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Results in Immunology
j o u r n a l h o m e p a g e : www.elsevier.com/locate/rinim
Complement regulator C4BP binds to
Staphylococcus
aureus
surfaceproteins SdrE and Bbp inhibiting bacterial opsonization and
killing
夽
Pamela S. Hair
a, Caitlin K. Foley
a, Neel K. Krishna
b, Julius O. Nyalwidhe
b,c, Joan A. Geoghegan
d, Timothy J.
Foster
d,
Kenji M. Cunnion
a,e,f,*aDepartmentofPediatrics,EasternVirginiaMedicalSchool,855WestBrambletonAvenue,P.O.Box1980,Norfolk,VA23501-1980,USA bDepartmentofMicrobiologyandMolecularCellBiology,EasternVirginiaMedicalSchool,700WestOlneyRoad,Norfolk,VA,USA cLeroyT.CanolesJr.CancerResearchCenter,EasternVirginiaMedicalSchool,651ColleyAvenue,Norfolk,VA,USA
dDepartmentofMicrobiology,TrinityCollege,TheUniversityofDublin,Dublin,Ireland eChildren’sSpecialtyGroup,601Children’sLane,Norfolk,VA,USA
fTheChildren’sHospitalofTheKing’sDaughters,601Children’sLane,Norfolk,VA,USA
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received31July2013
Receivedinrevisedform25October2013 Accepted29October2013
Keywords: Staphylococcusaureus
Complement Opsonophagocytosis C4BP
SdrE
a
b
s
t
r
a
c
t
Staphylococcusaureusisapremierhumanpathogenandthemostcommoncauseofosteoarticular,wound, andimplanteddeviceinfections.WerecentlydemonstratedS.aureusefficientlybindstheclassical comple-mentregulatorC4b-bindingprotein(C4BP)inhibitingantibody-initiatedcomplement-mediated opsoniza-tion.HereweidentifyS.aureussurfaceproteinSdrEasaC4BP-bindingprotein.RecombinantSdrEand re-combinantbonesialoprotein-bindingprotein(Bbp),anallelicvariantofSdrE,bothefficientlyboundtoC4BP inheat-inactivatedhumanserum.WepreviouslydescribedSdrEasbindingalternativepathwayregulator factorH.RecombinantSdrEandBbpefficientlyboundC4BPandfactorHinserumwithoutapparent interfer-ence.GainoffunctionstudiesutilizingLactococcuslactisclonesexpressingSdrEorBbpincreasedserumC4BP andfactorHbinding,comparedwithempty-vectorcontrol(WT)approximately2-fold.Correspondingly, classicalpathway-mediatedC3-fragmentopsonizationandbacterialkillingbyhumanneutrophilsdecreased byhalfforL.lactisclonesexpressingSdrEorBbpcomparedwithWT.Insummary,weidentifySdrEand allelicvariantBbpasS.aureussurfaceproteinsthatbindthecomplementregulatorC4BPinhibitingclassical pathway-mediatedbacterialopsonizationandkilling.
c
2013TheAuthors.PublishedbyElsevierB.V.Allrightsreserved.
1. Introduction
Staphylococcusaureusisthepreeminenthumanpathogencausing skinandskin-structureinfections[9]aswellaspost-operative in-fections[12],inadditiontobeingthemajorcauseofmanytypes of invasive bacterial infections [3,39]. Hospital- and community-associatedMRSAinfectionscontinuetorise[16]makingtreatment ofthispathogenevermorechallenging.Additionally,evidence sug-geststhatwild-type infectiondoesnot yield protectiveimmunity againstrepeatedinfectionwiththesamestrain[29]andallattempts todevelopasuccessfulvaccineagainstS.aureushavefailed[7].
Thelackofprotectiveimmunityfrominfectionorimmunization
夽Thisisanopen-accessarticledistributedunderthetermsoftheCreative
Com-monsAttribution-NonCommercial-NoDerivativeWorksLicense,whichpermits non-commercialuse,distribution,andreproductioninanymedium,providedtheoriginal authorandsourcearecredited.
* Correspondingauthorat:EasternVirginiaMedicalSchool,855WestBrambleton Avenue,P.O.Box1980,Norfolk,VA23501–1980,USA.Tel.:+17576687238;fax:+1 7576688275.
