Staphylococcus intermedius Produces a Functional agr Autoinducing Peptide Containing a Cyclic ...

12 Pages·2005·0.57 MB·English

JOURNALOFBACTERIOLOGY,May2005,p.3139–3150 Vol.187,No.9
0021-9193/05/$08.00(cid:1)0 doi:10.1128/JB.187.9.3139–3150.2005
Copyright©2005,AmericanSocietyforMicrobiology.AllRightsReserved.
Staphylococcus intermedius Produces a Functional agr Autoinducing
Peptide Containing a Cyclic Lactone
Guangyong Ji,1* Wuhong Pei,1 Linsheng Zhang,1† Rongde Qiu,1 Jianqun Lin,1 Yvonne Benito,2
Gerard Lina,2 and Richard P. Novick3*
DepartmentofMicrobiologyandImmunology,UniformedServicesUniversityoftheHealthSciences,4301JonesBridgeRd.,
Bethesda,Maryland208141;CentreNationaldeR´ef´erencedesStaphylocoques,INSERME0230,
IFR62Laennec,7rueGuillaumeParadin,69372LyonCedex08,France2;and
MolecularPathogenesisProgram,SkirballInstituteofBiomolecularMedicine,
NewYorkUniversityMedicalCenter,540FirstAve.,
NewYork,NewYork100163
D
o
Received31July2004/Accepted19January2005 w
n
The agr system is a global regulator of accessory functions in staphylococci, including genes encoding lo
a
exoproteinsinvolvedinvirulence.Theagrlocuscontainsatwo-componentsignaltransductionmodulethatis
d
activated by an autoinducing peptide (AIP) encoded within the agr locus and is conserved throughout the e
d
genus.TheAIPhasanunusualpartiallycyclicstructurethatisessentialforfunctionandthat,inallbutone

f
case,involvesaninternalthiolactonebondbetweenaconservedcysteineandtheC-terminalcarboxylgroup. r
o
The exceptional case is a strain of Staphylococcus intermedius that has a serine in place of the conserved m
cysteine.WedemonstrateherethattheS.intermediusAIPisprocessedbytheS.intermediusAgrBproteinto
h
generate a cyclic lactone, that it is an autoinducer as well as a cross-inhibitor, and that all of five other S. t
t
intermediusstrainsexaminedalsoproduceserine-containingAIPs. p
:
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jb
.
Theagrlocus,aglobalregulatorofgenesinvolvedinpatho- ring, essential for function, in which a conserved cysteine is a
s
genesisandotheraccessoryfunctions(27),iswidelyconserved attached to the C-terminal carboxyl by a thioester linkage. m
among staphylococci (2) and has homologs in other species AgrDhasbeensequencedforca.20othernon-S.aureusstaph- .o
(11).Itconsistsoftwodivergentoperons,drivenbypromoters ylococci,andallbutonearepredictedtocontainacysteineat rg
P2andP3,respectively(6,22,24,26).TheP2operoncontains thesameposition(2),suggestingthatthematureAIPswould /
o
four genes: agrA, -B, -C, and -D. AgrA and -C comprise a each contain the same thiolactone ring (8, 9, 16–18, 25). The n

two-componentsignalingmodule,ofwhichAgrCistherecep- exceptionisastrainofStaphylococcusintermediusthatispre- J
a
tor and AgrA the response regulator (22), which, when acti- dictedtocontainaserineinplaceoftheusualcysteine(2,20). n
u
vated,upregulatesthetranscriptionofboththeP2andtheP3 AIPs with a serine replacing cysteine and therefore having a a
operons.TheP3transcript,RNAIII,encodesdelta-hemolysin lactoneratherthanathiolactoneringhavebeensynthesizedin ry
andistheeffectoroftheagrresponse(5,24).TheAgrCligand vitro. These do not activate the cognate receptor, and they 1
isanautoinducingpeptide(AIP)(9,14,15)thatisproteolyti- inhibit agr activation in heterologous combinations (17, 18). 1,

cally processed by AgrB from a propeptide encoded by agrD Thisraisesthreequestions.(i)DootherS.intermediusstrains 2
0
and probably also secreted by AgrB (9, 37). Although struc- produceserine-containingAIPsanddotheyproduceanAIP, 1
turallyconserved,agrB,-D,and-Chavedivergedwidelyamong 9
oristheserinetheresultofamissensemutationresultinginan
b
staphylococci, giving rise to multiple specificity groups, in
inactive agrD gene? (ii) If a lactone AIP is produced, is it a y
which heterologous AIP-receptor interactions are inhibitory. functional autoinducer? (iii) Finally, can an S. aureus AgrB g
ThematureAIPsareseventonineaminoacidslongandhave u
that normally processes a cysteine-containing pro-AIP also e
variable amino acid sequences. All of the seven native staph- s
processaserine-containingpro-AIPandviceversa? t
ylococcal AIPs thus far analyzed, including the four known
We have analyzed here the serine-producing S. intermedius
Staphylococcus aureus variants plus strains of Staphylococcus
CCM5739plusfiveotherS.intermediusstrainsandhavefound
warneri, Staphylococcus epidermidis, and Staphylococcus lug-
that all have a serine in the conserved position in AgrD and
dunensis (7, 8, 23, 25), contain a five-amino-acid thiolactone
that culture supernatants of each of the six S. intermedius
strainsinhibitagractivationinS.aureus,suggestingthateach
*Correspondingauthor.MailingaddressforR.P.Novick:Depart- produces an AIP. We determined the specificity and the in-
ments of Microbiology and Medicine, Molecular Pathogenesis Pro- volvementofS.intermediusAgrB(AgrB-Si)intheprocessing
gram,SkirballInstituteofBiomolecularMedicine,NewYorkUniver-
of S. intermedius AgrD (AgrD-Si) to produce the mature lac-
sity Medical Center, 540 First Ave., New York, NY 10016. Phone:
[phone].Fax:[phone].E-mail:[email protected] toneAIP.ForstrainCCM5739,tandemmassspectroscopyhas
.edu. Mailing address for G. Ji: Department of Microbiology and confirmed that the AIP contains a lactone ring (10), and we
Immunology, Uniformed Services University of the Health Sciences, showthatthisAIPandalsothatproducedbyATCC29663are
4301JonesBridgeRd.,Bethesda,MD20814.Phone:[phone].
self-activatorsandthatallsixarecross-inhibitors.Inaddition,
Fax:[phone].E-mail:[email protected].
†Presentaddress:DivisionofPediatricOncology,TheJohnsHop- an S. aureus agr group I strain with a mutation in agrD that
kinsUniversity,1650OrleansSt.,Baltimore,MD21231. would replace the conserved cysteine with a serine does not
3139
3140 JI ET AL. J.BACTERIOL.
TABLE 1. Staphylococcalstrainsandplasmidsusedinthisstudy
Plasmidorstrain Genotypeanddescriptiona Sourceorreference
Plasmids
pRN5543 Cloningvector 24
pRN5548 VectorcarryingastaphylococcalinducibleP promoter 24
bla
pRN6441 Cloningvector 22
pRN6683 S.aureusgroupIagrP3-blaZfusion 22
pRN7062 Shuttleplasmid,AIP-Itester 14
pRN7105 Shuttleplasmid,AIP-IItester 14
pRN7106 Shuttleplasmid,AIP-IIItester 14
pRN7128 Shuttleplasmid,AIP-IVtester 14
pGJ2001 S.aureusagrB-IinpRN5548 37
pGJ2002 S.aureusagrB-IIinpRN5548 38
pGJ4002 S.aureusagrD-IinpRN6441 38
pLZ4001 S.aureusagrD-IIinpRN6441 38
pLZ4003 S.aureusagrD-IIIinpRN6441 Thisstudy D
pLZ4005 agrD-IwithdoubleHis tagsinpRN6441(HDH) 37 o
pLZ4006 agrD-IwithC27Smuta6tionanddoubleHis tagsinpRN6441 Thisstudy w
(HDHcs) 6 n
pWP1004 S.intermediuspromoterP3-blaZfusioninpRN5543 Thisstudy lo
a
pWP1101 S.intermediusagrB-SiinpRN5548 Thisstudy d
pWP1102 agrB-SiandS.intermediusagrD-SiinpRN5548 Thisstudy e
pWP1103 T7epitope-agrD-Si-His (TDsiH)inpRN5548 Thisstudy d
6
pWP1104 agrB-SiandTDsiHinpRN5548 Thisstudy fr
pWP1105 agrD-Simutation(S27C)inpWP1104 Thisstudy o
m
pWP1106 agrD-Simutation(S27A)inpWP1104 Thisstudy

