Elucidation of the Mode of Action of a New Antibacterial Compound Active against Staphylococcus

17 Pages·2016·1.53 MB·English

RESEARCHARTICLE
Elucidation of the Mode of Action of a New
Antibacterial Compound Active against
Staphylococcus aureus and Pseudomonas
aeruginosa
EvelienGerits1,ElineBlommaert1,AnnaLippell2,AlexJ.O’Neill2,BramWeytjens3,
DriesDeMaeyer3,AnaCarolinaFierro1,3,KathleenMarchal3,4,ArnaudMarchand5,
PatrickChaltin5,6,PieterSpincemaille7,KatrijnDeBrucker1,KarinThevissen1,BrunoP.
a11111 A.Cammue1,8,ToonSwings1,VeerleLiebens1,MaartenFauvart1,9,NatalieVerstraeten1,
JanMichiels1*
1 CentreofMicrobialandPlantGenetics,KULeuven,Leuven,Belgium,2 SchoolofMolecularandCellular
Biology,UniversityofLeeds,Leeds,UnitedKingdom,3 DepartmentofInformationTechnology(INTEC,
iMINDS),U.Ghent,Ghent,Belgium,4 DepartmentofPlantBiotechnologyandBioinformatics,U.Ghent,
Ghent,Belgium,5 CISTIMLeuvenvzw,Bio-Incubator,KULeuven,Leuven,Belgium,6 CentreforDrug
DesignandDiscovery(CD3),ResearchandDevelopment,KULeuven,Leuven,Belgium,7 Departmentof
OPENACCESS LaboratoryMedicine,UniversityHospitalsLeuven,Leuven,Belgium,8 DepartmentofPlantSystems
Biology,VIB,Ghent,Belgium,9 imec,SmartSystemsandEmergingTechnologiesUnit,DepartmentofLife
Citation:GeritsE,BlommaertE,LippellA,O’Neill ScienceTechnologies,Leuven,Belgium
AJ,WeytjensB,DeMaeyerD,etal.(2016)
ElucidationoftheModeofActionofaNew *[email protected]
AntibacterialCompoundActiveagainst
StaphylococcusaureusandPseudomonas
aeruginosa.PLoSONE11(5):e0155139. Abstract
doi:10.1371/journal.pone.0155139
Editor:MohamedN.Seleem,PurdueUniversity, Nosocomialandcommunity-acquiredinfectionscausedbymultidrugresistantbacteriarep-
UNITEDSTATES resentamajorhumanhealthproblem.Thus,thereisanurgentneedforthedevelopmentof
Received:October16,2015 antibioticswithnewmodesofaction.Inthisstudy,weinvestigatedtheantibacterialcharac-
teristicsandmodeofactionofanewantimicrobialcompound,SPI031(N-alkylated3,6-
Accepted:April25,2016
dihalogenocarbazol1-(sec-butylamino)-3-(3,6-dichloro-9H-carbazol-9-yl)propan-2-ol),
Published:May11,2016
whichwaspreviouslyidentifiedinourgroup.Thiscompoundexhibitsbroad-spectrumanti-
Copyright:©2016Geritsetal.Thisisanopen
bacterialactivity,includingactivityagainstthehumanpathogensStaphylococcusaureus
accessarticledistributedunderthetermsofthe
andPseudomonasaeruginosa.WefoundthatSPI031hasrapidbactericidalactivity(7-log
CreativeCommonsAttributionLicense,whichpermits
unrestricteduse,distribution,andreproductioninany reductionwithin30minat4xMIC)andthatthefrequencyofresistancedevelopmentagainst
medium,providedtheoriginalauthorandsourceare SPI031islow.ToelucidatethemodeofactionofSPI031,weperformedamacromolecular
credited.
synthesisassay,whichshowedthatSPI031causesnon-specificinhibitionofmacromolecu-
DataAvailabilityStatement:Theentiresetof larbiosynthesispathways.Liposomeleakageandmembranepermeabilitystudiesrevealed
RNAseqdataisavailablefromthepublicdatabase
thatSPI031rapidlyexertsmembranedamage,whichislikelytheprimarycauseofitsanti-
ArrayExpress(AccessionNumberE-MTAB-4691).
bacterialactivity.ThesefindingsweresupportedbyamutationalanalysisofSPI031-resis-
Funding:ThisworkwassupportedbytheEuropean
tantmutants,atranscriptomeanalysisandtheidentificationoftransposonmutantswith
Commission’sSeventhFrameworkProgramme(FP7/
2007-2013)underthegrantagreementCOATIM alteredsensitivitytothecompound.Inconclusion,ourresultsshowthatSPI031exertsits
(projectn°278425),theInteruniversityAttraction antimicrobialactivitybycausingmembranedamage,makingitaninterestingstartingpoint
PolesProgrammeinitiatedbytheBelgianScience
forthedevelopmentofnewantibacterialtherapies.