E-mailaddresses:[email protected],[email protected](K.M.Cunnion).
suggeststhatantibody-mediatedimmunityagainstS.aureusis in-adequate.Thisisdespitethefactthatawidevarietyofantibodies aregeneratedbythehumanhostduringS.aureusinfection[37]and manydifferentimmunogenshavebeentriedasvaccinesinavariety ofcombinations[33].AntibodyinteractionswithFcreceptorsare al-teredbyS.aureusexpressionofstaphylococcalproteinA(SpA)andSbi [23],butantibodybindingtothebacterialsurfacealsoinitiatespotent classicalcomplementpathway-mediatedhostdefensesincluding op-sonizationandanaphylatoxinproduction[24].Inhibitionofclassical complementpathwayactivationinthehostisprimarilyregulated byC4b-bindingprotein(C4BP)bydisplacingC2afromtheclassical C3convertaseandfacilitatingfactorI-mediatedcleavageofC4bto inactiveforms[6,13,14].SurfacerecruitmentofC4BPasanimmune evasionstrategyhasbeendemonstratedforseveralpathogenic bacte-riaincludingStreptococcuspyogenes[1,21],Streptococcuspneumoniae
[8],Borreliaburgdorferi [30],andNeisseriameningitidis[20]. Wehaverecentlyshownthatthehostcomplementregulatorof classical pathwayactivationC4BP isrecruited tothe surfaceofS. aureusincreasingdegradationoftheclassicalC3convertaseand con-tributingtoC4bcleavageresultingindecreasedopsonizationbyC3b
[18].TheseresultssuggestedthatS.aureusrecruitmentofC4BPtoits surfacelikelycontributestotheinhibitionofantibody-initiated clas-sicalcomplementpathwayactivation,amechanismthatmayresult inlackofprotectiveadaptiveimmunityafterinfectionor immuniza-tion.
Here weidentifyS. aureussurfaceproteins that bindto C4BP andshowthatbacterialexpressionoftheseproteinsaltersclassical complementpathway-mediatedopsonizationandbacterialkilling. IdentificationofC4BP-bindingsurfaceproteinsthatinhibit antibody-initiatedcomplementactivationandimmuneeffectorsprovidesan opportunitytodevelopnovelstrategiestoincreasevaccine effective-nessagainstS.aureus.
2. Materialandmethods
2.1. EthicsStatement
Humanbloodwasobtainedfromfourhealthyvolunteersfor gen-erating serum andneutrophils usedas reagentsin thesestudies. Eastern VirginiaMedicalSchoolIRB approvedthisstudyprotocol: 02–06-EX-0216.Writteninformedconsentwasprovidedby study participants.
2.2. Bacteriaandgrowthconditions
S.aureusReynoldsstrainwasgrowntomid-logarithmicphasein Columbia2%NaClmediaat37◦C,asdescribedelsewhere[5]. Lac-tococcuslactisstrainsthatconstitutivelyexpressthestaphylococcal surfaceproteinsSdrE,Bbp,orthatcontaintheemptyvectorpKS80are describedelsewhere[26,36].L.lactisweregrowninM17broth con-taining0.5%glucoseand5
μ
g/
mloferythromycinat30◦Cwithout shaking,aspreviouslydescribed[32].MRSAstrainsfromtheNebraska TransposonMutantLibrary,describedelsewhere[11],wereobtained fromNARSA.2.3. Buffers
BindingexperimentswereperformedwithGVBS++ buffer(VBS with 0.1% gelatin, 0.15mM CaCl2, and1.0mM MgCl2), or
EDTA-GVBS− −buffer(VBSwith0.1%gelatinand0.01MEDTA).
2.4. Humanserumandpurifiedcomplementproteins
Normalhumanserum(NHS)waspreparedaspreviouslydescribed [4]fromthebloodoffourhealthydonorsandpooled.Bloodwas ob-tainedwithconsentunderanIRB-approvedprotocol(EasternVirginia MedicalSchoolIRB02–06-EX-0216).Heat-inactivatedserawere gen-eratedbyincubatingNHSat56◦Cfor30min.CobraVenomFactor (CVF)treatedserawasgeneratedbyincubatingNHSwithCVF(Comp Tech)at20
μ
g/
mlfor1hat37◦C.Heat-inactivatedandCVF-treated serawereconfirmedtohavenocomplementactivityinaCH50assay. FactorBdepletedseraandpurifiedC4bBindingProtein(C4BP)were obtainedcommercially(CompTech).TotalIgGinourpooledserum wasmeasuredat16.5mg/
mlbyELISA,aspreviouslydescribed[10]. IgGbindingtoS.aureuswasestimatedbyincubating1010CFUofaSpa-deficientstrain(NebraskaTransposonMutantLibrary)with1ml ofheat-inactivatedpooledhumanserum,strippingboundantibody, andmeasurementbyELISA.Wemeasured0.0257mgofIgGbound 1010CFU,orabout0.15%oftotalIgGintheserum.Relativeamount
ofanti-SdrE antibodypresentintheserumwasperformedby se-rialdilutioncomparingantibodybindingtorSdrEoratotalcellwall preparation(Spa-deficientstrain)inamodifiedELISA-typeassay. Ap-proximately0.019%ofIgGintheserumboundtorSdrEcomparedto atotalcellwallpreparation.
