Staphylococcus epidermidis agr Quorum-Sensing System

12 Pages·2014·1.7 MB·English

Staphylococcus epidermidis agr Quorum-Sensing System: Signal
Identification, Cross Talk, and Importance in Colonization
MichaelE.Olson,aDanielA.Todd,bCarolynR.Schaeffer,cAlexandraE.Paharik,aMichaelJ.VanDyke,aHenningBüttner,e
PaulM.Dunman,dHolgerRohde,eNadjaB.Cech,bPaulD.Fey,cAlexanderR.Horswilla
DepartmentofMicrobiology,RoyJ.andLucilleA.CarverCollegeofMedicine,UniversityofIowa,IowaCity,Iowa,USAa;DepartmentofChemistryandBiochemistry,
UniversityofNorthCarolinaGreensboro,Greensboro,NorthCarolina,USAb;DepartmentofPathologyandMicrobiology,UniversityofNebraskaMedicalCenter,Omaha,
Nebraska,USAc;DepartmentofMicrobiologyandImmunology,UniversityofRochesterMedicalCenter,Rochester,NewYork,USAd;InstituteforMedicalMicrobiology,
VirologyandHygiene,UniversityMedicalCentreHamburg-Eppendorf,Hamburg,Germanye
Staphylococcusepidermidisisanopportunisticpathogenthatisoneoftheleadingcausesofmedicaldeviceinfections.Global
regulatorsliketheagrquorum-sensingsysteminthispathogenhavereceivedalimitedamountofattention,leavingimportant
D
questionsunanswered.TherearethreeagrtypesinS.epidermidisstrains,butonlyoneoftheautoinducingpeptide(AIP)signals
o
hasbeenidentified(AIP-I),andcrosstalkbetweenagrsystemshasnotbeentested.WestructurallycharacterizedallthreeAIP w
n
typesusingmassspectrometryanddiscoveredthattheAIP-IIandAIP-IIIsignalsare12residuesinlength,makingthemthe lo
largeststaphylococcalAIPsidentifiedtodate.S.epidermidisagrreporterstrainsweredevelopedforeachsystem,andwedeter- a
d
minedthatcross-inhibitoryinteractionsoccurbetweentheagrtypeIandIIsystemsandbetweentheagrtypeIandIIIsystems. e
Incontrast,nocrosstalkwasobservedbetweenthetypeIIandIIIsystems.TofurtherunderstandtheoutputsoftheS.epidermi- d

f
disagrsystem,anRNAIIImutantwasconstructed,andmicroarraystudiesrevealedthatexoenzymes(EcpproteaseandGeh ro
lipase)andlow-molecular-weighttoxinsweredownregulatedinthemutant.Follow-upanalysisofEcpconfirmedtheRNAIIIis m

requiredtoinduceproteaseactivityandthatagrcrosstalkmodulatesEcpactivityinamannerthatmirrorstheagrreporterre- h
t
sults.Finally,wedemonstratedthattheagrsystemenhancesskincolonizationbyS.epidermidisusingaporcinemodel.This tp
:
workexpandsourknowledgeofS.epidermidisagrsystemfunctionandwillaidfuturestudiesoncell-cellcommunicationinthis /
/
importantopportunisticpathogen. jb
.
a
s
m
Staphylococcusepidermidisisahumancommensaloftheskin Each of these systems is controlled by an extracellular peptide .o
r
andmucosalsurfacesandisoneofthebest-knownmembersof signal called an autoinducing peptide (AIP), which is secreted g
/
the coagulase-negative staphylococci (CoNS) (1). CoNS are the during growth and activates gene expression in a cell density- o
mostfrequentcauseofhospital-associatedinfections(HAIs)(2), dependentmanner.Amongthestaphylococci,theAIPsaretypi- n

A
includingcentral-line-associatedinfections,andthesecondmost cally 7 to 9 amino acids in length, and the last five residues are
p
commoncauseofsurgical-siteinfectionsandprosthetic-valvein- cyclizedintoalactoneorthiolactonestructurethroughaserineor r
fectiveendocarditis(3).Over150millionintravasculardevicesare cysteinesidechain,respectively.Muchoftheknowledgeonthe il 1
2
used each year in the United States alone (4), and a major risk mechanisticdetailsoftheagrsystemisbasedonstudiescarried
,

factorforCoNSdiseaseisthepresenceofamedicalimplant(5). outwithStaphylococcusaureus(reviewedinreferences14and15). 2
0
Formanyoftheseinfections,S.epidermidismakesupmorethan Briefly,theagrlocusiscomposedoftheagrBDCAoperon,which 1
80%oftheclinicalburdenofCoNS(3,6). encodes the core machinery for producing and detecting AIPs 9