PolicyOfficeandbytheFWO(grantsG.0413.10,
G.0471.12N,G0B2515NtoJM).KTacknowledges
thereceiptofamandateofthe‘IndustrialResearch
PLOSONE|DOI:10.1371/journal.pone.0155139 May11,2016 1/17
ModeofActionoftheNewAntibacterialSPI031
Fund’ofKULeuven(IOFm/05/022).imecprovided Introduction
supportintheformofsalariesforauthorMF.The
Bacterialinfectionsareamongthemostseriousthreatstohumanhealth.Millionsofpeople
fundersdidnothaveanyadditionalroleinthestudy
design,datacollectionandanalysis,decisionto acquiresuchinfectionseachyear,leadingtoincreasedmortalityratesworldwideandaneco-
publish,orpreparationofthemanuscript.Thespecific nomicburdenonsociety[1].TheGram-positivepathogenStaphylococcusaureusisoneofthe
rolesoftheseauthorsarearticulatedinthe‘author majorcausesofnosocomialandcommunity-acquiredinfections[2].Thisbacteriumaccountsfor
contributions’section.
12.3%ofallnosocomialinfectionsinEurope,resultinginbacteremiaandsurgicalwoundinfec-
CompetingInterests:MFisaresearcheratimec. tions,andisthemaincauseofimplant-relatedinfections[3,4].Infectionscausedbycommunity-
Thisdoesnotaltertheauthors'adherencetoPLOS acquiredS.aureuscanrangefromminorskinandtissueinfectionstoprogressivepneumonia
ONEpoliciesonsharingdataandmaterials.
[5].Theemergenceofmethicillin-resistantS.aureushasseverelycomplicatedthetreatmentof
suchinfections.Currently,glycopeptideantibioticssuchasvancomycinareoftenusedtotreat
bothmethicillin-susceptibleandmethicillin-resistantinfections[6,7].However,inthepastfew
years,therehavebeenseveralreportsofvancomycin-resistantS.aureusinfections[8–10].
TheGram-negativebacteriumPseudomonasaeruginosaisanotherimportantpathogenthat
isfrequentlyinvolvedinnosocomialandcommunity-acquiredinfections[11].Thispathogen
isresponsiblefor8.9%ofinfectionsinEuropeanhospitalsandismostcommonlyfoundin
patientswithcancer,cysticfibrosisorburnwounds[3,11].Inaddition,P.aeruginosaisthe
maincausativeGram-negativeagentinimplant-relatedinfections[4].Community-acquired
infectionscausedbythispathogenincludeulcerativekeratitis,otitisexternaandskin/softtissue
infections[11].P.aeruginosainfectionsareusuallytreatedwithantibioticssuchasβ-lactams,
aminoglycosides,orquinolones[12].Alarmingly,multiplemultidrugresistantstrainsofP.aer-
uginosahaveemergedduringthelastyears,someofthemdisplayingresistanceeventowards
thelast-resortantibioticpolymyxinB[12,13].
Lately,ithasbecomeclearthatmultidrugresistanceisspreadingataveryfastrate[1].
Unfortunately,only5newclassesofantibioticsweremarketedsince2000,andmostofthese
donotworkagainstGram-negativepathogens[14].Thus,thereexistsapressingneedforthe
developmentofnewantibacterialagents.Recently,weidentifiedanewantibacterialcompound
SPI031(N-alkylated3,6-dihalogenocarbazol1-(sec-butylamino)-3-(3,6-dichloro-9H-carba-
zol-9-yl)propan-2-ol)(Fig1)withactivityagainstavarietyofbacteria,includingEscherichia
coliandthehumanpathogensS.aureus,P.aeruginosa,StaphylococcusepidermidisandPor-
phyromonasgingivalis[15].Inaddition,weshowedthatthecompounddisplaysantifungal
activityagainstCandidaalbicans[16].Furthermore,wedemonstratedthatSPI031hastheclin-
icalpotentialtobeusedasanantibacterialcoatingforimplants,therebyreducingtheincidence
ofimplant-associatedinfections[17].Inthepresentstudy,wefurtherinvestigatedtheantibac-
terialcharacteristics,includingbactericidalactivityandspontaneousresistancefrequency,and
modeofactionofSPI031usingS.aureusandP.aeruginosaasmodelpathogens.
