Insight into the mechanism of action of temporin-SHa, a new broad-spectrum antiparasitic and

41 Pages·2017·8.79 MB·English

RESEARCHARTICLE
Insight into the mechanism of action of
temporin-SHa, a new broad-spectrum
antiparasitic and antibacterial agent
ZahidRaja1☯,SoniaAndre´1☯,FetenAbbassi1¤,VincentHumblot2,OlivierLequin3,4,
TaharBouceba5,IsabelleCorreia3,4,SandraCasale2,ThierryFoulon1,DenisSereno6,7,
BrunoOury6,7‡,AliLadram1‡*
1 SorbonneUniversite´s,UPMCUnivParis06,CNRS,InstitutdeBiologieParis-Seine(IBPS),Biogenèsedes
SignauxPeptidiques(BIOSIPE),Paris,France,2 SorbonneUniversite´s,UPMCUnivParis06,CNRS,
a1111111111
LaboratoiredeRe´activite´deSurface(LRS),Paris,France,3 SorbonneUniversite´s,UPMCUnivParis06,
a1111111111
EcoleNormaleSupe´rieure,CNRS,LaboratoiredesBiomole´cules,Paris,France,4 DepartmentofChemistry,
a1111111111 EcoleNormaleSupe´rieure,PSLResearchUniversity,UPMCUnivParis06,CNRS,Laboratoiredes
a1111111111 Biomole´cules,Paris,France,5 SorbonneUniversite´s,UPMCUnivParis06,CNRS,InstitutdeBiologie
a1111111111 Paris-Seine(IBPS),Plate-formeInteractionsMole´culaires,Paris,France,6 InstitutdeRecherchepourle
De´veloppement(IRD),UMR224IRD-CNRS-UnivMontpellier1et2MaladiesinfectieusesetVecteurs:
e´cologie,ge´ne´tique,e´volutionetcontroˆle(MiVegec),Montpellier,France,7 IRD,UMR177IRD-CIRAD,
InteractionsHoˆtes-Vecteurs-Parasites-Environnementdanslesmaladiestropicalesne´glige´esduesaux
Trypanosomatidae(InterTryp),Montpellier,France
OPENACCESS ☯Theseauthorscontributedequallytothiswork.
¤ Currentaddress:EcoleSupe´rieuredesSciencesetTechniquesdelaSante´deMonastir(ESSTSM),
Citation:RajaZ,Andre´S,AbbassiF,HumblotV,
Universite´deMonastir,Monastir,Tunisia.
LequinO,BoucebaT,etal.(2017)Insightintothe
‡Theseauthorsalsocontributedequallytothiswork.
mechanismofactionoftemporin-SHa,anew *[email protected]
broad-spectrumantiparasiticandantibacterial
agent.PLoSONE12(3):e0174024.https://doi.org/
10.1371/journal.pone.0174024
Abstract
Editor:SurajitBhattacharjya,Nanyang
TechnologicalUniversity,SINGAPORE Antimicrobialpeptides(AMPs)arepromisingdrugstokillresistantpathogens.Incontrastto
Received:December5,2016 bacteria,protozoanparasites,suchasLeishmania,werelittlestudied.Therefore,theanti-
parasiticmechanismofAMPsisstillunclear.Inthisstudy,wesoughttogetfurtherinsight
Accepted:February22,2017
intothismechanismbyfocusingourattentionontemporin-SHa(SHa),asmallbroad-spec-
Published:March20,2017
trumAMPpreviouslyshowntobeactiveagainstLeishmaniainfantum.Toimproveactivity,
Copyright:©2017Rajaetal.Thisisanopen
wedesignedanalogsofSHaandcomparedtheantibacterialandantiparasiticmechanisms.
accessarticledistributedunderthetermsofthe
[K3]SHaemergedasahighlypotentcompoundactiveagainstawiderangeofbacteria,
CreativeCommonsAttributionLicense,which
permitsunrestricteduse,distribution,and yeasts/fungi,andtrypanosomatids(LeishmaniaandTrypanosoma),withleishmanicidal
reproductioninanymedium,providedtheoriginal intramacrophagicactivityandefficiencytowardantibiotic-resistantstrainsofS.aureusand
authorandsourcearecredited.
antimony-resistantL.infantum.Multipassageresistanceselectiondemonstratedthattem-
DataAvailabilityStatement:Allrelevantdataare porins-SH,particularly[K3]SHa,arenotpronetoinduceresistanceinEscherichiacoli.Anal-
withinthepaperanditsSupportingInformation
ysisofthemodeofactionrevealedthatbacterialandparasitekillingoccurthroughasimilar
files.
membranolyticmechanisminvolvingrapidmembranepermeabilizationanddepolarization.
