TLR-2 Recognizes Propionibacterium acnes CAMP Factor 1 from Highly Inflammatory Strains

26 Pages·2016·2.87 MB·English

RESEARCHARTICLE
TLR-2 Recognizes Propionibacterium acnes
CAMP Factor 1 from Highly Inflammatory
Strains
CoralieLheure1☯,PhilippeAlainGrange1☯,GuillaumeOllagnier1,PhilippeMorand2,
NathalieDe´sire´3,4,SophieSayon3,4,Ste´phaneCorvec5,Jo¨elRaingeaud6,Anne-
GenevièveMarcelin3,4,VincentCalvez3,4,AmirKhammari7,Fre´de´ricBatteux1,8,
BrigitteDre´no7,NicolasDupin1,9*
1 Universite´SorbonneParisDescartes,Faculte´deMe´decine,INSERM,InstitutCochin,Laboratoirede
Dermatologie-CNRSyphilis,Paris,France,2 AP-HP,GroupeHospitalierParisCentreCochin-HoˆtelDieu-
Broca,ServicedeBacte´riologie-CNRStreptocoques,Paris,France,3 SorbonneUniversite´,UPMC
Universite´Paris06,INSERM,InstitutPierreLouisd’Epide´miologieetdeSante´Publique,Paris,France,4 AP-
HP,GroupehospitalierPitie´Salpêtrière,LaboratoiredeVirologie,Paris,France,5 ServicedeBacte´riologie-
a11111 Hygiènehospitalière,CHUdeNantes,Nantes,France,6 UMR8126,InstitutGustaveRoussy,Villejuif,
France,7 CHU,servicededermatologie,CIC,HoˆtelDieu,Nantes,HoˆtelDieu,Nantes,France,8 AP-HP,
GroupeHospitalierParisCentreCochin-HoˆtelDieu-Broca,Serviced’ImmunologieBiologique,Paris,France,
9 AP-HP,GroupeHospitalierParisCentreCochin-HoˆtelDieu-Broca,ServicedeDermatologie-Ve´ne´re´ologie,
Paris,France
☯Theseauthorscontributedequallytothiswork.
*[email protected]
OPENACCESS
Citation:LheureC,GrangePA,OllagnierG, Abstract
MorandP,De´sire´N,SayonS,etal.(2016)TLR-2
RecognizesPropionibacteriumacnesCAMPFactor
1fromHighlyInflammatoryStrains.PLoSONE11 Background
(11):e0167237.doi:10.1371/journal.
pone.0167237 Propionibacteriumacnes(P.acnes)isananaerobic,Gram-positivebacteriaencounteredin
inflammatoryacnelesions,particularlyinthepilosebaceousfollicle.P.acnestriggersa
Editor:HolgerBru¨ggemann,AarhusUniversitet,
DENMARK strongimmuneresponseinvolvingkeratinocytes,sebocytesandmonocytes,thetargetcells
duringacnedevelopment.Lipoteicoicacidandpeptidoglycaninducetheinflammatoryreac-
Received:July29,2016
tion,butnoP.acnessurfaceproteininteractingwithToll-likereceptorshasbeenidentified.
Accepted:November10,2016
P.acnessurfaceproteinshavebeenextractedbylithiumstrippingandshowntoinduce
Published:November30,2016 CXCL8productionbykeratinocytes.
Copyright:©2016Lheureetal.Thisisanopen
accessarticledistributedunderthetermsofthe
Methodologyandprincipalfindings
CreativeCommonsAttributionLicense,which
permitsunrestricteduse,distribution,and Far-westernblottingidentifiedtwosurfaceproteins,of24.5-and27.5-kDainsize,specifi-
reproductioninanymedium,providedtheoriginal
callyrecognizedbyTLR2.Theseproteinswerecharacterized,byLC-MS/MS,asCAMPfac-
authorandsourcearecredited.
tor1devoidofitssignalpeptidesequence,asshownbyN-terminalsequencing.Purified
DataAvailabilityStatement:Allrelevantdataare
CAMPfactor1inducesCXCL8productionbyactivatingtheCXCL8genepromoter,trigger-
withinthepaperanditsSupportingInformation
ingthesynthesisofCXCL8mRNA.AntibodiesagainstTLR2significantlydecreasedthe
files.