Fig1.StructureofcompoundSPI031.
doi:10.1371/journal.pone.0155139.g001
PLOSONE|DOI:10.1371/journal.pone.0155139 May11,2016 2/17
ModeofActionoftheNewAntibacterialSPI031
MaterialandMethods
Bacterialstrainsandchemicals
S.aureusSH1000[18],P.aeruginosaPA14[19],KlebsiellapneumoniaeLMG2095,Acinetobac-
terbaumanniiNCTC13423,EnterobacteraerogenesLMG2094,EnterococcusfaeciumLMG
8148andStaphylococcusaureusATCC33591weregrownin1/20dilutedtrypticasesoybroth
(TSB,BectonDickinsonBenelux),TSB,lysogenybroth(LB)oronsolidTSBmediumcontain-
ing1.5%agarat37°C.
SPI031wassuppliedbyCD3(Leuven,Belgium)andstocksolutionsof10mMwerepre-
paredindimethylsulfoxide(DMSO).Vancomycin,polymyxinB,ofloxacin,tobramycin,cipro-
floxacin,rifampicin,tetracycline,SDS,TritonX-100andmelittinwerepurchasedfromSigma-
Aldrich.
MinimumInhibitoryConcentration(MIC)Assay
MICvaluesweredeterminedin1/20TSBwithabrothmicrodilutionprocedureaspreviously
described[20].AllMICvaluescalculatedinthepresentstudyarelistedinS1Table.
Time-killassay
S.aureusandP.aeruginosacellswereculturedtoexponentialphaseat37°Cin1/20TSBand
challengedwithantibacterialcompoundsat1xand4xMIC(seeS1Table).Atdifferenttime
points(0,0.1,0.5,1,2,3,4,5,24h),a200μlaliquotwasremovedfromeachsample,washed,
seriallydilutedinMgSO4(10mM)andplatedonTSBagarforenumerationofcolonyforming
units(CFU).
Spontaneousmutationfrequencytoresistance
ThespontaneousmutationfrequencyofS.aureusandP.aeruginosatoresistanceagainstdif-
ferentantibioticswasdeterminedbyplatingbacterialinocula(107−109CFU)onagarplates
containingantibioticsat5xMIC(seeS1Table).Thenumberofviablecellspresentintheinoc-
ulumwasdeterminedbyserialdilutionsondrug-freeagarplates.Themutationfrequencywas
calculatedbydividingthenumberofcoloniesformedontheplatesafter48hofincubationby
thenumberofcoloniesonthedrug-freeplates.Toconfirmtheresultsofthissingle-stepmuta-
tionprocedure,MICtestswereperformedforallresistantmutants.
Wholegenomesequencingofspontaneousresistantmutants
SpontaneousSPI031-resistantmutantsofP.aeruginosaweregeneratedasdescribedaboveand
threeindependentmutantswereselectedforfurtheranalysis.Resistanceofselectedmutants
wasverifiedbyMICdeterminations.Subsequently,genomicDNAfromP.aeruginosawild-
type(WT)andthespontaneousresistantmutantswasisolatedusingtheDNeasyBlood&Tis-
sueKit(Qiagen)followingthemanufacturer’sprotocol.DNAquantityandpuritywere
assessedusingaNanoDropND-1000.SamplesweresenttotheGenomicsCoreFacilityof
EMBL(Heidelberg,Germany)forwholegenomesequencingonanIlluminaHiSeq2000plat-
form.AssemblyofthereadsandfurtheranalysiswereperformedusingtheCLCGenomics
Workbenchprogramv8.0.Genomesequencesofspontaneousresistancemutantswerealigned
withthegenomesequenceoftheWTtodetectmutations.Acoverageabove10xandcutofffre-
quencyof75%wereconsideredforthedetectionofthemutations.Detectedmutationswere
verifiedbyPCRamplificationandSangersequencing.