h
t
Strains t
p
RN4220 MutagenizedderivativeofS.aureusthatreadilyacceptsforeignDNA 21 :
RRNN66379304B S(cid:4).1a3ulryesuosgegnrooufpRINa6gr3(cid:1)90laboratorystrain 2T4hisstudy //jb.
RN6911 RN6390B(cid:5)agr1057–4546::tetM 24 a
s
RN7206 (cid:4)13lysogenofRN6911 Thisstudy m
RN8463 S.aureusgroupIIIagr(cid:1)strain 8 .
SA502A S.aureusgroupIIagr(cid:1)strain 8 or
GJ2035 RN6911(pI524) 37 g
/
ATCC29663 S.intermediusstrain AmericanTypeCultureCollection o
CCM5739 S.intermediusstrainfromtheCzechoslovakCollectionof 2 n
Microorganisms J
RN9515 S.intermediusstrain Novicklab a
n
RN9161 S.intermediusstrain W.Kloos u
RN9167 S.intermediusstrain W.Kloos a
RN9169 S.intermediusstrain W.Kloos ry

1
aagrgroups,includingindividualgenesandproducts,areindicatedbyromannumerals.Thus,agr-IImeanstheagrlocusfromagroupIIS.aureusstrain;AIP-III 1
meanstheAIPproducedbyagrgroupIIIstrains;AgrB-IreferstotheAgrBproteinofS.aureusagrgroupI,etc.Thesuffixorsubscript“Si”isusedtoindicatethat ,

S.intermediusisthesourceofthegeneorgeneproduct. 2
0
1
9

produce any detectable AIP, whereas an S. intermedius strain products.Thismaterialcouldthenbeusedforprotoplasttransformation(21). b
y
withcysteinereplacingtheAIPserineproducesamaturethio- ThesuccessofthismethoddependsontheuseofwhollyPCR-generatedDNA, g
lactone AIP that retains (weak) self-activation, as well as whichseemstobetakenupacrossspeciesbarriers,possiblyowingtolackof u
methylationand/orothermodifications. e
strongcross-inhibitionactivity. s
Nucleotide sequencing. The agr locus of S. intermedius ATCC 29663 was t
sequencedbytwomethods.First,aPCRproductwasmadewitholigonucleotides
MATERIALSANDMETHODS WP4andWP6asprimers(oligonucleotidesusedinthepresentstudyarelisted
inTable2),andchromosomalDNApreparedfromS.intermediusATCC29663
Bacterialstrainsandgrowthconditions.Staphylococcalstrainsandplasmids
asatemplate.ThePCRproductwasdirectlysequenced.TheDNAsequencewas
usedinthepresentstudyarelistedinTable1.EscherichiacolistrainsJM109and
DH5(cid:2)wereusedforcloning. usedtodesignprimersforthesecondsequencingmethodasfollows:chromo-
somalDNAwasdigestedwithbothHindIIIandEcoRIenzymes.Theresulting
S.aureuscellsweregrowninCYGPbroth(21)withshakingat37°C.Super-
DNA fragments were separated on agarose gels, followed by Southern blot
natants of cultures in late exponential phase or post-exponential phase were
centrifuged,filtersterilized,andusedasasourceofgroup-specificAIPs.Gen- hybridizationwitheitherPCRprobeAgeneratedwitholigonucleotidesWP4and
erally, a 1/10 volume of such supernatants contained sufficient AIP for full WP11asprimersandchromosomalDNAasatemplateorPCRprobeBam-
activation or inhibition. Overnight cultures on GL plates (21) were routinely plifiedfromchromosomalDNAwithprimersWP14andWP6.Southernblot
usedasinocula.CellgrowthwasmonitoredwithaKlett-Summersoncolorimeter hybridization was performed accordingly (28). Two chromosomal DNA frag-
withagreen(540-nm)filter(Klett,LongIslandCity,NY).Antibioticsusedfor ments (ca. 1.7 and 2.0 kb) were detected by using probe A and probe B,
plasmidmaintenancewereerythromycin(10(cid:3)g/ml)andtetracycline(5(cid:3)g/ml). respectively.DNAfragmentswithineither1.7-or2.0-kbregionswereexcised
TransformationofS.intermedius.AttemptstodirectlytransformS.interme- fromagarosegels,purified,andthenligatedintotheHindIIIandEcoRIsitesof
diuswithDNAisolatedfromS.aureusbyeitherelectroporation(30)orproto- anE.colicloningvector,pGEM-3zf(Promega).PCRproductswerethenmade
plasttransformation(21)failed.S.intermediuswassuccessfullytransformedby byusingoligonucleotidesWP7andT7promoterprimer(locatedonthevector)
firstamplifyingtheintactplasmidbyusingPCR,followedbyligationofthePCR asprimersandtheligationmixtures(pGEM-3zfplus1.7-kbDNAfragment)as
VOL.187,2005 S. INTERMEDIUS agr AUTOINDUCING PEPTIDE 3141
TABLE 2. Oligonucleotidesusedinthisstudy
Primer Nucleotidesequence(5(cid:9)33(cid:9))a Locationb
WP4 CATCAAATATGCACCAGCAGC agrB-Si(118–138)(cid:1)
WP6 CATAATCATGACGGAACTTGCGCA agrC-I(3201–3224)†
WP7 GTATGGTGGTGGTACGATGCATGC agrB-Si(796–809)‡
WP8 CGCAACGACTTTAACGATTGAACGTG agrC-Si(1246–1271)‡
WP11 GAAAAGGTAACAACCTAGCGC agrC-Si(1416–1436)‡
WP12 CCATCACCAATGTGATGATG P3-Si(109–129)‡
WP14 TAGCGTGCGGGTTCGTCA agrC-Si(1525–1542)‡
SINT1 GCTCTAGAAGGAGGAGAGCCGT SDofagrB-Si(317–330)‡(XbaI)
SINT2 GCGAATTCACACTTTCTAACTTT agrD-Si(1036–1050)‡(EcoRI)
SINT3 GCGAATTCACATGAGAATTTTAGAAG agrD-Si(897–914)‡(EcoRI)
SINT4 CGATTCATGATTAATGATGATGATGATGATGTTTTTCCTCTTCTAA agrD-Si(1010–1033)‡(His (cid:1)1stopcodon)
6
CAACTCAGC (BspHI)
SINT5 GCTCTAGATGTTAAACTTCCTCC agrB-Si(885–899)‡(XbaI)
SINT6 GCTAGAATCCCAACGTGTACTGGATTTTTCG agrD-Si(962–992)‡(AGT3TGT) D
SINT8 AAAAGTACCGATTGATTGG agrD-Si(943–961)‡ o
SINT11 CGGCTCTCCTCCTTGTTT P2-Si(312–329)‡ w
SINT15 GCTAGAATCCCAACGGCTACTGGATTTTTCG agrD-Si(962–992)‡(AGT3TGT) n
GJ#28 CTAATGATGATGATGATGATGTTCGTGTAATTGTGTAATTC agrD-I(2459–2479)†(His (cid:1)1stopcodon) lo
6 a
GJ#45 GTAAATGAAGTCCATGGAATAATAG SequencearoundtheNcoIsiteofpRN5548(NcoI) d
GJ#56 GCTCTAGAAGCTATTACATTATTACC BeforetheSDofagrD-I(2297–2314)†(XbaI) e
CS-1 GCTTATAGTACTAGTGACTTC agrD-I(2411–2431)†(TGT3AGT) d
SiB-F3 TATGCACCAGCAGCTACGAAAAAAAG agrB-Si(125–150)(cid:1) fr
SiC-R5 TTTCCAATTTTATACACAC agrC-Si(475–493)(cid:1) o
m
SaR3-F1 GGCCGCGAGCTTGGGAGGG agr-IrnaIII(1059–1077)†

