unmaskspotentantimicrobialactivityofhuman

Publisher:NPG;Journal:Nature:Nature;ArticleType:BiologyletterDOI:10.1038/nature09674Page1of16Reductionofdisulphidebondsunmaskspotentantimicrobialactivityofhumanβ-defensin1BjoernO.Schroeder1,2,ZhihongWu3,SabineNuding1,2,SandraGroscurth4†,MoritzMarcinowski5,JuliaBeisner1,2,JohannesBuchner5,MartinSchaller6,EduardF.Stange7&JanWehkamp1,2,71Dr.MargareteFischer-Bosch-InstituteofClinicalPharmacology,70376Stuttgart,Germany.2UniversityofTübingen,72076Tübingen,Germany.3DepartmentofDermatology,UniversityHospitalSchleswig-Holstein,CampusKiel,24105Kiel,Germany.4Department1ProteinEvolution,MaxPlanckInstituteforDevelopmentalBiology,72076Tübingen,Germany.5CenterforIntegratedProteinScienceMunich,DepartmentChemie,TechnischeUniversitätMünchen,85747Garching,Germany.6DepartmentofDermatology,UniversityHospitalTübingen,72076Tübingen,Germany.7DepartmentofInternalMedicineI,RobertBoschHospital,70376Stuttgart,Germany.†Presentaddress:BrukerBioSpinAG,8117Fällanden,Switzerland.Humanepitheliaarepermanentlychallengedbybacteriaandfungi,includingcommensalandpathogenicmicrobiota1,2.Inthegut,thefractionofstrictanaerobesincreasesfromproximaltodistal,reaching99%ofbacterialspeciesinthecolon3.Atcolonicmucosa,oxygenpartialpressureisbelow25%ofairborneoxygencontent,moreovermicrobialmetabolismcausesreductiontoalowredoxpotentialof−200mVto–300mVinthecolon4.Defensins,characterizedbythreeintramoleculardisulphide-bridges,arekeyeffectormoleculesofinnateimmunitythatprotectthehostfrominfectiousmicrobesandshapethecompositionofmicrobiotaatmucosalsurfaces5–8.Humanβ-defensin1(hBD-1)isoneofthemostprominentpeptidesofitsclassbutdespiteubiquitousexpressionbyallhumanepithelia,comparisonwithotherdefensinssuggestedonlyminorantibiotickillingactivity9,10.Whereasmuchisknownabouttheactivityofantimicrobialpeptidesinaerobicenvironments,dataaboutreducingenvironmentsarelimited.HereinweshowthatafterreductionofdisulphidebridgeshBD-1becomesapotentantimicrobialpeptideagainsttheopportunisticpathogenicfungusCandidaalbicansandagainstanaerobic,Gram-positivecommensalsofBifidobacteriumandLactobacillusspecies.ReducedhBD-1differsstructurallyfromoxidizedhBD-1andfreecysteinesinthecarboxyterminusseemimportantforthebactericidaleffect.Invitro,thethioredoxin(TRX)system11isabletoreducehBD-1andTRXco-localizeswithreducedhBD-1inhumanepithelia.HenceourstudyindicatesthatPublisher:NPG;Journal:Nature:Nature;ArticleType:BiologyletterDOI:10.1038/nature09674Page2of16reducedhBD-1shieldsthehealthyepitheliumagainstcolonisationbycommensalbacteriaandopportunisticfungi.Accordingly,anintimateinterplaybetweenredox-regulationandinnateimmunedefenceseemscrucialforaneffectivebarrierprotectinghumanepithelia.Wemodifiedtheradialdiffusionassay12toanalyseantimicrobialactivityofsynthetichBD-1againstanaerobicbacteriaofthenormalfloraunderanaerobicconditions.Increasingconcentrationsofthereducingagentdithiothreitol(DTT)wereaddedtoassaymedium.InmediumwithoutDTThBD-1didnotaffectgrowthofGram-positiveBifidobacteriumadolescentis(Fig.1a).Surprisingly,additionofincreasingamountsofDTTledtoanincreaseofinhibitionzonesinsizeandsharpness.Incontrast,humanβ-defensin3(hBD-3)andlysozymeshowedlessantimicrobialactivityuponadditionofDTT(Fig.1bandSupplementaryFig.1).Thus,increasedantimicrobialactivityofhBD-1inreducingenvironmentwasspecificforhBD-1andnotcausedbyimpairedbacterialfitness.Remarkably,atconcentrationsof2mMDTThBD-1becameaseffectiveashBD-3againstB.adolescentis(Fig.1c).ThisfindingiscrucialashBD-3isoneofthemostpowerfulantimicrobialpeptidesinoxygen-richenvironment,whereashBD-1wasthoughttobeoneoftheweakest.FortheGram-negativeanaerobeBacteroidesvulgatuswefoundnoantimicrobialeffectofhBD-1andlysozymeunderanyconditions,whereashBD-3showedconcentration-dependentinhibitionzones(SupplementaryFig.2).Becauseβ-defensinscontainthreeintramoleculardisulphide-bridges13weinvestigatedtheinvolvementofcystinesfortheobservedantimicrobialeffect.Therefore,weincubatedhBD-1withincreasingconcentrationsofDTTandanalysedsamplesusingmatrix-assistedlaserdesorption/ionisation(MALDI)-massspectroscopy(MS).AsinglesignalcouldbedetectedforoxidizedhBD-1(oxhBD-1)atm/z3,926.8(Fig.2a).DTT-treatmentandcarboxamidomethylationresultedinasecondsignal,correspondingtothecompletelyreducedform(redhBD-1)withallsixcysteineresiduesalkylated.Theseresultssuggestthatallthreecystinesareeitherpresentorabsent,withouttheexistenceofintermediatestatescontainingoneortwodisulphide-bridges.Reversed-phasehighperformanceliquidchromatography(RP-HPLC)showedthatoxhBD-1elutedafter30min(Fig.2b).IncubationwithincreasingconcentrationsofDTTresultedinashifttowardsapeakat33.5min,representingthecompletelyreducedhBD-1(confirmedbyMALDI-MS,datanotshown)andindicatinganincreaseofhydrophobicityofredhBD-1insolution.Publisher:NPG;Journal:Nature:Nature;ArticleType:BiologyletterDOI:10.1038/nature09674Page3of16Toelucidatestructuralchangesfurther,nuclearmagneticresonance(NMR)experimentsonrecombinant,uniformly15N-labelledreducedandoxidizedhBD-1wereperformed.oxhBD-1showedawell-dispersed15N-heteronuclearsinglequantumcoherence(HSQC)s