CXCL8response.Forthe27P.acnesstrainsusedinthisstudy,CAMP1-TLR2binding
Funding:ThisworkwassupportedbytheSocie´te´
intensitywasmodulatedandappearedtobestrongintypeIBandIIstrains,whichproduced
Franc¸aisedeDermatologieetdePathologie
SexuellementTransmissible. largeamountsofCXCL8,whereasmostofthetypeIA andIA strainspresentedlittleorno
1 2
CAMP1-TLR2bindingandlowlevelsofCXCL8production.Thenucleotidesequenceof
CompetingInterests:Theauthorshavedeclared
thatnocompetinginterestsexist. CAMPfactordisplaysamajorpolymorphism,definingtwodistinctgeneticgroups
PLOSONE|DOI:10.1371/journal.pone.0167237 November30,2016 1/26
P.acnesCAMPFactor1RecognitionbyTLR2
correspondingtoCAMPfactor1with14amino-acidchangesfromstrainsphylotypedIIwith
moderateandhighlevelsofCAMP1-TLR2bindingactivity,andCAMPfactor1containing0,
1or2amino-acidchangesfromstrainsphylotypedIA ,IA ,orIBpresentingno,weakor
1 2
moderateCAMP1-TLR2binding.
Conclusions
OurfindingsindicatethatCAMPfactor1maycontributetoP.acnesvirulence,byamplifying
theinflammationreactionthroughdirectinteractionwithTLR2.
Introduction
Propionibacteriumacnes(P.acnes)isananaerobicGram-positivebacteriumfrequentlypresent
inthenormalhumanskinmicrobiota,whereitaccumulatespreferentiallyinthepiloseba-
ceaousunitsinindividualswithandwithoutacne[1].Thisbacteriumhaslongbeenconsid-
eredtobecommensal,butthereisgrowingevidencethatitalsoactsasanopportunistic
pathogen,causinginfectionsassociatedwithdiverseimplants,includingbreastimplants,neu-
rosurgicalshunts,cardiovasculardevices,ocularimplantsandprostheticjoints,andthatspe-
cificclonesofP.acnesareassociatedwithacne[2,3,4,5,6,7].P.acnesis,indeed,bestknown
foritsassociationwithacne,acommoninflammatorydisorderofthesebaceousfolliclesaffect-
ingmorethan85%ofadolescentsbutalsopersistingoroccurringinsomeadults[8].Acneisa
multifactorialdiseasecharacterizedbyanincreaseinsebumsecretionassociatedwithchanges
insebumcompositioninducedbyandrogens,hyperkeratinizationleadingtotheobstruction
ofsebaceousfollicles,changesinP.acnesproteinproductionandanintenseinflammatory
reaction,buttheexactsequenceoftheseeventsremainsunclear[9,10,11].Studiesinvolving
MLSTapproacheshaveclassifiedP.acnesstrainsintosixphylotypes(IA ,IA ,IB,IC,IIand
1 2
III)accordingtotheirabilitytoinducetheproductionofproinflammatorymolecules[12],
theirassociationwithinfections,theirbiochemicalandmorphologicalcharacteristicsand
theirabilitytoaggregate[13,14,15,16,17,18].Avariablenumberoftandemrepeats-based
methodwasrecentlydeveloped,toimprovegenotypinganddiscriminatebetweenP.acnes
strains[19].ThecoregenesofP.acnesseemtobehighlyconservedbetweenstrains,butseveral
non-corelocihavebeenidentifiedthatinterferewithexpressionlevelsandarecorrelatedwith
thedifferentphylotypes[20].Indeed,differenceshavebeenobservedinCXCL8productionby
keratinocytesstimulatedwithdifferentP.acnesstrains[21],togetherwithdifferencesinpro-
teinsecretion[22].TheIA phylotypehasalsobeenshowntobestronglyassociatedwithacne
1
lesions,whereasthetypeIIIphylotypeisrarelyfoundintheselesionsbutaccountsfor20%of
isolatesfromnormalskin.TypesIBandIIareoverrepresentedinsoft-tissueandimplant–
associatedinfections,andinbacteremia[16,23].