b
TheabilityofS.epidermidistocolonizeimplanteddevicesand (Fig.1).AgrDservesasthepropeptideprecursorthatissecreted y
formabiofilmisitsprimarymechanismofpathogenesis(1,6). andprocessedbyAgrB,amultipassintegralmembranepeptidase g
u
Thepolysaccharideintercellularadhesin(PIA)producedbypro- thatworkswithsignalpeptidaseSpsBtosecreteAIP.Accumula- e
teinsencodedintheicaADBClocusisoneofthebest-character- tionofextracellularAIPisdetectedbythehistidinekinaseAgrC. s
t
ized biofilm determinants (7). More recently, protein-mediated WhenAIPbindstothisreceptor,asignaltransductioncascadeis
biofilmmechanismshavebeenidentified(reviewedinreference initiated, with AgrC phosphorylating the response regulator
6),withtheaccumulation-associatedprotein(Aap)beingoneof AgrA,resultingininductionofexpressionfromtheagrP andP
2 3
thebeststudied(8).Regulationofbiofilmformationhasbeena promoters(Fig.1),aswellasthepromoterscontrollingexpression
focusofnumerousstudies,andsomeoftheseinvestigationshave ofPSMtranscripts.TheP promoterdrivestheexpressionofthe
3
demonstratedthattheagrquorum-sensingsystemimpactsS.epi- transcriptRNAIII,whichistheprimaryeffectorofthesystem.
dermidisbiofilmdevelopment.Inthisregard,strainsdefectivein
agrhavebeenshowntodisplayenhancedadherenceandbiofilm
properties (9, 10) and reduced production of extracellular en- Received25May2014 Accepted18July2014
zymes,suchaslipasesandproteases(10–12).Theagr-regulated Publishedaheadofprint28July2014
low-molecular-weight toxins, termed phenol-soluble modulins AddresscorrespondencetoAlexanderR.Horswill,[email protected].
(PSM(cid:2)s),areknowntodisruptS.epidermidisbiofilmsandtobe M.E.O.andD.A.T.contributedequallytothiswork.
importantfordisseminationduringbiofilminfection(13). Copyright©2014,AmericanSocietyforMicrobiology.AllRightsReserved.
Thefundamentalfeaturesoftheagrsystemareconservedina doi:10.1128/JB.01882-14
numberofstaphylococcalspecies,includingS.epidermidis(14).
3482 jb.asm.org JournalofBacteriology p.3482–3493 October2014 Volume196 Number19
StaphylococcusepidermidisQuorumSensing
the S. epidermidis chromosome flanking the dihydrofolate reductase
(dhfr)genefromplasmidpGO558(27),whichconfersresistancetoTmp.
Thefirstfragmentwasa697-bppieceofthe5=regionofRNAIIIthatwas
PCRamplifiedfromS.epidermidisRP62Ausingoligonucleotides367and
378(Table2).ThePCRproductwasdigestedwithEcoRIandBamHIand
wasligatedintopUC19cutwiththesameenzymes.Thesecondfragment
wasa991-bp3=regionofRNAIIIamplifiedfromS.epidermidisRP62A
withprimers369and379,andthisPCRproductwasdigestedwithSalI
andPstIandclonedintothesamesitesonthepUC19-5=-RNAIIIplasmid.
ThedhfrgenewasexcisedfrompGO558withaSalIdigestandwasligated
intothesamesiteontheRNAIIIknockoutplasmidtoprovideTmpresis-
FIG 1Schematic of the staphylococcal agr locus and S. epidermidis AgrD tance.Finally,pROJ6448(28)waslinearizedwithPstIandinsertedinto
sequences.ThethreeAgrDtypesareshownandaredividedintotheleader theplasmidtoprovideatemperature-sensitivestaphylococcalreplicon
region,AIP-containingregion,andtailregion.Thehighlyconservedresidues
andErmresistance.
across staphylococcal AgrDs are indicated by asterisks. The previously de-
Constructionofanecpmutant.Roughly1,000-bpregionsflanking
ducedAIP-Istructureisboxed.Alsodiagramedisthedeletion/insertionofthe
dhfrcassettethatdisruptsRNAIIIinS.epidermidis1457toyieldthe(cid:4)rnaIII:: theecpgenewereamplifiedfromchromosomalS.epidermidis1457DNA
dhfrmutantusedinthisstudy. usingprimersEcp5_att_for,Ecprev_Eco,3_for_Eco,andEcp_att_rev3 D
(Table2).Ampliconswerepurified,cleavedusingEcoRI,andligatedby o
w
T4ligase.The2,000-bpligationproductwasclonedintopKOR1usingthe
n
There remain important unanswered questions about the S. BPClonasereaction(Invitrogen),resultinginpKOecp.pKOecpwasin- lo
troducedintoS.aureusRN4220byelectroporationand,fromthere,again a
epidermidis agr system. Following the initial description of the d
by electroporation into S. epidermidis mutant 1457-M12 (29). Using
e
locus(16,17),itwasseveralyearsbeforesequencingstudiesre-
phageA6C,pKOecpwasnexttransducedintoS.epidermidis1457(8). d
vealedthreedifferentclassesofS.epidermidisagrsystemsinclin- Allelic replacement was carried out essentially as described previously f
r
icalisolates(18),whicharereferredtohereasagrtypesI,II,and (30).PutativemutantswerescreenedbyPCR,andthelossofecpwas o
m
III.Althoughtherehasbeensomeefforttocorrelatetheseclasses verifiedbysequenceanalysis.

h
withS.epidermidisdisease(19–23),thepresenceofdifferentagr
t
t
classes,andtheiruniqueAIPsignals,hasremainedlargelyover- p
:
looked.Ofthese,onlytheAIP-Istructurehasbeencharacterized TABLE1Strainsandplasmids //
jb
(16). Many studies have focused on prototype biofilm-forming Sourceor .
a
strain1457,whichencodesanagrtypeIIsystem,butthestructure Strainorplasmid Description reference s
ofS.epidermidisAIP-IIisnotknown. E.coliDH5(cid:5) Cloningstrain NewEngland m
.
OurgoalinthisstudywastogainabetterunderstandingofS. Biolabs o
r
epidermidis agr system function and address several open ques- g
/
tions.Byusingbioactivity-guidedfractionationandhigh-resolu- S.aureus o
tion mass spectrometry, the AIP signal structures were deter- AH1263 LACErms 52 n

RN4220 Restrictiondeficientstrain 24 A
minedandcrosstalkbetweeneachofS.epidermidisagrsystems
p
was investigated. An RNAIII mutant was constructed in strain r
1457;theregulatorychangesinthismutantwereassessed,anda S.eApTidCeCrm1i2d2is28 Clinicalisolate(icaagrtypeI) 53 il 1
skincolonizationphenotypewasidentified.Collectively,thesere- 1457(AH2490) Clinicalisolate(ica(cid:6)agrtypeII) 54 2,
sults provide new insights into the molecular details, function, RP62a Clinicalisolate(ica(cid:6)agrtypeI) 55 2
0
andimportanceoftheS.epidermidisagrsystem. 4804 Clinicalisolate(agrtypeI) Feycollection 1
7237 Clinicalisolate(agrtypeI) Feycollection 9

