StrategiesforStudying

MicrobialPathogenesisBIOL533Lecture4MedicalMicrobiologyBIOL5331StrategiesforStudying

MicrobReporterGeneFusionsOperonfusion:transcriptionalPromoterfromforeigngeneTranslationalsequencefromreportergeneBIOL5332ReporterGeneFusionsOperonfuReporterGeneFusionsGenefusion:translationalPromoterandtranslationalsignalfromforeigngeneInitiationsignal/+NH2codingsequenceTranslationalsignalalsofromreportergeneInitiationsequenceandpartofNH2sequenceofreportergenemissing,butCOOHregionstillpresentTwocodingsequencesmustbeinframeBIOL5333ReporterGeneFusionsGenefusiTechniqueUsetransposonvectorcontaininglacfusionCarriespromoterlesslacZgeneandselectableantibioticmarkerSelectionforantibioticmarkergeneratessetofrandominsertionsinchromosomeColoniescarryingtransposonscreenedforexpressionunderconditionstestedBIOL5334TechniqueUsetransposonvectorForeignGeneExpressionAssayforproductionofβgalHighertemperature+—ironetc.LocateunknowngeneBIOL5335ForeignGeneExpressionAssayflacZFusionUsedtofindregulatorygenesthatcontrolexpressionofvirulencegenelacZfusionstrainismutagenized(chemicaloranothertransposon)ResultingcoloniesscreenedforaberrantregulationBIOL5336lacZFusionUsedtofindregulalacZFusionAberrantregulation:ColoniesarenotLac+inpresenceofinducingconditionsLossofactivatorColoniesareLac+underbothinducingandnon-inducingconditionsLossofrepressorBIOL5337lacZFusionAberrantregulationlacZFusionGenetaggedbytransposoncanbeclonedSegmentofDNAadjacenttotransposon:Canbeusedashybridizationprobetoclonewild-typelocusCanalsobeclonedbycomplementationClonedDNAcanbesequencedBIOL5338lacZFusionGenetaggedbytranphoAGeneFusionsVirulencegenesaresmallsubsetoftotalgenes,sorandommutagenesiswillaffectmanymorenon-virulencegenesWaytooptimizenumberofvirulencegenemutationstakesadvantageoffactthatmostvirulencegenesareonbacterialsurfaceorexcretedintomediumBIOL5339phoAGeneFusionsVirulencegenphoAGeneFusionsTranslationalproteinfusionAlkalinephosphataseisexpressedonlyinperiplasmForeigngeneproducthastobeexpressedoutsideofcytoplasmInvector,signalsequencemutationsblockexportofalkalinephosphataseandrenderitinactiveBIOL53310phoAGeneFusionsTranslationalphoAGeneFusionsExportandactivityrestoredbyfusinginframearestrictionfragmenttotruncatedphoAgene;i.e.,lackingsignalsequencesNoAUGstartcodonNoribosomebindingsiteRestrictionfragmenthasportionsofNH2-terminalgenesencodingsignalsequencesofheterologousproteins,suchasOmpForLamBBIOL53311phoAGeneFusionsExportandacTnphoATransposonCreatedTnphoAthatrandomlyinsertsphoAintotargetgenesHybridproteinsdisplayalkalinephosphataseactivityonlyiftargetgeneencodesaperiplasmicoutermembraneorextracellularproteinBIOL53312TnphoATransposonCreatedTnphoTnphoATransposonUseTnphoA:StudytopologyofinnermembraneproteinsβgalfusionsexpressedonlyincytoplasmIdentifygenesthatencodeexportproteinsExportedproteinsrepresentmajorclassofvirulencefactorsNumberofadditionaldeliverysystemsRememberanalysisofGroismanandHeffronBIOL53313TnphoATransposonUseTnphoA:BIAnalysisofVirulenceStudyTnphoAmutantsofS.typhimuriumidentifiedasPho+onL-agarplatesTestedforvirulencebyoralinfectionofBALB/CmiceFound15/150Pho+mutantswereavirulent9/15weredefectiveinLPSbiosynthesisNonehadinsertionsinvirulenceplasmidBIOL53314AnalysisofVirulenceStudyTnpAnalysisofVirulenceConclusion:samecaveatsasapplytonormaltransposonmutagenesis:ButuseofTnphoAlimitsidentificationofrandominsertionstothoseaffectingcellenvelopefactorsBIOL53315AnalysisofVirulenceConclusioAnalysisofVirulenceDisadvantage:Limitsselectiontogenesexpressedin