Theinnateimmuneresponseisthebody’sfirstlineofdefenseagainstinfectiousagents,and
itssuccessisreflectedinhealthandwell-being.Pathogenrecognitionbytheinnateimmune
systemreliesonalimitednumberofpatternrecognitionreceptors(PRR)thatrecognizecon-
servedproductsofmicrobialmetabolismproducedbymicrobialpathogensandknownas
pathogen-associatedmolecularpatterns(PAMPs).Thebest-knownPRRsaretheToll-like
receptors(TLRs).TenTLRshavebeendescribedinmammalsandhavebeenclassifiedinto
twogroups:TLRs1,2,4,5,and6,localizedonthecellularmembrane,areactivatedbyextra-
cellularPAMPS;andTLRs3,7,8,9,localizedonintracellularorganelles,suchaslysosomes
andendosomes.TogetherwithTLR1,TLR6andCD36,TLR2playsacrucialroleinthe
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P.acnesCAMPFactor1RecognitionbyTLR2
recognitionofpeptidoglycan(PGN,amoleculeexpressedbymanybacterialspecies),lipopro-
teins,andlipoteichoicacid(LTA)fromGram-positivebacteria,andoflipoarabinomannan
frommycobacteriaandzymosanfromfungal.TLR4actstogetherwithCD14andMD2inthe
recognitionofLPSfromGram-negativebacteria[24].
P.acnescontributestotheinflammatorylesionsofacnebyactivatinginnateimmunityvia
theTLR2expressedoncutaneouscells[25].Invitro,P.acnesstimulateskeratinocytesand
monocytes,leadingtotheproductionofproinflammatorycytokines(IL-1α/β,CXCL8,IL-12,
TNFα)viaTLR2activation[26,27,28],andthegenerationofreactiveoxygenspeciesthrough
theactivationofscavengerreceptorCD36[29].Invivo,thegenesencodingCXCL8,TLR2,and
β-defensin-4havebeenshowntobeupregulatedinacnelesions,togetherwithNF-κBandAP-
1,suggestinganactivationofTLR2byP.acnes[30,31,32,33,34].
ThesurfaceproteinsofP.acnesinvolvedintheactivationofTLR2onkeratinocytesand
monocytesandleadingtoinflammatorylesionsinacnehaveyettobeidentified.Theaimof
thisstudywastoidentifysurfaceproteinsofP.acnesrecognizedbyTLR2inseveralstrains
withdifferentinflammatoryprofiles.