PLOSONE|DOI:10.1371/journal.pone.0155139 May11,2016 3/17
ModeofActionoftheNewAntibacterialSPI031
Macromolecularsynthesisassay
SaturatedculturesofS.aureuswerediluted1/100inLBandgrowntoanOD of0.2at37°C.
600
withaeration.Cultureswerelabeledbytheadditionofeither[methyl-3H]thymidine,[5,6-3H]
uridineorL-[G-3H]glutamineat1μCi/mltomonitorsynthesisofDNA,RNA,orproteins,
respectively.After10minofincubationat37°C,100μlofeachculturewasmixedwith100μl
ofice-cold10%trichloroaceticacid(TCA)andstoredonice.Theremainderofeachculture
wasthentreatedwith4xMICofSPI031,ciprofloxacin,rifampicinortetracycline(seeS1
Table).After10minofincubation,100μlofthetreatedcultureswasmixedwithanequal
amountof10%TCAandkeptonicefor30min.TCAprecipitateswerecollectedundervac-
uumusinga96-wellfilterplate(96-wellUnifilterGF/B,Perkin-Elmer),andfiltersontowhich
[5,6-3H]uridine-labelledsampleshadbeendeposited,werewashedtwicewith100μlunlabeled
uridine.Individualfilterswerethenwashedtwicewith200μlof10%TCAeach,andtwicewith
200μlofaceticacid.Filterplatesweredried,25μlscintillant(Microscint20,Perking-Elmer)
wasaddedtoeachwell,andradiolabeledincorporationwasmeasuredusingaChameleonmul-
tilabelplatescintillationcounter(Hidex).
Carboxyfluoresceinleakageassay
Carboxyfluorescein(CF)loadedintoliposomesmatchingthelipidcompositionofthecyto-
plasmicmembraneofS.aureuswerepreparedasdescribedpreviously[21].Aliquots(5μl)of
theliposomesweremixedwith95μlliposomebuffer(10mMHEPES,107mMNaCl,pH7.4)
containingSPI031atafinalconcentrationequivalentto1xMICand4xMICagainstS.aureus
SH1000(seeS1Table).Sampleswereincubatedat37°CwhileshakingandCFrelease(λ =
ex
485nm,λ =520nm)wasmeasured10,60and180minafterincubationusingaFLUOstar
em
omegaplatereader(BMGLabtech).Thepercentageofliposomeintegrityaftertreatmentwas
determinedrelativetotheamountofCFreleaseaftertreatmentoftheliposomeswith0.5%Tri-
tonX-100(correspondingto0%liposomeintegrity).
Membranepermeability
SPI031-mediatedpermeabilizationofthecytoplasmicmembraneofbothS.aureusandP.aeru-
ginosawasmeasuredusingSYTOXgreen(Invitrogen,USA)aspreviouslydescribed[22],with
somemodification.Exponential-phasecellsofS.aureusandP.aeruginosagrownin1/20TSB
werewashedandresuspendedinphosphate-bufferedsalinetoanOD of0.5.Cellswere
595
stainedwith1μMofSYTOXgreendyeandtreatedwithSPI031(0x,0.25x,0.5xand1xMIC;
seeS1Table).Afteraperiodofincubation(30minforS.aureus,15minforP.aeruginosa),
fluorescencewasmeasured(λ =504nm,λ =523nm)usingaSynergyMXmultimode
ex em
reader.
OutermembranepermeabilityofP.aeruginosawasassessedusingthehydrophobicfluores-
centprobe1-N-phenylnaphthylamine(NPN,Sigma,USA)aspreviouslydescribed[23],with
minormodifications.Briefly,P.aeruginosacellsweregrowntoexponentialphasein1/20
TSB,washedandresuspendedtoanOD of0.5inbuffercontaining5mMHEPES,pH7.2.
595
CellsweremixedwithNPNtoafinalconcentrationof10μMandtreatedwithSPI031(0x,
0.25x,0.5xand1xMIC;seeS1Table).Increaseoffluorescencewasmeasuredimmediately
(λ =350nm,λ =420nm)usingaSynergyMXmultimodereader(Biotek,Winooski,VT)
ex em
at37°C.
