Activity of LL-37, CRAMP and antimicrobial peptide-derived compounds E2, E6 and CP26 against

ContentslistsavailableatSciVerseScienceDirect

InternationalJournalofAntimicrobialAgents

journalhomepage:http://www.elsevier.com/locate/ijantimicag

ActivityofLL-37,CRAMPandantimicrobialpeptide-derivedcompoundsE2,E6andCP26againstMycobacteriumtuberculosis

BrunoRivas-Santiagoa,∗,CesarE.RivasSantiagob,c,JulioE.Casta˜neda-Delgadoa,d,JuanC.León–Contrerasc,RobertE.W.Hancocke,RogelioHernandez-Pandoc

MedicalResearchUnit–Zacatecas,MexicanSocialSecurityInstitute,Zacatecas,Mexico

DepartmentofExperimentalPathology,NationalInstituteofMedicalSciencesandNutrition‘SalvadorZubirán’,MexicoCity,Mexicoc

UniversityofMedicineandDentistry,NewJersey(UMDNJ)–SchoolofPublicHealth,DepartmentofEnvironmentalandOccupationalHealth,CenterforGlobalPublicHealth,Piscataway,NJ,USAd

DepartmentofImmunology,SchoolofMedicine,AutonomousUniversityofSanLuisPotosi,SanLuisPotosi,Mexicoe

CentreforMicrobialDiseasesandImmunityResearch,UniversityofBritishColumbia,2259LowerMallResearchStation,Vancouver,BC,Canada

a

b

article

info

abstract

Articlehistory:

Received25June2012Receivedinrevisedform13September2012

Accepted26September2012

Keywords:TuberculosisTreatment

Antimicrobialpeptides

Tuberculosis(TB)isamajorworldwidehealthprobleminpartduetothelackofdevelopmentofnewtreatmentsandtheemergenceofnewstrainssuchasmultidrug-resistant(MDR)andextensivelydrug-resistantstrainsthatarethreateningandimpairingthecontrolofthisdisease.Inthisstudy,theefficacyofnaturalandsyntheticcationicantimicrobial(hostdefence)peptidesthathavebeenshownoftentopossessbroad-spectrumantimicrobialactivitywastested.ThenaturalantimicrobialpeptideshumanLL-37andmouseCRAMPaswellassyntheticpeptidesE2,E6andCP26weretestedfortheiractivityagainstMycobacteriumtuberculosisbothininvitroandinvivomodels.Thepeptideshadmoderateantimicrobialactivities,withminimuminhibitoryconcentrationsrangingfrom2␮g/mLto10␮g/mL.InavirulentmodelofM.tuberculosislunginfection,intratrachealtherapeuticapplicationofthesepeptidesthreetimesaweekatdosesofca.1mg/kgledtosignificant3–10-foldreductionsinlungbacilliafter28–30daysoftreatment.Thetreatmentsworkedbothagainstthedrug-sensitiveH37RvstrainandaMDRstrain.TheseresultsindicatethatantimicrobialpeptidesmightconstituteanoveltherapyagainstTB.

© 2012 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

1.Introduction

Tuberculosis(TB)[1],causedbythebacteriumMycobacteriumtuberculosis,remainsoneoftheleadingcausesofdiseaseandmor-talityduetoaninfectiousagent.AccordingtorecentdatafromtheWorldHealthOrganization(WHO),in2010therewere8.8millionactiveTBcasesworldwideandnearly1.5milliondeaths.Ithasbeenestimatedthatone-thirdofthehumanpopulationcarriesM.tuber-culosisand10%ofthesepeoplewilldevelopactivediseaseatsometimeintheirlives,creatinganenormousreservoir[2].

TreatmentofpulmonaryTBhasbecomeincreasinglychalleng-ingdueinparttotherequiredlongdurationoftherapyandtheadventofmultipledrugresistance.Oneofthemostimportantfac-torsistheemergenceofmultidrug-resistant(MDR)bacillithathasbeenassociatedwithinadequateuseofantibioticsandpooradher-encetorecommendedtreatmentregimens[3].Inrecentyears,new

∗Correspondingauthor.Presentaddress:UnidaddeInvestigaciónMédica–Zacatecas,InstitutoMexicanodelSeguroSocial(IMSS),InteriordelaAlameda#45,Zacatecas,Z.C.98000,Mexico.Tel.:+524929226019.

E-mailaddress:rondovm@yahoo.com(B.Rivas-Santiago).

strainshaveemerged,termedextensivelydrug-resistant(XDR),thatarealsoresistanttosecond-lineantibioticssuchasfluo-roquinolonesandeitherkanamycin,amikacinorcapreomycin.Thesestrainsleadtopoortreatmentoutcomesandaconsiderablyincreasedrateofmortality[4].RecentreportssuggestthepossibleexistenceofcasesofcompletelyresistantTBintheMiddleEast,raisingconcernsregardinghowtotreattheseTBcaseseffectively[5].

Inthepast40years,nobroadlysuccessfulnewTBdrughasbeendeveloped.Therefore,thereisastrongdrivetodevelopnewtreatmentsforTBand/ortoimprovethosecurrentlyinuse.Impor-tantadvanceshavebeenmadeandthereareseveralclinicaltrialsunderwaythathaveutilisedfluoroquinolonesinplaceofetham-butol,leadingtopreliminaryindicationsofasignificantreductioninthedurationoftherapyandencouragingthepossibilityofanimprovementinpatientsurvival[6].