SiB-R3 CTTTTTTTCGTAGCTGCTGGTGCATA ComplementarytoSiB-F3 h
t
RNAIII-F ACTGAGTCCAAGGAAACTAACT agr-IrnaIII(1259–1280)† t
p
RNAIII-R TAGATCACAGAGAT agr-IrnaIII(1558–1571)† :
/
T7primer TAATACGACTCACTATAGGG T7promotersequencingprimer(Promega) /jb
.
aAddedenzymesitesareunderlined.His plus1stopcodonareindicatedinitalics. a
b(cid:1),NucleotidenumberingofS.intermedi6usCCM5739(GenBankaccessionno.AF346723);†,nucleotidenumberingofS.aureusagr-I(GenBankaccessionno. s
m
X52543);‡,nucleotidenumberingofS.intermediusATCC29663agr(GenBankaccessionno.AY557375).
.
o
r
g
/

o
atemplateorbyusingoligonucleotidesWP8andT7promoterprimerasprimers activationandinhibitionassays,respectively),5(cid:3)lofsupernatantprepared(or n
andtheligationmixtures(pGEM-3zfplus2.0-kbDNAfragment)astemplates. dilutedinCYGPmedium)wasadded,andthemixtureswereincubatedat37°C J
Both PCR products were used for DNA sequencing. We note that all PCR for55min(activation)or80min(inhibition)inaVERSAmaxmicroplatereader a
n
products were amplified with PfuTurbo high-fidelity DNA polymerase (Strat- (MolecularDevices).Dilutedcultures(10%inCYGPplus5nMsodiumazide)
u
agene),andthePCRproductsfromthreeindependentPCRsweregenerated were mixed with equal volumes of nitrocefin solution, and the (cid:8)-lactamase a
andsequencedtoconfirmthelackofPCRerrors.FourotherS.intermediusagrD activitiesweremeasuredasdescribedpreviously(8,9). ry
genesweresequencedbyusingoligonucleotidesSiB-F3andSiC-R5,fromcon- AgrinductionbyS.intermediusculturesupernatants.Thestationary-phase 1
served positions in agrB and agrC, respectively, and rnaIII in S. intermedius filteredbacterialsupernatantfromanovernight25-mlcultureofS.intermedius 1
CCM5739wassequencedbyusingprimersSaR3-F1andSiB-R3. strainCCM5739waslyophilized.Lyophilizedbrainheartinfusionbrothwasused ,
2
agractivationandinhibitionreportergeneassays.Twomethodswereusedfor asacontrol.From50mlofa1-hculture,25mlwaspouredintoaflaskcontaining 0
theseassays,(i)Assayswereperformedwithbacterialculturesinearlyexponen- thelyophilizedsupernatantand25mlwaspouredintoanotherflaskcontaining 1
tialphase((cid:6)2(cid:7)108cells/ml)byusinga(cid:8)-lactamasereportergenereadout.For thelyophilizedbrainheartinfusion.Then,1-mlaliquotsweredrawnat0,30,60, 9
activationassays,80(cid:3)lofcells(9(cid:7)108cells/ml)weretreatedinduplicatewith and90min,andthecelldensitiesweremeasured.The1-mlsampleswereused b
y
variousamountsofculturesupernatantcontainingAIPatca.60nM,asesti- toprepareRNAforNorthernblothybridization.Equalmassesofcellswereused
g
matedbycomparingitsactivitywiththatofasampleofsyntheticAIPofknown foreachtimepoint.
u
concentration, and incubated with shaking at 37°C for 60 or 90 min in a Plasmid constructions. Plasmid pWP1004 was constructed as follows. The e
THERMOmaxmicroplatereader(MolecularDevices)withmonitoringofcell putativeP2/P3promoterregionofS.intermediuswasamplifiedbyPCRusing s
t
densityatanopticaldensityat650nm,followedbydeterminationofagractiva- oligonucleotides WP12 and SINT11 as primers and chromosomal DNA as a
tion by the (cid:8)-lactamase–nitrocefin assay (9). Nitrocefin was purchased from template. The PCR product was cloned into E. coli cloning vector pGEM-T
BectonDickinson(FranklinLakes,NJ).Forinhibitionassays,80(cid:3)lofcells(9(cid:7) (Promega)inbothorientations.TheresultingplasmidpGEM-T-P2P3wasdi-
108cells/ml)wastreatedinduplicatewithAIP-containingculturesupernatantsin gested with EcoRI and SpeI, and the DNA fragment containing the P2/P3
thepresenceofthegroup-specificwild-typeAIPagonistataconcentrationof promoterregionwasthenclonedintotheEcoRI/NheIsitesofpRN5543,fol-
100nMfor90min,followedbya(cid:8)-lactamaseassay.Anagonistconcentrationof lowedbyinsertionofanEcoRIfragmentfrompRN6683containingthestaph-
100nMisasaturatingbutnotoversaturatingdoseofactivator,whichgenerates ylococcalblaZgeneintotheEcoRIsiteoftheresultingplasmid.
maximalactivationagainstwhichtotestvariousconcentrationsofheterologous PlasmidpWP1101wasconstructedbycloningaPCRproductamplifiedfrom
AIPs.(ii)AssayswithS.aureusreportercellscontainingplasmidpRN6683(22) thechromosomalDNAusingprimersSINT1andSINT5intotheXbaIsiteof
withtheS.aureusP3-blaZfusionweredoneaccordingtothemethoddescribed pRN5548.PlasmidpWP1102wasmadebycloningaPCRproductamplifiedfrom
previously(8,9).ThesamemethodwasusedfortheAIPactivityassayswithS. thechromosomalDNAusingprimersSINT1andSINT2intotheXbaI/EcoRI
intermediusreportercellsharboringplasmidpWP1004(S.intermediusP3-blaZ) sitesofpRN5548.ToconstructpWP1103,aPCRproductwaspreparedbyusing
(Table1).Inbrief,culturesupernatantswerepreparedfromeitherS.intermedius oligonucleotidesSINT3andSINT4asprimersandS.intermediuschromosomal
wild-typecellsgrowninCYGPmediaat37°Cfor6horS.aureuscellscontaining DNAasatemplate.TheEcoRI/BspHI-digestedPCRproductwasthencloned
theclonedS.intermediusagrgene(s)underthecontrolofP promotergrown intotheEcoRI/BspHIsitesofpLZ4012(38).PlasmidpWP1104wasmadeby
bla
at37°Cto70Klettunits,followedbyinductionwith0.5mMmethicillinat37°C cloning a PCR product amplified from chromosomal DNA by using primers
for5h.Thesupernatantswerefilteredwith0.22-(cid:3)m-pore-sizefilters.To45(cid:3)lof SINT1andSINT5intotheXbaIsiteofpWP1103.
cultureofS.intermediusATCC29663(pWP1004)((cid:6)40and(cid:6)100Klettunitsfor Site-directedmutagenesisoftheagrD-SigenewasdonebyusingPCRprimers
3142 JI ET AL. J.BACTERIOL.
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FIG. 1. ComparisonoftheAgrDaminoacidsequencesfromS.aureusandS.intermedius.Theaminoacidsequenceswereanalyzedbythe o
CloneManagersoftware(Sci-EdSoftware,Durham,NC).Dashlinesrepresentgapsgeneratedbytheanalysisprogram.Theidenticalaminoacid n
residuesaredarkshaded,andtheAIPsequencesinAgrDsareinboldface.TheaminoacidsequencesofAgrDproteinscanbeaccessedthrough J
the NCBI protein database under NCBI accession no. CAA36782 (S. aureus group I, Sa I) (22), AAB63265 (S. aureus group II, Sa II) (8), a
n
AAB63268(S.aureusgroupIII,SaIII)(8),AAG03056(S.aureusgroupIV,SaIV)(7),AY871105(RN9161),AY871106(RN9167),AY871107 u
(RN9169),AAL65836(CCM5739)(2),AF346723(RN9515),andAAS66746(ATCC29663).TheaminoacidsequencesofAgrDproteinscanbe a
accessedthroughtheNCBIproteindatabaseunderNCBIaccessionno.CAA36782(S.aureusgroupI,SaI)(22),AAB63265(S.aureusgroupII, ry
SaII)(8),AAB63268(S.aureusgroupIII,SaIII)(8),AAG03056(S.aureusgroupIV,SaIV)(7),AAC38294(S.epidermidis,Se)(35),AAA71977 1
(S.lugdunensis,Sl)(34),andAAS66746(S.intermediusATCC29663,Si). 1
,