S.typhimuriumonL-agarGenesexpressedatlowlevelsorrepressedonL-agarplateswouldbemissedBIOL53316AnalysisofVirulenceDisadvantAnalysisofVirulenceExpressionproblemmayexplainwhy9/15Pho+avirulentmutantstrainswereLPS—ratherthanbeingdefectiveinothervirulencegenesBIOL53317AnalysisofVirulenceExpressioAnalysisofVirulenceTnphoAnarrows,butdoesn’tsolvealldetectionproblemsWhiledisruptinggene,theTnphoAmutationsproducealkalinephosphatasehybridproteinsCouldbephenotypenotduetolossofgene,butpresenceofdeleterioushybridprotein;Therefore,tobesuremutationinvirulencegene,mustconductothergeneticanalysesBIOL53318AnalysisofVirulenceTnphoAnaInVivoExpressionTechnologyMahanet.al.,1993.Science259:686-688Basedongenethat,whenmutated,causesorganismtobeattenuatedUsedSalmonellatyphimuriumΔpurA—lacZYbothlackpromoter (insuicideplasmid)BIOL53319InVivoExpressionTechnologyMInVivoExpressionTechnologySau3AdigestedchromosomalDNAelectroporatedSalmonellaPurA—strainInjectedintomicetwiceAftereach3daysharvestspleenPlateonartificialmediatodetectLac—strainsBIOL53320InVivoExpressionTechnologySInVivoExpressionTechnologyBasedsolelyonfactthativigenesarehighlyexpressedinanimaltissues,butarenotexpressedinlabmediaSelectionstrategy:BacterialstrainwithmutationthatattenuatedgrowthinvivoComplementedbyoperonfusionstosameattenuatedgeneBIOL53321InVivoExpressionTechnologyBInVivoExpressionTechnologyCriteriaforIVETsystem:FusionsinsinglechromosomalcopyNoplasmidcomplicationsFusionsconstructedwithoutdisruptionofgeneLossofgenecouldbelethaltobacteriumMonitoredbothinanimalandonlabmediaBIOL53322InVivoExpressionTechnologyCpIVETVectorpIVET1plasmidSuicidevectorReplicationdependentonPireplicationproteinSalmonelladoesnotproducePromoterlesspurAgeneandlacZoperonCanmonitorlevelortranscriptionofentireoperonUniquecloningsite5’topurAgeneClonerandomfragmentsofDNAblageneallowsplasmidtobeselectedBIOL53323pIVETVectorpIVET1plasmidBIOLpIVETVectorConstructionofclonesCloneSau3AfragmentsintovectorPutfragmentpoolintoPurAdeletionstrainofSalmonella(—Pireplicationprotein)SelectionforApRplasmid:integratesathomologousSalmonellageneNonaturallacZgeneDeletedpurAgeneBIOL53324pIVETVectorConstructionofclpIVETVectorClonesofinterestProductofplasmidintegrationgeneratesaduplicationOnepromoterdrivespurAlacZwhileotherdriveswild-typecopyofvirulencegeneIntegrateatunknowngenebecausepurAandlacZarefromE.coli;notenoughhomologytoSalmonellaBIOL53325pIVETVectorClonesofinterestPropertiesofClonesTosurviveandpropagateinhost:purAlacZfusionhastobeexpressedatsufficientleveltoovercomedeletionofpurAIfgeneofinterestcodesforvirulencefactor,thenexpressionalsorequiredforinfectionMonitorexpressioninanimalandlabbymeasuringβgalfromstrainsthatsurvivethreedaysandarerecoveredfromspleenBIOL53326PropertiesofClonesTosurviveAnalysisofVirulenceToidentifygenescodeforvirulence,junctionsoffusionsclonedandsequencedPreparedinactivatedgenesAnyeffectonpathogenesis?Transposoninsertionsin3/5genesBALB/CmiceinfectedorallyhaveincreasedLD50(200to20,000fold)Therefore,IVETselectsforvirulenceAlso,miceimmunizedwith1mutantwereprotectedwhenchallengedwithwild-typeBIOL53327AnalysisofVirulenceToidentiIdentityofGenesResults:Mostgenesfoundsofararehousekeepinggenes,perhapsbecauseofscreeningconditionsScreenedonlyforexpressionofgenesonaerobicmedia,butmostbodypartsareanaerobicCrudeapproach;atbest,identifiesasetofgenesthatareexpressedatsomepointofinfectionBIOL53328IdentityofGenesResults:BIOLIdentityofGenespheSThimAoperonencodes2suphenylalanylt-RNAsynthetaseand1suhostintegrationfactor(IHF)AntisenseRfbO-antigensynthesisO-antigensynthesismutantsattenuated-orally,butvirulentIProuteMutationsthatincreaseLD50doaffectvirulenceBIOL53329IdentityofGenespheSThimAopProsandConsDetailedanalysisofgenesandgeneproductsexpressedinanimalnecessarytodeterminewhereandwhenduringinfectionthegenewasexpressedMoreseriousproblem—focusesontranscriptionalregulationBIOL53330ProsandConsDetailedanalysisProsandConsGenesthatareregulatedoractivatedpost-transcriptionallywillbemissedAlso,manygenesexpressedonlabmediamaybeimportantinvivoIfsomeaspectofinvitrogrowthconditionissimilaroridenticaltoinvivocondition(e.g.,37°C),certaingenesmaybediscardedBIOL53331ProsandConsGenesthatarereResearchStrategyTogetmaximalvariabilityingenesselected,usedifferent:ReportergenesInfectionmodelsinvitrogrowthconditionsLikelythatinfectionbydifferentroutesorhostscharacterizedbyuniqueH-PinteractionsleadtodifferentgenesBIOL53332ResearchStrategyTogetmaximaAnalysisofVirulenceHeithoffet.al.,1999(Mahan’sgroup)Previouslyfoundover100differentgenes25%unidentifiedAllaffectvirulenceLookedatregulationofgenesinintestine,liver,andspleen,plusculturedmurinemacrophageBIOL53333AnalysisofVirulenceHeithoffAnalysisofVirulenceRegulationinresponsetoMg+2,pH,andFeproceededsameasinvitroatapproximatelythesamelevelsNottrueinspleen;hastobedifferentsignalsThinkthatresponsetoMg+2andpHcorrelateswithassumingmacrophageexistenceBIOL53334AnalysisofVirulenceRegulatioUsesExpectedtoprovideAgforvaccineGenesexpressedonlyinvivoAssistinconstructionlive-attenuatedvaccines(inactivatedgenes)AlsocanestablishinvivoregulatedexpressionofheterologousAginlivevaccinesBIOL53335UsesExpectedtoprovideAgforUsesIdentificationofivigenesallowsdetectionofchangesinmetabolism,generegulation,andcellsurfacepropertiesduringgrowthinanimaltissuesAllowsnewantimicrobialtargetstobeidentifiedBIOL53336UsesIdentificationofivigeneLecture4Questions?Comments?Assignments...BIOL53337Lecture4Questions?BIOL53337StrategiesforStudying

MicrobialPathogenesisBIOL533Lecture4MedicalMicrobiologyBIOL53338StrategiesforStudying

MicrobReporterGeneFusionsOperonfusion:transcriptionalPromoterfromforeigngeneTranslationalsequencefromreportergeneBIOL53339ReporterGeneFusionsOperonfuReporterGeneFusionsGenefusion:translationalPromoterandtranslationalsignalfromforeigngeneInitiationsignal/+NH2codingsequenceTranslationalsignalalsofromreportergeneInitiationsequenceandpartofNH2sequenceofreportergenemissing,butCOOHregionstillpresentTwocodingsequencesmustbeinframeBIOL53340ReporterGeneFusionsGenefusiTechniqueUsetransposonvectorcontaininglacfusionCarriespromoterlesslacZgeneandselectableantibioticmarkerSelectionforantibioticmarkergeneratessetofrandominsertionsinchromosomeColoniescarryingtransposonscreenedforexpressionunderconditionstestedBIOL53341TechniqueUsetransposonvectorForeignGeneExpressionAssayforproductionofβgalHighertemperature+—ironetc.LocateunknowngeneBIOL53342ForeignGeneExpressionAssayflacZFusionUsedtofindregulatorygenesthatcontrolexpressionofvirulencegenelacZfusionstrainismutagenized(chemicaloranothertransposon)ResultingcoloniesscreenedforaberrantregulationBIOL53343lacZFusionUsedtofindregulalacZFusionAberrantregulation:ColoniesarenotLac+inpresenceofinducingconditionsLossofactivatorColoniesareLac+underbothinducingandnon-inducingconditionsLossofrepressorBIOL53344lacZFusionAberrantregulationlacZFusionGenetaggedbytransposoncanbeclonedSegmentofDNAadjacenttotransposon:Canbeusedashybridizationprobetoclonewild-typelocusCanalsobeclonedbycomplementationClonedDNAcanbesequencedBIOL53345lacZFusionGenetaggedbytranphoAGeneFusionsVirulencegenesaresmallsubsetoftotalgenes,sorandommutagenesiswillaffectmanymorenon-virulencegenesWaytooptimizenumberofvirulencegenemutationstakesadvantageoffactthatmostvirulencegenesareonbacterialsurfaceorexcretedintomediumBIOL53346phoAGeneFusionsVirulencegenphoAGeneFusionsTranslationalproteinfusionAlkalinephosphataseisexpressedonlyinperiplasmForeigngeneproducthastobeexpressedoutsideofcytoplasmInvector,signalsequencemutationsblockexportofalkalinephosphataseandrenderitinactiveBIOL53347phoAGeneFusionsTranslationalphoAGeneFusionsExportandactivityrestoredbyfusinginframearestrictionfragmenttotruncatedphoAgene;i.e.,lackingsignalsequencesNoAUGstartcodonNoribosomebindingsiteRestrictionfragmenthasportionsofNH2-terminalgenesencodingsignalsequencesofheterologousproteins,suchasOmpForLamBBIOL53348phoAGeneFusionsExportandacTnphoATransposonCreatedTnphoAthatrandomlyinsertsphoAintotargetgenesHybridproteinsdisplayalkalinephosphataseactivityonlyiftargetgeneencodesaperiplasmicoutermembraneorextracellularproteinBIOL53349TnphoATransposonCreatedTnphoTnphoATransposonUseTnphoA:StudytopologyofinnermembraneproteinsβgalfusionsexpressedonlyincytoplasmIdentifygenesthatencodeexportproteinsExportedproteinsrepresentmajorclassofvirulencefactorsNumberofadditionaldeliverysystemsRememberanalysisofGroismanandHeffronBIOL53350TnphoATransposonUseTnphoA:BIAnalysisofVirulenceStudyTnphoAmutantsofS.typhimuriumidentifiedasPho+onL-agarplatesTestedforvirulencebyoralinfectionofBALB/CmiceFound15/150Pho+mutantswereavirulent9/15weredefectiveinLPSbiosynthesisNonehadinsertionsinvirulenceplasmidBIOL53351AnalysisofVirulenceStudyTnpAnalysisofVirulenceConclusion:samecaveatsasapplytonormaltransposonmutagenesis:ButuseofTnphoAlimitsidentificationofrandominsertionstothoseaffectingcellenvelopefactorsBIOL53352AnalysisofVirulenceConclusioAnalysisofVirulenceDisadvantage:Limitsselectiontogenesexpressedin S.typhimuriumonL-agarGenesexpressedatlowlevelsorrepressedonL-agarplateswouldbemissedBIOL53353AnalysisofVirulenceDisadvantAnalysisofVirulenceExpressionproblemmayexplainwhy9/15Pho+avirulentmutantstrainswereLPS—ratherthanbeingdefectiveinothervirulencegenesBIOL53354AnalysisofVirulenceExpressioAnalysisofVirulenceTnphoAnarrows,butdoesn’tsolvealldetectionproblemsWhiledisruptinggene,theTnphoAmutationsproducealkalinephosphatasehybridproteinsCouldbephenotypenotduetolossofgene,butpresenceofdeleterioushybridprotein;Therefore,tobesuremutationinvirulencegene,mustconductothergeneticanalysesBIOL53355AnalysisofVirulenceTnphoAnaInVivoExpressionTechnologyMahanet.al.,1993.Science259:686-688Basedongenethat,whenmutated,causesorganismtobeattenuatedUsedSalmonellatyphimuriumΔpurA—lacZYbothlackpromoter (insuicideplasmid)BIOL53356InVivoExpressionTechnologyMInVivoExpressionTechnologySau3AdigestedchromosomalDNAelectroporatedSalmonellaPurA—strainInjectedintomicetwiceAftereach3daysharvestspleenPlateonartificialmediatodetectLac—strainsBIOL53357InVivoExpressionTechnologySInVivoExpressionTechnologyBasedsolelyonfactthativigenesarehighlyexpressedinanimaltissues,butarenotexpressedinlabmediaSelectionstrategy:BacterialstrainwithmutationthatattenuatedgrowthinvivoComplementedbyoperonfusionstosameattenuatedgeneBIOL53358InVivoExpressionTechnologyBInVivoExpressionTechnologyCriteriaforIVETsystem:FusionsinsinglechromosomalcopyNoplasmidcomplicationsFusionsconstructedwithoutdisruptionofgeneLossofgenecouldbelethaltobacteriumMonitoredbothinanimalandonlabmediaBIOL53359InVivoExpressionTechnologyCpIVETVectorpIVET1plasmidSuicidevectorReplicationdependentonPireplicationproteinSalmonelladoesnotproducePromoterlesspurAgeneandlacZoperonCanmonitorlevelortranscriptionofentireoperonUniquecloningsite5’topurAgeneClonerandomfragmentsofDNAblageneallowsplasmidtobeselectedBIOL53360pIVETVectorpIVET1plasmidBIOLpIVETVectorConstructionofclonesCloneSau3AfragmentsintovectorPutfragmentpoolintoPurAdeletionstrainofSalmonella(—Pireplicationprotein)SelectionforApRplasmid:integratesathomologousSalmonellageneNonaturallacZgeneDeletedpurAgeneBIOL53361pIVETVectorConstructionofclpIVETVectorClonesofinterestProductofplasmidintegrationgeneratesaduplicationOnepromoterdrivespurAlacZwhileotherdriveswild-typecopyofvirulencegeneIntegrateatunknowngenebecausepurAandlacZarefromE.coli;notenoughhomologytoSalmonellaBIOL53362pIVETVectorClonesofinterestPropertiesofClonesTosurviveandpropagateinhost:purAlacZfusionhastobeexpressedatsufficientleveltoovercomedeletionofpurAIfgeneofinterestcodesforvirulencefactor,thenexpressionalsorequiredforinfectionMonitorexpressioninanimalandlabbymeasuringβgalfromstrainsthatsurvivethreedaysandarerecoveredfromspleenBIOL53363PropertiesofClonesTosurviveAnalysisofVirulenceToidentifygenescodeforvirulence,junctionsoffusionsclonedandsequencedPreparedinactivatedgenesAnyeffectonpathogenesis?Transposoninsertionsin3/5genesBALB/CmiceinfectedorallyhaveincreasedLD50(200to20,000fold)Therefore,IVETselectsforvirulenceAlso,miceimmunizedwith1mutantwereprotectedwhenchallengedwithwild-typeBIOL53364AnalysisofVirulenceToidentiIdentityofGenesResults:Mostgenesfoundsofararehousekeepinggenes,perhapsbecauseofscreeningconditionsScreenedonlyforexpressionofgenesonaerobicmedia,butmostbodypartsareanaerobicCrudeapproach;atbest,identifiesasetofgenesthatareexpressedatsomepointofinfectionBIOL53365IdentityofGenesResults:BIOLIdentityofGenespheSThimAoperonencodes2suphenylalanylt-RNAsynthetaseand1suhostintegrationfactor(IHF)AntisenseRfbO-antigensynthesisO-antigensynthesismutantsattenuated-orally,butvirulentIProuteMutationsthatincreaseLD50doaffectvirulenceBIOL5