Results
IdentificationofP.acnessurfaceproteinsrecognizedbyTLR2
WeinvestigatedtheproteinrecognizedbyTLR2,byextractingproteinsinthepresenceofa
highconcentrationoflithium,toensuretheselectiveremovalofP.acnessurfaceproteins.We
checkedthatallthestrainsusedinthisstudywereabletoinduceproinflammatoryresponses,
bymeasuringCXCL8productionbykeratinocytes,andalsothattheHaCaTkeratinocytecell
lineusedintheinvitroassaycouldproduceTLR2.Totalcelllysateswereanalyzedbywestern
blotting,whichrevealedthepresenceofaproteinwithamolecularweightofabout90-kDain
theHaCaTlysate,andinthe293TandThP1lysatesusedaspositivecontrols(S1Fig).Theker-
atinocyteswerethenstimulatedwithbothwholebacteriaandlithiumproteinextract(LiE),
andCXCL8productionwasmeasuredafter18hofstimulation.Representativeresultsare
shownfortheevaluationoftheCXCL8productionfromthe27P.acnesstrainsusedinthis
study(Fig1).WholeP.acnesbacteriainducedCXCL8mRNAandproteinproductionina
dose-dependentmanner,ataMOIof10to1000(Fig1Aand1B).LiEfromthecorresponding
bacteriaweretestedandshowntohaveasimilarcapacitytoinduceCXCL8mRNAandpro-
teinproductioninadose-dependentmanner(Fig2Aand2B).BothwholebacteriaandLiEof
CHR(typeIA )andPIE(typeIB)strains,and,toalesserextent,RON(typeIA )strains,
2 1
inducedlargeamountsofCXCL8atthelowerMOIof10(Fig1B)orforaLiEproteincontent
of3.1μg/ml,whereastheotherstrainsdidnot(Fig2B).TheseresultsindicatethatP.acnessur-
faceproteinsinducetheinflammatoryreactionandareasuitablestartingmaterialforidentifi-
cationofthesurfaceproteinrecognizedbyTLR2.Far-westernblottingwasperformedontotal
LiE,andtwoproteinbandsrecognizedbyTLR2,withapparentmolecularmassesof24.5-and
27.5-kDa,weredetected(Fig3B).Accordingtothenumberofproteinbandsrecognizedby
TLR2,andtheintensityofthesignal,werankedstrainsasfollows:(+++)for2stronglyrecog-
nizedbands(Fig3B,lane7);(++)for2moderatelyrecognizedbands(lane4);(+)1moderately
recognizedband(lane6);(+/-)1veryfaintband(lane3);and(-)nobandrecognizedbyTLR2.
Theresultsforthe27strainsarepresentedinS2Fig.Nobandsweredetectedinnegativecon-
trolexperimentsusingonlyrecombinantTLR2/detectionsubstrateorbiotinylatedanti-TLR2
antibody/detectionsubstrate(datanotshown).Weassessedthespecificityofrecognitionby
TLR2,byincubatingthesurfaceproteinswithrecombinantTLR4;onlytwofaintbandswere
detected,atanapparentmolecularweightofabout60-kDa(Fig3C,lanes5,7)inonlytwo
strains,andneitherthe24.5-kDanorthe27.5-kDaproteinwasrecognizedbyTLR4.Asthe
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P.acnesCAMPFactor1RecognitionbyTLR2
Fig1.InductionofCXCL8productioninkerationocytesstimulatedwithwholeP.acnesbacteria.
HaCaTcellswereincubatedfor18hwithwholeP.acnesbacteria(6919,RON,CHR,GUE,TRI,PIE,Table1)
ataMOIof10(A)andatMOIof10,100,1000(B).TotalRNAwasextractedandCXCL8mRNAlevelswere
determinedbyreal-timeRT-PCRandcomparedthoseofGAPDHmRNA(usedasthecontrol).Thedifference
isexpressedasafold-change.CXCL8productionwasanalyzedbyELISAonculturesupernatants.Control
experimentswererunwithunstimulatedcells.Thedataarepresentedasthemean±standarddeviationof
threeindependentexperiments.Statisticalsignificanceisindicatedby*P(cid:20)0.05,**P(cid:20)0.01,***
P(cid:20)0.001,****P(cid:20)0.0001.
doi:10.1371/journal.pone.0167237.g001
24.5-kDaand27.5-kDaproteinsappearedtoberecognizedbyTLR2in48%ofthestrains
tested,wechosetofocusonthecharacterizationofthesetwoproteins.