MATERIALSANDMETHODS 5183 Clinicalisolate(agrtypeII) Feycollection b
y
Growthconditionsandreagents.Bacterialstrainsusedinthisstudyare 7022 Clinicalisolate(agrtypeII) Feycollection g
listedinTable1.EscherichiacoliwasgrowninLBmedium,andS.epider- 8595 Clinicalisolate(agrtypeII) Feycollection u
8099 Clinicalisolate(agrtypeIII) Feycollection e
midisculturesweregrownintrypticsoybroth(TSB).S.epidermidiscul- s
5794 Clinicalisolate(agrtypeIII) Feycollection t
turesweregrownat37°Cwiththeappropriateantibioticsatconcentra-
tionsof10(cid:3)g/mlforchloramphenicol(Cam),erythromycin(Erm),and 8247 Clinicalisolate(agrtypeIII) Feycollection
trimethoprim (Tmp) and 50 (cid:3)g/ml for kanamycin (Kan), as needed. AH2673 1457/pCM40(Ermr) Thiswork
AH2491 1457(cid:4)rnaIII::dhfr Thiswork
ChemicalreagentswerepurchasedfromSigma-Aldrich(St.Louis,MO)
AH2589 1457(cid:4)ica::dhfr 27
unlessotherwisenoted.S.epidermidisAIP-IIwascustomsynthesizedby
AH2924 1457(cid:4)ecp Thiswork
AmericanPeptideCompany(Vista,CA).Alloligonucleotidesusedinthis
AH3408 ATCC12228/pCM40(Ermr) Thiswork
studyarelistedinTable2.
AH3409 8247/pCM40(Ermr) Thiswork
Strainandplasmidconstructions.Plasmidconstructionswereper-
formedinE.coliusingstandardtechniques.DNAsequencingwasper-
Plasmids
formedattheUniversityofNebraskaMedicalCenter(UNMC)corese-
pCM11 P -sGFPErmr 50
quencinglaboratory.Followingpassagethroughtherestriction-deficient sarA
pCM40 agrP3-sGFPErmr Thiswork
S.aureusstrainRN4220(24),plasmidDNAwaselectroporatedintoS.
pGO558 Sourceofdhfrcassette;Tmpr 27
epidermidis strains as previously described (25). Plasmids were moved
pKOR1 Knockoutvector 30
between S. epidermidis strains using bacteriophage 71 transduction as
pROJ6448 Temp-sensitivereplicon 28
previouslydescribed(26).
pUC19 Cloningvector NewEngland
ConstructionofanRNAIIImutant.TheRNAIIIallelicreplacement
Biolabs
vector(pNF41)wasdesignedtohavetworegionsofsequencesimilarityto
October2014 Volume196 Number19 jb.asm.org 3483
Olsonetal.
TABLE2Oligonucleotidesusedinthisstudy
Name Sequence
367(3=rnaIII-F) GGAATTCCCGACTAATGCCATAGATAAAAG
378(3=rnaIII-R) CGGGATCCCACCGATTGTAGAAATGATATC
369(5=rnaIII-F) ACGCGTCGACGCCGTGAGTCTCTCCCAAG
379(5=rnaIII-R) AACTGCAGGTAACTAAAGCTTTACCCTAAGC
ARH132 GTTGTTAAGCTTCTGTCATTATACGATTTAGTACAATC
ARH133 GTTGTTGGTACCTTAAACAACTCATCAACTATTTTCC
2298(ecp-F) TGTGCTTAAAACGCCACGTA
2299(ecp-R) GTATAGCCGGCACACCAACT
2301(gyrB-F) CTCGAAGCGGTTCGTAAAAG
2302(gyrB-R) TACCACGGCCATTGTCAGTA
2547SERP0736(psm(cid:2)1)-F CGGCCTCATTTAGGAGTG
2548SERP0736(psm(cid:2)1)-R CGATTCACCATATCAACGC
Ecp5_att_for GGGGACAAGTTTGTACAAAAAAGCAGGCTTAGCGTGGTGAAGTTAATGATG
Ecprev_Eco CAACACATGAATTCGCTAGCTTTTGCAACTCTTTCAAATCG D
3_for_Eco CAACACATGAATTCGCGGCCGCTATTAATATAGAAAGGTGTGCTTATGC o
Ecp_att_rev3 GGGGACCACTTTGTACAAGAAAGCTGGGTAGAAGATATTCATATTAGTGGTGCTG w
n
lo
a
d
e
ConstructionofS.epidermidisagrreporterstrains.TheagrP3pro- izedtoanS.epidermidisGeneChip.Signalintensityvaluesforeachqual- d
moterwasPCRamplifiedfromS.aureusstrainAH1263usingoligonucle- ifier(predictedopenreadingframe[ORF]andintergenicregion)were f
r
otidesARH132andARH133(Table2).ThePCRproductwasdigested normalizedtothemediansignalintensityvalueforeachGeneChip.Sam- o
m
withHindIIIandKpnIandclonedintothesamesitesonplasmidpCM11. plevalueswerethenaveraged.Genesforwhichtherewasatleasta2-fold

The new plasmid was confirmed for agr responsiveness and called difference(P(cid:2)0.05,ttest)inRNAtiterbetween1457and1457(cid:4)rnaIII:: h
t
pCM40. Plasmid pCM40 was transformed into S. epidermidis 1457 to dhfr were considered differentially expressed in an rnaIII-dependent tp
constructreporterstrainAH2673.Thereporterplasmidwastransduced manner. :/
/
tootherstrainsusingbacteriophage71toconstructAH3408andAH3409 (cid:2)-Toxinimmunoblots.Forthe(cid:7)-toxinimmunoblots,strain1457, jb
reporters. strain1457supplementedwith40nMAIP-II,andthe1457(cid:4)rnaIII::dhfr .a
agrP3-sGFPreporterassays.FortheagrP3-sGFPtimecourse,an mutantweregrowninTSBfor21or24hat37°C.Cellswereremovedby s
m
overnight culture of reporter strain AH2673 was diluted to an optical filtrationthrough0.22-(cid:3)mfilters,andspentmediumwasusedforthe
.
densityat600nm(OD )of0.05in20mlofTSBin125-mlflaskssup- immunoblots.Immunoblottingfor(cid:7)-toxinwasperformedbycross-reac- o
600 r
plemented with 10 (cid:3)g/ml Erm. For exogenous AIP-II addition, spent tivitywithrabbitpolyclonalantiseratoS.aureus(cid:7)-toxin(Abgent,San g
/
mediumwascollectedfromS.epidermidis1457bycentrifuging24-hcul- Diego,CA),diluted1:1000,followedbysecondarygoatanti-rabbitIgG o
turesat3,750rpmandfilteringthrough0.22-(cid:3)mfilters.Thespentme- (1:1,000)conjugatedtohorseradishperoxidase(ToxinTechnology,Sara- n