Fig.1. IdentificationofSdrEasabindingpartnerforC4BP.(A)S.aureuscellwall preparationfractionsseparatedbysize-exclusionchromatographythenassayedfor C4BPbindingbydotblot.(B)Cellwallproteinsfromconcentratedfractionswere separatedbySDS-PAGEandvisualizedbytotalproteinstain(left).Blottedproteins wereoverlayedwithserum(CVF-treated)andthenprobedforboundC4BP(right). Threebandswereexcisedandanalyzedbymassspectrometry.(C)Peptidecoverage mapforSdrEpeptidesidentifiedbymassspectrometryinboldandunderlined.
2.5. Recombinantproteins
Recombinant SdrE (rSdrE), recombinant bone sialoprotein-bindingprotein(rBbp),andrecombinantClfA(rClfA)wereexpressed asHis-taggedproteinscontainingtheirrespectiveuniqueAregions in an Escherichia coli expression system, as described elsewhere [26,27,36]. Briefly, recombinant proteins were purified from cell lysatesbynickelcolumnchromatographyandverifiedby anti-His tagdotblotsandSYPRORuby(Invitrogen)stainedSDS-PAGE.
2.6. IsolationofcellwallproteinsbindingC4BP
Fig.2. ELISA-typebindingassaysforC4BPandfactorHwithrecombinantSdrEandrecombinantBbp.(A)C4BPinheat-inactivatedserumbindingrSdrE,rBbp,orrClfA(n=5).(B) Time-courseofC4BPinheat-inactivatedserumbindingrSdrEorrBbp(n=4).(C)C4BPinnormalhumanserum(NHS)orheat-inactivatedserumbindingrSdrE.(D)PurifiedC4BP bindingrSdrEorrBbp(n=4).(E)FactorHinheat-inactivatedserumbindingrSdrEorrBbp(n=5).(F)Time-courseofFactorHinheat-inactivatedserumbindingrSdrEorrBbp(n
=3).Dataarethemeansofresultsofindependentexperiments.ErrorbarsdenoteSEM.
preparationsweregeneratedfrommid-logarithmicphaseS.aureus
Reynoldsusinglysostaphininaprotoplaststabilizingraffinosebuffer withDNAseandproteaseinhibitors.Separationbysize-exclusion col-umnchromatographywasperformedusingaHiPrep16
/
60Sephacryl columnequilibratedwithPBS.Fractionsweredotblottedandblocked with3%BSA/
TBSandwashedwithTBS-Tween.Blotoverlaywas per-formedwith10%CVF-NHSfor2handthenprobedwithamouse anti-humanC4BPantibody(Quidel)followedbyagoatanti-mouseHRP an-tibody(Sigma-Aldrich).FractionsthatboundserumC4BPwerethenFig.3. SerumC4BPbindingtoL.lactisexpressingSdrE,orBbp,oremptyvectorcontrol.(A)SdrEandBbpexpressionbyL.lactisstrainsassayedbymodifiedELISA.(B)C4BPin 10%heat-inactivatedserumbindingtoL.lactisstrainsat30minassayedbydotblotofstrippedsurfaceproteins(n=4).(C)FactorHin10%heat-inactivatedserumbindingL. lactisstrainsat30minassayedbydotblotofstrippedsurfaceproteins(n=4).(D)C4BPandfactorHin20%heat-inactivatedserumbindingtoSdrE-expressingL.lactisover30 minassayedbydotblotofstrippedsurfaceproteins(n=4).(E)C4BPin10%heat-inactivatedserumbindingtoS.aureusstrainsincludingwildtype(WT),SdrE-deficient(SdrE−), Sbi-deficient(Sbi−),CHIPS-deficient(Chs−),andcapsule-deficient(Cap50−)assayedbydotblotofstrippedsurfaceproteins(n=4).Dataarethemeansofresultsofindependent experiments.ErrorbarsdenoteSEM.
