修磊31408053微生物AbstractIntroductionMaterialsandMethodsResultsDiscussionTableSupplementalEthicsStatement

PrimaryBovineMammaryEpithelialCellCulture

Photomicrographsoftheprocessobtainedbovineepithelialcellsandmorphologyofepithelialcells.(A-F).InpanelA,singlecellsandsmallepithelioidislandsofcellswereapparent,spreaduponthesubstrateafter24hlater,singlecellswiththegranularappearanceofmacrophageswerepresent.whichnotedbyredcolor(100×);InpanelB,alargeepithelioidislandsgeneratedfromasinglecellafter3days,Occasionalmacrophageswerepresent,whichmadeup<5%ofallattachedcells(100×);.InpanelC,manylargeepithelioidislandsgeneratedwerepresentafter9days,andphagocytesweredisappeared(100×);InpanelD,areasoflargecellsarosewithinislandsaftertwoweeksinculture(100×);InpanelE,theemulsiondropletssecretedtotheoutsideofthecells(100×);InpanelF,thepurifiedepithelialcellsobtainedoverthreepassages(100×).Immunofluorescenceanalysisofbovinemammarycells(G-H).InpanelG,firstantibody(anti-cytokeratin8)replacedbyPBSasnegativecontrol,whichpicturedbyfluorescencemicroscope.Thecellnucleuswereblue(DAPI)(100×);InpanelH,fluorescentimageofcellsincubatedwithanti-cytokeratin8monoclonalantibody,picturedbyfluorescence(100×).InvasionAssay

PBMECswerewashedthreetimes.Thereafter,1mlofasuspensionofS.aureusinDMEMwasaddedperwell.Cellmonolayersinfectedwithbacteriawereincubatedat37℃in5%CO2for4h.Afterincubation,monolayerswerewashedthreetimeswith1ml/wellofphosphatebufferedsaline(PBS,pH7.4),supernatantswerereplacedwithtissue-growthmediumcontaininggentamicin(100mg/ml)andincubatedfor2hat37℃.Aftercell-culturemediumcontainingantibioticswasremoved,pBMECswerewashedthreetimeswithPBS,treatedwith1ml/welllysisbuffercontaining0.25%trypsinand0.25%TritonX-100inPBS.Suspensionsofbacteriaforinoculation,cell-culturesupernatantstreatedwithantibioticsandPBMEClysateswereplatedinduplicateontrypticasesoyagarusingserial10-folddilutionsandincubatedovernightat37℃.Thenumberofbacterialcellswasdeterminedbycolonycount.DGETagPreparationDGETagPreparationTherawdata(tagsequencesandcounts)weredepositedintheSRA(:///sra/)databaseundersubmissionnumberSRP029201.Thesequencing-obtainedrawimagedataweretransformedbybasecallingintosequencedata,calledrawdataorrawreads.Therawsequenceshad39adaptorfragments,low-qualitysequences,andseveraltypesofimpurities.Rawsequencesweretransformedintocleantagsaftervariousdatprocessingsteps.ProcedureofbioinformaticsanalysisfordigitalgeneexpressionprofilingGeneExpressionAnnotation

TomaptheDGEtagsofthetotalRNA,wecreatedavirtuallibrarythatcontainedallpossibleCATGsitesand17-basereferencegenesequences[29].Allcleantagsweremappedtothereferencesequences,andonlya1-bpmismatchwasconsidered.Cleantagsthatmappedtoreferencesequencesfrommultiplegeneswerefiltered.Theremainingcleantagsweredesignatedunambiguouscleantags.ThenumberofunambiguouscleantagsforeachgenewascalculatedandnormalizedtotheTPM(numberoftranscriptspermillioncleantags)[28,30–31].

ScreeningofDifferentiallyExpressedGenes(DEGs)

Tocomparethedifferencesingeneexpressionbetweensamples(C/S56,C/S36,C/S178,S56/S36,S56/S178,S36/S178),thenumberofrawcleantagsineachlibrarywasanalyzedstatisticallyasdescribed[32].WithreferencetoClaverieetal.[32],wehavedevelopedarigorousalgorithmtoidentifydifferentiallyexpressedgenesbetweentwosamples.Thepvaluecorrespondedtothedifferentialgeneexpressiontest,andthefalsediscoveryrate(FDR)wasusedtodeterminethethresholdofpvaluesinmultipletestsandanalysisthroughmanipulatingtheFDRvalue.Ap-value＜0.005,FDR≤0.01,andestimatedabsolutelog2-foldchange≥1weresetasthethresholdofsignificantdifferenceingeneexpression[33].ClusterAnalysisofDEGsPatternsGeneswithsimilarexpressionpatternsusuallycorrelatefunctionally.Weperformaclusteranalysisofgeneexpressionpatternswiththecluster[34]andJavaTreeview[35]programs.GeneOntologyFunctionalEnrichmentAnalysisforDEGs

GOisaninternationalstandardizedfunctionalclassificationsystemthatprovidesadynamic,updated,controlledvocabularyandastrictlydefinedconcepttodescribethepropertiesofgenesandtheirproductsinanyorganismcomprehensively.GOhas3ontologies:molecularfunction,cellularcomponent,andbiologicalprocess.Duringgeneontologyanalysis,thegenesetswithP≤0.05shouldbedeemedsignificantlyenriched.PathwayEnrichmentAnalysisofDEGs

Differentgenesusuallycooperatewitheachothertoexercisetheirbiologicalfunctions.Pathway-basedanalysishelpstofurtherunderstandgenesbiologicalfunctions.KyotoEncyclopediaofGenesandGenomes(KEGG)isthemajorpublicpathway-relateddatabase.PathwayenrichmentanalysisidentifiessignificantlyenrichedmetabolicpathwaysandsignaltransductionpathwaysinDEGscomparedwiththeentiregenomicbackground.ThecalculatingformulaofpathwayenrichmentanalysisisthesameasthatintheGOanalysis.PathwayswithQ≤0.05wereconsideredsignificantlyenriched.QuantitativeReal-timePCRAnalysisThecDNAofpBMECswasreversetranscribedusingtheTRIzolreagentaccordingtothemanufacturer’sprotocol(Tiangen,Dalian,China).Real-timeRT-PCRreactionswereper-formedusingSYBRPremixExTaq(Takara,Dalian,China)onanABIPrism7500SequenceDetectionSystem(AppliedBiosystems,FosterCity,USA).Glyceraldehyde-3-phosphatede-hydrogenase(GAPDH)wasusedfornormalization[36].SenseandantisenseprimersforthegenesareshowninTable2.Eachreactionvolumewas20ul,andthereactionconditionswereasfollows:95℃for5min,followedby40cyclesat94℃for15s,56℃for15s,and72℃for30s.Relativeexpressionlevelswerenormalizedtoglyceraldehyde-3-phosphatedehydrogenase(GAPDH).ReturnCharacterizationofstaphylococcalStrainsAnalysisofDGELibraries

ToconfirmthetranscriptionalchangesinpBMECs,weanalyzedtheglobalchangesingeneexpressionusingtheSolexa/IlluminaDGEsystem,atag-basedtranscriptomesequencingmethod.Wegenerated4DGElibraries:1DGElibraryfromthecontrolgroupand3DGElibrariesfromeveryS.aureus-infectedcellsgroups.Thechiefcharacteristicsoftheselibrariesaresummarizedintable3.IdentificationofDEGsThereweredifferentiallyexpressedgenesfromthecomparisonofeachinfectedpBMECslibrarywiththecontrol(i.e.,CvsS36,Cvs