Funding:Thisworkwassupportedbythe
Thiswasconfirmedbyhigh-resolutionimaging(atomicforcemicroscopyandfieldemission
UniversityPierreandMarieCurie(UPMC),Institute
ofResearchforDevelopment(IRD–AAPLeishmed gun-scanningelectronmicroscopy).Multiplecombinedtechniques(nuclearmagneticreso-
2010),FrenchstatefundsmanagedbytheANR nance,surfaceplasmonresonance,differentialscanningcalorimetry)allowedustodetail
(Investissementsd’Avenirprogram,reference peptide-membraneinteractions.[K3]SHawasshowntointeractselectivelywithanionic
ANR-11-IDEX-0004-02,withintheframeworkof
modelmembraneswitha4-foldhigheraffinity(K =3x10−8M)thanSHa.Theamphipathic
theClusterofExcellenceMATISSE),andbyfunds D
fromtheConvergenceMECV2011programof α-helicalpeptideinsertsin-planeinthehydrophobiclipidbilayeranddisruptstheacylchain
PLOSONE|https://doi.org/10.1371/journal.pone.0174024 March20,2017 1/41
Antiparasitic/antibacterialmechanismoftemporin-SHa
UPMC.ZRandSAweresupportedbyafellowship packingviaadetergent-likeeffect.Interestingly,cellularevents,suchasmitochondrial
fromtheFrenchMinistèredel’Enseignement membranedepolarizationorDNAfragmentation,wereobservedinL.infantumpromasti-
Supe´rieuretdelaRecherche,allocatedbythe
gotesafterexposuretoSHaand[K3]SHaatconcentrationsaboveIC .Ourresultsindicate
EcoleDoctoraleiViv(ED387,UPMC,Paris, 50
France).Thefundershadnoroleinstudydesign, thatthesetemporinsexertleishmanicidalactivityviaaprimarymembranolyticmechanism
datacollectionandanalysis,decisiontopublish,or butcanalsotriggerapoptotis-likedeath.Themanyassetsdemonstratedfor[K3]SHamake
preparationofthemanuscript.
thissmallanaloganattractivetemplatetodevelopnewantibacterial/antiparasiticdrugs.
Competinginterests:Theauthorshavedeclared
thatnocompetinginterestsexist.
Introduction
Antimicrobialpeptides(AMPs)areaubiquitousgroupofnaturalcompoundsthatplaya
majorroleintheinnateimmunesystem[1,2].Becauseoftheirabilitytorapidlykillawide
rangeofmicroorganismsbyinducingthelysisoftheirmembranesand/oractingonintracellu-
lartargets[3],thesepeptidesarelesssusceptibletoinducemicrobialresistance.Naturally
occurringAMPs,suchasthosefromamphibians,areconsideredpromisingcandidatesforthe
developmentoftherapeuticdrugs,includinganti-infectiveagentstotreatresistantpathogens
aswellasanticancer,antidiabeticandimmunomodulatoryagents[4,5].
AmphibianAMPsofthetemporinfamily[6–10]representparticularlyattractivepotential
therapeuticcandidates.Thesepeptidesaresynthesizedfromprecursorsofthedermaseptin
superfamilyanddisplaythecharacteristicstructuralfeatures:ahighlyconservedN-terminal
region(signalpeptidefollowedbyanacidicpropiece)andahypervariableC-terminalregion
encodingtheprogenitorsequenceoftheAMP[11,12].Maturetemporinsshareuniqueprop-
ertiesthatdistinguishthemfromotherAMPs.Thesepeptideshaveasmallsize,usually13–14
residues[13].However,werecentlyisolatedanatypicalmemberofthetemporinfamilycon-
tainingonly8aminoacidresidues,namedtemporin-SHf(FFFLSRIF ),whichisthesmall-
NH2
estnaturallinearAMPfoundtodate,withthehighestpercentageofPheresidues(50%)for
anyknownpeptideorprotein[14].IncontrasttomostAMPs,temporinshavealownetposi-
tivecharge(0to+3).AlltemporinsareC-terminallyamidatedandadoptanamphipathicα-
helicalstructureinapolarmediaorinmembranemimeticenvironments[15–17].Recently,
suchstructureforatemporinwasalsoobservedinamediacontainingbacterialcells[18],
usingapreviouscirculardichroismprotocolthatwasusedforthefirsttimetostudythesec-
ondarystructureofAMPs(cecropinAandmagainin-2)inthepresenceofE.colicells[19].