diumwasaddedtothereportercultureatafinalconcentrationof10% sota,FL)aspreviouslydescribed(32). A
p
(vol/vol).Culturesweregrownat37°Cwithshaking(250rpm).Measure- Ecpactivityanalysis.AproteaseassaywasdevelopedtofollowEcp r
mentsoftheopticaldensityat600nm(OD600)andsuperfoldergreen activity using FRET substrate [5-carboxyfluorescein (FAM)-Lys-Leu- il 1
fluorescentprotein(sGFP)fluorescence(excitation,490nm;emission, Leu-Asp-Ala-Ala-Pro-(QXL520)-OH;AnaSpec,Fremont,CA].Thissub- 2
520nm)wereobtainedhourlyfor27husingaTecanInfiniteM200plate strateisbasedontheCXCR2substratedevelopedforS.aureusstaphopain ,
2
reader.Foreachmeasurement,3samplesof200(cid:3)lwereremovedfrom A(33),anditwassuspendedto50(cid:3)Musing20mMTris(pH7.4).ForEcp 0
eachflaskandtransferredtoa96-wellmicrotiterplate(Corning3096). activityanalysis,strainsweregrowninTSBfor12hat37°C.Cellswere 1
9
The values are reported as relative fluorescence after normalization to removedbyfiltrationthrough0.22-(cid:3)mfilters,andspentmediumwas
b
OD600readings.Atleastthreebiologicalreplicateswereperformedfor savedfortheanalysis.A50-(cid:3)lportionofcollectedmediumwasmixed y
eachcondition.Ofnote,theAH2673reporterstrainwasreconstructedfor with25(cid:3)loftheFRETsubstrateinamicrotiterplate,andfluorescence g
thetimecourse. measurements(excitation,490nm;emission,520nm)wereobtainedat u
e
Forthecrosstalktests,S.epidermidisstrainsrepresentingdifferentagr 37°CinaTecanInfiniteM200platereader.Reactionrateswererecorded s
t
systemsweregrownfor20hinTSB,andspentmediumwascollectedand asunitsoffluorescenceperminute.
passedthrough0.22-(cid:3)mfilters.TheagrreportersfortypeI(AH3408), For the agr cross talk impact on Ecp activity, spent medium from
typeII(AH2673),andtypeIII(AH3409)weregrownovernightin5mlof differentS.epidermidisstrainswaspreparedasdescribedabovefortheagr
TSBwithErmandsubcultured1:200into18-mmtubeswithfreshculture reportermethods.FortheEcpmeasurements,cultureswerepreparedas
medium.ThefilteredspentmediumcollectedfromS.epidermidisstrains describedaboveexceptthatcollectedspentmediumwasaddedto10%
wasaddedto10%(vol/vol).Thereporterculturesweregrownfor24h, (vol/vol)ofthetestingstrainatthetimeofinoculation.Finally,cellswere
andgrowth(OD )andsGFPmeasurementswereobtainedasdescribed removed,andEcpactivitywasmeasuredinthesamples.
600
above. Reverse transcriptase PCR. Overnight cultures were diluted 1:100
Transcriptionalprofiling.OvernightculturesofS.epidermidis1457 andgrownmicroaerobically(5:1[vol/vol],flask-to-mediumratio)inTSB
and1457(cid:4)rnaIII::dhfrwerediluted1:100intofreshTSBandgrownat toanOD of(cid:8)7.6.Sampleswerespunat6,000(cid:9)gfor5minat4°C.
600
37°CtoanOD of8.2(flask-to-volumeratio,5:1;shakingat200rpm). PelletswereresuspendedinRLTbuffer(Qiagen)with0.01%(cid:2)-mercap-
600
RNAwasconvertedtocDNA,andmicroarrayanalysiswasperformed toethanol.Cellswerelysedbymechanicaldisruptionandthecelldebris
according to the manufacturer’s instructions (Affymetrix expression was removed by centrifugation at 21,000 (cid:9) g for 15 min at 4°C. The
analysistechnicalmanual;Affymetrix,Inc.,SantaClara,CA)forantisense supernatantwasremovedtoanewtubewith0.7volumeofethanoland
prokaryotic arrays as described previously by Beenken and colleagues vortexed.RNAwaspreparedusingtheRNeasykit(Qiagen)accordingto
(31). To ensure reproducibility, cDNA samples from each strain were the manufacturer’s instructions. cDNA was synthesized from approxi-
preparedfromtwoseparateexperiments.EachcDNAsamplewashybrid- mately1.5(cid:3)gofRNAusingtheTranscriptorfirst-strandcDNAsynthesis
3484 jb.asm.org JournalofBacteriology
StaphylococcusepidermidisQuorumSensing
FIG2DevelopmentofagrreportersforS.epidermidis1457.PlasmidpCM40(agrP-GFP)wastransformedintoWT1457,andreporterstrainsweregrownin
3
TSB(opensymbols)orTSBwithspentmediumaddedto10%oftotalvolume(filledsymbols).Absorbance(dashedlines)andfluorescence(solidlines)readings
weretakenattheindicatedpointsthroughoutthetimecourse.
D
o
w
n
kit(Roche)accordingtothemanufacturer’sinstructionsusingtheran- lated using the slope of the best-fit line obtained by linear regression lo
domhexamerprimer.RT-PCRmixturescontainedQuik-Load2(cid:9)Midas analysisofthecalibrationcurvedata. a
PCRmix(MonserateBiotech,SanDiego,CA),10(cid:3)Mprimers,and2(cid:3)lof S.epidermidiscolonizationofporcineskin.S.epidermidiscoloniza- d
e
cDNA.ReactionswereruninaMyCyclerthermocycler(Bio-Rad)for20 tionofporcineskinwasperformedasdescribedpreviously(34).Biopsy d
cycleswitha15-sextension.Followingagarosegelelectrophoresis,DNA punchesof8mmweretakenfromtheearsofnewbornpiglets(tissue f
r
bandswerevisualizedbyethidiumbromidestaining. kindlyprovidedbyD.Stoltz).Explantedtissuewaswashedpriortobiopsy o
m
FractionationofS.epidermidisspentmedium.S.epidermidisstrain withethanol,andbiopsypuncheswereplacedindividuallyonapieceof

5183wasgrowninTSBat37°Cwithshakingat200rpmovernight.Cells sterilegauzein12-wellcellcultureplates.Theskinbiopsyspecimenswere h
t
werepelletedbycentrifugationat6,000(cid:9)gfor5min.andremovedby culturedinDulbecco’smodifiedEaglemedium(DMEM)containinghy- tp
0.22-(cid:3)mfiltration.Thefilteredmediumwasseparatedbyautomatedflash drocortisone,10%fetalbovineserum(FBS),andpenicillin-streptomycin :/
/
chromatographyusingaTeledyneIscoCombiflashRfsystem.ARediSep (35).Thegrowthmediumwasaddedtothewells,bringingtheheightnear jb
RfGoldC columnwasusedwithalinearmethanol-watergradientstart- theapicalsurfaceofthebiopsyspecimen.Thesesampleswereincubatedat .a
18
ingat10%methanolandendingat100%methanolover100column 37°Cwith5%CO forthedurationoftheexperiment,andtheculture s
2 m
volumes,andthefractionswerecollectedin25-mltesttubes. mediumwaschangeddaily.After15hofincubation,10(cid:3)lofbacterial
.
LC-MSidentificationandquantificationofS.epidermidisAIP.All suspensioninsalinecontaining1.5(cid:9)104to2(cid:9)104CFUofwild-type o
r
liquidchromatography-massspectrometry(LC-MS)analyseswereper- (WT) 1457 or 3.8 (cid:9) 104 to 7.5 (cid:9) 104 CFU of 1457 (cid:4)rnaIII::dhfr was g
/
formed on a Waters Acquity ultraperformance liquid chromatograph spottedontheapicalskinsurface.Thecultureswereallowedtodryonto o
(UPLC)coupledtoaThermoFisherScientificLTQOrbitrapXLmass theskinsurfaceinalaminarflowhoodfor1h,andtheplateswerere- n