2
0
1
containingdesiredmutationsandtheExSitePCR-basedsite-directedmutagen- incubatedonicefor30minwithlysostaphin(150(cid:3)g/ml)inTES-sucrosebuffer 9

esismethodaccordingtothemanufacturer’sinstruction(Stratagene).PCRprod- (20%sucrose,20mMTris[pH7.6],10mMEDTA,50mMNaCl)andshaken b
uctsweregeneratedbyusingT4polynucleotidekinase(MBIFermentas)phos- for1hwithproteinaseK(50(cid:3)g/ml;Sigma)and2%sodiumdodecylsulfate y
g
phorylated oligonucleotides SINT6 and SINT8, or SINT15 and SINT8, as (SDS)at4°C.ForNorthernblotting,thesameamountofcelllysatewaselec-
u
primers and pWP1104 as a template. The PCR products were purified from trophoresedthrougha0.66Mformaldehyde–1%agarosegelinmorpholinepro- e
agarose gels and then ligated, resulting in plasmids pWP1105 and pWP1106, panesulfonicacidbuffer(28).Nucleicacidsweretransferredtoanitrocellulose s
respectively.pLZ4003carryingtheS.aureusgroupIIIagrDgenewasconstructed membrane(Amersham)withaVacuGeneapparatus(Pharmacia)in20(cid:7)SSC(3 t
bycloningaClaIfragmentofpRN6963(8)intotheClaIsiteofpRN6441. M NaCl plus 0.3 M sodium citrate [pH 7]) and fixed under UV light. The
PlasmidpLZ4006thatcarriestheS.aureusgroupIagrD(agrD-I)genewiththe membranewaspreincubatedfor2hat52°Cin2(cid:7)Denhardtsolution(0.02%
cysteinecodonintheAIPcodingregionchangedtoaserinecodonandwithsix bovineserumalbumin,0.02%Ficoll,0.02%polyvinylpyrrolidone,0.05MEDTA
histidinecodonsfusedatbothits3(cid:9)and5(cid:9)endswasconstructedasfollows.A [pH 8], 0.2% SDS, and 5(cid:7) SSC, with sonicated and heat-denatured salmon
PCRproductwasgeneratedbyusingprimersGJ#56andGJ#28,withpRN6852 spermDNAat100(cid:3)g/ml)and10%dextran(Sigma)andthenhybridizedover-
(9)asatemplate.ThePCRproductwasthendigestedwithXbaIandclonedinto night with a 32P-labeled DNA probe in hybridization solution. 32P-labeled
pRN5548XbaIandEcoRI(bluntedwithKlenow)sites.Theresultingplasmid RNAIIIprobeswerepreparedbyPCRusingoligonucleotideprimersRNAIII-F
wasthenusedasatemplatetogenerateaPCRproductusingoligonucleotides andRNAIII-R,andpRN6735DNAasatemplate.Inlabelingreactions,dATP
GJ#45andCS-1asprimers.ThePCRproductsweredigestedwithScaIand concentrationwasreducedto2(cid:3)M,andthereactionmixturecontained50(cid:3)Ci
NcoIandclonedintotheScaI/NcoIsitesofpGJ4004(37). of[(cid:2)-32P]dATP(Amersham;1Ci(cid:10)37GBq).Theblotwasexposedtoastorage
AllPCRproductsusedforplasmidconstructionwereamplifiedwithPfuTurbo phosphorscreen(MolecularDynamics).
high-fidelityDNApolymerase(Stratagene).TheDNAsequencesofthecloned Westernblothybridization.S.aureuscellsexpressingvariousgenesunderthe
wild-typeandmutatedgenesintheconstructedplasmidswereconfirmed. controlofP weregrownandinducedwith0.5mMmethicillinfor4hat37°C.
bla
Whole-celllysatesandNorthernblottingforRNAIIIexpression.Whole-cell Thecultureswerethenmixedwithanequalvolumeofice-coldsolution(50%
lysateswerepreparedasdescribedpreviously(13).Cultureswerecentrifuged, acetoneand50%ethanol).Thefixedcellswerewashedwith1(cid:7)SMM(21)and
fixed in acetone-ethanol (1:1), and washed in N-tris(hydroxymethyl)methyl-2- suspendedin1(cid:7)SMMpluslysostaphin(Sigma).After1hofincubationat37°C,
aminoethanesulfonic acid (TES)–sucrose buffer. Equalized cell samples were cellswerelysedbysonication.Whole-celllysateswereseparatedby16%poly-
VOL.187,2005 S. INTERMEDIUS agr AUTOINDUCING PEPTIDE 3143
These are shown in Fig. 1A with the four groups of the S.
aureusAgrDsforcomparison.AgrD-Siishighlyconservedand
therearetwovariantsamongthesixstrains.Itisnotknownat
present whether this variation affects activation specificity.
ShowninboldfacearethepredictedsequencesoftheS.inter-
medius AIPs, one of which, that of CCM5739, has been con-
firmed as a cyclic lactone with the above sequence by mass
spectroscopy (10). Note that the C-terminal processing site is
absolutelyconservedamongthesestrains,aswellasamong30
otherstaphylococci,whereastheN-terminalprocessingsiteis
not conserved among other staphylococci (2). The latter is
clearlyconserved,however,amongtheseS.intermediusstrains.
TheAgrB-Sisequence.Sequencingofa2,084-bpsegmentof
the S. intermedius ATCC 29663 agr locus revealed that this
D
portion of the locus was identical to the published partial
o
sequence of the same region of S. intermedius CCM5739 (2). w
n
ComparisonoftheAgrB-Sisequencetothatofotherstaphy-
lo
lococcishowedthatAgrB-Siwasclearlyrelatedtootherstaph- a
ylococcalAgrBs(Fig.1B).Interestingly,theN-terminalregion, d
e
tworegionsthatarehighlyhydrophilicandareproposedtobe d