Thisamphipathicα-helicalstructureenablesthetemporinstointeractwithanddestabilize
microbialcytoplasmicmembrane,therebypromotingmembranepermeabilizationand/ordis-
ruptionviaa“carpet-like”mechanismthatcaninvolvetheformationoftoroidalpores,chan-
nelaggregatesormorecomplexstructuresdependingontheconcentration,lengthand
sequenceofthepeptide[13,15,20,21].Atveryhighpeptideconcentrations,themembrane
canbedisintegratedinadetergent-likemanner.
Temporinshavearelativelynarrowspectrumofactivity,predominantlyagainstGram-pos-
itivebacteria[7–9].However,afewmembersofthisfamilydisplayawiderspectrum,with
potentactivityagainstGram-negativebacteriaandyeasts[22–25].Moreover,asmallnumber
oftemporinsareabletokillparasites.Currently,onlysixtemporinshavebeenreportedto
haveleishmanicidalactivity(Table1).OtheramphibianAMPswithalargersizewerealso
showntoactonLeishmaniaparasites,suchasdermaseptin-S1(34residues),thefirstdiscov-
eredleishmanicidalpeptide,orbombininsH2(20residues)andH4(21residues)[26,27].
DifferentAMPantiparasiticactivitiesareobserveddependingonthenatureoftheAMP
andtheparasiteandalsoonthestageoftheparasite.Severalantiparasiticmechanismshave
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Antiparasitic/antibacterialmechanismoftemporin-SHa
Table1. Temporinsreportedtohaveleishmanicidalactivity.
Temporin Sequence Netchargea Reference
Ta FLPLIGRVLSGIL +2 [28]
NH2
Tb LLPIVGNLLKSLL +2 [28]
NH2
SHa FLSGIVGMLGKLF +2 [22]
NH2
SHd FLPAALAGIGGILGKLF +2 [23]
Tl FVQWFSKFLGRIL +3 [29]
NH2
Tf FLPLIGKVLSGIL +2 [29]
NH2
apH7.4.
https://doi.org/10.1371/journal.pone.0174024.t001
beendescribed[30–32],whichinvolvedisruptionoftheparasitemembraneinthecaseof
temporin-TaandTb[28]oractivityagainstintracellulartargetsinthecaseofhistatine-5[33].
Thefactthatamastigotes(theintracellularforminthevertebratehost)aregenerallymore
resistanttoAMPscomparedtopromastigotes(theextracellularformintheinsectvector)sug-
geststhatthesurfacecompositionoftheLeishmaniaparasitesisimportantandthatthenega-
tivelychargedglycocalyxofpromastigotes,composedmainlyoflipophosphoglycan(LPG)and
proteophosphoglycan(PPG),playsasignificantroleinAMPactivity.ArecentstudybyEggi-
mannandcollaboratorsindicatedthatPPGisamajorfactorfortheactivityoftemporin-SHa
againstL.mexicanapromastigotesandthatthelackofPPGandLPGonthesurfaceincreases
theresistanceofthisLeishmaniaspeciestotemporins[29].Temporinsareamongthesmallest
naturalantiparasiticpeptidesreportedtohaveactivityagainstbothpromastigotesandamasti-
gotes.Moreover,itappearsthatthesmallsizeandlowchargeoftemporinsfavordiffusion
acrosstheglycocalyxintotheplasmamembrane.Therefore,thesepeptidesmaybeusefultools
toelucidatetheantiparasiticmechanismandarealsoattractivecandidatestoreinforcethelim-
itedarsenalofchemotherapeuticagentsforwhichthereisevidenceofemergingresistance,
suchaspentavalentantimonials[34–36]ormiltefosine[37].
Wepreviouslyshowedthattemporin-SHa(SHa)hasabroad-spectrumactivitytoward
Gram-positiveandGram-negativebacteria,yeastsandLeishmaniaparasites[22].Thispeptide
inhibitedthegrowthofL.infantumpromastigotesandaxenicamastigotes.Basedonthecom-
plexbalanceofstructuralandphysicochemicalparameters(length,secondarystructure,net
positivecharge,hydrophobicity,helicityandamphipathicity)thatgoverntheantimicrobial
activityofAMPs[4,38–41],wedesignedsubstitutedanalogsofSHa([K3]temporin-SHa:[K3]
SHa;[A2,6,9]temporin-SHa:[A2,6,9]SHa;[A2,6,9,K3]temporin-SHa:[A2,6,9,K3]SHa)toimprove
theantibacterial/antiparasiticactivityoftheparentpeptide.