Antimicrobialpeptides(AMPs)aregene-encoded,amphipathic,cationicpeptidesthatareproducedbyseveralspeciesofmammals,birds,reptilesandamphibians.Thesepeptidescaninhibitmicrobialgrowththroughavarietyofoftencomplexmechanisms,includingmembraneinteractionsthatleadtopermeabilisationofcells,inhi-bitionofcellwallsynthesis,andentryintocellsleadingtoinhibition

0924-8579/$–seefrontmatter© 2012 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.http://dx.doi.org/10.1016/j.ijantimicag.2012.09.015

144B.Rivas-Santiagoetal./InternationalJournalofAntimicrobialAgents41 (2013) 143–148

ofmacromolecularsynthesis[7–9].Inaddition,thesepeptides,alsotermedhostdefencepeptides,canprofoundlyandfavourablymod-ulateinnateimmunity,upregulatingprotectiveimmunitysuchasincreasingtheproductionofchemokinestorecruitimmunecellswhilstdampeningpotentiallyharmfulinflammation[9,10].ThemajorgroupsofAMPsinhumansarethedefensinsandasin-glecathelicidin,LL-37.Ithasbeenreportedthatalterationsintheproductionofthesemoleculesincreasesusceptibilitytoinfectiousdiseases,includingTB[11].Conversely,upregulationofcathelicidinLL-37throughuseofvitaminDsupplementationhasbeenconsid-eredtobeapotentialstrategytoimproveTBinfectionoutcomes,althoughcurrentdatadonotnecessarilyfavourthispossibility[12].

PreviousstudiesbyourgrouphavereportedthatduringM.tuberculosisinfectionoflungepithelialcells,therewasahighproductionof␤-defensins-3and-4,andbothwereassociatedwithmycobacteriainthelung,suggestingtheirpossiblepartici-pationinclearanceofM.tuberculosis[13,14].Subsequently,itwasreportedthatinmurineTBmodels,BALB/cmiceproducedlowquantitiesofmurine␤-defensins-3and-4duringlateprogressiveTB,andwhenbothdefensinswereoverproducedbyintratrachealadministrationofisoleucine(adefensininducer)theseanimalsefficientlycontrolledinfectionbothbydrug-sensitiveanddrug-resistantbacilli[14,15].Inaddition,ithasbeenshownthattheinteractionofa19-kDalipopeptideofM.tuberculosiswithToll-likereceptor-2onthemacrophagesurfaceupregulatedtheexpressionofvitaminDreceptorleadingtotheinductionofcathe-licidinLL-37,promotingthekillingofintracellularM.tuberculosis[16,17].

Recently,methodologieshavebeendevelopedtoenabletheenhanceddesignofAMPs(e.g.[18]).Rationalsubstitutionstudiesledtoanenhanced26-amino-acid␤-helicalpeptideCP26derivedfromahybridpeptidecomprisingtheamphipathic␣-helicalN-terminalregionofcecropinAandthehydrophobicN-terminal␣-helixofthebeevenompeptidemelittin[17].Peptidearraymeth-odsandsubstitutionstudies,startingfromthesmallestknownbroad-spectrumnaturalAMPbactenecin(alsoknownasbovinedodecapeptide),ledtopeptidesE2(alsoknownasBac8c),an8-amino-acidpeptide,aswellasE6(alsocalledSub3),a12-amino-acidpeptide,bothofwhichdemonstratedenhancedactivityagainstarangeofpathogenicGram-positiveandGram-negativebacteriaandtheyeastCandidaalbicans[18,19].

Inthisstudy,theantimicrobialactivityoffivenaturalandsyn-theticpeptidesagainstM.tuberculosiswasevaluatedinaninvitrosetting.

2.Materialsandmethods

2.1.Peptidesynthesisanddesign

PeptidesweresynthesisedbythePeptideSynthesisFacility,BiomedicalResearchCentreattheUniversityofBritishColumbia(Vancouver,Canada)usingtertiarybutyloxycarbonyl(tBOC)solid-phasesynthesis.Peptideswerepurifiedbyhigh-performanceliquidchromatographyto>95%purityandwereconfirmedbymassspec-trometry.

Thefollowingpeptideswereutilised:mouseCRAMP(GLL-RKGGEKIGEKLKKIGQKIKNFFQKLVPQPEQ)[20];humanLL-37[20,21](LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES);E2(alsotermedBac8c;RIWVIWRR-NH2)[18,19];E6(alsotermedSub3;RRWRIVVIRVRR-NH2)[18,19];andCP26(KWKSFIKKLT-SAAKKVVTTAKPLISS)[22].Briefly,thesepeptideswereselectedaseithernaturalpeptideswithmoderateantimicrobialactivity(LL-37andCRAMP)orasbroad-spectrumsyntheticpeptideswithmoderatetogoodantimicrobialactivity(E2,E6andCP26).

2.2.Mycobacteriumtuberculosisstraingrowth

Thedrug-sensitiveM.tuberculosisstrainH37Rv(ATCC)andaMDRstrain(clinicalisolate,resistanttofirst-lineantibi-otics)weregrowninMiddlebrook7H9broth(DifcoLaborato-ries,Detroit,MI)supplementedwith0.2%glycerol,10%oleicacid–albumin–dextrose–catalase(OADCenrichmentmedia;Bec-tonDickinson,FranklinLakes,NJ)and0.02%Tween80at37◦C.Mid-logphasecultureswereusedforallexperiments.