two transmembrane segments (37), and a small C-terminal fr
o
regionweresignificantlysimilar.ThedivergentN-terminalhalf m
of AgrC (data not shown) was also clearly a member of the
h
AgrCreceptorfamily,havingsignificantsimilaritieswithother t
t
p
AgrC receptor domains (2, 20), but with little overall conser- :
/
vation, providing no obvious indication of how it would be /jb
activatedbyalactoneAIP. .a
AgrautoactivationbyanS.intermediusculturesupernatant. s
m
The supernatant of a 6-h (postexponential) CYGP culture of
.
o
FIG. 2. agrself-activationbyS.intermediusCCM5739supernatant. strain CCM5739 was tested for agr activation by Northern r
(A) Northern blot analysis. The lyophilized residue from an equal blottingfortheagreffectortranscript,RNAIII.Expressionof g/
volumeofpostexponentialS.intermediussupernatant(plusS.interme- RNAIII in an untreated culture was compared to that in a o
dius)orbroth((cid:1)BH)wasaddedtoanearly-exponential-phaseculture n
ofCCM5739,andsamplesweredrawnevery30minforNorthernblot culture in which the residue from 25 ml of lyophilized J
hybridization analysis with an RNAIII-specific probe. (B) Growth CCM5739culturesupernatantwasaddedtoa25-mlcultureof a
n
curves.Celldensitymonitoredturbidimetricallyduringthe4-hexper- thesamestrainduringearlyexponentialphase.Ascanbeseen u
imentisplottedversustime. a
in Fig. 2, there was clear induction of RNAIII transcription
r
y
aftertheadditionofthepostexponentialsupernatantwellbe-
1
fore RNAIII expression was initiated in the parallel control 1
,
acrylamideTris-TricineSDS-polyacrylamidegelelectrophoresis(29)andelec- culture.Theamountofpostexponentialsupernatantusedhere 2
trophoreticallytransferredontoaProtrannitrocellulosemembrane(Schleicher was10-foldgreaterthanthatordinarilyusedforagrinduction, 0
&Schuell).Afterincubationat4°Covernightinblockingbuffer(Tris-buffered 1
suggesting that agr induction is weak in this strain, either be- 9
salineplus0.05%Tween20and5%bovineserumalbumin[TBST])(28),the causetheAIPconcentrationwasloworbecausetheresponse b
membraneswereincubatedinblockingbuffercontaininganti-T7-tagmonoclonal
y
antibody(1:5,000dilution;Novagen)for1hatroomtemperature.Themem- totheAIPwasweak.Atthisconcentrationthepostexponential
g
branes were washed extensively with TBST buffer, probed with horseradish supernatanthadamoderateinhibitoryeffectonthegrowthof u
peroxidase-conjugatedgoatanti-mouseantibody(Amersham),anddetectedwith thebacteria,owingtotherelativelyhighconcentrationofpost- e
s
ECLPlusWesternblottingdetectionkit(Amersham),followedbyexposureto exponential products in the added supernatant. Similar self- t
KodakBiomaxMRfilm.
TheS.intermediusagrnucleotidesequence.ThenucleotidesequencesoftheS. activationwasseenwithaculturesupernatantofS.intermedius
intermedius ATCC 29663 agr locus (GenBank accession number AY557375), ATCC29663(datanotshown).InS.aureus,however,previous
agrDgenes(RN9161,GenBankaccessionnumberAY87105;RN9167,GenBank studies with synthetic lactone analogs of the agr-I and agr-II
accessionnumberAY87106;RN9169,GenBankaccessionnumberAY87107;and
thiolactone AIPs have shown that neither of these is a self-
RN9515,GenBankaccessionnumberAF346723),andhldcodingsequenceofS.
activator but that both are potent cross-inhibitors of heterol-
intermediusCCM5739(GenBankaccessionnumberAY860843)havebeende-
positedintheGenBank. ogousagractivationinvolvingagr-I,-II,and-III(agr-IVwasnot
tested). Thus, S. intermedius represents the first example of
activationbyalactoneAIPinthestaphylococci.Theonlyother
RESULTS
knowncaseofalactone-AIPisthatrecentlydescribedforan
The S. intermedius agrD sequences. We determined the agr analog in Enterococcus faecalis, which is unrelated to the
DNA sequence of agrD genes for three uncharacterized S. staphylococcalAIPs(19).
intermediusstrainskindlyprovidedbyWesleyKloos,twomore Inhibitoryactivities.Postexponentialsupernatantsoffiveof
fromourstraincollection,andasixthstrain,ATCC29663,by theS.intermediusstrainswerethentestedfortheireffectson
using the PCR methods described in Materials and Methods. rnaIIIexpressionforeachofthefourS.aureusagrgroups.For
3144 JI ET AL. J.BACTERIOL.
these experiments, an agr-P3-blaZ transcriptional fusion was
introducedintofourdifferentderivativesoftheagr-nullstrain,
RN6911, each containing a tester plasmid specific for activa-
tionbyoneofthefouragrgroup-specificAIPs.Eachofthese
testerplasmidscontainstheagrACsegmentsderivedfromone
of the four groups, driven by the respective agr-P2 promoter,
and can be activated only by the cognate AIP supplied exog-
enously. Activation of the agr-P3-blaZ fusion is not itself
group-specific but depends on activation of the coresident
agrAC unit, which is group specific. For each test, a fixed
amount of an S. aureus culture supernatant containing an
AIPwasaddedtoanearly-exponential-phasecultureofthe
correspondingtesterstrain,alongwithanaliquotofapost-
exponential-phase supernatant from one of the S. interme-
D
dius strains. These cultures were incubated for a further 90
o
min and then assayed for (cid:8)-lactamase activity. For each w
strain, the (cid:8)-lactamase activity obtained in the presence of n
lo
an S. intermedius supernatant was normalized to that ob- a
tained with the cognate activator alone, and the results are d
e
plotted in Fig. 3A. As can be seen, all five S. intermedius d