Inthisstudy,thestructureofthepeptidesandtheircytotoxicityagainstseveralmammalian
cellsweredetermined,andadetailedcomparisonoftheantibacterialandantiparasiticactivi-
tiesofSHaanditsanalog[K3]SHawascarriedout.WescreenedalargepanelofGram-nega-
tiveandGram-positivebacteriaofclinicalinterestandalsovarioustrypanosomatidparasites
(LeishmaniaandTrypanosoma),includingantibiotic-resistantstrainsofS.aureusandanti-
mony-resistantL.infantum.Multipassageresistanceselectionwasperformedtodetermine
whetherbacterialresistancecouldoccuragainstSHaanditsanalogs.Biochemicalandbio-
physicalstudiesallowedustocomparetheantiparasiticandantibacterialmechanismsofthe
temporinsindetail.Wefirstcarriedoutmembranepermeabilization,depolarizationand
time-killassays.Thetemporin-inducedmembranedamageswerethenvisualizedbyatomic
forcemicroscopy(AFM)andfieldemissiongun-scanningelectronmicroscopy(FEG-SEM)
imagingofbacteriaandparasites.WealsoanalyzedthetypicalhallmarksofapoptosisinL.
infantumpromastigotes,suchasmitochondrialdepolarizationandDNAfragmentation.
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Antiparasitic/antibacterialmechanismoftemporin-SHa
Finally,peptide-membraneinteractionswerestudiedbysurfaceplasmonresonance(SPR)and
differentialscanningcalorimetry(DSC)usingmodelsofeukaryoticandprokaryoticcellmem-
branesandalsobynuclearmagneticresonance(NMR)spectroscopyusingbicellesasamem-
branemimetic.
Results
DesignofSHaanalogs
TheSHaanalogsweredesignedandsynthetizedbymodifyingthestructuralandphysicochem-
icalparametersknowntocontroltheantimicrobialactivityofAMPs,suchasnetpositive
charge,hydrophobicity,helicityandamphipathicity.Specificaminoacidpositionsinthe
sequenceofSHaweresubstitutedtoimprovetheantibacterialandantiparasiticactivityofSHa
whilereducingitscytotoxicactivity.First,thenetpositivechargeofSHawasincreasedtoa
valueof+3,yieldingtheanalog[K3]SHa(Table2).ThisanaloghasaLysresidueinposition3,
whichislocatedonthepolarfaceoftheα-helix(Fig1),insteadofaSerresidue.Becausehigh
hydrophobicitywasshowntoincreasecytotoxicactivity[4],wealsodesignedtwoanalogswith
reducedhydrophobicityontheapolarfaceoftheα-helix(Fig1).TheresiduesLeu2,9andVal6
werereplacedwithAla,yieldingtheanalog[A2,6,9]SHaandalso[A2,6,9,K3]SHa(Table2).As
shownbytheSchiffer-Edmundsonhelicalwheelprojections(Fig1),allanalogswerepredicted
toconservetheamphipathiccharacterofSHa.
[K3]SHadisplaysmorepotentantimicrobialactivitythanSHa
WefirstinvestigatedtheantimicrobialactivityofSHaandanalogsbydeterminingminimal
inhibitoryconcentrations(MICs)againstawidepanelofGram-negativeandGram-positive
bacteriaandyeasts/fungi(Table3).Thetwoanalogs[A2,6,9]SHaand[A2,6,9,K3]SHawereinac-
tiveagainstallstrains(MIC>200μM).Thesetwopeptideswerethenusedasnegativecon-
trols.Incontrast,theanalog[K3]SHawasfoundtobehighlyactiveagainstallthetested
bacteria(MIC=1–6μM)andyeasts/fungi(MIC=3–25μM),includingthemultidrug-resis-
tantstrainsoftheclinicallyrelevantpathogenicspeciesS.aureus,ATCC43300andATCC
BAA-44.Asignificantincreaseinactivitywasobservedforthisanalogcomparedtotheparent
peptide,especiallyforGram-negativebacteriaandyeasts/fungi(Table3).Indeed,forGram-
negativestrains,MICvaluesdecreasedbyapproximately8-foldforP.aeruginosa,aspecies
thatfrequentlydevelopshighintrinsicresistancetomanyconventionalantibiotics,4-foldfor
S.enterica(serotypeEnteritidis)andK.pneumoniae,3-foldforE.coli,and2-foldforA.bau-
mannii.Moreover,[K3]SHawas4-foldmorepotentagainstyeasts/fungi,eventowardthe
Table2. Sequenceandphysicochemicalpropertiesoftemporin-SHaanditsanalogs.