2.3.Microdilutioncolorimetricreductionassay

Susceptibilitytestingutilisingresazurin(TrekDiagnostic,West-lake,OH)asanindicatorofresidualbacterialviabilitywasperformedinCostar®96-wellflat-bottomplates(CorningInc.,Corning,NY)asdescribedpreviously[23].Briefly,alltestwellscon-tained100␮LofOADC-supplementedMiddlebrook7H9growthmedium.Then,100␮Lofthedilutedpeptideatthehighestconcen-trationstartingat12.8␮g/mLwasaddedtoonewell.Thecontentsofthewellsweremixedthoroughlyand100␮Lwastransferredintothenextwell;theprocesswasthenrepeated,thuscreatingserialtwo-folddilutions.Inadditiontothetestedpeptides,rifampicin(8.0␮g/mL)wasusedasapositivecontrol,andmediumwithoutanycompoundwasusedasanegativecontrolineachplate.Pep-tidesweretestedintheconcentrationrange0.4–12.8␮g/mL.

Plateswereincubatedat37◦Cfor5days.OnDay5,20␮Lof0.01%resazurinsolutionand12␮Lofsterile10%Tween80solu-tionwereaddedtoseveralcontrolwellscontainingM.tuberculosisbutnoantibacterialagentandplateswereincubatedagainfor24hunderthesameconditions.IftheM.tuberculosisviabilitycontrolstestedpositiveforresazurinreduction,resazurinwasaddedtoallwells.Theminimuminhibitoryconcentration(MIC)wasdefinedasthelowestpeptideconcentrationthatpreventedthereductionofresazurinandthereforeacolourchangefrombluetopink.PreviousstudiesbyourgroupsuggestthatsomeAMPsmayinducedormancyorabacteriostaticstateinM.tuberculosis[24].Toexaminethis,10␮Lfromthelowestconcentrationthatdidnotreduceresazurinwasseriallydilutedandseededonto7H10agarplatessupple-mentedwithMiddlebrookOADCenrichmentmediaandincubatedfor≥21daysat37◦CtoobservewhetherM.tuberculosisre-growthoccurred.

2.4.ExperimentalmodelofprogressivepulmonarytuberculosisinBALB/cmice

TheexperimentalmodelofprogressivepulmonaryTBhasbeendescribedindetailelsewhere[25].Briefly,maleBALB/cmiceaged6–8weekswereanaesthetisedinagaschamberusing0.1mLpermouseofsevoflurane,andeachmousewasinfectedbyendotra-chealinstillationwith2.5×105livebacilli.Miceweremaintainedintheverticalpositionuntiltheyunderwentspontaneousrecovery.Infectedmiceweremaintainedingroupsoffiveincagesfittedwithmicro-isolators.AnimalworkwasperformedinaccordancewithMexicannationalregulationsonAnimalCareandExperimentation(NOM062-ZOO-1999).

2.5.Treatmentofinfectedmicewithpeptides

After60daysofinfection,animalswerearbitrarilyallocatedintofourgroups.Peptidetreatmentstarted60daysafterinfection,whenadvancedprogressivediseasewaswellestablished.InthefirstexperimentsconductedtodeterminetheinvitroMICofeachpeptide,itwasdeterminedthatdosesatornearto3.2␮g/mLforallpeptideswereabletokillM.tuberculosis.Thus,adoseof32␮gin100␮Lofsalinesolution(ca.1mg/kg)wasusedforthetherapeuticexperiments.Threeindependentexperimentswereperformed.All

B.Rivas-Santiagoetal./InternationalJournalofAntimicrobialAgents41 (2013) 143–148

145

groupsofanimalsreceivedthecorrespondingdosethreetimesaweekforupto4weeksbyintratrachealinstillation,sinceprelim-inarystudiesindicatednoefficacyviatheintraperitonealdeliveryroute.Sixanimalsineachgroupweresacrificedat7,14and28daysafterstartingtreatment.Theefficiencyofeachpeptidetreat-mentwasdeterminedbyquantifyingthelungbacillaryloadsbyassessingCFUsandtheextentoftissuedamagebyhistopathology.

2.6.DeterminationofCFUsininfectedlungs

LungswerehomogenisedwithaPolytron®homogeniser(Kine-matica,Lucerne,Switzerland)insteriletubescontaining1mLof0.05%Tween80inphosphate-bufferedsaline(PBS).Fivedilutionsofeachhomogenatewerespreadontoduplicateplatescontain-ingBactoMiddlebrook7H10agar(DifcoLaboratories)enrichedwithOADC-enrichedmedium(BectonDickinson,Sparks,MD).Thenumberofcolonieswascountedafter21daysofincubation.

2.7.Preparationoflungtissueforhistology

Thelungsfromeachofthreedifferentanimalspertimepointandgroupwereperfusedintratracheallywithethylalcohol(J.T.Baker,MexicoCity,Mexico).Lungswerethendehydratedandembeddedinparaffin(OxfordLabware,StLouis,MO),sectionedandstainedwithhaematoxylinandeosin.Thepercentageofthelungsurfaceaffectedbypneumoniawasdeterminedusinganauto-matedimageanalyser(AxiovertM200;CarlZeiss,Oberkochen,Germany).