FluorescencevaluesweredividedbycorrespondingOD valuestocorrectforcelldensi-
595
ties.Inaddition,valueswerecorrectedforbackgroundfluorescencebysubtractingthevalues
oftheuntreatedcontrolculturesstainedwithNPNorSYTOXgreen.
PLOSONE|DOI:10.1371/journal.pone.0155139 May11,2016 4/17
ModeofActionoftheNewAntibacterialSPI031
Fluorescencemicroscopy
Exponential-phasecellsofS.aureusandP.aeruginosagrownin1/20TSBweretreatedfor5
minwith0.5%DMSO(solventcontrol)orwith1xMICofSPI031(seeS1Table),centrifuged
andstainedwith10μg/mlN-(3-triethylammoniumpropyl)-4-(p-diethylaminophenyl-hexa-
trienyl)pyridiniumdibromide(FM4–64,MolecularProbes).Sampleswerespottedon2%
agarosepadsforimaging.ImageswerecapturedusingaZeissAxioimagerZ1fluorescence
microscopeequippedwithaECPlan-Neofluar100xobjective,usingtheFM4–64channel
(λ =540–580nm;λ =593–668nm).
ex em
RNAsequencinganddataanalysis
OvernightculturesofP.aeruginosawerediluted1/100in1/20TSBandweregrownfor3h
untillate-exponentialphase.Next,cellsweretreatedfor5minwith0.2xMICofSPI031(seeS1
Table)or1%DMSO(solventcontrol).Foreachcondition,threebiologicalrepeatsweresam-
pled.TotalRNAisolationwasperformedaspreviouslydescribed[20].Subsequently,rRNA
wasdepletedusingtheRibo-ZerorRNARemovalKit(Illumina)aspermanufacturer’sinstruc-
tions.RNAintegritywasanalyzedusingExperionRNAStdSensChips(Bio-Rad).RNAquan-
tityandpuritywasassessedusingaNanoDropND-1000spectrophotometer.Sampleswere
senttotheGenomicsCoreFacilityofEMBL(Heidelberg,Germany)forlibraryconstruction
andRNAsequencingusingtheIlluminaHiSeq2000platform.TheRNAsequencingdatasets
reportedinthisarticleareavailableintheArrayExpressdatabase(ArrayExpressaccession:
E-MTAB-4691;http://www.ebi.ac.uk/arrayexpress).
Fordataanalysis,FastQCwasusedtoverifythequalityoftherawsequencingreads[24].
Next,sequenceswerealignedtothereferenceP.aeruginosaUCBPP-PA14genome
(NC_008463.1)withBowtie2usingstandardsettings.Onlysamplesinwhichmorethan90%
ofthereadsalignedexactlyoncewerewithheldforfurtheranalysis(seeS2Table).Subse-
quently,readcountspergenewerecalculatedforeachsamplewithHtseq-count[25].The
DESeq2package[26]wasusedtonormalizethedataandtodetectdifferentialexpression
betweentreatedandcontrolsamplesusingaFalseDiscoveryRate(FDR)<0.05.Next,anet-
workanalysiswascarriedoutusingPheNetic[27].Thisanalysislooksforbothregulatorypro-
gramscommontomultipledifferentiallyexpressedgenesandfordownstreamprocesseswhich
areactivatedbydifferentiallyexpressedgenes,thusvisualizingimportantmolecularprocesses
underlyingtheobservedphenotypeofthetestedorganism.Inputdataconsistofthreetypesof
information:aninteractionnetworkofthetestedorganism,thedifferentialexpressiondataof
allgenesinthenetworkandagenelistofsignificantlydifferentiallyexpressedgenes.Currently,
nointeractionnetworkexistsforP.aeruginosaPA14.Forthisreason,aninteractionnetwork
forP.aeruginosaPAO1wascreated,usingdifferentpubliclyavailabledatasets(STRING[28],
KEGG[29,30]).Subsequently,thegenesofPA14weremappedtothegenesofPAO1usingthe
RapidAnnotationusingSubsystemTechnology(RAST)server[31].Significantlydifferentially
expressedgenesweredefinedasthe100geneshavingthehighestlog2-foldchange.PheNetic
wasrunusingthestandardparametersonthewebserver(22).GeneOntology(GO)enrich-
mentwasperformedontheresultingsubnetworkusingtheCytoscape[32]pluginBiNGO[33]
(hypergeometrictest,α<0.05,FDRcorrection).GOannotationsforP.aeruginosaPAO1were
retrievedfromUniprot[34].