spectrometerwithelectrospraysource.Samples(5(cid:3)l)wereinjectedonto turnedtotheincubator.After48hofincubation,theskinbiopsyspeci- A
p
yasheisghw-esrterecnognthdusciltiecda(aHtSaSfl)oTw3Cra1t8ecooflu0m.2n50(Wmal/temrsinCworipthortahteiofno)ll.oAwnianlg- matemnawxaimspulmacesdpeined1mforlo1fmphino.spThhaetec-eblulsffwereerdessaerliinaelly(PdBilSu)teadn,dpvlaotretdexoend ril
1
binarygradient,wheresolventAis0.1%formicacidinH2O,andsolvent high-salt tryptic soy agar (TSA) (supplemented with 50 g/liter NaCl), 2
Bis0.1%formicacidinacetonitrile:0to6min,from80%Ato20%A;6.0 incubatedovernightat37°C,andenumerated. ,
2
to6.5min,from20%Ato80%A;6.5to7.0min,80%A(isocratic). 0
Analyses were conducted in both the positive- and negative-ion RESULTS 1
9
modes.Inpositive-ionmode,theOrbitrapwasoperatedatascanrangeof S. epidermidis agr systems. S. epidermidis clinical isolates have
b
m/z300to2,000withthefollowingsettings:tubelensvoltage,110V; anyoneofthreeclassesofagrsystems(Fig.1)(18),andofthese, y
sourcevoltage,4.50kV;sourcecurrent,100(cid:3)A;heated-capillaryvoltage, onlythestructureandactivityoftheAIP-Isignalhavebeenchar- g
20.0V;heated-capillarytemperature,300.0°C;sheathgasflowrate,20.0; u
acterizedtodate(16).Wesoughttoexpandthisknowledgeand e
auxiliarygasflowrate,0.Fornegative-ionmodeanalyses,theOrbitrap s
defineeachofthethreeS.epidermidisAIPmolecules.Inparticu- t
wasoperatedatascanrangeofm/z300to2,000withthefollowingset-
lar,wefocusedontheunknownAIP-IIstructurefoundinproto-
tings:tubelensvoltage,100V;sourcevoltage4.00kV;sourcecurrent,100
(cid:3)A; heated-capillary voltage, 20.0 V; heated-capillary temperature, typestrain1457(agrtypeIIsystem),whichisoneofthemodel
275.0°C;sheathgasflowrate,25.0;auxiliarygasflowrate,0.ForMS-MS strainsformoleculargeneticstudiesinS.epidermidis.Toachieve
analysis in the negative-ion mode, the precursor masses of 873.34, thisgoal,wedevelopedbioassaystrainsinthe1457background
1,355.60,and1296.60(thepredictedmassesofdeprotonatedAIP-I,AIP- thatcontainedanagrP -sGFPreporter(pCM40)thatrespondsto
3
II,andAIP-III,respectively)weresubjectedtocollision-induceddissoci- quorum-sensingactivityinamannersimilartothatofS.aureus
ationwithactivationenergyof35%.ThesameMS-MSconditionswere agrreporters(36).Asexpected,theS.epidermidisagrsystemwas
usedinthepositiveionmode,butprecursorm/zvaluesforprotonated activated,andsGFPaccumulatedovertime(Fig.2).Theexoge-
AIP-I,API-II,andAIP-III(873.34,1,355.60,and1,296.60,respectively)
nousadditionof1457spentmediumcontainingAIP-IItriggersa
wereselected.
positive-feedbackloop,causinganaccelerationoftheagractiva-
TheAIP-IIconcentrationwascalculatedusingan8-pointcalibration
curveofsyntheticAIP-IIranginginconcentrationfrom30.0(cid:3)Mto0.3 tionkinetics.
(cid:3)Mby2-folddilutions.Thecalibrationcurvewasgeneratedbyplotting AIPidentification.Usingthenew1457agrreporterstrain,we
log(peakarea)versuslog(concentration).Peakareawasselectedforam/z testedvariousS.epidermidisagrtypeIIisolatestoenableselection
of678.38(themostintenseproductpeakformass1,355.60)fromthe ofthemostactivestrainforfractionationpurposes.Clinicaliso-
MS-MSselectedionchromatogram.AIP-IIconcentrationswerecalcu- late5183waschosenasthebestproducer;spentmediumfromthis
October2014 Volume196 Number19 jb.asm.org 3485
Olsonetal.
strainhadthestrongestenhancementofagractivationkineticsin IIIreporter(Fig.5C).InagrtypeI,theadditionofspentmedium
the reporter assay. To characterize the signal, strain 5183 spent containingAIP-Iminimallyenhancedoutput,whiletheaddition
mediumwasfractionatedbyreverse-phaseflashchromatography. of AIP-II or AIP-III markedly repressed GFP production (Fig.
Fractionsweretestedfortheirinfluenceonthe1457agrreporter, 5A). The weak positive feedback from the cognate signal is not
and one fraction was identified that enhanced reporter output unusual.ThecloselyrelatedS.aureusagrsystemdoesnotrespond
(datanotshown).TheAgrDtypeIIsequence(Fig.1)wasusedas stronglytoexogenoussignaladditionunlessspecificgrowthcon-
aguideforpredictingthem/zvalueofthetypeIImolecularion, ditionsareused(38).InS.epidermidisagrtypeII,theadditionof
startingwiththepredicted8-residuesequenceYNPCSNYL,which spentmediumcontainingAIP-IIsignificantlyenhancedoutput,
would represent a 5-residue cyclic thiolactone (CSNYL) and supporting our previous observations (Fig. 2), while AIP-I re-
3-residue amino-terminal extension. The masses that resulted pressedGFPproduction(Fig.5B).Interestingly,AIP-IIIneither
from adding or removing amino-terminal residues were calcu- enhancednorrepressedagroutput.InagrtypeIII,similarobser-
lated,andthemass-spectraldatawerefilteredtofindanm/zvalue vations were made, with spent medium containing AIP-III en-
matchingtheexactmass(within5ppm)ofapredictedpeptide. hancing GFP expression (Fig. 5C), and the medium containing
Usingthisstrategy,thepeptideNASKYNPCSNYL(Fig.3B),m/z AIP-Iinhibitingit.TheAIP-IIsignalhadnosignificantimpacton
1353.5879[M-H](cid:10)(calculatedforC H N O S(cid:10),1,353.5903; theagrtypeIIIreporter.Takentogether,thesedatademonstrate
59 85 16 19 D
1.8ppmmassaccuracy),wasdetected.Thisfindingsuggestedthat thatagrinterferencedoesoccuramongS.epidermidisstrains.Spe-
o
AIP-IIisa12-residuepeptidethatcontainsa5-residuecyclicthio- cifically,interferenceoccursbetweentheagrtypeIandII/IIIsys- w
lactoneringanda7-residueaminoterminalextension.Asfurther temsbutnotbetweenthetypeIIandIIIsystems(Fig.5D). n
lo
supportforthisidentification,strain1457wasanalyzedusingthe S.epidermidisagrmutantprofiling.Toassesstheregulatory a
outlined method, and the same results were obtained (data not profile of the agr system in S. epidermidis, an RNAIII allelic re- d
e
shown).Theproposed12-residuestructuremakestheS.epider- placementmutantwasconstructedinstrain1457(theregionre- d