Proinflammatoryactivityoftheproteinsofinterest
Weassessedtheproinflammatorypropertiesofthe24.5-and27.5-kDaproteins,byelectro-
phoreticseparationoftheseproteinsfromtheLiEoftheCHR(typeIA )andPIE(typeIB)
2
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P.acnesCAMPFactor1RecognitionbyTLR2
Fig2.InductionofCXCL8productioninkeratinocytesbyP.acnessurfaceproteins.HaCaTcellswere
incubatedfor18hwithlithiumextractsofbacterialproteinsatconcentrationsof50μg/ml(A)and3.12,12.5,
and50μg/ml(B).TotalRNAwasextractedandCXCL8mRNAlevelsweredeterminedbyreal-timeRT-PCR
andcomparedwithGAPDHmRNAlevels(usedasthecontrol),withexpressionasafold-change.CXCL8
productionwasassessedbyELISAonculturesupernatants.Controlexperimentswereperformedwith
unstimulatedcells.Dataarepresentedasthemean±standarddeviationofthreeindependentexperiments.
Statisticalsignificanceisindicatedby*P(cid:20)0.05,**P(cid:20)0.01,***P(cid:20)0.001,****P(cid:20)0.0001.
doi:10.1371/journal.pone.0167237.g002
strains,elutionfromthegelandassessmentoftheirabilitytoinduceCXCL8production(Fig
4).WeshowedinNF-κBandCXCL8promoterassaysthattheseproteinswereabletoactivate
bothpromoters(Fig4A).Thedirectstimulationofepidermalkeratinocytesbythesetwopro-
teinsalsoledtothe10-to100-foldinductionofCXCL8transcription(Fig4B)andanincrease
intheamountofCXCL8proteinto10pg/ml(Fig4C).Controlexperimentswereperformed
oncellsaloneandoncellsstimulatedwithasolutionelutedfromtheacrylamidegelinthe
sameconditions;noactivitywasobservedineithercase.Bothproteinsofinterestwereableto
stimulatetheproductionofCXCL8mRNAandproteininkeratinocytes.
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P.acnesCAMPFactor1RecognitionbyTLR2
Fig3.TLR2bindingtoP.acnessurfaceproteins.P.acnessurfaceproteinswereextractedfroma5-dayculturebacterialpelletand
separatedbyelectrophoresisina4–12%NuPAGELDSBisTrisgel(50μg)withdetectionbyCoomassiebluestaining(A).Separated
proteinsweretransferredontonitrocellulosemembranes,whichwereincubatedwithrecombinantTLR2(B)andTLR4(C)(0.1μg/ml).TLR
bindingactivitywasdetectedwithspecificbiotinylatedantibodiesagainstTLR2andTLR4,respectively,asdescribedintheMaterialsand
Methods.Lane1containsthemolecularmassmarkers.Lanes2to7containproteinsfromstrains6919,RON,CHR,GUE,TRI,andPIE,
respectively.Arrowsindicatethepositionsofthe24.5-and27.5-kDabandsofinterest.
doi:10.1371/journal.pone.0167237.g003
Characterizationoftheproteinsofinterest
Afterelectrophoreticseparation,theproteinsofinterestwereexcisedfromthegelandcharac-
terizedbyLC-MS/MSofthepeptidemixtureobtainedafterin-geldigestion.Proteinsequence
databasesearchesidentifiedthetwoproteinsofinterestasproductsofgeneencodingahypo-
thetical285-aminoacidproteinfromtheCAMPfactorsuperfamily(Accessionnumbergi|
488487765),covering66%oftheamino-acidsequenceforthe27.5-kDaproteinand61%of
thesequenceforthe24.5-kDaprotein(Table1).Thesequenceofthe27.5-kDawas99%,38%,
33%,50%and51%identicaltotheP.acnesCAMPfactors1,2,3,4,and5,respectively(Fig5),
andbothproteinscorrespondedtothesameP.acnessurfaceprotein.Thetheoreticalmolecu-
larmassofCAMPfactor1is30.411-kDa.WethereforeanalyzedtheN-terminalsequencesof
thetwoelutedproteins(Fig6),comparingthemwiththeexpectedsequenceforCAMPfactor
1.Thefirstfiveaminoacidsofthe27.5-kDaproteinwereAPVAP,locatedattheputativesignal
sequencecleavagesitestartingatposition29[35].Thissequenceisconsistentwiththelossof
2476DafromtheN-terminusoftheprotein,resultinginatruncatedproteinwithacalculated
molecularmassof27.935-kDa.Forthe24.5-kDaCAMP-1,thefirstfiveaminoacidswere
SLLDT,atposition64,correspondingtothelossof6322Da,resultinginatruncatedprotein
withacalculatedmolecularmassof24.089-kDa(Fig6).Basedonthesefindings,weconcluded
thatthesurfaceproteinofP.acnesrecognizedbyTLR2wasCAMPfactor1.