Peptide Sequencea M b(Da) Netchargec <H>d <μH>e
w
SHa FLSGIVGMLGKLF 1380.76 +2 0.91 0.69
NH2
[K3]SHa FLKGIVGMLGKLF 1421.86 +3 0.84 0.74
NH2
[A2,6,9]SHa FASGIAGMAGKLF 1268.55 +2 0.63 0.51
NH2
[A2,6,9,K3]SHa FAKGIAGMAGKLF 1309.64 +3 0.56 0.57
NH2
aThesubstitutedresiduesareindicatedinboldinthesequenceoftheanalogs.
bTheoreticalaveragemolecularmassusingPeptideMassCalculatorv3.2(http://rna.rega.kuleuven.ac.be/masspec/pepcalc.htm).
cpH7.4.
dHydrophobicityand
ehydrophobicmomentwerecalculatedusingHeliQuest(http://heliquest.ipmc.cnrs.fr/).
https://doi.org/10.1371/journal.pone.0174024.t002
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Antiparasitic/antibacterialmechanismoftemporin-SHa
Fig1.Schiffer-Edmundsonhelicalwheelrepresentationoftemporin-SHaanditsanalogs.SHa,temporin-SHa;[K3]SHa,
[K3]temporin-SHa;[A2,6,9]SHa,[A2,6,9]temporin-SHa;[A2,6,9,K3]SHa,[A2,6,9,K3]temporin-SHa.Apolarresiduesarerepresented
inblackandpolar/basicresiduesingray/white.Aminoacidmodificationsareinbold.Anamphipathiccharacter,withtwowell-
separatedpolarandapolarfaces,isobserved.AdaptedfromHeliquest.
https://doi.org/10.1371/journal.pone.0174024.g001
SHa-insensitivespeciesC.parapsilosis(MIC=25μM).Table3alsoindicatesthat[K3]SHa
retainedthehighpotencyoftheparentpeptideagainstS.aureusATCC25923(MIC=3μM)
butwasslightlymoreactivetowardotherGram-positivebacteria(2-fold)andsignificantly
moreactivetowardS.pyogenes(6-fold).ThegeometricmeanoftheMICvalues(MIC )was
GM
calculatedforseveralstrainsoftheTable3toprovideanoverallevaluationoftheantimicrobial
activityagainstGram-negativebacteria,Gram-positivebacteriaandyeast/fungi,andthento
useitforthedeterminationofthetherapeuticindexofSHaand[K3]SHa.MIC valuesindi-
GM
catedinTable3(inbold)underlinethehigherantimicrobialpotencyof[K3]SHa,particularly
againstGram-negativebacteriaandyeasts/fungi.Ataconcentration2-foldabovetheMIC,
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Antiparasitic/antibacterialmechanismoftemporin-SHa
Table3. Antibacterialactivityoftemporin-SHaanalogscomparedtotheparentpeptide.
MICa,MIC b(μM)
GM
SHa [K3]SHa [A2,6,9]SHa [A2,6,9,K3]SHa
Gram-negativebacteria
E.coliATCC25922 10 3 >200 >200
E.coliML-35p 10 3 >200 >200
P.aeruginosaATCC27853 50 6 >200 >200
S.entericac 25 6 >200 >200
A.baumanniiATCC19606 6 3 >200 >200
K.pneumoniaeATCC13883 12 3 >200 >200
14.4 3.8 - -
Gram-positivebacteria
S.aureusATCC25923 3 3 >200 >200
S.aureusST1065 6 3 >200 >200
S.aureusATCC43300d 6 3 - -
S.aureusATCCBAA-44e 6 3 - -
S.pyogenesATCC19615 6 1 >200 100
L.ivanovii 6 3 >200 >200
E.faecalisATCC29212 12 6 >200 >200
6 2.8 - -
Yeasts/fungi
C.albicansATCC90028 25 6 >200 >200
C.parapsilosisATCC22019 100 25 >200 >200
S.cerevisiae 12 3 >200 >200
31.1 7.7 - -
aValuesrepresentthemeansofthreeindependentexperimentsperformedintriplicate.
bMIC denotesthegeometricmeanoftheMICvalues(indicatedinbold)forallGram-negative,Gram-positiveandyeast/fungalstrains.