midisAIP-IIthelargeststaphylococcalAIPidentifiedtodate. placedwiththedhfrcassetteisshowninFig.1).Microarraytran- fr
TheAIPidentificationapproachdescribedforS.epidermidis scriptionalprofilingwasperformedonthe(cid:4)rnaIII::dhfrmutant om
AIP-IIwasalsoappliedtotheotherS.epidermidisagrsystems.For andrevealed21genesthatweredownregulatedcomparedtothe
h
theagrtypeIsystem,theclinicalisolate4804wastested,andan WT(Fig.6A).Theseresultsincluded(cid:7)-toxin,whichwasexpected t
t
ion matching the peptide DSVCASYF, m/z 873.3453 [M(cid:6)H](cid:6) given that this toxin is encoded in the RNAIII transcript. The p:
(calculated for C39H53N8O13S(cid:6), 873.3447; 0.8 ppm mass accu- PSM(cid:2),agrC,andgehgeneswerealsoidentifiedasbeingdown- //jb
racy),wasdetected(Fig.3A).Thisisconsistentforthesequence regulated,confirmingapreviousstudythatdemonstratedtheex- .
a
previouslyproposedforS.epidermidisAIP-I(16).Fortheagrtype pression of these genes to be agr dependent (11). The cysteine s
m
IIIsystem,wetestedclinicalisolate5794anddetectedanionat proteinase, Ecp, was identified as one of the most significantly
.
o
m/z1,296.6011,consistentwiththepredictedmassoftheproto- regulatedgenes(19-foldreductioninthemutant),whichisalsoin
r
g
nated form of the peptide NAAKYNPCASYL (measured, linewithpreviousstudies(11).ThestaphostatinEcpB,whichis /
1,296.6011;calculatedforC H N O S(cid:6),1,296.6041;2.3ppm encodedinadicistronicoperonwithEcp,showedasimilarlevelof o
58 86 15 17 n
mass accuracy) (Fig. 3C). AIP-III likewise is an unusually large downregulation(Fig.6A).
A
structure,similarinsizetotheAIPpeptidedetectedinthespent SubsequenttoAIPidentification,wesoughttoconfirmsome p
r
mediumofS.epidermidisAIP-II. oftheregulatoryobservationsfromthetranscriptionalprofiling il
ToconfirmtheAIPidentification,theproposedAIP-IIpeptide usingreversetranscriptasePCR(RT-PCR)andexogenousaddi- 1
2
was synthesized and used in verification studies. The retention tionofsyntheticAIP-II.ThedownregulationofthePSM(cid:2)andecp ,

2
time (Fig. 4A) and MS-MS fragmentation pattern (Fig. 4C ) of transcriptswasconfirmedbyusinggyrBasareference(Fig.6B).
0
syntheticAIP-IImatchedthoseoftheputativeAIP-IIionfromS. Forthesignaladditionexperiment,40nMAIP-IIwasaddedto 1
9
epidermidisAIP-IIstrains(Fig.4BandD).SyntheticAIP-IIwas brothculturesof1457,andmediumwasharvestedat21and24h
b
alsousedtoquantifythelevelsofAIP-IIinthespentmedium,and ofgrowth.(cid:7)-Toxinaccumulationinthemediumwasassessedby y
itwasdeterminedthatstrain1457produced1(cid:3)MAIP-IIinsta- protein immunoblotting, and as anticipated, the addition of g
u
tionaryphaseafter19hofgrowthinTSB.Finally,thesynthetic AIP-IItothemediumledtoincreasedproductionof(cid:7)-toxin(Fig. e
AIP-IIwasaddedtothe1457agrreporterstrain,anditaccelerated 6C).The(cid:4)rnaIII::dhfrmutant,whichdoesnotproduce(cid:7)-toxin, st
activationkineticsinamannerthatmirroredthosein1457spent wasincludedasanegativecontrol.
medium(Fig.4E),indicatingthattheproposedAIP-IIstructureis agrregulationofthecysteineproteaseEcp.Basedonourecp
functional.Collectively,thedatapresentedinthissectiondemon- regulatoryfindings,weanalyzedEcpactivitytoassesstheimpact
strate that the fractionation-and-MS approach was effective for oftheagrsystemontheextracellularproteome.Werecentlyde-
accuratelyidentifyingthebiologicallyactiveAIPsignalsfromS. veloped assays for S. aureus staphopain A based on the CXCR2
epidermidis. receptor(33,39),andwetestedwhethertheseassayscouldtrans-
S. epidermidis agr cross talk. Considering the large size of latetoS.epidermidis.1457spentmediumcontainingEcpcleaved
AIP-IIandAIP-III(12residues)versusthesmallerAIP-I(8resi- theCXCR2substrate(datanotshown),andthisactivitywasin-
dues), we reasoned that agr cross talk between S. epidermidis hibited with E-64, as expected for this protease (40). We have
strainsmightoccur,ashasbeenobservedwithS.aureus(37).To adaptedtheCXCR2cleavagesubstratetoasmallerFRETpeptide
evaluate this, agr reporters in type I and III strains were con- (39), and 1457 spent medium containing Ecp also cleaved this
structedandvalidated,similartowhatwasdonewiththetypeII substrate (Fig. 7A). In support of the transcriptional profiling,
strain(Fig.2).Toassessinterference,spentmediumfromselected spent medium from the 1457 (cid:4)rnaIII::dhfr mutant showed an
agrtypeI,II,andIIIstrainswascollectedandaddedtoculturesof 8.6-foldreductioninEcpactivity.Asacontrol,a1457(cid:4)ecpmu-
theagrtypeIreporter(Fig.5A),typeIIreporter(Fig.5B),andtype tant was constructed, and this mutant did not possess activity
3486 jb.asm.org JournalofBacteriology
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FIG3S.epidermidisAIPidentification.ThisfigureshowsMS-MSspectrathatresultfromcollisionallyinduceddissociationofthe[M(cid:6)H](cid:6)ionsforAIP-I(m/z
873.34)(A),AIP-2(m/z1,355.60)(B),andAIP-3(m/z1,296.60)(C).PredictedstructuresforallthreeAIPsandfragmentassignmentssupportingthese
predictionsareincluded.
October2014 Volume196 Number19 jb.asm.org 3487
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1
2
,