2.7. Massspectrometryidentification
ProteinbandswereexcisedfromSYPRO-RubystainedSDS-PAGE gelsandprocessedforliquidchromatographyelectrospray ioniza-tion tandem mass spectrometry (LC-ESI-MS
/
MS) ona linear trap quadrupole ion trap(LTQ) massspectrometer(Thermo Fisher) as previouslydescribed[17,19,38].Theacquireddatawasprocessedby2.8. Alignmentanalysis
Amino acid sequences of SdrE (GenBank accession #
YP001331559.)andBbp(GenBankAccession#Q14U76.1)aswell ashumanC4BP (GenBankAccession# AAA36507.1)andfactor H (GenBankAccession#P08603.4)werealignedusingClustalOmega [15].
2.9. Bindingassays
RecombinantBbporSdrEwasboundto96-wellImmulonIIplates at10
μ
g/
mlin a carbonatecoating buffer pH 9.6, andincubated overnight at4◦C. The plateswere then washed with2 × PBS/
1%Tween-20threetimesandblockedwith0.5%gelatininPBSfor 2hatroomtemperature.Theplateswerewashedagain,andNHS, heat-inactivatedsera,orpureC4BPweredilutedinblockingbuffer, titrated,andincubatedforthetimeindicatedatroomtemperature. Afterwashing,theplateswereprobedwithchickenanti-humanfactor Hantibody(AccurateChemical),orthemouseanti-humanC4BP anti-body;antibodiesweredilutedto1:1000inblockingbufferand incu-batedfor1h.Secondaryprobewasperformedwithgoatanti-chicken HRPantibody(Genway)orgoatanti-mouseHRPantibody; antibod-iesweredilutedto1:1000inblockingbufferandincubatedfor1h. PlateswerewashedanddevelopedwithTMBsubstrate(Thermo Sci-entific)andstoppedwith2.5NH2SO4.Absorbancevalueswerereadat450nm.Valuesfromcontrolwellsperformedwithoutprimary an-tibodiesyieldedlowbackgroundsignals,whichweresubtractedout.
2.10. L.lactisexpressionofSdrEandBbp
LogphaseL.lactisincoatingbufferwereusedtocoatan Immu-lonIIplatestartingat1.3× 108cells
/
mlthenserialdilutions.Afterovernightat4◦C,wellswerewashedandblockedwith0.5%gelatin inPBSfor2h.Afterwashing,wellswereincubatedwithachicken anti-SdrE
/
Bbpantibodydilutedinblockbuffer,at1:1000for1h.The wellswerethenwashedandincubatedwithanti-chickenHRP anti-body(Genway)dilutedinblockbufferat1:1000for1h .Wellswere washedanddevelopedwithTMB,asdescribedabove.2.11. FactorHandC4BPbindingL.lactisstrains
L.lactisstrainsweresuspendedatOD600=4.0,ofwhich0.25ml
wasdilutedwithheat-inactivatedseraandGVBS++ buffertoatotal volumeof0.5mlandincubatedfor30minat37◦C[32].Bacteriawere washedwithGVBS−−buffer,andsurfaceproteinswerestrippedwith 50
μ
lof2%SDSandboiledfor5min.Samplesweresedimentedand supernatantsrecovered.2.12. FactorHandC4BPquantitativedotblot
C4BPdotblothasbeenpreviouslydescribed[18].Briefly,after addingsamplesandwashing,themembranewasblockedwith3% BSA,probedwithamonoclonalmouseanti-C4BPprimaryantibody, andthena HRP-labeledgoatanti-mousesecondary antibody.The factorHdotblotwasprobedwithagoatanti-humanfactorHantibody primarilyandarabbitanti-goatHRPantibodysecondarily.Purified factorHwasusedtogenerateastandardcurveanddetectionwasvia ECL.QuantityOnesoftware(Bio-Rad)wasusedtoassigngreyscale valuestothestandardcurvetitrationsandthesamplessothatalinear regressioncouldbeusedtoquantifytheamountofC4BPorfactorH ineachsample.
2.13. C3bindingtoL.lactis
L.lactisstrainswereincubatedwith10%factorB-depletedserain
GVBS++ bufferfor10minat37◦C.Thesecellswerewashed thor-oughlywithGVBS−−buffer,andthecellpelletswereresuspended in50
μ
Lof25mMmethylaminetostrip offcovalentlybound C3-fragments.QuantityoftotalC3-fragmentswasmeasuredviaC3ELISA aspreviouslydescribed[17].C3-fragmentswerequalitatively ana-lyzedbyWesternblotanalysis,aspreviouslydescribed[17].2.14. L.lactiskillingassay
The complement-mediatedkillingassaywasperformed as de-scribedpreviously[32].Briefly,in1.0mltotalvolume,6×106CFUL. lactis,5× 106neutrophilsand10%factorB-depletedseradilutedin
HBSSwithCa++ weretumbledat37◦C.At15and30min,samples weredilutedinsterilewaterandplatedonGM17agarsupplemented witherythromycin.Afterovernight growthat30◦C,colonieswere counted.Thenumberofbacteriakilledwascalculatedbysubtracting thebacterialcountofthesamplewithneutrophilsfromthebacterial countofamatchedbase-linesamplewithoutneutrophils.Additional controlsperformedwithoutserumyieldedzerokilling.