Screeningofatransposonmutantlibrary
AP.aeruginosaPA14transposonlibrary[35]wasscreenedforincreasedsensitivityorresis-
tancetoSPI031.Mutantswereallowedtogrowin1/20TSBin96-wellplates.Afterovernight
incubation,thecultureswerediluted100-foldin1/20TSBcontainingeither0.2xMICor2x
PLOSONE|DOI:10.1371/journal.pone.0155139 May11,2016 5/17
ModeofActionoftheNewAntibacterialSPI031
MICofSPI031(seeS1Table)todetecthypersensitiveorresistantmutants,respectively.Next,
theplateswereincubatedwhileshakingfor24hat37°C.Growthwasmonitoredbymeasuring
theOD .MutantswereidentifiedashypersensitiveiftheirOD waslessthan0.05after
595 595
incubationinthepresenceof0.2xMICofSPI031.Ontheotherhand,mutantswereidentified
asresistantiftheirOD wasmorethan0.1afterincubationinthepresenceof2xMICof
595
SPI031.Subsequently,thesusceptibilityoftheselectedmutantswasvalidatedbydetailedmoni-
toringofgrowthinthepresenceofeither0.2xor2xMICofSPI031usinganautomatedOD
platereader(BioscreenC,OyGrowthcurvesAbLtd).
Statisticalanalysis
Allexperimentswererepeatedindependentlyatleast3times.Statisticalsignificanceofdata
wasdeterminedbyapplyingastudent’st-testusingGraphPadPrismversion5(GraphPadSoft-
ware,USA).Differenceswereconsideredsignificantif(cid:1)p(cid:3)0.05.
Results
SPI031displaysrapidkillingactivityagainstpathogenicbacteria
Recently,wedemonstratedthatSPI031exhibitsantibacterialeffectsagainstdifferentbacterial
pathogens(MICbetween4.63and18.5μg/ml)[15].Inthepresentstudy,time-killassayswere
performedtoanalyzethekillingrateofSPI031andtocompareitwiththatofconventionalanti-
bioticsfrequentlyusedinclinicalsettings.MICvaluesforSPI031andselectedantibioticswere
determinedandarelistedinS1Table.Fig2AshowsthekillingcurvesofSPI031andvancomycin
forS.aureus.At1xMICofSPI031,thebactericidalendpointwasachievedafter24h,witha
reductionofCFUbyalmost6logunits.Vancomycinhadalowbactericidalactivityat1xMIC
andsubstantialregrowthwasobservedafter2h.At4xMICofSPI031,morethan99%ofthebac-
teriawerekilledwithin30min.Incomparison,thekillingactivityofvancomycinat4xMICwas
muchslower,withareductionoftheinitialinoculumbyonly2logunitswithin24h.
ThekillingkineticsofSPI031andpolymyxinBagainstP.aeruginosaareshowninFig2B.At
1xMICofSPI031,a4-logreductionofviablecountswasachievedandregrowthwasobserved
after24h.At1xMICofpolymyxinB,a5-logreductionofthebacterialinoculumwasobtained
within10min,butfastregrowthwasobservedafter30min.At4xMIC,thebactericidalactivity
ofbothSPI031andpolymyxinBwasveryfast(7-logreductionwithin30min),indicatingthatat
highertherapeuticconcentrations,SPI031andpolymyxinBdisplaysimilarkillingactivities.
FrequencyofresistancedevelopmentagainstSPI031islow
ToinvestigatethepotencyofspontaneousresistancedevelopmentagainstSPI031,thefre-
quencybywhichP.aeruginosamutantsresistanttoSPI031emergewasdetermined,andcom-
paredtothoseassociatedwithconventionalantibiotics.ThemutationfrequencyofP.
aeruginosaforSPI031(6.09±2.10x10−8)wassignificantlylowerthanforpolymyxinB
(2.20±0.44x10−7)andnotsignificantlydifferentthanthatforofloxacin(4.03±1.10x10−8).
Interestingly,nospontaneousSPI031-resistantmutantsofS.aureuscouldbegenerated(limit
ofdetection<10−9).