supernatantsinhibitedmostofthetesterstrainstoagreater fr
o
or lesser extent. agr-I was generally the most susceptible, m
and agr-IV was the least susceptible. The strength of the
h
observedinhibitionsuggeststhattheS.intermediusAIPwas t
t
p
producedinamountscomparabletothoseproducedbystan- :
/
dardS.aureuscultures,andthereforetheweakresponseof /jb
S. intermedius to its own AIP probably represents weak .a
activation. Similar results were obtained for the sixth S. s
m
intermedius strain, ATCC 29663, using a slightly different
.
o
reportersystem(datanotshown).AlthoughtwooftheAIPs r
g
have an N-terminal arginine and the other four have an /

N-terminal lysine, there was no obvious difference in activ- o
n
itiesthatcouldbeattributedtotheN-terminalvariation.In
J
addition, there was apparent strain-dependent variation in a
n
theAIPinhibitoryactivitiesdespitetheidentityoftheAIPs, u
a
perhaps owing to differing amounts of AIP produced or to
r
y
other strain-specific factors. This result adds to previous
1
observations that non-S. aureus AIPs generally inhibit agr 1
,
activation in S. aureus. Finally, as shown in Fig. 3B, the
2
AIP-containingsupernatantsfromS.aureusgroupsI,II,and 0
1
III had weak inhibitory activities for S. intermedius ATCC 9
29663. b
y
ThespecificityoftheS.intermediusAgrB-Si.Itiswellestab-
g
lishedthatAgrBdeterminesthespecificityofAgrDprocessing u
e
inS.aureus(8).Accordingly,wedeterminedthespecificityof
s
the interaction between S. intermedius AgrB-Si and AgrD-Si, t
FIG. 3. (A)agrinhibitionbyS.intermediussupernatants.To50-(cid:3)l bycloningthesetwogenesonseparateplasmids.Variouscom-
aliquotsofearly-exponential-phasecultures(atcelldensitiesof(cid:6)100 binationsofAgrBsandAgrDsfromS.aureusgroupI,II,and
Klett)ofthefouragrgroup-specifictesterstrainswereadded10(cid:3)lof
III,aswellasfromS.intermedius,wereexpressedinS.aureus
culturesupernatantsfromtherespectiveagrwild-typestrainsplus0,
10,or30(cid:3)lofaculturesupernatantoftheS.intermediusstraintobe GJ2035, and the AIP activities were then measured (Fig. 4).
tested for inhibitory activity. Total volumes were made up to 100 (cid:3)l Neither S. aureus AgrB-I nor AgrB-II could process S. inter-
with CYGP broth, and the plates were incubated for 90 min with medius AgrD-Si, and the S. intermedius AgrB-Si could only
shakingat37°C.Then,50(cid:3)lwastransferredtoanewmicrotiterplate,
processitscognateAgrD-SitogeneratematureAIP-Si.These
50(cid:3)lofsaturatednitrocefinwasadded,andthe(cid:8)-lactamasereaction
wasmonitoredkinetically,withtheslopeofthereactionoverthefirst
5minusedtorepresenttheenzymeactivity.Theratesinthepresence
oftheS.intermediussupernatantswerethennormalizedtotheratein
thepresenceofactivatoralone.Hatchedbars,10(cid:3)lofS. intermedius
supernatant; black bars, 30 (cid:3)l ofS. intermedius supernatant; shaded
bars, control, activator only. (B) S. intermedius agr inhibition by S. groupIII,RN8463).AIPactivityassayswithS.intermediusharboring
aureusgroupI,II,andIIIAIPs.Culturesupernatantswereprepared pWP1004asreportercellswereperformedasdescribedinMaterials
from S. aureus strains (group I, RN6390B; group II, SA502A; and andMethods.
VOL.187,2005 S. INTERMEDIUS agr AUTOINDUCING PEPTIDE 3145
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FIG. 4. Interaction between AgrB and AgrD. Conditioned media .
o
werepreparedfromS.aureusGJ2035expressingvariouscombinations r
g
ofAgrBandAgrD,andtheAIPactivitiesweremeasuredbyusingS.
/
intermediuscontainingpWP1004asreportercells.(AandB)Testfor o
activationofS.intermediusagrbyAIPspreparedfromcellscoexpress- n
FIG. 5. Inability of AgrB-I to process a serine-containing AgrD-I
ingS.aureusAgrB-IandAgrB-IIorS.intermediusAgrB-SiandtheS. J
mutant. Using a cloned agrD-I derivative, the cysteine codon, TGT, a
intermediusAgrD-Si(A)andtestforinhibitionofS.intermediusagrby
wasreplacedbyaserinecodon,AGT,andthemutantagrDwascloned n
AIPspreparedfrombacteriacoexpressingtheS.intermediusAgrB-Si u
andS.aureusAgrD-I,AgrD-II,orAgrD-III(B).Reportercellsgrown into a vector between N- and C-terminal His6 tags. The resulting a
constructwastestedinvivointhepresenceorabsenceofanagrB-I- r
intheabsenceofAIPwereusedascontrols.Valuesaremeansfrom y
threeindependentexperimentswithstandarderrorsasindicated. containingplasmidandcomparedtothenativeagrD-I,alsocontaining 1
theN-andC-terminalHistags,fortheproductionofagr-activatingor 1
-inhibitingsubstancesbyusingagrreporterstrainswitha(cid:8)-lactamase ,