GM
cSerotypeEnteritidis.
dResistanttomethicillinandoxacillin.
eResistanttoamoxicillin/clavulanicacid,cephalothin,ciprofloxacin,erythromycin,gentamicin,imipenem,oxacillin,penicillin,tetracycline,ampicillin,
doxycycline,methicillin,azithromycin,ceftriaxone,clindamycin,lincomycin,perfloxacin,rifampin,andtobramycin.
https://doi.org/10.1371/journal.pone.0174024.t003
temporin-SHa(12μM)and[K3]SHa(6μM)werebothabletocompletelykillGram-positive
bacteria,suchasS.aureus,within5min(Fig2A).However,atsimilarconcentrations,[K3]SHa
wasmoreefficientthanSHainkillingGram-negativebacteria(Fig2B).Whilea120-minexpo-
surewasneededforapproximately93%killingbySHa,only15minwasneededforthe[K3]
SHaanalogtoachievecompletelethalityinE.colicells.
Leishmanicidalactivitywasanalyzedonbothpromastigotesandamastigotes(axenicand
intramacrophagic).PotentandrapidantiprotozoalactivitieswereobtainedforSHaandits
analog[K3]SHa(Table4andFig3).Theeffectoftemporin-SHaagainstL.infantumcan
bevisualizedinrealtime(S1Movie).AsshowninTable4,agrowthinhibitoryeffectwas
observedwithpromastigotesofseveralspeciesofLeishmaniaresponsibleforvisceral(L.infan-
tum),cutaneous(L.majorandL.amazonensis)andmucocutaneous(L.braziliensis)leishmani-
ases,withslightlyhigheractivityfor[K3]SHa(IC valuesintherangeof5–10μM)compared
50
totheparentpeptideSHa(IC =7–18μM).Wenoticeda2-foldimprovementofleishmanici-
50
dalactivity(IC =8μMforSHaand5μMfor[K3]SHa)whenpeptideswereincubatedwith
50
L.infantumpromastigotesinserum-freemedium.Moreover,time-killcurvesrevealedclear
differencesintheparasite-killingefficiencyoftemporin-SHa(Fig3A)and[K3]SHa(Fig3B).
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Antiparasitic/antibacterialmechanismoftemporin-SHa
Fig2. Time-killingcurvesofSHaanditsanalog[K3]SHaagainstS.aureusST1065(A)andE.coli
ML-35p(B).Bacteria(106cfu/ml)wereincubatedinphosphate-bufferedsaline(PBS)withtemporinsat
concentrations2-foldabovetheMICobtainedforS.aureusST1065(6μMfor[K3]SHaand12μMforSHa).
ThenegativecontrolcorrespondstobacteriaincubatedinPBSwithoutpeptide(w/opeptide).Thedataare
shownasthemeans±SEMfromasingleexperimentcarriedoutintriplicateandarerepresentativeofthree
independentexperiments.
https://doi.org/10.1371/journal.pone.0174024.g002
WhilebothpeptideswereactiveagainstL.infantumpromastigotesat3μM(38%and49%kill-
ingforSHaand[K3]SHa,respectively,at180min),[K3]SHainduceddeathinstantlyat12μM
(withinthefirstmin)comparedto5minforSHa.However,at6μM,[K3]SHawasabletorap-
idly(15min)andcompletelykillpromastigotes,whereasasignificantreductioninthenumber
ofparasites(~91%)wasobservedonlyaftera180-minincubationwithtemporin-SHa.Con-
sistentwiththeirlackofantibacterialeffect,[A2,6,9]SHaand[A2,6,9,K3]SHashowednoanti-
parasiticactivityagainstL.infantumpromastigotes(IC >200μM,Table4).AsshowninFig
50
3C,nokillingwasobservedwhenparasiteswereincubatedwith96μM[A2,6,9,K3]SHa.As
observedforantibiotic-susceptibleandantibiotic-resistantS.aureusstrains,[K3]SHaandSHa
retainedtheabilitytokillL.infantumparasitessusceptibleandresistanttoantimonywiththe
sameefficiency(Table4).Interestingly,leishmanicidalactivitywasdemonstratedfor[K3]SHa
(IC =5μM)andSHa(IC =9μM)againstintramacrophagicamastigotesaswellasagainst
50 50
theaxenicforms(IC =20μM)(Table4).Othertrypanosomatids,suchasT.bruceigambiense
50
(responsibleforsleepingsickness)andT.cruzi(theetiologicalagentofChagasdisease),were
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Antiparasitic/antibacterialmechanismoftemporin-SHa
Table4. Antiprotozoalactivityoftemporin-SHaand[K3]temporin-SHa.