2.8.UltrastructuralanalysisoftreatedMycobacteriumtuberculosis

DeterminationoftheultrastructuraldamagetoM.tuberculosiscausedbytreatmentwiththedifferentAMPswasevaluatedusingtransmissionelectronmicroscopy.Briefly,bacilliwereculturedinMiddlebrook7H9broth(DifcoLaboratories)supplementedwithMiddlebrookOADCenrichmentmedia(BBL;BD,FranklinLakes,NJ)untillogarithmicphasewasachieved.Viablebacilli(1×107)wereplacedinthewellsof96-wellplatesandwereexposedtothecorrespondingAMPfor18hattheMICsdeterminedpreviouslyusingtheresazurinassay.Subsequently,fixationwasperformedwith4%paraformaldehydeinPBSandthefixedbacillisuspensionwastreatedwith0.05mMNH4ClinPBStoblockfreealdehydegroups.ThebacterialsuspensionwasthencentrifugedtoformapelletthatwaslaterdehydratedwithgradedethylalcoholsolutionsandembeddedinLRWhitehydrosolubleresin(LondonResinCom-pany,London,UK).Thinsectionsof70–90nmwidthwereplacedonnickelgridsandwerecontrastedwithuraniumsalts(ElectronMicroscopySciences,FortWashington,PA)andwereexaminedwithaZeissM-10electronmicroscope(CarlZeiss).

2.9.Statisticalanalysis

Datawereanalysedbyparametrictwo-wayanalysisofvariance(ANOVA)withTukey’spost-testoranon-parametricKruskal–WallismultiplecomparisonstestwithDunn’spost-test.GraphPad5.02software(GraphPadInc.,LaJolla,CA)wasusedtoperformtheanalysis.Forallanalyses,aP-valueof<0.05wascon-sideredstatisticallysignificant.

3.Results

3.1.AntimicrobialactivityofCRAMP,LL-37,E2,E6andCP26invitro

Forpre-clinicaltestingofantimycobacterialdrugs,themostversatileandefficienttechniqueutilisesresazurinfordeterminingresidualM.tuberculosisviability[23].

15P.aeruginosaM.tuberculosisLm10/gµ sCIM50P7266M32-EEALPRLCCFig.1.Theeffectofantimicrobialpeptides(AMPs)humanLL-37,mouseCRAMP,E2,E6andCP26onthegrowthofMycobacteriumtuberculosis.Mycobacteriumtuberculo-sisstrainH37RvwasincubatedwithincreasingconcentrationsoftheindicatedAMPtodeterminetheminimuminhibitoryconcentration(MIC).Dataareexpressedasthemean±standarddeviationofthreeindependentexperiments,eachperformedinduplicate.

ThisassaywasperformedheretoevaluatethecapacityofselectedAMPstoinhibitthegrowthofM.tuberculosisstrains.Fig.1showsthatallpeptideshadstrongantimi-crobialactivityagainstM.tuberculosis,withCP26beingthemostefficient(MIC=2.1±0.33␮g/mL),followedbyE2andE6(MICs=2.6±0.34␮g/mLand3.2±0.10␮g/mL,respectively).Inter-estingly,thesethreeoptimisedsyntheticpeptidesallshowedhigheractivitythanthenaturalhuman(LL-37)andmice(CRAMP)cathelicidins,with1.5–2-foldlowerMICs.Thefast-growingbacte-riaPseudomonasaeruginosawasincludedasacontrolunderthesameconditions,showingsimilarresultsasthoseobtainedforM.tuberculosis.

3.2.UltrastructuralchangesinMycobacteriumtuberculosisinresponsetoantimicrobialpeptides

ToexaminethecytotoxiceffectoftheseAMPsagainstM.tuberculosis,theultrastructureofbacilliaftertreatmentwiththesepeptideswasexamined.Inpreviousstudies,itwasdemon-stratedthatantimicrobialcationicpeptidesbindtonegativelychargedmoleculesofthemembraneandcellwallcomponentssuchaslipoarabinomannaninM.tuberculosisandleadtomem-branedisruption[26].ToinvestigatetheeffectsofsemisyntheticpeptidesE2,E6,LL-37,CRAMPandCP26onM.tuberculosis,bacilliincubatedwiththesepeptideswerestudiedusingelectronmicroscopy.

Controluntreatedbacillishowedawell-defined,homogeneousandslightlyelectron-lucentcellwall,whilstthecytoplasmwasgenerallyelectrondensewithsomemedium-sizedvacuoles(Fig.2).IncubationwithpeptideE2producedsubstantialabnormalitiesinthecellwall,includingthinning,buddingandthickeningofthewallaswellascondensationofthecytoplasmproducinganelectron-lucentareaunderthewallthatwasmorepromi-nentatonepole(Fig.2).IncubationwithpeptideCP26ledtoanalmostcompletedisappearanceofthecellwall,withonlyathinsuperficialrimofelectron-densematerialevidentaroundthebacteria,whilstthecytoplasmexhibitedlargevacuolesorwascondensedleadingtoashrinkingofthewholebacillus(Fig.2).IncubationwithpeptideE6alsoinducedsignificantabnormal-itiesinthecellwall,includingextremethinningofthewallwhichalternatedwiththickenedareasandregionsofvesicularbuddingthatcouldbevisualisedasanirregularsurfacecoex-istingwithextremecytoplasmiccondensationleadingtoalarge