readout.(A)Foractivationtests,theculturewasgrownfor90minin 2
0
the presence of the supernatant to be tested for activation and then 1
results indicated that the interaction between AgrB-Si and assayed. (B) For inhibition tests, a sample of a cognate supernatant 9
AgrD-Siwasspecific. (activator) (i.e., group II supernatant for the group II reporter and b
Cross-processing of S-to-C and C-to-S mutants of AgrD. group III supernatant for the group III reporter) was added at the y
same time as the supernatant to be tested for inhibition, and the g
SinceAgrBdeterminesthespecificityofAIPprocessing(8),it culturethengrownfor90minandassayedfor(cid:8)-lactamase.Foreach ue
is very likely that AgrB catalyzes the formation of the cyclic
ofthefivesetsoftestsshown,thecolumnsarelabeledasfollows:A, s
thiolactonebondintheS.aureusAIPsand,therefore,alsothe wild-type AgrD; B, AgrD, C28S; C, wild-type AgrD plus AgrB-I; D, t
cyclic lactone bond in the S. intermedius AIPs. We therefore AgrD,C28SplusAgrB-I[thereporterswereRN6390B(pRN6683)for
testedthepossibilitythatAgrB-Icancatalyzetheformationof groupI,SA502A(pRN6683)forgroupII,andRN8463(pRN6683)for
groupIIIasdescribedpreviously(8,9)].
theAIP-Ilactoneanalogbymutationallyreplacingthecysteine
codon (TGT) in agrD-I with a serine codon (AGT), yielding
agrD-I (C28S). Since the synthetic AIP-I lactone is a potent
cross-inhibitor,andpossiblyaveryweakself-activator,culture wild-type agrD-I and agrB-I plasmids. No activation was seen
supernatantsofthemutantstrainwerethentestedforinhibi- witheitherthegroupIIorIIIreporters.Conversely,inhibition
tionofagractivationintheotherS.aureusagrgroups,aswell ofeitherthegroupIIorgroupIIIreporters(tracksC)wasseen
asforself-activationofagr-I.AsshowninFig.5,noinhibitory only with the wild-type agrD-B combination. We therefore
activitywasdetected,nordidthissupernatantactivatetheagr-I conclude that AgrB-I cannot process a serine-containing
reporter or either of the other two. For these tests, we used AgrD-I.
onlythegroupI,II,andIIIreporterstrains,sincethesewould We next prepared and tested the corresponding S27C mu-
have detected any AIP produced. Note that activation of the tantinAgrD-Si,inwhichtheserinewasreplacedbyacysteine
group I reporter was seen only in track C, containing the andalsoanS27Amutation.Asnoted,S.intermediusAgrB-Si
3146 JI ET AL. J.BACTERIOL.
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J
a
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FIG. 6. Processingofthewild-typeandtheserine-to-cysteinemu- u
a
tantAgrD-SibyAgrB-Si.(AtoD)AIPactivityassays.S.aureuscells
r
were grown and induced. After centrifugation, the culture superna- y

tants were used as the conditioned media (either concentrated or 1
1
dilutedwithCYGPmedium)toperformeitherAIPactivationassays ,
withS.intermediuscontainingpWP1004asreportercells(A)orAIP 2
inhibition assays with RN6390B(pRN6683) (S. aureus group I) (B), 0
1
SA502A(pRN6683)(S.aureusgroupII)(C),andRN8463(pRN6683) 9
(S.aureusgroupIII)(D)reportercells.Undilutedconditionedmedia
b
were equal to 100% AIP. Conditioned media were prepared from y
culturesofcellscoexpressingAgrB-Siandthewild-typeAgrD-Si(F), g
AgrD-Si(S27C) (Œ), or AgrD-Si(S27A) (■). Values are means from u
e
threeindependentexperimentswiththestandarderrorsasindicated.
s
(E) Western blot hybridization analysis. S. aureus cells coexpressing t
AgrB-Si and wild-type AgrD-Si, AgrD-Si(S27C), or AgrD-Si(S27A)
weregrownandinduced,andthecellculturesweremixedwithetha-
nol-acetone(1:1).Themixtureswerecentrifuged,andthecellswere
washedandlysed.Whole-celllysateswereseparatedbySDS-polyacryl-
amide gel electrophoresis and transferred to nitrocellulose mem-
branes.Themembraneswerethenprobedwithananti-T7tagmono-
clonalantibody.
is quite similar to the other known AgrBs (2), so that its determined by AIP activity assays (Fig. 6A to D). However,
divergentsitesmaybeinformativewithrespecttothepro-AIP activation of the S. intermedius agr response by the mutant
processingmechanismand,especially,withregardtotheabil- AIP-Si was much weaker than that by wild-type AIP-Si. The
itytoprocessaserine-containingpro-AIP.AsshowninFig.6, AgrD-Si serine-to-alanine mutant, AgrD-Si(S27A), did not
theAgrD-Siserine-to-cysteinemutant,AgrD-Si(S27C),could generateanyfunctionallydetectableAIP(Fig.6).Theinhibi-
beprocessedbyAgrB-SitogenerateamaturemutantAIP,as toryactivitiesoftheS27CmutantAIP-Si(presumablyathio-
VOL.187,2005 S. INTERMEDIUS agr AUTOINDUCING PEPTIDE 3147
TABLE 3. Alignmentofdelta-hemolysinsequences The results of a simple test for the production of delta-
Strain Sequence hemolysin and other hemolysins, involving cross-streaking on
sheepbloodagaragainstRN4220,areshowninFig.7.Atleast
S.intermedius.................................MAGDIISTIVDFIKLIAETVKKFTK
four of the five S. intermedius strains shown—RN9161,
S.simulans.....................................MAGDIVGTIGEFVKLIIETVQKFTQK
S.aureus.........................................MAQDIISTIGDLVKWIIDTVNKFTK RN9167, 9168, and 9515—produce delta-hemolysins that are
S.epidermidis.................................MMAADIISTIGDLVKWIIDTVNKFKK detectable by this test. The fifth strain, CCM5739 (shown as
S.warneriI.....................................MAADIISTIGDLVKLIINTVKKFQK RN9423 in the picture), is also likely to produce delta-hemo-
S.warneriII....................................MTADIISTIGDFVKWILDTVKKFTK
lysin,sinceagrisautoinducibleinthisstrainandencodesdelta-
hemolysin,althoughitisnotidentifiableintheCCM5739he-
molyticpattern.Hemolysinsaretypicallyagrupregulated,but
lactonemolecule)ontheagrresponsesingroupIIbutnotin the group of S. intermedius strains is rather heterogeneous with
groupIandIIIS.aureusstrainswerestrongerthanthoseofthe respecttohemolyticactivities.Thus,threeproducebeta-hemoly-
wild-typeAIP-Si(Fig.6BtoD). sin, although in considerably different amounts, and none pro-
TodemonstratethatAgrB-Siwasinvolvedintheproteolytic ducesdetectableamountsofalpha-hemolysin,butCCM5739has
D
processingofAgrD-Si,weperformedWesternblothybridiza- thestrongesthemolyticactivityofanyofthefive,probablypro- o
tionanalysiswithananti-T7monoclonalantibodyasaprobe. ducingatleasttwodifferenthemolysins,neitherofwhichistypical w
n
We note that AgrD-Si used in these experiments was doubly of the S. aureus hemolysins as characterized by this simple test. lo
taggedwithaT7epitopeattheNterminusandasix-histidine Theseresultsareconsistentwithagrfunctionalitybutcertainlydo a
stretch (His ) at the C terminus, which would facilitate the notproveit.Experimentsareinprogresstoisolateanagrknock- d
6 e
detection of AgrD-Si, as well as of its potential processing out to obtain a definitive view of what is regulated by agr in S. d

intermediate(s), by Western blot analyses with commercially intermedius. fr
o
availableantibodies.Wenotethatcellsexpressingthedoubly m
tagged AgrD-Si and AgrB-Si produced an amount of AIP-Si
h
comparable to those expressing the wild-type AgrD-Si and DISCUSSION tt
p
AgrB-Si, indicating that the addition of these tags had no :
/
effects on the AgrD-Si processing and AIPSi secretion (data Since S. intermedius was classified as a new staphylococcal /jb
notshown).AsshowninFig.6E,abandwithamolecularmass speciesin1972,thisbacteriumhasbeenisolatedfromvarious .a
ofca.9kDacorrespondingtothecalculatedmolecularmassof animals(12).S.intermediusproducestoxins,hemolysins,coag- s
m
thedoublytaggedAgrD-Si(9,205Da)wasdetectedinthelane ulase and maybe other virulence factors that have been sug-
.
containingalysateofcellslackingagrB-SiorcontainingAgrD- gested to cause food poisoning in humans (1) and diseases or
g
Si(S27A) and producing the tagged AgrD-Si. In the presence rangingfromabscessestomastitisandendocarditisindogs(4, /

