BacterialToxinsBIOL533Lecture9MedicalMicrobiologyBIOL5331BacterialToxinsBIOL533MedicaBacterialToxins:

GeneralAspectsDefinitionSolublesubstancesthatalternormalmetabolismofhostcellswithdeleteriouseffectsonthehostHostrangeKnownforbacteria,butpossiblethattheyplayaroleindiseasescausedbyfungi,protozoa,andwormsBIOL5332BacterialToxins:

GeneralAspBacterialToxins:

GeneralAspectsToxintypeExotoxin—proteinproducedbybacteriaeitherexcretedorboundtobacterialsurfaceandreleasedwhenlysedEndotoxin—lpsoftheoutermembraneofGram—bacteriaActsastoxinonlyunderspecialcircumstancesBIOL5333BacterialToxins:

GeneralAspBacterialToxins:

GeneralAspectsSpecificitySomeactoncertaincelltypesOtheraffectwiderangeofcellsandtissuesNumbersproducedbysinglebacteriumSomeproducenonePneumococciBIOL5334BacterialToxins:

GeneralAspIsToxinImportantinInfection?Questionstoask:Isvirulencequantitativelycorrelatedwithtoxinproduction?Doesthepurifiedtoxinproducedamage?Canaspecificantibody(antitoxin)preventoralleviatethemanifestationsofthedisease?Iftoxinproductionisimpairedbyamutation,isthediseaseprocessaffected?BIOL5335IsToxinImportantinInfectioIfSo,WhatareToxinProperties?Questionstoask:Whatisthemechanismofaction?Whyisitspecificforcertaincellsortissues?Doesthepathogenmakeothertoxins,andifso,dotheyinteractwithoneanother?Somemakenone:pneumococciSomemakeonlyone:agentsthatcausecholera,diphtheria,tetanus,andbotulismOthermakemany:staphylococci,streptococciBIOL5336IfSo,WhatareToxinPropertiToxinProductionPropertiesDispensable,butessentialundercertainsituationswheresurvivalandspreadareatstakeGenesfrequentlycarriedonplasmidsandtemperatebacteriophageBIOL5337ToxinProductionPropertiesBIOLToxinProductionFoundonphage;toxingenesfor:DiphtheriaBotulismScarletfeverToxicstreptococci(“flesh-eating”)Foundonplasmids:E.colitoxincausesdiarrheaS.aureustoxincauses“scaldedskinsyndrome”E.coli0157:H7BIOL5338ToxinProductionFoundonphageToxinProductionPropertiesMobileelementsensurethatgenescanbespreadtonontoxigenicderivativesorbelostfromcellExperimentallycalled“curing”—getnontoxigenicderivativesPhaseofproductionSomeproducedcontinuouslybygrowingbacteriaOthersynthesizedwhencellsenterstationaryphase(truealsoformanyantibiotics)BIOL5339ToxinProductionPropertiesBIOLToxinProductionExplanationCertaintoxinsmayhelpbacteriagetscarcenutrientsExample:highlevelsofdiphtheriatoxinproducedwhencelldepletedofironVerylittlefreeironinnormaltissueIsthisawayfororganismstoobtainitfromdeadtissue?BIOL53310ToxinProductionExplanationBIOToxinProductionSporulatingbacteriasometimesreleasetoxinsduringsporeformationBacterialcellseventuallylyseandliberatecytoplasmicproteins,includingtoxinsExamples:organismsthatcausebotulism,gasgangrene,ortetanusIncontaminatedwound,someorganismsaregrowingandsomearesporulatingEndresultiscontinualproductionBIOL53311ToxinProductionSporulatingbaMechanismofActionGeneralaspectsSphereofinfluenceSomeactlocally,killingwbcnearbyOthershelporganismtospreadinhostitssuesbydegradingconnectivetissueStillothersaredisseminatedveryfarfromsitewheresynthesizedDiphtheriatoxinmadeinthroat,butactsonheartandbrainBIOL53312MechanismofActionGeneralaspMechanismofActionLeveloftoxicityWorkatextremelylowlevels;includestrongestpoisonsknown1gtetanus,botulinus,orShigatoxinisenoughtokill10millionpeople100-foldmoreisrequiredfordiptheria1000-foldmoreforPseudomonasABIOL53313MechanismofActionLeveloftoMechanismofActionMechanismsofdamageLysisofhostcellsStoporinterferewithcellgrowthExaggeratenormalphysiologicalmechanismsBydepressingoraugmentingparticularfunctions,toxinscankillwithoutdamaginganycellsTetanustoxinparalyzesbodywithoutaffectingtargetneuronsCholeratoxinspeedsupnormalexcretoryprocess,resultinginmassivelossofwaterBIOL53314MechanismofActionMechanismsMechanismofActionToxinsthatassistbacterialspreadintissuesPropertiesDonottargetanytypeofcellIncludedegradativeenzymesthatallowspreadingBIOL53315MechanismofActionToxinsthatMechanismofActionExamples:StreptococciSomesecreteHyaluronidase—breaksdownhyaluronicacid(connectivetissue)DNase—thinsoutpusmadeviscousbyDNAfromdeadwhitebloodcellStreptokinase(protease)—cleavesprecursorofplasminogenactivatortoactiveformConvertsplasminogentoplasmin(serumproteasethatdissolvesfibrinclots)BIOL53316MechanismofActionExamples:BIMechanismofActionExamples:SimilarrolessuggestedforelastasesandcollagenasesofotherorganismsInthiscase,areunregulatedformsofenzymesthatalsoexistinuninfectedhost(activityisnormallyundercontrol)BIOL53317MechanismofActionExamples:BIMechanismofActionToxinsthatlysecellsGeneralaspectsLargeclasskillhostcellsbydestroyingtheirmembranes;actaslipasesExampleoflipasetype:Clostridiumperfringens(gasgangrene)lecithinaseLysescellsindiscriminatelybecausephosphatidylcholine(lecithin)isubiquitousinmammalianmembranesAlsohemolysinsareofthistype;lysebothredbloodcellsandwhitebloodcellsBIOL53318MechanismofActionToxinsthatMechanismofActionActbyinsertingthemselvesinmembraneformingporesMechanism:makemembranemorepermeable,waterpoursintocytoplasm,cellbeginstoswell,andeventuallyburstsAtverylowconcentrations(notenoughtocauselysis),cellfunctionsmaybeseverelydamaged.Slightperturbationsofpermeabilitycause:LeakageofpotassiumionsneededforproteinsynthesisandcellviabilityLowlevelsinhibitphagocytefunctioningBIOL53319MechanismofActionActbyinseMechanismofActionExamples:Staphylococci-toxin(homogeneousporeformer)Receptorsexist—cellsshow100-foldrangeinsensitivityConsequencesofaction:aggregationofplateletsandnarrowingofbloodvesselsleadstonecrosisBIOL53320MechanismofActionExamples:BIMechanismofActionExamples:Streptococcalstreptolysin0(heterogeneousporeformer)BindstocholesterolincellmembraneFreetoxincanbeinactivatedbycholesterol,butonceboundbymembrane,itisimperviousConsequencesoftheaction:lysesredbloodcells,butnotneutrophilsormacrophageWhitebloodcellsarekilledbylowlevelsoftoxinbecauseitactspreferentiallyonmembranesoflysosomes,releasinghydrolyticenzymesBIOL53321MechanismofActionExamples:BIMechanismofActionToxinsthatblockproteinsynthesisStructureandmodeofactionToxinsthatworkoutsidethecellarevariableinstructureandmodeofactionToxinsthatworkinsidehaveanumberofsimilaritiesBIOL53322MechanismofActionToxinsthatMechanismofActionSimilaritiesMosthavetwoportions(A-Btoxins)SubunitsToxicactivity(A)Bindingtocellmembrane(B)CanbeonepolypeptidechainormanyBindingtomembranemaybefollowedbyreceptor-mediatedendocytosisandinternaliztionofthetoxin(someinvestigatorsproposedirectpassagethroughpore)BIOL53323MechanismofActionSimilaritieMechanismofAction“A”moityisoftenlatent,evenafterengulfmentMaybeactivatedbyproteoyticcleavageandreductionofdisulfidebridgesToxinsofdiphtheria,cholera,tetanus,andShigellaaresynthesizedasinactiveprecusorsBIOL53324MechanismofAction“A”moityiMechanismofActionMayhavecommonmodeofactionCatalyzetransferofadenosine-diphosphategroupfromNADtotargetproteinsExamplesofADP-ribosyltransferases—toxinsof:DiphtheriaCholeraExotoxinA(Pseudomonasaeruginosa)BIOL53325MechanismofActionMayhavecoDiphtheriaToxinHowdoestoxinentercell?AandBaresinglepolypeptidechainHydrophobicBportionbindstoreceptoronmembraneBythistime,moleculeiscleavedatsensitivesitebetweenAandBportions,butisstillcovalentlyassociatedbydisulfidelinkageEntirereceptor-toxincomplexenterscellbyreceptor-mediatedendocytosisBIOL53326DiphtheriaToxinHowdoestoxinDiphtheriaToxinOncetoxinenters,reductionS-SbondseparatesAandBportionAcidicconditionswithinendosomalvesiclespromoteinsertionofBchainintoendosomalmembraneSomehow,thisfacilitatespassageofAintocytosolResistanttodenaturationandislong-livedwithincellsAccountsinpartforpotency(singlemoleculecankillcell)BIOL53327DiphtheriaToxinOncetoxinentDiphtheriaToxinMechanismofkillingADP-ribosylationofEF2(proteinthatcatalyzeshydrolysisofGTPthatdrivesmovementofribosomesoneucaryoticmRNA)Reactionis: EF-2+NAD+ADPR-EF2+H+BIOL53328DiphtheriaToxinMechanismofkDiphtheriaToxinEF2isonlyknownsubstratefordiphtheriatoxinEF2containsraremodificationofoneofhistidineresiduesandthisissiterecognizedbytoxinMutantcellsthatcannotmodifysiteareresistantAdditionofADP-riboseinactivatesEF2KillscellsbyirreversibleblockofproteinsynthesisP.aeruginosaexotoxinAworkssameasdiphtheriatoxinBIOL53329DiphtheriaToxinEF2isonlyknMechanismofActionPhamacologicaltoxins(elevationofcAMP-cholera)ExcessofcAMPinterfereswithphagocytefunctioning(chemotaxisandphagocytosis)Methodsofincreasing:SecretionofcAMPSecretionofadenylcyclasetomakemorecAMPSecretionoftoxinaltersactivityofhostadenylcyclase(cholera)BIOL53330MechanismofActionPhamacologiCholeraToxinTargettissueissmallintestineepitheliumStructureandmechanismoftoxinToxinhasseparateAandBsubunitsBhasaffinityforintestinalepithelialmucosaAADP-ribosylatesGTPase(partofcomplexthatmakescAMP)SynthesisofcAMPbecomesunregulated;madeinlargeamountsProvokeslossoffluidsandcopiousdiarrheaBIOL53331CholeraToxinTargettissueisCholeraToxinStructureofsubunitsFiveBsubunitsandoneAsubunitAsubunitissynthesizedassinglechainThen,aftersecretion,cleavedintotwofragments(A1andA2;heldtogetherbydisulfidebonds)BIOL53332CholeraToxinStructureofsubuCholeraToxinMechanismWholetoxinbindsto5gangliosidereceptorsonsurfaceofintestinalepithelialcellsA1-A2portionenterscellandiscleavedintoA1andA2pieces(byreductionofdisulfidebonds)A1fragmentinenzymaticallyactiveBIOL53333CholeraToxinMechanismBIOL533CholeraToxinRegulationNormalAdenylatecyclasecomplexismembraneboundandiscomposedofthreeproteins(Gs,R,cyclase)GsproteinisGTPaseproteinwithtwoconformationalstatesBindsGTP—stimulatesadenylcyclasetomakecAMPGTPasethatcleavesGTPtoGDPBIOL53334CholeraToxinRegulationBIOL53CholeraToxinBalanceisdeterminedbybindingofRproteinBindingofGTPbyGsstimulatedbybindingRproteinRisreceptorforseveraldifferenthormones(adenergics)Wholepicture—whenRproteinbindswithhormone,interactswithGsproteintoincreaseitsbindingofGTPGsremainsinactivestatetostimulateadenylcyclaseBIOL53335CholeraToxinBalanceisdetermCholeraToxinAbnormal(cholera)normalactionofRproteinmimickedbycholeratoxinPromotesactivestateofGsproteinbydifferentmechanismADP-ribosylatesGsatoneofitsarginineresidues(Gsproteinlockedintoactiveconformation)BIOL53336CholeraToxinAbnormal(choleraMechanismofActionOthertoxinsthatactivateadenylatecyclaseNumberofenterotoxinsthatproducediarrheaLT(labile)—E.coliBordetellapertussisadenylatecyclaseRaiselevelcAMPinleucocytesBIOL53337MechanismofActionOthertoxinMechanismofActionToxinsthatblocknervefunctionMostlethaltoxinsknownaretetanusandbotulinumtoxinsTetanustoxinproducesirreversiblemusclecontractionBotulinumtoxinblocksmusclecontractionBIOL53338MechanismofActionToxinsthatMechanismofActionGeneralmechanismofbothConsistofsinglepolypeptidechainswithAandBregionsBindingtogangliosidereceptorsspecificfornervetissueActivatedbyproteolysisanddisulfidereduction,andtheyfunctionintracellularlyBIOL53339Mechanismof