BlockingofCAMP-1/TLR-2interaction
WeinvestigatedtheabilityofablockingagenttooutcompetetheinteractionofCAMPfactor
1withTLR2.Anti-TLR2antibodieshavebeenshowntoblockCXCL8productionbykeratino-
cytes[18,21].ThestimulationofkeratinocyteswithelutedCAMP-1proteinaftertheirprior
treatmentwithanti-TLR2antibodiesresultedinsignificantlylowerlevelsofNF-κB(P=0.008)
andCXCL8(P=0.0012)promoteractivation(Fig7Aand7B),and,consequently,significantly
lowerlevelsofCXCL8mRNA(P=0.00047)(Fig7C)andCXCL8proteinproductioninkerati-
nocytes(P=0.0024)(Fig7D).Nosucheffectwasobservedwhenthisexperimentwas
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P.acnesCAMPFactor1RecognitionbyTLR2
Fig4.P.acnessurfaceproteinsrecognizedbyTLR2haveproinflammatoryactivityinvitro.Surface
proteinextractswereelectrophoreticallyseparatedin10%SDS-PAGEgels(13x13cm),withdetectionby
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P.acnesCAMPFactor1RecognitionbyTLR2
Coomassiebluestaining.Proteinsofinterestwereexcisedfromthegelandelutedasdescribedinthe
MaterialsandMethods.(A)HaCaTcellsweretransfectedwithCXCL8(-173bp)(lightgraybar)andNF-κB
(darkgraybar)insertedintoaconstructupstreamfromtheluciferasegene,for24h,afterwhich,aninternal
control(theRenillaluciferaseexpressionplasmid)wasaddedtothetransfectionmixture.Cellswerestimulated
byincubationwiththeelutedproteins(10μg)for24hat37˚C.RelativeNF-κBandCXCL8promoteractivities
weredeterminedastheratiooffireflyandRenillaluciferaseactivities.HaCaTcellswerestimulatedwiththe
elutedproteins(20μg)for24hat37˚CandCXCL8mRNAlevelswereevaluatedbyRT-qPCR(B),andCXCL8
proteinlevelswereassessedbyELISA(C).Dataarepresentedasthemean±standarddeviationoftwo
independentexperiments.Statisticalsignificanceisindicatedby*P(cid:20)0.05,**P(cid:20)0.01,***P(cid:20)0.001,
****P(cid:20)0.0001.
doi:10.1371/journal.pone.0167237.g004
performedwiththeIgGisotypecontrol.Thus,CAMPfactor1isolatedfromP.acnesinteracts
withTLR2,triggeringadownstreamsignalingpathwayleadingtoCXCL8production.
Comparisonofphylotype,TLR2bindingactivityandCXCL8production
UsingapreviouslydescribedrapidmultiplexPCRassay[36],wephylotypedalltheisolates.