of the wild-type AgrB-Si or AgrD-Si(S27C), an anti-T7 anti- 31). Occasionally, S. intermedius causes infection in humans o
n
body-respondingbandwithamolecularmassofca.6kDawas (32). Although S. intermedius is, in most respects, a typical
J
detected. Although this band probably corresponds to a pro- staphylococcal species, it differs strikingly in one respect: it a
n
cessing intermediate previously observed with S. aureus contains a serine in place of the cysteine that is absolutely u
AgrD-I(18),wehavenotbeenabletoobtainsufficientmate- conserved in the AIPs of all of the 14 other staphylococcal a
r
rialforconfirmationbymassspectroscopy.Theseresultssug- species thus far analyzed. All of the six S. intermedius strains y
1
gested that AgrB-Si could process both the serine and the studiedproduceanonomericagrDlactoneAIP.Thepredicted 1
cysteine containing AgrD-Si to generate AIPs that had the AgrB-Si,AgrD-Si,andtheN-terminalportionofAgrC-Sifrom ,
2
ability to activate the S. intermedius and inhibit the S. aureus thesequencedS.intermediusagrlocuswereclearlymembersof 0
1
agrresponses. thewidespreadfamilyofagrgeneproducts.TheAgrB-Sispe- 9
Hemolytic activities and agr function. agr is widely con- cifically processed AgrD-Si to produce mature AIP-Si, and it b
y
served among the staphylococci (2), although its function has alsohadoneinterestingfeaturethatwasdifferentfromother
g
beendefinedinonlytwospecies,S.aureusandS.epidermidis AgrBs, i.e., its abilities to process both the wild-type and a u
(9, 36). Given that the S. intermedius lactone AIP is an agr mutant AgrD-Si, in which the serine residue in the AIP-Si e
s
autoinducer, it follows that agr is likely to be functional in S. region was replaced by a cysteine residue, to produce mature t
intermedius,whichwouldmakeS.intermediusthethird.Avery AIPs. In contrast, the AgrB-I could process only the native
preliminarypictureofthehemolyticactivitiesofS.intermedius cysteinecontainingAgrD-Ibutnotcysteine-to-serinemutants
supports this, although a definitive conclusion would require togeneratematureAIP.Wenotethatchemicallysynthesized
theconstructionofanagrknockout.Theproductionofdelta- linearAIPshaveneitheractivationnorinhibitionactivities(9,
hemolysin, which is encoded by rnaIII, is generally an indica- 17). These results imply that AgrB-Si can catalyze the forma-
tion of agr self-activation. We have determined the DNA se- tionofbothesterandthioesterbondsinAIP-Si.
quenceofagrrnaIIIinanS.intermediusstrain,CCM5739,and Theagrautoinducingactivityhasbeendemonstratedfortwo
identified a typical staphylococcal delta-hemolysin reading ofthestrains,andtheproductionofacycliclactonewascon-
frame (3). An alignment of delta-hemolysin sequences from firmed. Cross-inhibition has been demonstrated for all six
staphylococci is shown in Table 3. The NCBI accession num- strains.Fourofthesixstrainsclearlyproducedelta-hemolysin,
bersareasfollows:AAW55662(S.intermedius),CAA11542(S. andthefifthprobablydoessoaswell,andtheoverallhemolytic
simulans)(33),LESAD(S.aureus)(3),CAA11541(S.epider- patterns are consistent with agr functionality. Given that the
midis)(33),CAA11543(S.warneri1)(33),andCAA11544(S. onlyagrautoinducingandcross-inhibitingsubstancesyetiden-
warneri2)(33). tified are the cyclic AIPs, we suggest that S. intermedius pro-
3148 JI ET AL. J.BACTERIOL.
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,

FIG. 7. Hemolyticpatterns.CulturestobetestedweregrownovernightonGLagar,cross-streakedonsheepbloodagaragainstacultureof 2
RN4220,incubatedovernightat37°C,andthenincubatedfor6hat4°C.Thepatternscanbeinterpretedasfollows:the“hot-cold”beta-hemolysin 0
1
appearsasapartiallyturbidzone,asseenwithRN4220,whichproducesonlybeta-hemolysin.Delta-hemolysinissynergisticwithbeta-hemolysin 9
and is seen as a clearing where the two hemolysins intersect; this is best seen with RN9515 (top left). Coproduction of beta-hemolysin and
b
delta-hemolysinisseenasaclearingnexttothestreakwithinawiderbeta-hemolysinzone(bestillustratedwithRN9169,bottomleft)and,less y
strongly,withRN9167(bottomright).RN9161producesonlydelta-hemolysinandquiteweakly.RN6734producesaverystrongalpha-hemolysin g
zone,asshownbythecharacteristicantagonismbetweenalpha-hemolysinandbeta-hemolysin.Italsoproducesdelta-hemolysin,asshownbythe u
e
clearerzonewherethebeta-hemolysinanddelta-hemolysinzonesintersect.RN7206producesaveryweakalpha-hemolysinzone,asshownbyits
s
inhibitionbybeta-hemolysin.TheCCM5739(labeledRN9423)patterncouldrepresentabeta-hemolysinzoneequivalenttothatofRN9169plus t
a very strong delta-hemolysin zone, which obscures that of beta-hemolysin. The interaction of CCM5739 (labeled RN9423) with the RN4220
beta-hemolysinzoneisveryatypicalandisnotinterpretableaccordingtoourunderstandingoftheactivitiesoftheS.aureushemolysins.
duces lactone AIPs that are active as autoinducers and cross- hasnotbeenruledout(14).Ithasalsobeensuggestedthatthe
inhibitorsandthatagrhasafunctionalroleinS.intermedius. cyclicconfigurationcouldconferstabilitytowardenvironmen-
An enduring question is why evolution has favored the de- talproteases;thiswouldhavetobetestedbyacomparisonof
velopment of cyclic AIPs in staphylococci and enterococci, linear peptides in other systems with the staphylococcal and
since cyclic peptides are not used in any of the other known enterococcal cyclic ones. Since lactone- and thiolactone-con-
peptide-based receptor-ligand signaling systems described to tainingpeptideswouldhaveverydifferentintrinsicstabilities,it
date.Wedonothaveanycompellinghypothesisforthis.Itwas isdifficulttoarguethatintrinsicstabilityisrelevanthere.
initially thought that activation of the receptor would involve There is increasing evidence that AgrB is necessary and
theformationofacovalentbondwiththeAIP.Althoughthis sufficientfortheprocessingandsecretionoftheagrAIPs(8,9,
hasbeenruledout,thepossibilityofatransientcovalentbond 37–39)andthatthethiolactoneconfigurationisnecessaryfor