FIG4ConfirmationofstructurebycomparisonwithsyntheticAIP-II.Selectedionchromatogramsforthefragmentatm/z678.33obtainedfromcollisionally 2
induceddissociationofthe[M(cid:6)H](cid:6)ionatm/z1,355.60forbothsyntheticAIP-II(A)andspentmediumfromS.epidermidisstrain1457(B).Thesechromato- 01
gramsshowexcellentagreementinretentiontime(RT)betweenthetwoions.Asfurtherconfirmation,panelsCandDdemonstratethattheMS-MSspectrum 9
forsyntheticAIP-II(C)matchesthatoftheputativeAIP-IIionfromS.epidermidisstrain1457.TheMS-MSspectrawereobtainedbycollisionallyinduced b
dissociationofaprecursorionatm/z1,355.60(the[M(cid:6)H](cid:6)ionofAIP-II).(E)AsabiologicalconfirmationofthesyntheticAIP-II,WT1457withtheagrP-GFP y
3
reporterwasgrownwithincreasingdosesofAIP-II,andabsorbanceandfluorescencereadingsweretakenafter24h.Relativefluorescencewasplottedatthe g
indicatedAIP-IIconcentrations. u
e
s
t
againsttheFRETsubstrate(Fig.7A),demonstratingthatnoother previouslydevelopedskincolonizationmodel(34),wecompared
proteases contributed to the activity measurements. To further theabilityofWT1457andthe(cid:4)rnaIII::dhfrmutanttocolonize
assess the role of quorum-sensing regulation in Ecp regulation, freshlyisolatedporcineskinfor2days.Atthistimepoint,asig-
spent-mediumcrosstalktestswereperformedusinginformation nificantdefectintheabilityofthe(cid:4)rnaIII::dhfrmutanttocolonize
gleanedfromtheresultsoutlinedinFig.5.ForeachS.epidermidis wasobserved(Fig.8),indicatingthatS.epidermidisdoesrequire
agrsystem,spentmediumfromthatagrtypeaddedbacktothe theagrsystemforoptimalgrowthduringskincolonization.
samestraininducedEcpactivity(Fig.7B).Incontrast,spentme-
diumfromaknowninterferingstraininhibitedactivity,resulting DISCUSSION
in significantly reduced Ecp activity. Taken together, these Ecp S.epidermidisisthemostfrequentcauseofdevice-relatedinfec-
findingssupporttheagrtranscriptionalreporter(Fig.5)andmi- tions(6).Oneoftheimportantregulatorsthatcontrolthepro-
croarrayresults(Fig.6). ductionofS.epidermidisvirulencefactorsistheagrquorum-sens-
S.epidermidiscolonizespigskininanagr-dependentman- ingsystem.Despitethesignificanceofagr,keymoleculardetails
ner. We hypothesized that S. epidermidis retains the agr system aboutthissystemhaveremainedunknown,andinthiswork,we
becauseitfacilitatessuccessfulcolonizationofthehost.Usinga addressedsomeoftheseopenquestions.Usingamassspectrom-
3488 jb.asm.org JournalofBacteriology
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FIG5IdentificationofagrinterferenceinS.epidermidis.SpentmediumfromS.epidermidisstrainsrepresentingeachagrtypewascollectedandaddedto /jb
reporterstrainsto10%ofthetotalvolume.Thereportersweregrownfor24h,andGFPfluorescencewasmeasured(***,P(cid:11)0.001,**,P(cid:11)0.01,and*,P(cid:11)0.005, .
a
relativetotheno-additionreporter[noadd],asdeterminedbypairedttest).(A)agrtypeIreporter.(B)agrtypeIIreporter.(C)agrtypeIIIreporter.Thestrains s
usedforspentmediumadditionwereasfollows:1,ATCC12228(agrtypeI);2,7237(agrtypeI);3,1457(agrtypeII);4,5183(agrtypeII);5,8247(agrtypeIII); m
6,8099(typeIII).(D)SchematicofidentifiedcrosstalknetworkbetweenAIP-I,AIP-II,andAIP-III. .
o
r
g
/

etryapproach,weelucidatedthestructuresofAIP-IIandAIP-III approach that tested synthetic AIP-Is with various possible o
n
andconfirmedthepreviousdeductionofAIP-I.Wealsodiscov- lengthsoftheN-terminalextension.Throughprocessofelimina-
A
eredthatagrinterferenceoccursbetweenthethreeS.epidermidis tion,itwasdeterminedthatDSVCASYFwasthemostactive;thus, p
r
agr systems and, through transcriptional profiling, discovered itwasinferredthatitwasthecorrectstructure(16).Inthepresent il
strikingregulationofthecysteineproteaseEcp.Finally,wedem- study, the AIP was purified from agr type I spent medium and 1
2
onstrated that S. epidermidis retains the agr system for optimal demonstratedtohavetheidenticalstructure(Fig.3A).Usingan ,

2
skincolonization. alternativeapproachandmediumcollecteddirectlyfromS.epi-
0
ThemassspectrometryassignmentofAIP-IIandAIP-IIIindi- dermidis,ouranalysisconvergedonthesamestructure,support- 1
9
catedthatbothofthesestructuresare12residuesinlength,mak- ingtheinitialAIP-Ireport.
b
ingthemthelargeststaphylococcalAIPsidentifiedtodate(14). ThisisthefirststudytoidentifycrosstalkbetweentheS.epi- y

ThereasonforthislargeAIPsizeisnotclear.BothAIPsequences dermidis agr systems. The phenomenon of agr interference was g
u
start on the C-terminal side of the highly conserved IG motif, firstidentifiedinS.aureusandworksascross-inhibitionbetween e
s
whichisfoundinmostAgrDsequences(Fig.1),andthisjunction theagrtypeI,II,andIIIsystems(37),whilethesimilaritiesbe- t
couldbeimportantforreleaseofAIPfromthecytoplasmicmem- tweentypeIandIVsignalsmakethemessentiallyinterchangeable
brane.ThehousekeepingtypeIsignalpeptidaseisknowntore- (14).ByconstructingagrreportersineachS.epidermidissystem,
leaseS.aureusAIP-Iinthismanner(41),butitispossiblethat we determined that agr interference also occurs in this species.
otherresidentproteaseshavethisfunctionamongthestaphylo- ThereisstronginterferencebetweentheagrtypeIandIIsystems
cocci.ConsideringthattheidenticalsequenceispresentattheS. andbetweenthetypeIandIIIsystems(Fig.5).Insomestudies,the
epidermidiscleavagesiteinAgrD-IIandAgrD-III(VIG2NA),it typeIstrainswerethemostfrequentlyidentifiedinS.epidermidis
seemslikelythatthesameproteaseisresponsiblefortherelease. infections(19,21,22),andperhapstheseinterferenceproperties
WhilethisisnotatypicaltypeIsignalpeptidasecleavagesite,these give these strains a competitive advantage. However, the high
residuesareintheallowedwobbleforsignalpeptidaserecognition prevalenceofagrtypeIstrainsisnotuniversal,asarecentlarge
(42).TheAgrD-IcleavagesiteisalsoC-terminaltoasimilarse- studyof200S.epidermidisisolatesfromhospitalizedpatientsin
quencewithaglycineresidue(VAG2DSV),anditmaybethat Germanyfoundovertwo-thirdstobeagrtypeIIandIII(23).
releaseofAIPisduetothesamepeptidaseintheseS.epidermidis WedidnotobserveinterferencebetweentheagrtypeIIandIII
strains. systems. Although these signals are similar, they do not signifi-
Ourmassspectrometryapproachconfirmedthestructureof cantlycross-activatetheagrtypeIIIandtypeIIsystems,respec-
AIP-I.Thisstructurewaspreviouslyidentifiedusingasystematic tively(Fig.5D).Thereasonforthisisnotclear,butitisknownthat
October2014 Volume196 Number19 jb.asm.org 3489
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FIG6TranscriptionalprofilingofS.epidermidis(cid:4)rnaIIImutant.(A)HeatmapofthemicroarrayresultsofWT1457versusthe(cid:4)rnaIIImutant.(B)RT-PCR il 1
resultsoftheWTversusthe(cid:4)rnaIIImutantforPSM(cid:2)andecpgenes,alongwithgyrBasacontrol.(C)Proteinimmunoblotof(cid:7)-toxininWT1457andthe(cid:4)rnaIII 2
mutant.TheadditionofAIP-II(40nM)induces(cid:7)-toxinexpression. ,
2
0
1
9
signal activation is more difficult to achieve than inhibition in thefactthatproteasesareimportantforprocessingtheaccumu-
b
staphylococcalagrsystems(14).Withanactivatingsignal,thereis lation-associatedprotein(Aap)(8).Aapisacriticalplayerinas- y