146B.Rivas-Santiagoetal./InternationalJournalofAntimicrobialAgents41 (2013) 143–148

Fig.2.Representativemicrographsofconventionalelectronmicroscopyandimmu-noelectronmicroscopyofMycobacteriumtuberculosisstrainH37Rvtreatedwithantimicrobialpeptides.Incontrasttotheuntreatedcontrolbacteria,bacilliincu-batedwithaminimuminhibitoryconcentrationofpeptidesE2,CP26,E6,LL-37orCRAMPshowedcellwallthinning,buddingordisruption(arrows),andcytoplas-miccondensationproducingabroadelectron-lucenthalobetweenthewallandthecondensedcytoplasm(*).

electron-lucentspacebetweenthecellwallandthecytoplasm(Fig.2).E6inducedthemoststrikingsubcellularabnormalities,howeverthegeneralthemeofcellwalldestruction/modificationandcytoplasmiccondensationwasevidentforallpeptides,includ-ingLL-37andCRAMP,whichinducedahomogeneousincreaseoftheelectron-lucentcellwallsurroundedbyathinelectron-denserim(Fig.2).

Overall,theseobservationsindicatedthatthecellwallandmembraneareimportanttargetsofthesepeptides,whilsttheobservationofacondensedcytoplasmisconsistentwithosmoticactivityandperhapsalsoDNAbinding.Overall,theseobservationsmirroredthoseforotherpeptidesinGram-positivebacteria[22].

3.3.EffectofintratrachealadministrationofLL-37,CRAMP,E2,E6andCP26duringlateprogressivetuberculosisproducedbythedrug-sensitivestrainH37Rv

ThemeanofthehighestMICforallpeptidesagainstM.tuber-culosisinvitrowas3.2␮g/mL,thuswedecidedtouseadoseof32␮gin100␮Lofsalinesolution(ca.1mg/kg),whichwasadmin-isteredintratracheallythreetimesaweek.Treatmentwasstarted60dayspostinfection,whenadvancedactiveTBwaswellestab-lished.Incomparisonwithcontrolmiceinwhichbacillinumbers

A)

Control8E2E6**LL-376CrampCP26**601**x s4UFC200748DD12DDB)

80ControlE2E6ae60LL-37ra cCRAMPinoCP26m40uenp %2000748DD12DDFig.3.Effectofantimicrobialpeptide(AMP)treatmenton(A)pulmonarybacillibur-denand(B)tissuedamage(pneumonia)duringadvancedexperimentaltuberculosis.(A)Micewereinfectedwiththedrug-sensitiveH37RvMycobacteriumtuberculosisstrainandafter60daysweretreatedthreetimesperweekwith32␮goftheindi-catedAMPin100␮Lofsalinesolution.AllAMPsdecreasedthelungbacillaryloadsincomparisonwithnon-treatedmice.(B)Percentageoflungsurfaceaffectedbypneumoniadeterminedbyautomatedmorphometry.Resultsareexpressedasthemean±standarddeviation.P<0.05wasconsideredstatisticallysignificant.Aster-isksshowthekineticpointswheretherewasstatisticaldifferenceinCFUsbetweenthecontrolgroupandtherestofthegroups.NoneoftheAMPsinducedastatisticallysignificantdecreaseorincreaseinthepneumonicareacomparedwiththecontrolgroup.

increasedprogressivelyoverthe28dayswithanetdoublingoverthistime,animalstreatedwitheachofthetestedpeptidesshowedasignificantreductioninbacilliloadsduringtheentiretreatment(Fig.3A).ForthemouseandhumancathelicidinpeptidesCRAMPandLL-37,therewasasimilarinitialdecreaseinbacterialloadafter7daysbutthebacteriaappearedtogrowthereafter,albeitataslowerratethantheuntreatedcontrol,consistentwiththeweakerantimicrobialactivityofthesepeptides.Forthethreesyn-theticpeptides,thebacillididnotre-grow,andforE6andCP26therewasanapparentlyincreasingtherapeuticeffectbetween7daysand28days.

Consistentwiththesefindings,after4weeksoftreatmentwithE2,CRAMPorCP26,histologicalexaminationrevealedthatthelungareaaffectedbypneumoniatendedtobesmallerthanincontrolmicebutnotsignificantly.Incontrast,micetreatedwithLL-37showedamodestbutnon-significantincreaseinlungareaaffectedbypneumonia,whilstthosetreatedwithE6showedaslightornoincreaseinthepneumonicarea(Fig.3B).

B.Rivas-Santiagoetal./InternationalJournalofAntimicrobialAgents41 (2013) 143–148

147

A)

656041 X 3UFC2*1**0Control E2E6CP-26CRAMPLL-37B)

100aer80a cin60omue40np %200lo6267r2EEPtM3--nPALoCCRLC30 Days

Fig.4.Effectofantimicrobialpeptides(AMPs)inthetreatmentofmiceinfectedwithamultidrug-resistant(MDR)strainofMycobacteriumtuberculosis.(A)AnimalswereinfectedwithMDRstrainandafter60daysweretreatedthreetimesperweekwith32␮goftheindicatedAMPin100␮Lofsalinesolution.Incomparisonwiththenon-treatedcontrolanimals,allofthesemisyntheticpeptidesinducedasignifi-cantdecreaseinthelungbacillaryloads,whilstcathelicidinsbothfromhumanandmouseinducedanon-significantreductionofbacilliburdens.(B)Incontrast,simi-larlungconsolidationdeterminedbyautomatedmorphometrywasseenbetweentreatedandcontrolgroups.Resultsareexpressedasthemean±standarddeviation.*P<0.05wasconsideredstatisticallysignificant.

3.4.EffectofintratrachealadministrationofLL-37,CRAMP,E2,E6andCP26duringlateprogressivetuberculosisproducedbyamultidrug-resistantstrain

OwingtotheemergenceofMDRstrainsworldwideaswellastheresultsobservedinmicetreatedwiththedifferentpeptidesduringinfectionwiththedrug-sensitiveH37Rvstrain,westudiedwhetherthistherapyhadtheabilitytoinducesimilarbeneficialeffectsduringlateactivediseaseinmiceinfectedwithaclinicalisolateresistanttoallfirst-lineantibiotics.Incomparisonwithcon-trolanimals,MDR-infectedmicetreatedwithallthreesyntheticpeptidesdemonstratedasignificant2.5–4.5-foldreductioninCFUcounts(Fig.4A).Incontrast,whilsttherewasatrendtowardsactivityforthenaturalpeptidesLL-37andCRAMP,thiswasnotsignificant.Althoughtreatmentwiththedifferentpeptidesledtoaslightdecreaseinpneumonicarea,thesedifferenceswereagainnotstatisticallysignificantwhencomparedwithmicethatreceivedonlysalinesolution(Fig.4B).

4.Discussion

Inthepastdecade,anincreasingnumberofpublicationshavesuggestedAMPsasmoleculeswithgreatpotentialforthetreatment

ofTB[11,27–29].Thepresentstudydemonstratesthatseveraldif-ferentAMPsshowedanotableantimicrobialeffectagainstthedrug-sensitiveM.tuberculosisstrainH37Rv,insomecasesevenmorethanobservedforaP.aeruginosaclinicalisolate.Sinceprevi-ousstudiesbyourgroupshowedthat␤-defensin-2and-3mightbeinvolvedinthemaintenanceoflatencyusingamurinemodel[24],wewantedtoassesswhethertreatmentwiththesepeptideswouldledtoadecreaseinmetabolicactivityinM.tuberculosis,makingthemycobacteriumunabletoreduceresazurin.However,bacte-riacouldnotbere-grownfromthewellscontainingthelowestinhibitoryconcentrationsintheMICassays,indicatingthatthesepeptideswereinfactbactericidalratherthanmerelyinducingareductionofthemetabolicactivityinM.tuberculosis.

AMPshavecomplexmultimodalmechanismsofactionthathavebeenproposedtoinvolveseveraltargets,includingcellmembrane-associatedandintracellulartargets[8].Thesemechanismshaveincommontheinitialinteractionofpositivelychargedpeptideswiththenegativelychargedcytoplasmicmembraneandtheinsertionofpeptidesinthemembraneowingtotheiramphipathicnature,leadingtoeithermembraneperturbationortranslocationtocyto-plasmictargets.Theelectronmicroscopystudy,whichshoweddisruption,thinningandbuddingofthebacterialcellwallafterincubationofbacilliwiththedifferentpeptides,suggeststhattheirinteractionwiththemembraneand/orcellwallmightbeanimpor-tantmechanismtoproducebacterialdamage.Thisdamagecouldinturnrelatetotriggeringofautolyticmechanismsorinterferencewithcellwallbiosynthesis,bothofwhichhavebeenreportedtobemechanismsbywhichpeptidescanact[8,9].Inaddition,abnormal-itiessuchasbacterialcytoplasmicshrinkageareconsistentwiththepeptidebeingtakenupbycells[8].Overall,wecanconcludethatpeptidesutilisecomplexmechanismstoproduceM.tuberculosisdamage,asobservedforseveralpeptidesinotherbacteria[22].Thesepeptidesdidnotcauselysisofredbloodcellsatveryhighconcentrations[18,19,22].

Treatmentwiththedifferentpeptideswasinitiatedafter8weeksofinfection,whenactivediseasewasoccurring,mimickingacommonclinicalsituationindevelopingcountries.Intratra-chealinstillationofthedifferentpeptidesledtodecreasedlungbacillaryloads.TheactivityofthepeptidesdidnotseemtobestronglyrelatedtotheirorigininthathumanLL-37,mouseCRAMP,bovine-derivedE2andE6andinsect-derivedCP26allhadrathersimilarinitialactivities,withthenaturalpeptidesallowingslightre-growthofthebacillus.Thisindicatesthatneitherthespecificsequencenottheoriginofthepeptidesdeterminedtheiractiv-ity,butrathertheirantimicrobialproperties.Althoughnoneoftheresultswerestatisticallysignificant,forthreepeptides(mouseCRAMP,E2andCP26)slightlydecreasedpneumoniawasobserved,whilstLL-37ledtoamodestbutinsignificantincreaseinpneumo-niaandE6showedaslightornoeffect.ThemodestsuppressionofpneumoniabymouseCRAMP,E2andCP26cannotbejustduetotheantimicrobialactivity,whichwassimilarforboththenaturalpeptidesandallthreesyntheticpeptides.Indeed,itmightsuggestthatthereisanotherpropertyofpeptidesthatcontributestothesuppressionofpneumonia,suchasanimmunomodulatory,anti-inflammatoryactivity[9,10]thatmightdifferamongthepeptides.Cationicpeptideslikethesehaveavarietyofrelevantprop-erties,includingsuppressionofinflammation,enhancementofcellularrecruitmentandawoundhealingfunction.Thus,whilstthepeptideswereselectedfortheirantimicrobialactivities,otherpropertiesmayassistinthecontrolofM.tuberculosisinfection.Intriguingly,theenhancedAMPsdemonstratedapparentlysupe-rioractivitiestothenaturalpeptides,whilstLL-37showedanincreased(butnotsignificantly)pneumonicarea,perhapsduetothefactthatittendstobemorecytotoxic.Toevaluatethepoten-tialroleofimmunemodulation,wearecurrentlyinvestigatingpeptideswithenhancedimmunomodulatoryactivities[30]tosee