2.15. Statisticalanalysis
Meansandstandarderrorofthemeans (SEM)werecalculated from‘n’independentexperimentsperformedonseparatedays.Data wereanalyzedbystudent’sttest;calculatedPvaluesof≤0.05were consideredstatisticallysignificant.
3. Results
3.1. IdentificationofS.aureuscellwallproteinSdrEasaC4BPbinding protein.
InordertoidentifyS.aureuscellwallproteinsthatbindC4BPwe utilizedmethodologywehavepreviouslyreported[19,32].Briefly,S. aureuscellwallpreparationsweregenerated,fractionatedbycolumn chromatographyandanalyzedbyoverlayC4BPdotblot(Fig.1A).Cell wallfractionsdemonstratingthehighestC4BPbindingwere concen-tratedandassayedbyserumC4BPoverlayWesternblotidentifying threebandsofinterest(Fig.1B).ControlWesternblotsperformed withoutserumoverlayorwithoutprimaryantibodywerenegative. Correspondingbandswereexcisedfromtotalproteinstained SDS-PAGEgelsandsubjectedtoingeldigestionandmassspectrometry analysis(LC-ESI-MS
/
MS)inordertoidentifypeptidesequences.All threebandsofinterestyieldedmultiplesignificantidentificationsof peptidesfromtheS.aureuscellwallproteinSdrE(Table1).Peptide scores>
30andExpectscores<
0.05wereconsideredsignificant. TheseidentifiedpeptidesyieldedapeptidemapforSdrEwith58% coverageofthenon-SD-repeatregion(Fig.1C).WehavepreviouslyreportedidentificationofSdrEasaS.aureus
cellwallreceptorforthehostcomplementregulatorfactorHthat decreasescomplementalternativepathway-mediatedopsonization andbacterialkilling[32].ThemostcommonconfigurationofC4BP issevenalphachainsandonebetachain[2].The1231aminoacid factorHandthe597aminoacidalpha-chainofC4BParecomprised ofcomplementcontrolprotein(CCP)domains[22]withregionsof limitedhomology(SupplementalFig.S1A).
3.2. BindingofC4BPorfactorHwithSdrEorBbp.
Table1
MassspectrometryanalysisofS.aureuscellwallproteinbindingC4BP.
Mr(expt) Mr(calc) Peptidescore Expectscore Peptidesequence
1213.3464 1213.5727 49 0.014 K.YRFDNLDSGK.Y
1242.8066 1242.6860 48 0.018 R.LTLYSYIDKK.T
1252.6867 1252.6340 51 0.0089 K.FETPTGYLPTK.V
1385.2488 1384.6695 72 9.1e-05 K.VNSDQQLPQSNR.I
1435.3694 1434.7090 78 2e-05 K.TATEDTSVILEEK.K
1440.9608 1440.7460 78 2.3e-05 K.VIGTTTTDASGKYK.F
1443.6196 1443.7933 79 1.8e-05 K.GIKDVTVTLQNEK.G
1492.2922 1492.6028 69 0.00018 K.DADNMTLDSGFYK.T
1594.4430 1593.7522 73 8.4e-05 K.VDIAGSQVDDYGNIK.L
1603.4596 1602.8101 62 0.0011 K.LGNGSTIIDQNTEIK.V
1764.2652 1763.8941 48 0.029 K.TVPNETSLNLTFATAGK.E
1775.5260 1774.8962 69 0.00023 K.VYKVNSDQQLPQSNR.I
1895.0746 1894.8756 62 0.0012 K.VNGTTDGEKDSNGSSVTVK.I
1900.6288 1899.9003 78 3.4e-05 K.GHYEFGGLKDGETYTVK.F
1931.1484 1931.0211 87 3.6e-06 K.DVTVTLQNEKGEVIGTTK.T
1943.5350 1943.0000 96 5e-07 R.FAVAQPAAVASNNVNDLIK.V
1957.3312 1956.8735 78 3.5e-05 K.YTPTSDGELDIAQGTSMR.T
1964.6628 1963.9698 106 4.7e-08 K.NTTAEDKDSNGLTTTGVIK.D
1992.8768 1992.0528 77 3.8e-05 K.LGNGSTIIDQNTEIKVYK.V
2098.5814 2097.9167 82 1.6e-05 R.IYDFSQYEDVTSQFDNK.K