SPI031exhibitsnon-specificinhibitionofmacromolecularbiosynthesis
pathways
ToinvestigatetheeffectofSPI031onmacromolecularpathways,amacromolecularsynthesis
assaywasperformed.Thepercentageofincorporationofradiolabeledprecursorsintomacro-
moleculeswasdetermineduponexposuretoSPI031andwascomparedwithresultsobtained
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ModeofActionoftheNewAntibacterialSPI031
Fig2.Time-killkineticsofSPI031againstS.aureusandP.aeruginosa.(A)Concentration-dependentkillingofS.aureusby
SPI031andvancomycin(VAN).(B)Concentration-dependentkillingofP.aeruginosabySPI031andpolymyxinB(PMB).Alldata
representmeans±standarderrorofthemean(SEM)from3independentexperiments(*p<0.05;**p<0.01;***p<0.001).The
blackdottedlinesindicatethelowerlimitofdetection.
doi:10.1371/journal.pone.0155139.g002
aftertreatmentwithknowninhibitorsofDNA,RNAorproteinsynthesis:ciprofloxacin,rifam-
picinandtetracycline,respectively(Fig3).At4xMICofSPI031,nopreferentialinhibitionof
testedmacromolecularprocesseswasobserved,aprofilethatisobservedforantibacterial
agentsthatactbydisruptingtheintegrityofthecytoplasmicmembrane[36–39].
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ModeofActionoftheNewAntibacterialSPI031
Fig3.EffectofSPI031onmacromolecularsynthesisinS.aureus.Incorporationof[methyl-3H]thymidine(A),[5,6-3H]uridine(B)andL-[G-3H]glutamine
(C)byS.aureusaftertreatmentwithSPI031,ciprofloxacin,rifampicinortetracyclineat4xMIC.Incorporationwasexpressedaspercentageofuntreated
control.Valuesshownaremeans±SDoftriplicatedeterminations.
doi:10.1371/journal.pone.0155139.g003
SPI031permeabilizesthemembrane(s)ofbothS.aureusandP.
aeruginosa
Basedontheresultsofthemacromolecularsynthesisanalysis,wefurtherinvestigatedmem-
branepermeabilisation.PermeabilizationofthemembraneofS.aureusaftertreatmentwith
SPI031wasassessedusingthenucleicacidstainSYTOXgreen.Thisstainonlyentersthecyto-
plasmifthemembraneiscompromised.Uponuptake,SYTOXgreenbindstonucleicacids,
whichinturnresultsinincreasedfluorescence[40].AdditionofSPI031toS.aureuscells
causedaconcentration-dependentincreaseinSYTOXgreenfluorescence,indicatingthat
SPI031permeabilizesthemembrane(Fig4A).At1xMICofSPI031,membranepermeabiliza-
tionwascomparabletothataftertreatmentwiththetoxinmelittinfrombee-venom,which
wasusedasapositivecontrol.InnermembranepermeabilityofP.aeruginosaaftertreatment
withSPI031wasalsoinvestigatedusingSYTOXgreen(Fig4B).SPI031wasabletorapidly
Fig4.EffectofSPI031onmembranepermeability.(A)EffectofincreasingconcentrationsofSPI031onthemembranepermeabilityofS.aureus,
monitoredbytheuptakeofSYTOXgreen.Cellstreatedwithmelittin(MEL)(1xMIC)servedasapositivecontrol.(B)InnermembranepermeabilizationofP.
aeruginosaaftertreatmentwithdifferentconcentrationsofSPI031,determinedbymeasuringSYTOXgreenuptake.Melittin(MEL)(1xMIC)wasusedasa
positivecontrol.(C)OutermembranepermeabilizationofP.aeruginosaaftertreatmentwithdifferentconcentrationsofSPI031,assessedbyquantifyingNPN
uptake.CellstreatedwithpolymyxinB(PMB)(1xMIC)wereusedasapositivecontrol.Datarepresentthemeansofthreeindependentreplicates±SEM
(*p<0.05;**p<0.01;***p<0.001comparedtountreatedcontrol).
doi:10.1371/journal.pone.0155139.g004
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ModeofActionoftheNewAntibacterialSPI031
induceSYTOXgreenuptake,suggestingthatthecompoundalsopermeabilizestheinnermem-
braneofP.aeruginosa.
