英文版-Indiana-University-Purdue-University-Fort-Wayne微生物学授课讲义-lecture-09课件

BacterialToxinsBIOL533Lecture9MedicalMicrobiologyBIOL5331BacterialToxins:

GeneralAspectsDefinitionSolublesubstancesthatalternormalmetabolismofhostcellswithdeleteriouseffectsonthehostHostrangeKnownforbacteria,butpossiblethattheyplayaroleindiseasescausedbyfungi,protozoa,andwormsBIOL5332BacterialToxins:

GeneralAspectsToxintypeExotoxin—proteinproducedbybacteriaeitherexcretedorboundtobacterialsurfaceandreleasedwhenlysedEndotoxin—lpsoftheoutermembraneofGram—bacteriaActsastoxinonlyunderspecialcircumstancesBIOL5333BacterialToxins:

GeneralAspectsSpecificitySomeactoncertaincelltypesOtheraffectwiderangeofcellsandtissuesNumbersproducedbysinglebacteriumSomeproducenonePneumococciBIOL5334IsToxinImportantinInfection?Questionstoask:Isvirulencequantitativelycorrelatedwithtoxinproduction?Doesthepurifiedtoxinproducedamage?Canaspecificantibody(antitoxin)preventoralleviatethemanifestationsofthedisease?Iftoxinproductionisimpairedbyamutation,isthediseaseprocessaffected?BIOL5335IfSo,WhatareToxinProperties?Questionstoask:Whatisthemechanismofaction?Whyisitspecificforcertaincellsortissues?Doesthepathogenmakeothertoxins,andifso,dotheyinteractwithoneanother?Somemakenone:pneumococciSomemakeonlyone:agentsthatcausecholera,diphtheria,tetanus,andbotulismOthermakemany:staphylococci,streptococciBIOL5336ToxinProductionPropertiesDispensable,butessentialundercertainsituationswheresurvivalandspreadareatstakeGenesfrequentlycarriedonplasmidsandtemperatebacteriophageBIOL5337ToxinProductionFoundonphage;toxingenesfor:DiphtheriaBotulismScarletfeverToxicstreptococci(“flesh-eating”)Foundonplasmids:E.colitoxincausesdiarrheaS.aureustoxincauses“scaldedskinsyndrome”E.coli0157:H7BIOL5338ToxinProductionPropertiesMobileelementsensurethatgenescanbespreadtonontoxigenicderivativesorbelostfromcellExperimentallycalled“curing”—getnontoxigenicderivativesPhaseofproductionSomeproducedcontinuouslybygrowingbacteriaOthersynthesizedwhencellsenterstationaryphase(truealsoformanyantibiotics)BIOL5339ToxinProductionExplanationCertaintoxinsmayhelpbacteriagetscarcenutrientsExample:highlevelsofdiphtheriatoxinproducedwhencelldepletedofironVerylittlefreeironinnormaltissueIsthisawayfororganismstoobtainitfromdeadtissue?BIOL53310ToxinProductionSporulatingbacteriasometimesreleasetoxinsduringsporeformationBacterialcellseventuallylyseandliberatecytoplasmicproteins,includingtoxinsExamples:organismsthatcausebotulism,gasgangrene,ortetanusIncontaminatedwound,someorganismsaregrowingandsomearesporulatingEndresultiscontinualproductionBIOL53311MechanismofActionGeneralaspectsSphereofinfluenceSomeactlocally,killingwbcnearbyOthershelporganismtospreadinhostitssuesbydegradingconnectivetissueStillothersaredisseminatedveryfarfromsitewheresynthesizedDiphtheriatoxinmadeinthroat,butactsonheartandbrainBIOL53312MechanismofActionLeveloftoxicityWorkatextremelylowlevels;includestrongestpoisonsknown1gtetanus,botulinus,orShigatoxinisenoughtokill10millionpeople100-foldmoreisrequiredfordiptheria1000-foldmoreforPseudomonasABIOL53313MechanismofActionMechanismsofdamageLysisofhostcellsStoporinterferewithcellgrowthExaggeratenormalphysiologicalmechanismsBydepressingoraugmentingparticularfunctions,toxinscankillwithoutdamaginganycellsTetanustoxinparalyzesbodywithoutaffectingtargetneuronsCholeratoxinspeedsupnormalexcretoryprocess,resultinginmassivelossofwaterBIOL53314MechanismofActionToxinsthatassistbacterialspreadintissuesPropertiesDonottargetanytypeofcellIncludedegradativeenzymesthatallowspreadingBIOL53315MechanismofActionExamples:StreptococciSomesecreteHyaluronidase—breaksdownhyaluronicacid(connectivetissue)DNase—thinsoutpusmadeviscousbyDNAfromdeadwhitebloodcellStreptokinase(protease)—cleavesprecursorofplasminogenactivatortoactiveformConvertsplasminogentoplasmin(serumproteasethatdissolvesfibrinclots)BIOL53316MechanismofActionExamples:SimilarrolessuggestedforelastasesandcollagenasesofotherorganismsInthiscase,areunregulatedformsofenzymesthatalsoexistinuninfectedhost(activityisnormallyundercontrol)BIOL53317MechanismofActionToxinsthatlysecellsGeneralaspectsLargeclasskillhostcellsbydestroyingtheirmembranes;actaslipasesExampleoflipasetype:Clostridiumperfringens