Weidentifiedfourdifferentphylotypes:IA (15strains),IA (2strains),IB(9strains)andII(2
1 2
Table1. ProteinsidentifiedbyLC-MS/MSinthetrypsin-digestedfragmentfromthe24.5-and27.5-kDaP.acnesproteinrecognizedbyTLR-2
Protein Accession Name Scoreb MW pI Matched Sequence
banda number (kDa) peptide coverage(%)c
27.5 gi|488487765 MULTISPECIES:hypotheticalprotein 1159 30379 9.88 28 66
[Propionibacterium]
gi|488472745 MULTISPECIES:hypotheticalprotein 1070 30394 9.77 25 66
[Propionibacterium]
gi|495875308 hypotheticalprotein[Propionibacterium 1010 30408 9.77 23 64
sp.5_U_42AFAA]
gi|488491325 hypotheticalprotein[Propionibacteriumacnes] 967 30448 9.81 25 66
gi|695301769 CAMPfactor[Propionibacteriumacnes] 813 30257 9.74 19 49
gi|56244570 Camp4,partial[Propionibacteriumacnes] 446 27466 9.4 10 52
gi|313823890 hypotheticalproteinHMPREF9605_00761,partial 245 35838 10.04 6 32
[PropionibacteriumacnesHL036PA2]
gi|313763866 Tatpathwaysignalsequencedomainprotein 71 32585 9.83 1 12
[PropionibacteriumacnesHL013PA1]
gi|455647494 hypotheticalproteinH114_24280[Streptomyces 56 28817 5.4 1 2
gancidicusBKS13–15]
24.5 gi|488487765 MULTISPECIES:hypotheticalprotein 1159 30379 9.88 32 61
[Propionibacterium]
gi|488472745 MULTISPECIES:hypotheticalprotein 1058 30394 9.77 28 61
[Propionibacterium]
gi|488491325 hypotheticalprotein[Propionibacteriumacnes] 984 30448 9.81 27 61
gi|495875308 hypotheticalprotein[Propionibacterium 952 30408 9.77 25 59
sp.5_U_42AFAA]
gi|695301769 CAMPfactor[Propionibacteriumacnes] 705 30257 9.74 16 42
gi|328907812 cAMPfactor[PropionibacteriumhumerusiiP08] 302 27322 9.7 6 24
gi|663150685 transportintegralmembraneprotein[Streptomyces 60 62204 9.8 0 1
violaceorubidus]
gi|492266975 phasinfamilyprotein[Acidovoraxdelafieldii] 55 18911 6.3 1 19
gi|746357861 hypotheticalprotein,partial[Pandoraeasputorum] 53 160357 6.07 1 0
aElectrophoreticallyseparatedproteinbandswereexcisedandsubjectedtoin-geltrypsinedigestionfollowedbyLC-MS/MSanalysisasdescribedin
MaterialsandMethods.
bMASCOTscore.
cCoverageoftheproteinsequencebythepeptidesusedforidentification.
doi:10.1371/journal.pone.0167237.t001
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P.acnesCAMPFactor1RecognitionbyTLR2
Fig5.Comparisonofthesequencesofthe27.5kDaP.acnessurfaceproteinandP.acnesCAMPfactors.The
peptidesequenceofthe27.5kDaproteinwasobtainedafterLC-MS/MSanalysiscorrespondingto66%coverage(in
bold),asdescribedintheMaterialsandMethods.ItwascomparedwiththesequencesofP.acnesCAMPfactors1to
5(ReferencestrainNCTC737,GenBankaccessionnumberAY527218.1).Differencesbetweensequencesare
highlightedingray.
doi:10.1371/journal.pone.0167237.g005
strains)(Table2,S3Fig).WeanalyzedtherecognitionofCAMPfactor1byTLR2andfound
thatmostofthestrainswithstrongTLR2bindingactivitywereoftypesIBandII(Table2).
WethenmeasuredCXCL8productionbykeratinocytesstimulatedwiththe27strains.We
foundthattheP.acnesstrainstestedcouldbeseparatedintotwogroups,onewithlowtomod-
eratelevelsofCXCL8production(93to400pg/ml)andtheotherwithhighlevelsofCXCL8
production(>400pg/ml)(Table2).MostofthestrainsproducinghighlevelofCXCL8were
Fig6.N-terminalsequencingoftheproteinofinterest.Surfaceproteinextractswereelectrophoreticallyseparatedin10%
SDS-PAGEgels(13x13cm),withdetectionbyCoomassiebluestaining.Proteinsofinterestwereexcisedfromthegel,elutedand
subjectedtoN-terminalsequencingbyEdmandegradationtoobtainthefirstfiveamino-acidresiduesofeachprotein,asdescribedin
MaterialsandMethods.N-terminalsequenceswerecomparedtotheP.acnesCAMPfactor1sequence(referencestainNCTC737,
GenBankaccessionnumberAY527218.1).ThepeptidesignalcleavagesiteofCAMPfactor1isshowninbold.