specificity built into the AgrC receptor for proper interactions pectsofS.epidermidisbiofilmdevelopment,buttheexpressionof g
u
withtheAIPstructuretoinitiatethecascade,andthefewresidue this protein appears to be agr independent. The study reported e
s
differencesbetweentheseS.epidermidisAIP-IIandAIP-IIIsignals hereandothers(11,12)demonstratethatproteasesareunderagr t
maycontainthenecessarystructuralinformationforactivation. quorum-sensing control, and it is possible that these proteases
Withthislackofbothcross-activationandinhibition,theAgrC modulatebiofilmstructureinsomecapacity.Ofthemajorextracel-
receptorinatypeIIstrainisessentiallysignalblindtoAIP-III,and lularproteasesproduced,Espproteasewasfoundtobeimportantin
asimilarconclusioncouldbemadeforAgrCtypeIIIinteracting polymicrobialinteractionswithS.aureus(44),demonstratingthat
withAIP-II.Thus,theonlysignificantS.epidermidiscrosstalkis S. epidermidis needs to produce extracellular proteases to effec-
betweentheagrtypeIandII/IIIsystems. tivelycompeteduringcolonization.TheimportanceoftheS.epi-
Giventhatcolonizationandbiofilmformationarekeyaspects dermidisagrsysteminevasionofinnateimmunityhasalsobeen
ofS.epidermidispathogenesis,itissurprisingthatsomereports demonstrated(11),andthemetalloproteaseSepAwasshownto
indicate that agr-defective strains are better biofilm formers (9, inactivate the antimicrobial peptide dermcidin (45). Taken to-
43) and adhere more effectively to human skin epithelia (43). gether,theseobservationshighlightaclearneedforpathogenicS.
However,timecoursestudiesindicatethatagrisnecessaryincer- epidermidis strains to retain an active agr system, which would
tainbiofilmdevelopmentalstages(12),andourfindingsindicate makequorumsensinginhibitionthroughagrinterferenceanef-
thatagrisnecessaryforoptimalskincolonization(Fig.8),sug- fectivecompetitionstrategy.Thiscouldexplaininparttheevolu-
gesting that the contributions of the agr system are likely more tionofintraspeciesquorum-sensinginhibitionthatweidentified
importantthanoriginallyprojected.Inpart,thiscouldbedueto inthisstudy(Fig.5).TheS.aureusagrsystemisaprimetargetfor
3490 jb.asm.org JournalofBacteriology
StaphylococcusepidermidisQuorumSensing
FIG7Quorum-sensingcontrolofEcpproteaseactivity.(A)EcpactivityinspentmediumcollectedfromWT1457andstrainswith(cid:4)rnaIII::dhfror(cid:4)ecp
mutations.ActivitywasmeasuredbyFRETsubstrate(***,P(cid:11)0.001relativetoWT1457,asdeterminedbypairedttest).(B)S.epidermidisstrainsrepresenting
agrtypeI(ATCC12228),typeII(1457),andtypeIII(8247)weregrownwithoutadditionorinthepresenceofinducingspentmedium(mediumadded)or
inhibitingspentmedium(agrinterference;1457fortypeI,12228fortypeIIorIII).Ecpactivitywasmeasuredandplotted(***,P(cid:11)0.001relativetonoaddition, D
asdeterminedbypairedttest). o
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developmentofantivirulencetherapeutics(46–48),andasimilar results from reported and ongoing investigations. For instance, a
d
strategycouldbeappliedtoS.epidermidis. crosstalkbetweenstaphylococcihasbeenobservedinthenasal
e
OurregulationstudiesrevealedthatthecysteineproteaseEcp colonization environment (44, 49), and the availability of AIP d

isoneofthemosthighlyregulatedfactorsunderthecontrolofthe structural information and defined interference patterns could fr
o
S.epidermidisagrsystem.Insupportofthisobservation,expres- improve our understanding of these observations. In terms of m
sionoftheecpgenewasobservedinapreviousarraystudy(11). treatmentapproaches,itisknownthatafunctionalagrsystemis
h
Surprisingly,ourmicroarrayanalysesdidnotrevealregulationof important for hematogenous spread of S. epidermidis in device tt
p
the serine protease Esp or the metalloprotease SepA, although infections(13),andtheadditionofsyntheticAIPscouldbeusedto :
/
there are previous reports that these enzymes are agr regulated modulatethesephenotypesinamannersimilartothoseusedin /jb
(11,12).Thereasonforthesedifferencesarenotclearandcouldbe previousS.aureusstudies(50,51).WhileS.epidermidisandother .a
duetothenatureoftheagrmutationsusedortostrain-dependent CoNSdonotgarnerasmuchattentionasS.aureus,theclinical s
m
variances.Inthisstudy,onlytheRNAIIItranscriptwasdisrupted, burdenoftheseinfectionsissignificant,andthisnewinformation .
o
whileinotherstudies,completeagrdeletionswereused.Interest- ontheS.epidermidisagrsystemprovidesimportantinsightsfor r
g
ingly,theEcphomologinS.aureus,staphopainA,wasrecently futurestudies. /

showntohaveadramaticimpactonbiofilmstructure(39),sug- o
n
gestingthattheeffectEcphasonS.epidermidisbiofilmswarrants ACKNOWLEDGMENTS A
furtherinvestigation. WethankC.MalonefortechnicalassistanceandD.Stoltzforproving p
r
Theknowledgegainedfromourstudywillaidinterpretationof porcineskintissue. il
1
This work was funded in part by NIH public health service grants
2
AI049311toP.D.F.andAI083211(Project3)toA.R.H.Massspectrome- ,

2
try data were collected in the Triad Mass Spectrometry Facility at the
0
UniversityofNorthCarolinaGreensboro. 1
9

b
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