Next,permeabilizationoftheoutermembraneofP.aeruginosawasdeterminedaftertreat-
mentwithdifferentconcentrationsofSPI031,usingthehydrophobicprobeNPN.Thisprobe
isnormallyexcludedfrombacterialmembranesduetothepresenceoflipopolysaccharidemol-
eculesontheoutermembrane[41].However,whentheoutermembraneispermeabilized,
NPNcanenterthemembranes,leadingtoastrongincreaseinfluorescence[42].Asshownin
Fig4C,SPI031rapidlypermeabilizedtheoutermembraneofP.aeruginosainaconcentration-
dependentmanner.At1xMICofSPI031,theNPNuptakewassimilartothatofthepositive
controlpolymyxinB.
SPI031targetsthephospholipidcomponentofbacterialmembranes
Inadditiontolipids,thebacterialmembranecontainsmanyotherbiologicalmolecules.To
investigatethedirectinteractionofSPI031withthephospholipidbilayer,acarboxyfluorescein
(CF)leakageassaywasperformed.Forthisassay,CF-loadedliposomeswereusedthatmimic
thecompositionofthephospholipidbilayerofS.aureus.AsshowninFig5,SPI031causedsub-
stantialreleaseofCFat4xMIC,andanintermediatereleaseat1xMIC.Theseresultsindicate
thatSPI031isabletointeractwiththephospholipidbilayer,whichinturnresultsindestabili-
zationofthecytoplasmicmembrane.
MembranedamagecausedbySPI031canbevisualized
microscopically
TofurtherinvestigatetheeffectofSPI031onthemembrane,themembranestainFM4–64was
used(Fig6).TreatmentofexponentiallygrowingcellsofS.aureusandP.aeruginosawithsol-
ventcontrolDMSOresultedinuniformlystainedmembranes.Incontrast,treatmentofthe
cellswithSPI031at1xMICresultedinspecificmembraneaccumulations.Heterogeneityin
membranestainingwasalsoseeninP.aeruginosacellstreatedwith1xMICofpolymyxinB.In
contrast,P.aeruginosacellsexposedto1xMICoftobramycinshowednomembranedefects
(datanotshown).Overall,theseresultsfurthercorroborateacleardirecteffectofSPI031on
thebacterialmembrane.
Fig5.IntegrityofS.aureusliposomesaftertreatmentwithSPI031at1xMICand4xMIC.SDS(5%)and
tetracycline(TET)(4xMIC)wereusedaspositiveandnegativecontrols,respectively.Meansandstandard
deviation(SD)of3independentexperimentsareshown(*p<0.05;**p<0.01;***p<0.001comparedto
treatmentwithtetracycline).
doi:10.1371/journal.pone.0155139.g005
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ModeofActionoftheNewAntibacterialSPI031
Fig6.MicroscopicanalysisofthecellmembraneaftertreatmentwithSPI031.FluorescentimagesofS.aureuscells(upper
row)andP.aeruginosacells(lowerrow)stainedwithFM4–64intheabsenceorpresenceof1xMICofSPI031.Scalebar
correspondsto2μm.ImageswereprocessedwithunsharpmaskofZen2.0.
doi:10.1371/journal.pone.0155139.g006
WholegenomesequencingofSPI031-resistantmutantsreveals
mutationsingenescodingforproteinsinvolvedinoutermembrane
synthesisandeffluxmechanisms
Mutationalanalysisofspontaneousresistantmutantsisanapproachcommonlyusedtogain
moreinformationaboutprimarytargetsofnewantibacterialcompounds[43–46].Therefore,
threeindependentspontaneousSPI031-resistantmutantsofP.aeruginosawereselectedand
weresubjectedtowholegenomesequencing.AllidentifiedmutationsarelistedinS3Table.
OnemutantcarriedmutationsinnfxB,whichcodesfortherepressoroftheMexCD-OprJmul-
tidrugeffluxpump[47].ThesefindingsindicatethatSPI031maybeasubstrateforthisefflux
pump.Thetwoothermutantshadmutationsingenescodingforproteinsinvolvedinouter
membranesynthesis(htrBandPA14_23400)[48,49].Forthesemutants,wehypothesizethata
changeinthestructureoftheoutermembraneisresponsiblefortheobservedresistance.In
addition,twoP.aeruginosamutantsacquiredmutationsinintergenicregions.
PLOSONE|DOI:10.1371/journal.pone.0155139 May11,2016 10/17