doi:10.1371/journal.pone.0167237.g006
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P.acnesCAMPFactor1RecognitionbyTLR2
Fig7.TLR2blockadeinhibitsP.acnesCAMPfactor1-inducedCXCL8expression.Surfaceproteinextracts
wereelectrophoreticallyseparatedin10%SDS-PAGEgels(13x13cm),withdetectionbyCoomassiebluestaining.
Proteinsofinterestwereexcisedfromthegel,elutedandpooledasdescribedintheMaterialsandMethods.HaCaT
cellsweretreatedfor2hwithhumananti-TLR2(dashedbar)orwithgoatanti-IgGaantibodies(darkgraybar)and
transfectedbyincubationfor24hwithNF-κBtranscriptionfactor(A)andCXCL8(-173bp)(B)insertedintoa
constructupstreamfromtheluciferasegene,afterwhich,aninternalcontrol(theRenillaluciferaseexpression
plasmid)wasaddedtothetransfectionmixture.CellwerestimulatedwithelutedCAMPfactor1(50μg/ml)for24hat
37˚C.RelativeNF-κBandCXCL8promoteractivitiesweredeterminedastheratiooffireflyandRenillaluciferase
activities.PretreatedHaCaTcellswerestimulatedbyincubationwithelutedCAMPfactor1(50μg/ml)for24hat
37˚C,CXCL8mRNAlevelsweredeterminedbyRT-qPCR(C),andCXCL8productionwasmeasuredbyELISA(D).
Controlexperimentswereperformedwithcellsalone(darkbar)andwithstimulatedcellsnotpreviouslytreatedwith
CAMPfactor1(lightgraybar).Dataarepresentedasthemean±standarddeviationoftwoindependent
experiments.Statisticalsignificanceisindicatedby*P(cid:20)0.05,**P(cid:20)0.01,***P(cid:20)0.001,****P(cid:20)0.0001.
doi:10.1371/journal.pone.0167237.g007
oftypesIBandII.Acomparisonofphylotypewithabilitytoinduceinflammationforthe27P.
acnesstrainsstudiedshowedthatmostofthestrainswithhighlevelsofCAMP1-TLR2binding
activitybelongedtotypesIBandII,whereasthetypeIA andIA strainsproducedCAMPfac-
1 2
tor1moleculesthatwereonlymoderatelystronglyrecognized,ifatall,byTLR2(Table2).
CAMP-1nucleotideandpeptidesequencesanalysis
CAMP1-TLR2bindingprofiledifferedbetweenP.acnesstrains(Fig3).Wethereforeinvesti-
gatedthenucleotideandpeptidesequencesofthe27P.acnesstrains.The858bpcorrespond-
ingtothesequenceofCAMPfactor1reportedelsewhere[35]wassequencedandcompared
withthesequenceobtainedforCAMPfactor1referencestrainNCTC737(ATCC6919).
Sequenceswerethenclassifiedintofourgroupsaccordingtothenumberofnucleotidepoly-
morphisms(Fig8,Table2).ThefirstgroupcorrespondedtoCAMPfactor1geneswithno
nucleotidepolymorphism(Fig8A).ThesecondgroupcorrespondedtoCAMPfactor1genes
with1nucleotidepolymorphism,eitherA31>GorG361>T,correspondingtoamino-acid
changesatpositionsI11>VandV121>L,respectively(Fig8Band8C;Table2).Athirdgroup
consistedofCAMPfactor1genesdisplayingtwosinglenucleotidepolymorphismsfromthe
PLOSONE|DOI:10.1371/journal.pone.0167237 November30,2016 10/26