2141.7474 2141.0205 69 0.0003 K.QITYTFTDYVDKYEDIK.S
2146.6474 2146.0470 76 6.4e-05 K.DGETYTVKFETPTGYLPTK.V
2173.6574 2173.1266 80 2.4e-05 K.LDEDKQTIEQQIYVNPLK.K
2195.7294 2195.0706 98 3.9e-07 K.SATNTKVDIAGSQVDDYGNIK.L
2226.5414 2225.9965 70 0.00028 K.IGDYVWEDVDKDGVQGTDSK.E
2226.6934 2226.9957 84 1.1e-05 R.IYDFSQYEDVTSQFDNKK.S
2246.0554 2246.1365 71 0.00022 K.MRFAVAQPAAVASNNVNDLIK.V
2290.5614 2290.1005 93 1.3e-06 K.SFSNNVATLDFGDINSAYIIK.V
2302.7994 2302.2056 59 0.0031 K.LDEDKQTIEQQIYVNPLKK.S
2384.7054 2384.1536 69 0.00034 K.QITYTFTDYVDKYEDIKSR.L
2554.9747 2554.2762 75 0.0001 K.VDNQVTDATNPKEPVNVSKEELK.N
2576.0263 2576.2202 66 0.00078 K.SSTQQQQNNVTATTETKPQNIEK.E
2679.6994 2679.2228 75 9.4e-05 K.FTDLDNGNYTVEFETPAGYTPTVK.N
2766.0661 2766.3811 56 0.0086 K.NVIPSDLTDKNDPIDITDPSGEVIAK.G
2971.6754 2971.3651 65 0.0011 K.YKFTDLDNGNYTVEFETPAGYTPTVK.N
3038.1532 3038.3629 75 0.00011 K.VGDGKDNVAAAHDGKDIEYDTEFTIDNK.V
3130.4722 3130.4830 64 0.0014 K.FETPTGYLPTKVNGTTDGEKDSNGSSVTVK.I
3178.8622 3178.5517 85 1e-05 K.VDIAGSQVDDYGNIKLGNGSTIIDQNTEIK.V
3439.2682 3439.5461 60 0.0038 K.NTTAEDKDSNGLTTTGVIKDADNMTLDSGFYK.T
3482.7922 3482.5292 94 1.4e-06 K.QDDATTSDNKEVVSEAENNSTTENDSTNPIKK.E
3571.1422 3570.7464 70 0.00038 K.VDIAGSQVDDYGNIKLGNGSTIIDQNTEIKVYK.V
3593.4952 3593.6546 65 0.0012 K.VNSDQQLPQSNRIYDFSQYEDVTSQFDNKK.S
3780.7162 3780.8541 57 0.0085 K.SATNTKVDIAGSQVDDYGNIKLGNGSTIIDQNTEIK.V
3799.4212 3798.6697 116 1e-08 R.TTDKYGYYNYAGYSNFIVTSNDSGGGDGTVKPEEK.L
preventC4BPorfactorHfrombindingwithdepositedC4borC3b, re-spectively.C4BPinheat-inactivatedserumboundrecombinantSdrE andBbpinadose-dependentmannercomparedwithminimal bind-ingforrClfA(Fig.2A).Time-courseexperimentswereperformedwith 25%heat-inactivatedserumallowingC4BPtobindtorecombinant proteinsinELISA-typeassays.C4BPinheat-inactivatedserum demon-stratedtime-dependentbindingtorSdrEandrBbp(Fig.2B).Testing C4BPbindingtorSdrEusingeithernormalhumanserum(NHS)or heat-inactivatedserumyieldedsimilarbinding(Fig.2C).Inpreviously publishedexperimentstestingC4BPbindingtotheS.aureussurface, heat-inactivatedserumdemonstrateddecreasedC4BPbinding com-paredwithNHS[18].PurifiedC4BPdemonstrateddose-dependent bindingtorSdrEaswellasrBbp(Fig.2D).Half-maximalbindingfor pureC4BPwasobservedat15nmol
/
lforrSdrEand30nmol/
lforrBbp, wheretherecombinantproteinswerecoatedat10μ
g/
ml.These find-ingssuggestthatC4BPmayhaveahaveahigheraffinityforSdrEthan Bbp.Absorbancevaluesfor50%heat-inactivatedserumshoweda 3-foldincrease(p=0.02)forfactorHbindingtorBbpcomparedwith fHbindingtorSdrE(Fig.2E);howeverhalf-maximalbindingvalues weresimilar.Time-courseexperimentsalsosuggestedincreased ab-sorbance valuesforfactorHinheat-inactivated serumbindingto rBbpcomparedwithrSdrE(Fig.2F).ThesefindingssuggestthatBbp, inadditiontoSdrE,mayalsobeafactorH-bindingprotein.3.3. BindingofC4BPandfactorHtoL.lactisexpressingSdrEorBbp.