IC (μM)
50
SHa [K3]SHa
Leishmaniapromastigotes
L.infantuma 18(8)b 10(5)b
L.infantum(antimonyresistant) 14 9
L.major 13 10
L.braziliensis 7 5
L.amazonensis 13 8
Leishmaniaamastigotes
L.infantum(axenic) 20 20
L.infantum(intramacrophagic) 9 5
Trypanosomaepimastigotes
T.bruceigambiense 14 16
T.cruzi 17 10
a[A2,6,9]SHaand[A2,6,9,K3]SHawereinactiveagainstL.infantumpromastigotes(IC >200μM).
50
bvaluesinparentheseswereobtainedinserum-freemedium.
https://doi.org/10.1371/journal.pone.0174024.t004
susceptibletotemporins(Table4).TheactivityagainstT.bruceigambiensewasquitesimilar
forSHaand[K3]SHa(IC ~15μM)butT.cruziwasslightlymoresusceptibletothepeptide
50
analog(~2-fold).Thetrypanocidaleffectoftemporin-SHaisshownintheS2Movie.
Asapositivecontrol,wealsoevaluatedtheleishmanicidaleffectofdermaseptinB2,apotent
33-residueAMPisolatedfromfrogskin(Phyllomedusabicolor)thatkillsmicroorganismsby
thecarpetmechanism[42].AhighleishmanicidalactivityagainstL.infantumpromastigotes
wasobservedforthispeptide(IC =2.5μM).
50
[K3]SHadisplaysabettertherapeuticindex
Thecytotoxicityoftemporinswasevaluatedtowardratandhumanerythrocytes,andseveral
humancelllines(Table5).Atantimicrobialconcentrations(3–6μMforbacteriaandvirtually
allyeaststrains;5–15μMforparasites),lowcytotoxicity(humanerythrocytes,THP-1mono-
cytesandTHP-1-derivedmacrophages)ornocytotoxicity(HepG2cellsandfibroblasts)was
observedfor[K3]SHa.ForSHa,similarresultswereobtained,witha2-foldhigherhemolytic
activityonhumanerythrocytes(25μM)comparedto[K3]SHa.However,this2-foldhigher
activitywasalsofoundforbothpeptidesonraterythrocytes.Theseresultsindicatethatintra-
macrophagicamastigotescanbekilledbytemporinswithoutdamagetothehostcellatleish-
manicidalconcentrations.Asforbacteriaandparasites,theanalogs[A2,6,9]SHaand[A2,6,9,K3]
SHawereinactivetowarderythrocytes,andnocytotoxicityhasbeendetectedwhen[A2,6,9]
SHawasalsotestedonTHP-1monocytes(IC >60μM)andHepG2cells(IC >600μM)
50 50
(Table5).Incontrast,thepositivecontrol,dermaseptinB2,washighlycytotoxictowardTHP-
1monocytes(IC ~7μM).
50
Toevaluatethecellselectivity(microorganismsversushumancells),thetherapeuticindices
of[K3]SHaandSHawerecalculatedastheratioofIC orLC valuesforthedifferenthuman
50 50
cellsovertheMIC valuesobtainedforGram-negativebacteria,Gram-positivebacteriaand
GM
yeasts/fungi.TheresultsshowedinTable6revealthat[K3]SHadisplaysabettertherapeutic
indexwithvalues1.37-foldto8.12-foldhigherthanthoseofSHadependingonthetypeof
microorganismsandcellsconsidered.Thetherapeuticindicesof[K3]SHawerealsobetter
whentheywerecalculatedwithLC valuesagainstraterythrocytes(indicatedinTable5)and
50
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Antiparasitic/antibacterialmechanismoftemporin-SHa
Fig3.Time-killcurvesoftemporinsagainstL.infantum.Parasites(2x106cells/ml)wereincubatedin
HBSSwithvariousconcentrations(3,6and12μM)ofsyntheticSHa(A)and[K3]SHa(B).HBSSwithout
peptide(w/opeptide)orcontaining96μM[A2,6,9,K3]SHawasusedasanegativecontrol(C).Thedataare
shownasthemeans±SEMofonerepresentativeexperimentobtainedfromthreeindependentexperiments
carriedoutinduplicate.
https://doi.org/10.1371/journal.pone.0174024.g003
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Antiparasitic/antibacterialmechanismoftemporin-SHa
Table5. CytotoxicactivityoftemporinsSHaandanalogsagainsthumancellsandraterythrocytes.