148B.Rivas-Santiagoetal./InternationalJournalofAntimicrobialAgents41 (2013) 143–148

whetherthesearemoresuccessfulatreducingthepneumonicarea.

WithregardtotheMDRstrain,thecurrentresultsshowedthatintratrachealadministrationofpeptidesE2,E6andCP26inmiceinfectedwiththisstrainduringtheadvancedphaseofinfectioncouldsignificantlyreducelungbacillaryloads.However,thereduc-tionofpneumoniadidnotdemonstratesignificantdifferenceswhencomparedwithcontrolmice.Thus,similartotheH37Rv-infectedmice,theseresultsindicatethatthesepeptideshaveaneffectiveantimicrobialeffectagainstMDRinfectionwithoutaffect-ingpneumonia.

Inconclusion,theseresultsshowthatrepeatedintrapulmonaryadministrationofAMPspermitsanefficientmethodofsuppress-ingthegrowthofbacilliwhentheyareadministeredduringthelateprogressivediseaseinducedbydrug-sensitiveordrug-resistantvir-ulentmycobacteria.Althoughthistreatmentwasnotcompletelycurative,theseresultssuggestthat,inconjunctionwithothermoreconventionaltreatments,inhalationtherapywithAMPswouldbeafeasibletreatmentoptionindevelopingcountrieswherethereisanurgentneedfornewtreatmentoptions.

Funding:REWHacknowledgesfundingfromtheGrandChal-lengesinGlobalHealthResearchprogrammethroughtheFoundationoftheNationalInstitutesofHealthandtheCanadianInstitutesforHealthResearch,andholdsaCanadaResearchChairinantimicrobialsandgenomics.BR-SacknowledgestheMexicanSocialSecurityInstitutegrantprojectFIS/IMSS/PROT/G10/832.RH-PacknowledgesCONACyT(contract:84456).Competinginterests:Nonedeclared.

Ethicalapproval:AnimalworkwasperformedinaccordancewithMexicannationalregulationsonAnimalCareandExperimen-tation(NOM062-ZOO-1999).

References

[1]DauletbaevN,GroppR,FryeM,LoitschS,WagnerTO,BargonJ.Expression

ofhuman␤defensin(HBD-1andHBD-2)mRNAinnasalepitheliaofadultcysticfibrosispatients,healthyindividuals,andindividualswithacutecold.Respiration2002;69:46–51.

[2]WorldHealthOrganization.Globaltuberculosiscontrol:WHOreport2011.

Geneva:WHO;2011.

[3]SarkarS,SureshMR.Anoverviewoftuberculosischemotherapy—aliterature

review.JPharmPharmSci2011;14:148–61.

[4]TabarsiP,ChitsazE,BaghaeiP,ShamaeiM,FarniaP,MarjaniM,etal.Impactof

extensivelydrug-resistanttuberculosisontreatmentoutcomeofmultidrug-resistanttuberculosispatientswithstandardizedregimen:reportfromIran.MicrobDrugResist2010;16:81–6.

[5]VelayatiAA,MasjediMR,FarniaP,TabarsiP,GhanaviJ,ZiazarifiAH,etal.

Emergenceofnewformsoftotallydrug-resistanttuberculosisbacilli:superextensivelydrug-resistanttuberculosisortotallydrug-resistantstrainsinIran.Chest2009;136:420–5.

[6]TakiffH,GuerreroE.Currentprospectsforthefluoroquinolonesasfirst-line

tuberculosistherapy.AntimicrobAgentsChemother2011;55:5421–9.

[7]KalitaA,VermaI,KhullerGK.Roleofhumanneutrophilpeptide-1asapossible

adjuncttoantituberculosischemotherapy.JInfectDis2004;190:1476–80.[8]HaleJD,HancockRE.Alternativemechanismsofactionofcationicantimicrobial

peptidesonbacteria.ExpertRevAntiInfectTher2007;5:951–9.

[9]HancockRE,SahlHG.Antimicrobialandhost-defensepeptidesasnewanti-infectivetherapeuticstrategies.NatBiotechnol2006;24:1551–7.

[10]AfacanNJ,YeungAT,PenaOM,HancockRE.Therapeuticpotentialof

hostdefensepeptidesinantibiotic-resistantinfections.CurrPharmDes2012;18:807–19.

