James A Imlay

JamesAImlayProfessorofMicrobiologyRomanoScholarAssociateDirector,MCBGraduateProgramResearchTopicsBioenergeticsandPhotosynthesis,Enzymology,MicrobialPhysiologyEducationB.S.(Chemistry,English),DukeUniversity,1981Ph.D.(Biochemistry),UniversityofCalifornia,Berkeley,1987Postdoctoral(Biochemistry),DukeUniversity,1987-1992TeachingInterestsMCB501-AdvancedBiochemistryMolecularmechanismsofoxidativedamage;cellulardefensesagainstoxidants;obligateanaerobiosisOxygenisareactivechemical,anditsgradualaccumulationintheenvironmentmusthavebeenaproblemforearlylifeforms.Forkineticreasonsmuchofitstoxicityislikelytobemediatedbyitspartiallyreducedforms,superoxide(O2-)andhydrogenperoxide(H2O2).Virtuallyallaerobicorganismscontainenzymesdevotedtoeliminatingthesespeciesfromthecell:superoxidedismutaseforO2-,andcatalasesandperoxidasesforH2O2.ThisimpliesthatO2-andH2O2areformedsomehowduringaerobicmetabolismandthat,unlessscavenged,theywillharmthecell.Indeed,mutantsthatareunabletodetoxifytheseoxygenspecieshavedifficultygrowinginthepresenceofoxygen.YetthemostfundamentalquestionsaboutO2-andH2O2toxicityremainunanswered:Howdothesespeciesariseinthecell?Whatdamagecantheydo?Whatdocellsdotoavoidorrepairthatdamage?Andwheninnaturedoesoxidativedamagedeterminethefateofthecell?OurlaboratorypursuesthesequestionsprimarilyinE.coli,amodelorganismwhosemetabolismiswell-understoodandwhichisamenabletoeasygeneticmanipulation.However,themechanismsbywhichoxidantsdamageE.coli,andthestrategiesbywhichE.colidefendsitself,arewidelysharedbyotherbacteria—and,indeed,byhigherorganisms.Howaresuperoxideandhydrogenperoxideformedinsidecells?MutantstrainsofE.colithatlacksuperoxidedismutaseorcatalases/peroxidasesareverysick.YetE.colihasnoenzymesthatproduceO2-orH2O2asdeliberateproducts.SowhatisthesourceoftheO2-andH2O2thatdisablethesemutants?Wehavefoundthatsomeflavoenzymesinadvertentlytransferelectronstodissolvedoxygenwhenitbouncesintotheirreducedcofactors.O2-isformedifoneelectronistransferred,andH2O2isformediftwoelectronsaretransferred.Notsurprisingly,thisbehaviorisparticularlymarkediftheflavinissolvent-exposed,hasahighelectrondensity,andhasalowreductionpotential.Severalrespiratorydehydrogenaseshavethesetraits,andtheygeneratesignificantO2-andH2O2duringrespiration.However,geneticanalysesshowthattheprimarysource(s)ofoxidantsinsideE.colilieoutsidetherespiratorychain.Theidentificationofthesesourcesiscurrentlyanimportantgoalofourlab.Whatbiomoleculesdoessuperoxidedamage?Superoxideandhydrogenperoxideareusedasweaponsthroughoutthebiologicalworld:microbesandplantssecreteantibioticswhich,wheningestedbycompetitors,redox-cycletogeneratecripplingdosesofintracellularO2-andH2O2,andmammalsblastinvasivebacteriawithO2-andH2O2thatisgeneratedbyspecializedenzymesonthephagosomalmembranesofneutrophilsandmacrophages.Yetwestillaretryingtoidentifythebiologicalmoleculestheseoxidantsdamage.Superoxidedoesnotreactdirectlywithpolypeptides,sugars,ornucleicacids,anditisunlikelytotriggerlipidperoxidationbecausebacterialmembraneslackpolyunsaturatedfattyacids.IthasbecomeclearthatO2-disablestheTCAcycleandbranched-chainbiosyntheticpathwaysbydestroyingtheiron-sulfurclustersofcriticalenzymes.Thismustnotbeitsonlytoxicaction,however,sinceSOD-deficientE.colialsoexhibitothermetabolicdefects.WeareanalyzingthesephenotypesinordertopinpointadditionalbiomoleculesthatO2-damages.Whatbiomoleculesdoeshydrogenperoxidedamage?H2O2oxidizestransitionmetals.Thisbehaviorhasseveralconsequencesforcellhealth.First,likeO2-,H2O2directlydamagestheiron-sulfurclustersofkeyenzymes.H2O2inactivatesenzymesthatusesingleironatomstobindsubstrates,suchasribulose-5-phosphateepimerase.H2O2alsoreactswiththeintracellularpoolofunincorporatedferrousironandtherebygenerateshydroxylradicals.Thelatterspeciesreactavidlywithvirtuallyallbiomolecules—includingDNA.Thisinjurycausesmutations,afactthathasledmanyscientiststopostulatethatinmammalschronicoxidativeDNAdamageisarootcauseofcarcinogenesisand,perhaps,aging.Finally,asaconsequenceofitsreactionswithiron,H2O2generallydisruptsironhomeostasisandmetabolism.Thismakesitdifficultforcellstodeliverironintonewmetalloproteins.Weareworkingtoilluminatedetailsofthesedamageprocesses(seefigure).HowdoesE.colidefenditselfagainstoxidants?Whilesuperoxidedismutase,catalases,andperoxidasesscavengeO2-andH2O2,theycanneverdiminishtheintracellularlevelstozero.Thereforemanyadditionaldefensivemeasuresaretaken.E.colirepairstheiron-sulfurclustersofdamagedenzymesbyre-reducingtheirclustersandreplacingtheirlostironatoms.Iftherateofenzymedamageexceedstherepaircapacity,E.coligoesfurther,replacingsomeofthedamagediron-sulfurenzymes--aconitaseandfumarase—withisozymesthatareresistanttooxidation.DuringperiodsofoxidativestressE.colialsosynthesizesDps,aniron-sequesteringproteinthatminimizestheamountofunincorporatedironthatisavailabletoreactwithH2O2.ThisactionsubstantiallysuppressesDNAdamage.Sincethisactionwouldleaveiron-dependentenzymeswithoutacofactormetal,thecellsimultaneouslyactivatestheimportofmanganeseasareplacementmetal.Eachoftheseadjustmentsisnecessary—ifanyoneofthemisblocked,H2O2-stressedcellsdonotsurvive.GeneticanalyseshaveidentifiedmanyothergenesthataredeliberatelyinducedinresponsetoO2-orH2O2.Wearestillworkingtoidentifytheirroles.Whycan'tanaerobesgrowaerobically?Howdophagocyteskillbacteria?Andotherquestions....Oxidativestressdeterminesthefateofbacteriainseveralsituations.Obligateanaerobescannotgrowinthepresenceofoxygen,afeaturethatprofoundlyconstrainstheirlifestylebutisnotwellunderstood.Mostbacteriaareoverpoweredbytheoxidativeburstofphagocytes—butaselectfewpathogenssomehowtolerateit.WearehopefulthatE.coliwillprovideinsightsintotheseproblems,too.AtthispointweknowthatsomeoftheenzymesthatoxygeninactivatesinobligateanaerobesarethesameonesthatsuperoxidedamagesinE.coli.WealsoknowthatsomeoftheoxidativedefensesthatwereidentifiedinE.coliareessentialtothesuccessofinvadingpathogens.ThusthelessonsfromE.coliprovidedirectionforinvestigationsofoxidativestressintheseotherorganismsaswell.RepresentativePublicationsSobota,JasonM.,andJamesA.Imlay.2011.Theironenzymeribulose-5-phosphate3-epimeraseinE.coliisrapidlydamagedbyhydrogenperoxidebutcanbeprotectedbymanganese.Proc.Natl.Acad.Sci.USA,inpress.Liu,Yuanyuan,SarahC.Bauer,andJamesA.Imlay.2011.TheYaaAproteinoftheEscherichiacoliOxyRregulonlessenshydrogenperoxidetoxicitybydiminishingtheamountofintracellularunincorporatediron.J.Bacteriol.,inpress.Martin,JuliaE.,andJamesA.Imlay.ThealternativeaerobicribonucleotidereductaseofEscherichiacoli,NrdEF,isamanganese-dependentenzymethatenablescellreplicationduringperiodsofironstarvation.Mol.Microbiol.,inpress.Gu,Mianzhi,andJamesA.Imlay.2011.TheSoxRSresponseisdirectlyactivatedbyredox-cyclingdrugsratherthanbysuperoxide.Mol.Microbiol.79:1136-1150.Jang,Soojin,andJamesA.Imlay.2010.HydrogenperoxideinactivatestheEscherichiacoliIsciron-sulfurassemblysystem,andOxyRinducestheSufsystemtocompensate.Mol.Microbiol.78:1448-1467.