IC orLC (μM)
50 50
Cells SHa [K3]SHa [A2,6,9]SHa [A2,6,9,K3]SHa
Raterythrocytes 25 26 >200 >200
Humanerythrocytes 25 50 >100 ND
HumanTHP-1monocytes >60 48 >60 ND
HumanTHP-1-derivedmacrophages 60 55 ND ND
HumanHepG2 560 358 >600 ND
Humanfibroblasts >100 >100 ND ND
ValuesweredeterminedwithGraphPadPrism6.0softwareandarethemeansofthreeindependentexperimentsperformedintriplicate.IC ,halfmaximal
50
inhibitoryconcentration;LC ,halfmaximallyticconcentration(erythrocytesandmacrophages).
50
ND:notdetermined.
https://doi.org/10.1371/journal.pone.0174024.t005
MIC :Gram-negativebacteria,T.I.(SHa)=1.7,T.I.([K3]SHa)=6.8(4-foldhigher);Gram-
GM
positivebacteria,T.I.(SHa)=4.2,T.I.([K3]SHa)=9.3(2.21-foldhigher);yeasts/fungi,T.I.
(SHa)=0.8,T.I.([K3]SHa)=3.4(4.25-foldhigher).Therefore,theoverallresultsindicate
greaterantimicrobialspecificityfortheanalog[K3]SHacomparedtotheparentpeptide.
[K3]SHaaltersmoreefficientlytheintegrityofthebacterialandparasite
plasmamembrane
Theeffectsoftemporinsonthemembraneintegrityofbacteriaandparasiteswereinvestigated
bytwocomplementaryapproaches(ONPGandSYTOXGreenassays)toanalyzemembrane
permeabilization.First,weincubatedSHaand[K3]SHawithE.coliML-35p,abacterialstrain
constitutivelyexpressingcytoplasmicβ-galactosidase,andwemeasuredthetime-dependent
hydrolysisofthesmallchromogenicsubstrateo-nitrophenyl-β-D-galactopyranoside(ONPG)
intoo-nitrophenol(ONP)bycytoplasmicβ-galactosidase(Fig4).AsindicatedinFig4Aand
4B,bothpeptideswereabletopermeabilizethebacterialcytoplasmicmembrane.However,
[K3]SHainducedamorerapidandpotentpermeabilizationoftheGram-negativestrainE.coli
ML-35pcomparedtotheparentpeptide.Interestingly,at10μM,this13-residueanalogwasas
efficientasthetwopositivecontrolsusedinourstudy,dermaseptinB2(33residues)and
Table6. Therapeuticindices(T.I.)of[K3]SHaandSHaa.
T.I.b Human THP1- THP1-derived HepG2 Fibroblasts
[K3]SHa(SHa) erythrocytes monocytes macrophages
Gram-negativestrains 13.1(1.7) 12.6(8.3) 14.5(4.2) 94.2(38.9) 52.6(13.9)
7.70c 1.52 3.45 2.42 3.78
Gram-positivestrains 17.8(4.2) 17.1(20) 19.6(10) 127.8(93.3) 71.4(33.3)
4.24 0.85 1.96 1.37 2.14
Yeast/fungalstrains 6.5(0.8) 6.2(3.8) 7.1(1.9) 46.5(18) 26(6.4)
8.12 1.63 3.37 2.58 4.06
aTherapeuticindicesofSHaareindicatedinparentheses.
bRatioofIC orLC values(forhumancellsfromTable5)overthegeometricmeanofMICvalues(fromTable3andcorrespondingtoGram-negative,
50 50
Gram-positive,andyeast/fungalstrains).WhenIC orLC valueswerehigherthanthemaximumconcentrationtested,aminimal2-foldconcentration
50 50
valuewasusedtocalculatethetherapeuticindexbecauseIC orLC valuesweredeterminedbycarryingoutserial2-folddilutions(forexample,LC >
50 50 50
100wasconsideredas200).
cValuesinboldrepresentthefoldimprovementinthetherapeuticindexof[K3]SHacomparedtoSHa.
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