[11]Rivas-SantiagoB,SerranoCJ,Enciso-MorenoJA.Susceptibilitytoinfec-tiousdiseasesbasedonantimicrobialpeptideproduction.InfectImmun2009;77:4690–5.

[12]YamshchikovAV,KurbatovaEV,KumariM,BlumbergHM,ZieglerTR,RaySM,

etal.VitaminDstatusandantimicrobialpeptidecathelicidin(LL-37)con-centrationsinpatientswithactivepulmonarytuberculosis.AmJClinNutr2010;92:603–11.

[13]Rivas-SantiagoB,SchwanderSK,SarabiaC,DiamondG,Klein-PatelME,

Hernandez-PandoR,etal.Human␤-defensin2isexpressedandassociatedwithMycobacteriumtuberculosisduringinfectionofhumanalveolarepithelialcells.InfectImmun2005;73:4505–11.

[14]Rivas-SantiagoCE,Rivas-SantiagoB,LeonDA,Casta˜neda-DelgadoJ,Hernández

PandoR.Inductionof␤-defensinsbyl-isoleucineasnovelimmunotherapyinexperimentalmurinetuberculosis.ClinExpImmunol2011;164:80–9.

[15]Rivas-SantiagoB,SadaE,TsutsumiV,Aguilar-LeonD,ContrerasJL,Hernandez-PandoR.␤-Defensingeneexpressionduringthecourseofexperimentaltuberculosisinfection.JInfectDis2006;194:697–701.

[16]LiuPT,StengerS,LiH,WenzelL,TanBH,KrutzikSR,etal.Toll-likerecep-tortriggeringofavitaminD-mediatedhumanantimicrobialresponse.Science2006;311:1770–3.

[17]LiuPT,StengerS,TangDH,ModlinRL.Cuttingedge:vitaminD-mediatedhuman

antimicrobialactivityagainstMycobacteriumtuberculosisisdependentontheinductionofcathelicidin.JImmunol2007;179:2060–3.

[18]HilpertK,Volkmer-EngertR,WalterT,HancockRE.High-throughputgener-ationofsmallantibacterialpeptideswithimprovedactivity.NatBiotechnol2005;23:1008–12.

[19]SpindlerEC,HaleJD,GiddingsJrTH,HancockRE,GillRT.Decipheringthe

modeofactionofthesyntheticantimicrobialpeptideBac8c.AntimicrobAgentsChemother2011;55:1706–16.

[20]DorschnerRA,PestonjamaspVK,TamakuwalaS,OhtakeT,RudisillJ,Nizet

V,etal.Cutaneousinjuryinducesthereleaseofcathelicidinanti-microbialpeptidesactiveagainstgroupAStreptococcus.JInvestDermatol2001;117:91–7.

[21]ZanettiM,GennaroR,ScocchiM,SkerlavajB.Structureandbiologyofcatheli-cidins.AdvExpMedBiol2000;479:203–18.

[22]FriedrichCL,MoylesD,BeveridgeTJ,HancockRE.Antibacterialactionofstruc-turallydiversecationicpeptidesonGram-positivebacteria.AntimicrobAgentsChemother2000;44:2086–92.

[23]Luna-HerreraJ,Martinez-CabreraG,Parra-MaldonadoR,Enciso-MorenoJA,

Torres-LopezJ,Quesada-PascualF,etal.UseofreceiveroperatingcharacteristiccurvestoassesstheperformanceofamicrodilutionassayfordeterminationofdrugsusceptibilityofclinicalisolatesofMycobacteriumtuberculosis.EurJClinMicrobiolInfectDis2003;22:21–7.

[24]Rivas-SantiagoB,ContrerasJC,SadaE,Hernandez-PandoR.Thepotentialroleof

lungepithelialcellsand␤-defensinsinexperimentallatenttuberculosis.ScandJImmunol2008;67:448–52.

[25]Castaneda-DelgadoJ,Hernandez-PandoR,SerranoCJ,Aguilar-LeonD,Leon-ContrerasJ,Rivas-SantiagoC,etal.Kineticsandcellularsourcesofcathelicidinduringthecourseofexperimentallatenttuberculousinfectionandprogressivepulmonarytuberculosis.ClinExpImmunol2010;161:542–50.

[26]MiyakawaY,RatnakarP,RaoAG,CostelloML,Mathieu-CostelloO,LehrerRI,

etal.InvitroactivityoftheantimicrobialpeptideshumanandrabbitdefensinsandporcineleukocyteprotegrinagainstMycobacteriumtuberculosis.InfectImmun1996;64:926–32.

[27]NizetV,OhtakeT,LauthX,TrowbridgeJ,RudisillJ,DorschnerRA,etal.

Innateantimicrobialpeptideprotectstheskinfrominvasivebacterialinfection.Nature2001;414:454–7.

[28]ZasloffM.Antimicrobialpeptidesofmulticellularorganisms.Nature

2002;415:389–95.

[29]SharmaS,VermaI,KhullerGK.Therapeuticpotentialofhumanneutrophil

peptide1againstexperimentaltuberculosis.AntimicrobAgentsChemother2001;45:639–40.

[30]AchtmanAH,PilatS,LawCW,LynnDJ,JanotL,MayerML,etal.Effectiveadjunc-tivetherapybyaninnatedefenseregulatorypeptideinapreclinicalmodelofseveremalaria.SciTranslMed2012;4:135ra64.