(gasgangrene)lecithinaseLysescellsindiscriminatelybecausephosphatidylcholine(lecithin)isubiquitousinmammalianmembranesAlsohemolysinsareofthistype;lysebothredbloodcellsandwhitebloodcellsBIOL53318MechanismofActionActbyinsertingthemselvesinmembraneformingporesMechanism:makemembranemorepermeable,waterpoursintocytoplasm,cellbeginstoswell,andeventuallyburstsAtverylowconcentrations(notenoughtocauselysis),cellfunctionsmaybeseverelydamaged.Slightperturbationsofpermeabilitycause:LeakageofpotassiumionsneededforproteinsynthesisandcellviabilityLowlevelsinhibitphagocytefunctioningBIOL53319MechanismofActionExamples:Staphylococci-toxin(homogeneousporeformer)Receptorsexist—cellsshow100-foldrangeinsensitivityConsequencesofaction:aggregationofplateletsandnarrowingofbloodvesselsleadstonecrosisBIOL53320MechanismofActionExamples:Streptococcalstreptolysin0(heterogeneousporeformer)BindstocholesterolincellmembraneFreetoxincanbeinactivatedbycholesterol,butonceboundbymembrane,itisimperviousConsequencesoftheaction:lysesredbloodcells,butnotneutrophilsormacrophageWhitebloodcellsarekilledbylowlevelsoftoxinbecauseitactspreferentiallyonmembranesoflysosomes,releasinghydrolyticenzymesBIOL53321MechanismofActionToxinsthatblockproteinsynthesisStructureandmodeofactionToxinsthatworkoutsidethecellarevariableinstructureandmodeofactionToxinsthatworkinsidehaveanumberofsimilaritiesBIOL53322MechanismofActionSimilaritiesMosthavetwoportions(A-Btoxins)SubunitsToxicactivity(A)Bindingtocellmembrane(B)CanbeonepolypeptidechainormanyBindingtomembranemaybefollowedbyreceptor-mediatedendocytosisandinternaliztionofthetoxin(someinvestigatorsproposedirectpassagethroughpore)BIOL53323MechanismofAction“A”moityisoftenlatent,evenafterengulfmentMaybeactivatedbyproteoyticcleavageandreductionofdisulfidebridgesToxinsofdiphtheria,cholera,tetanus,andShigella