InordertoevaluatethebindingofC4BPtoSdrEorBbpexpressed onabacterialsurfaceweusedgain-of-functionL.lactisclones ex-pressingSdrE,orBbp,orcontaininganemptyvector.L.lactisclones weremeasuredforSdrEandBbpexpressionusingwholecellsina modifiedELISA-typeassay(Fig.3A)demonstratingsignificant expres-sioncomparedwithemptyvector.L.lactiscloneswereincubatedin heated-seraandthenstrippedofsurface-boundproteinswhichwere measuredbydotblotassays.Controldotblotswereperformedfor
L.lactiswithoutserumorwithoutprimaryantibodydemonstrating minimalbackground.L.lactisclonesin10%heated-seraover30min boundmoreC4BPwhenexpressingSdrE(p=0.04)orBbp(p=0.03) comparedwiththeemptyvectorstrain(Fig.3B).Similarly,L.lactis
clonesboundmorefactorHwhenexpressingSdrE(p=0.01)orBbp(p
heat-inactivatedserumfor30min,washedandstrippedof surface-boundproteinswereassayedforC4BPbydotblot.TheSdrE-deficient strainshoweda30%(p=0.05)decreaseinC4BPbindingcompared withthewild-type.SimilarC4BPbindingtowild-typewasfoundfor theSbi-deficient,CHIPS-deficient,andcapsule-deficientstrains.
3.4. Classicalcomplementpathwayopsonizationandbacterialkilling.
InordertoevaluatetheeffectofbacterialexpressionofBbpor SdrEonclassicalpathwaymediatedopsonization,wemeasured C3-fragmentbindingtoL.lactisstrainsincubatedin10%factorB-depleted sera(Fig.4A).FactorBdepletedserumwasusedtoisolateactivation totheclassicalandlectinpathways intheabsenceofthe alterna-tivepathway.At10min,L. lactisexpressingBbpbound47%fewer C3-fragmentscomparedwithemptyvector(p=0.05)andL.lactis ex-pressingSdrEbound33%fewerC3-fragmentscomparedwithempty vector(p= 0.03).C3-fragment binding comparing SdrE andBbp cloneswerenotstatisticallydifferent.Anti-C3Westernbotanalysis ofstrippedC3-fragmentsdemonstratedthattheboundC3-fragments wereopsonicformsC3bandiC3b(Fig.4B).Anti-C3Westernblot anal-ysisalsosuggestedthattheC3-fragments75kDa(
β
),68kDa(α
1),and42kDa(
α
2)appearedmoredensefortheemptyvectorcomparedwiththeSdrE
/
Bbp-expressingclones,consistentwithquantitative ELISAresults.TheseresultssuggestthatSdrEorBbpmediated recruit-mentofC4BPtothebacterialsurfacesignificantlydecreasedclassical complementpathway-mediatedopsonizationbyC3-fragments.InordertoevaluatetheeffectofbacterialexpressionofBbpof SdrEonclassicalcomplementpathwaymediatedbacterialkillingby neutrophils,weassayedsurvivalorL.lactisstrainsincubatedin10% factorB-depletedserumincubatedwithhumanneutrophils(Fig.4C). BaselinecontrolswereperformedbyincubatingL.lactisin10%factor B-depletedserumwithoutneutrophils.At15min,L.lactisexpressing Bbpshoweda44%decreaseinbacterialkillingcomparedwithempty vector(p
<
0.01)andL.lactisexpressingSdrEdemonstrateda50% decreaseinkillingcomparedwithemptyvector(p<
0.01).At30min,L.lactisexpressing Bbpshoweda29%decreaseinbacterialkilling comparedwithemptyvector(p=0.04)andL.lactisexpressingSdrE demonstrateda49%decreaseinkillingcomparedwithemptyvector (p=0.01).Thesedatasuggestthat bacterialexpressionofBbpor SdrEsignificantlyincreasebacterialsurvivalofclassicalcomplement pathway-mediatedneutrophilkilling.
