就是對(duì)基因組（genome）進(jìn)行研究的基因組學(xué)（genomics）和對(duì)蛋白組（proteome）進(jìn)行研究的蛋白組學(xué)（proteomics）技術(shù)生物信息學(xué)（bioinformatics）基因組學(xué)：成千上萬(wàn)個(gè)基因的表達(dá)水平可以用DNA芯片的技術(shù)同時(shí)進(jìn)行測(cè)量和分析，而且還可以用基因芯片技術(shù)進(jìn)行測(cè)序，發(fā)現(xiàn)單核苷酸多態(tài)性，以及基因剪接的鑒定蛋白組學(xué)則側(cè)重于決定盡可能多的蛋白的結(jié)構(gòu)、表達(dá)、定位、生化活性及相互作用等目前常用的蛋白組學(xué)技術(shù)有雙向電泳和質(zhì)譜相結(jié)合的方法（如雙向電泳與飛行質(zhì)譜MALDI-TOF的結(jié)合）也有高效液相色譜與質(zhì)譜相結(jié)合的方法（如LC-MS/MS）等應(yīng)用這些技術(shù)已經(jīng)得到了大量的生物學(xué)數(shù)據(jù)，并且已經(jīng)被系統(tǒng)的組織成幾個(gè)在線(xiàn)的數(shù)據(jù)庫(kù)，如：TheNationalCenterforBiotechnologyInformation()ProteinDataBank(http://www.rcsb.rog/pdb)KyotoEncyclopediaofGenesandGenomes(http://www.genome.ad.jp/kegg)BiomolecularInteractionNetworkDatabase(/products/BIND)EncyclopediaofEscherichiacoliGenesandMetabolism()等包含了諸如核苷酸序列、氨基酸序列、分子特性、相互作用和通路等多種信息。這些數(shù)據(jù)庫(kù)對(duì)于生物系統(tǒng)模型的建立是必不可少的在研究中的應(yīng)用：舉例：共調(diào)控基因的機(jī)理研究共調(diào)控基因可能參與一些緊密相關(guān)的生理功能可能由共同的轉(zhuǎn)錄因子激活環(huán)境致癌物誘導(dǎo)的細(xì)胞應(yīng)激反應(yīng)基因芯片分析GOanalysesofsomeoftheresultsTranscriptionfactorsInvolvedincellcycleregulationKinases蛋白組學(xué)分析為什么？許多信息僅從基因的研究是無(wú)法得到的蛋白質(zhì)，而非基因，才決定了細(xì)胞的表型僅僅研究基因無(wú)法闡明疾病、衰老、環(huán)境影響等的機(jī)理部分被誘導(dǎo)的蛋白是否有共同的激活方式？基因表達(dá)的調(diào)控可在多環(huán)節(jié)上，但最重要的一個(gè)環(huán)節(jié)在轉(zhuǎn)錄的起始：轉(zhuǎn)錄因子（TF）結(jié)合到轉(zhuǎn)錄因子結(jié)合位點(diǎn)（TFBS）這些蛋白的基因上是否有共同的TFBS？傳統(tǒng)方法：構(gòu)建缺失體，DNaseI超敏感位點(diǎn)，DNA印跡，凝膠阻滯等缺點(diǎn)：長(zhǎng)時(shí)間，人力，物力生物信息學(xué)：避免浪費(fèi)大量人力、物力可行性：人類(lèi)基因組全序列，TFBS預(yù)測(cè)軟件缺點(diǎn)：假陽(yáng)性如何解決假陽(yáng)性？進(jìn)化足跡法在DNA序列非功能區(qū)的突變率應(yīng)高于功能區(qū)有功能的DNA序列一般比較保守如一段DNA序列在幾個(gè)物種中都保守，則應(yīng)有功能在非保守序列（即非功能序列）中的TFBS可去除軟件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數(shù)據(jù)庫(kù)GenBank/entrez/query.fcgi?db=NucleotideTRANSFAC5.0(transcriptionfactors)/cgi-bin/pub/databases/transfac/search.cgi?SWISS-PROT&tremble/sprot/

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

程序確定人和鼠中的同源基因?qū)Υ_定5’端用軟件確定啟動(dòng)子區(qū)比較人和鼠基因發(fā)現(xiàn)保守序列在人和鼠中分別尋找TFBS比較，選擇那些只在保守區(qū)出現(xiàn)的TFBS最后選擇8個(gè)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

the

conservedregion

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

AP1C/EBPCMYBELK1GATANFY

USFAREB6CAATNKX25HAND1E47Experimentalvalidation:gelmobilityshiftassay

GATA

NFY

NEfromMNNGtreatedcell

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NEfromDMSOtreatedcell

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Unlabeledmutatedoligonucleotides

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Unlabeledconsensusoligonucleotides

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labeledcontrololigonucleotides

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labelledconsensusoligonucleotides

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NFY

binding

FreeProbe

GATAbinding

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?……在疾病研究中的應(yīng)用：發(fā)現(xiàn)前列腺癌的新標(biāo)記比較正常前列腺組織和一個(gè)前列腺腫瘤細(xì)胞系的mRNA表達(dá)，發(fā)現(xiàn)554個(gè)表達(dá)的轉(zhuǎn)錄因子中，有112個(gè)改變前列腺組織與其他組織mRNA比較，發(fā)現(xiàn)有近300個(gè)僅在前列腺組織表達(dá)其中60個(gè)含有信號(hào)肽，可能為分泌蛋白針對(duì)其中一個(gè)蛋白在血液中進(jìn)行檢測(cè)，可識(shí)別5/10早期前列腺癌和5/10晚期前列腺癌，而經(jīng)典的PSA不能識(shí)別早期，但識(shí)別大部分晚期前列腺癌患者CancerOutlierProfileAnalysis(COPA)假設(shè)：一個(gè)癌基因的過(guò)度表達(dá)可以在基因芯片的結(jié)果中體現(xiàn)出來(lái)對(duì)132組基因表達(dá)數(shù)據(jù)，共10486基因芯片實(shí)驗(yàn)結(jié)果()進(jìn)行分析對(duì)已知的兩個(gè)在腫瘤(myeloma)中高表達(dá)的癌基因的驗(yàn)證：鑒定得到兩個(gè)前列腺癌中高表達(dá)的基因：ETV1和ERG不同數(shù)據(jù)組可得到相同的結(jié)果部分通過(guò)COPA分析得到的結(jié)果ERG與ETV1高表達(dá)的原因前列腺特異基因TMPRSS2與ERG或ETV1的融合FISH驗(yàn)證系統(tǒng)生物學(xué)的新方向、新技術(shù)脂類(lèi)組學(xué)糖類(lèi)組學(xué)微流控芯片納米機(jī)械生物分子檢測(cè)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