aresynthesizedasinactiveprecusorsBIOL53324MechanismofActionMayhavecommonmodeofactionCatalyzetransferofadenosine-diphosphategroupfromNADtotargetproteinsExamplesofADP-ribosyltransferases—toxinsof:DiphtheriaCholeraExotoxinA(Pseudomonasaeruginosa)BIOL53325DiphtheriaToxinHowdoestoxinentercell?AandBaresinglepolypeptidechainHydrophobicBportionbindstoreceptoronmembraneBythistime,moleculeiscleavedatsensitivesitebetweenAandBportions,butisstillcovalentlyassociatedbydisulfidelinkageEntirereceptor-toxincomplexenterscellbyreceptor-mediatedendocytosisBIOL53326DiphtheriaToxinOncetoxinenters,reductionS-SbondseparatesAandBportionAcidicconditionswithinendosomalvesiclespromoteinsertionofBchainintoendosomalmembraneSomehow,thisfacilitatespassageofAintocytosolResistanttodenaturationandislong-livedwithincellsAccountsinpartforpotency(singlemoleculecankillcell)BIOL53327DiphtheriaToxinMechanismofkillingADP-ribosylationofEF2(proteinthatcatalyzeshydrolysisofGTPthatdrivesmovementofribosomesoneucaryoticmRNA)Reactionis:

EF-2+NAD+ADPR-EF2+H+BIOL53328DiphtheriaToxinEF2isonlyknownsubstratefordiphtheriatoxinEF2containsraremodificationofoneofhistidineresiduesandthisissiterecognizedbytoxinMutantcellsthatcannotmodifysiteareresistantAdditionofADP-riboseinactivatesEF2KillscellsbyirreversibleblockofproteinsynthesisP.aeruginosaexotoxinAworkssameasdiphtheriatoxinBIOL53329MechanismofActionPhamacologicaltoxins(elevationofcAMP-cholera)ExcessofcAMPinterfereswithphagocytefunctioning(chemotaxisandphagocytosis)Methodsofincreasing:SecretionofcAMPSecretionofadenylcyclasetomakemorecAMPSecretionoftoxinaltersactivityofhostadenylcyclase(cholera)BIOL53330CholeraToxinTargettissueissmallintestineepitheliumStructureandmechanismoftoxinToxinhasseparateAandBsubunitsBhasaffinityforintestinalepithelialmucosaAADP-ribosylatesGTPase(partofcomplexthatmakescAMP)SynthesisofcAMPbecomesunregulated;madeinlargeamountsProvokeslossoffluidsandcopiousdiarrheaBIOL53331CholeraToxinStructureofsubunitsFiveBsubunitsandoneAsubunitAsubunitissynthesizedassinglechainThen,aftersecretion,cleavedintotwofragments(A1andA2;heldtogetherbydisulfidebonds)BIOL53332CholeraToxinMechanismWholetoxinbindsto5gangliosidereceptorsonsurfaceofintestinalepithelialcellsA1-A2portionenterscellandiscleavedintoA1andA2pieces(byreductionofdisulfidebonds)A1fragmentinenzymaticallyactiveBIOL53333CholeraToxinRegulationNormalAdenylatecyclasecomplexismembraneboundandiscomposedofthreeproteins(Gs,R,cyclase)GsproteinisGTPaseproteinwithtwoconformationalstatesBindsGTP—stimulatesadenylcyclasetomakecAMPGTPasethatcleavesGTPtoGDPBIOL53334CholeraToxinBalanceisdeterminedbybindingofRproteinBindingofGTPbyGsstimulatedbybindingRproteinRisreceptorforseveraldifferenthormones(adenergics)Wholepicture—whenRproteinbindswithhormone,interactswithGsproteintoincreaseitsbindingofGTPGsremainsinactivestatetostimulateadenylcyclaseBIOL53335CholeraToxinAbnormal(cholera)normalactionofRproteinmimickedbycholeratoxinPromotesactivestateofGsproteinbydifferentmechanismADP-ribosylatesGsatoneofitsarginineresidues(Gsproteinlockedintoactiveconformation)BIOL53336MechanismofActionOthertoxinsthatactivateadenylatecyclaseNumberofenterotoxinsthatproducediarrheaLT(labile)—E.coliBordetellapertussis

adenylatecyclaseRaiselevelcAMPinleucocytesBIOL53337MechanismofActionToxinsthatblocknervefunctionMostlethaltoxinsknownaretetanusandbotulinumtoxinsTetanustoxinproducesirreversiblemusclecontractionBotulinumtoxinblocksmusclecontractionBIOL53338MechanismofActionGeneralmechanismofbothConsistofsinglepolypeptidechainswithAandBregionsBindingtogangliosidereceptorsspecificfornervetissueActivatedbyproteolysisanddisulfidereduction,andtheyf