4. Discussion
Theseexperiments identifySdrEandBbpasS.aureuscellwall proteinsthatbindthehostclassicalcomplementpathwayregulator C4BP.SdrEandBbpbelongtothestructurallyrelatedSdrfamilyofS. aureusproteinsnamedforarepeatingregionofserineandaspartic acid(SD)dipeptidesproximaltotheirLPXTGcellwalllinkage[25]. SdrEhasbeenimplicatedinplateletactivation[26]andrecently iden-tifiedasaligandforfactorH[32].Bbp,anallelicvariantofSdrE[28], haspreviouslybeennotedtobindfibrinogenandbonesialoprotein, forwhichitwasnamed[35].
ThedistributionofsdrEhaspreviouslybeenreportedtobeashigh as90%,includingnasalcarriage,invasivestrains,andMRSAstrains [31].AnotherstudyevaluatingsdrEandbbpamonginvasiveisolates found56%and38%prevalence,respectively,foracombined94%[28]. ThesestudiesdemonstratethatSdrEandBbparehighlyprevalent amongclinicalisolatesandsuggesttheylikelyplayanimportantrole ininfection.
Immunization studies were previously conducted in mice by Stranger-Jonesetal.using19differentrecombinantlyexpressedS. aureussurfaceproteins[34].ThesestudiesshowedthatrSdrEwas oneofthemosteffectiveimmunogensinreducingS.aureuscolony counts(
>
4logs)inmousekidneys.Oftheeffectivesurfaceprotein immunogens,rSdrEimmunizationyieldedserumwiththehighestFig.4. Functionaltestingofcomplement-mediatedopsonizationand opsonophago-cytickillingofL.lactisexpressingSdrE,orBbp,oremptyvectorcontrol.(A)C3-fragment opsonizationofL.lactisstrainsincubatedin10%factorB-depletedserafor30min as-sayedbyELISAofstrippedsurfaceproteins(n=4).(B)Anti-C3Westernblotofstripped surfaceproteinsfromL.lactisclonesincubatedwith10%factorB-depletedserum.(C) NeutrophilkillingofL.lactisstrainsincubatedin10%factorB-depletedseraand pu-rifiedhumanneutrophilsforthetimesindicatedandthenplatedforcolonycounting (n=4).Dataarethemeansofresultsofindependentexperiments.Errorbarsdenote SEM.
levelofcomplement-mediatedopsonophagocytickillingofS.aureus. ThesestudiessuggestthatinmiceSdrEisanexcellentimmunogen againstS.aureusandthiseffectislikelymediated,atleastinpart,by increasingcomplement-mediatedopsonophagocytosis.Ourfindings showthatbacterialsurfaceexpressionofSdrE
/
Bbpincreases recruit-mentofC4BPanddecreasesclassicalcomplementpathway-mediated opsonizationandbacterialkilling.Thus,ourresultsare congruent withthoseofStranger-Jonesetal.andsuggestthattheeffectiveness ofSdrEasaprotectiveimmunogenmayhavebeenmediated,inpart, viaalteringitsinteractionwithC4BP.fromhumanserumwithoutevidenceofinterference.Thissuggests thatSdrEmayplayavitalroleinthemodulationofclassicaland al-ternativepathwayactivationontheS.aureussurfaceviaC4BPand factorH,respectively.Thus,SdrE
/
Bbpappearstobeattractive tar-getstoinhibitthesemechanismsofimmuneevasionandoptimize complement-mediatedopsonophagocytosisandkillingofS.aureus.OurfindingsshowthatSdrE
/
Bbp-mediatedbindingofC4BP in-hibitsclassicalcomplementpathwayactivationandsubsequent op-sonizationandbacterialkilling.Inhibitionofantibody-initiated com-plementactivationlikelycontributes,inpart,tolackofeffective pro-tectiveimmunityafterS.aureusinfectionorimmunization. Disrup-tionofSdrE/
Bbp-mediatedrecruitmentofC4BPwilllikelyimprove antibody-initiatedcomplementeffectorsandcouldproveaneffective strategytoimproveanti-S.aureusvaccinedevelopment.Acknowledgements
AccesstothemassspectrometersoftheLeroyT.CanolesJr.Cancer ResearchCenterwaskindlyprovidedbyDr.O.JohnSemmes
SupplementaryMaterial
Supplementarymaterialassociatedwiththisarticlecanbefound, intheonlineversion,atdoi:10.1016
/
j.rinim.2013.10.004.References
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