The virulence of Streptococcus pneumoniae partially depends on dprA

h tt p : / / w w w . b j m i c r o b i o l . c o m . b r /

Medical

Microbiology

The

virulence

of

Streptococcus

pneumoniae

partially

depends

on

dprA

Yi

Yu

a,1

_,

_De

_Chang

a,b,1

_,

_Huiwen

_Xu

c,1

_,

_Xuelin

_Zhang

a

_,

_Lei

_Pan

a

_,

_Chou

_Xu

a

_,

Bing

Huang

a

_,

_Hong

_Zhou

a

_,

_Jia

_Li

a

_,

_Jun

_Guo

a

_,

_Changting

_Liu

a,∗

a_{ChinesePLAGeneralHospital,NanlouRespiratoryDiseasesDepartment,Beijing,China}

b_{GeneralHospitalofChinesePeople’sArmedPoliceForces,DepartmentofRespiratoryMedicine,Beijing,China}

c_{NationalInstituteforFoodandDrugControl,InstituteforMedicalDeviceStandardizationAdministration,Beijing,China}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received5March2016 Accepted5October2016 Availableonline6December2016 AssociateEditor:AgnesFigueiredo

Keywords: Streptococcuspneumoniae DprA Transformation Virulence

a

b

s

t

r

a

c

t

Streptococcuspneumoniaeisoneofthemostfrequentopportunisticpathogensworldwide.

DNAprocessingproteinA(DprA)isanimportantfactorinvolvedinbacterialuptakeandDNA integrationintobacterialgenome,butitsroleinS.pneumoniaevirulenceremainsunclear. TheaimofthisstudywastocharacterizetheeffectsofthepneumococcaldprAgeneonthe pathogenesisofS.pneumoniae.ToconstructadprA-deficientpneumococcalstrain,thedprA

geneoftheS.pneumoniaestrainD39wasinactivated.ThevirulenceofthisdprA-deficient

strain,designatedD39,wascomparedwiththatofthewild-typestrainbyevaluatingtheir respectivecapabilitiestoadheretohumanpulmonaryepithelialcells(PEC-A549)andby analyzingtheircholine-bindingproteinexpressionlevels.Inaddition,theexpression pro-filesofgenesassociatedwithvirulenceandhostsurvivalassayswerealsoconductedwith themutantandthewild-typestrain.OurresultsindicatethatthecapabilityofD39to adheretothePEC-A549airwaycellswassignificantlylower(p<0.01)comparedwithD39. Additionally,the100-KDcholine-bindingproteinwasnotdetectedinD39.Theaddition ofcompetence-stimulatingpeptide(CSP)leadtoasignificantlyreductionofpsaAmRNA expressioninthedprA-deficientmutantandanincreasedlevelofpsaAtranscriptsinthe wild-typestrain(p<0.01).Themediansurvivaltimeofmiceintraperitoneallyinfectedwith D39wassignificantlyhigher(p<0.01)thanthatofmiceinfectedwithD39.Theresults ofthisstudysuggestthatDprAhasasignificanteffectonvirulencecharacteristicsofS.

pneumoniaebyinfluencingtheexpressionofcholine-bindingproteinandPsaA.

©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/ licenses/by-nc-nd/4.0/).

∗ _{Correspondingauthorat:DepartmentofNanlouRespiratoryDiseases,ChinesePLAGeneralHospital,28FuxingRoad,Beijing100853,}

China.

E-mail:liuchangt@foxmail.com(C.Liu).

1 _{Theseauthorscontributedequallytothiswork.}

http://dx.doi.org/10.1016/j.bjm.2016.10.019

1517-8382/©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Streptococcus pneumoniae is a Gram-positive, facultative

anaerobic, alpha-hemolytic streptococci. This pathogenic bacteria iswidely foundthroughout natureand can cause differenttypesinfections,suchasbronchitis,rhinitis,acute sinusitis,otitismedia,conjunctivitis,meningitis,bacteremia, sepsis,osteomyelitis,septicarthritis,endocarditis, peritoni-tis, pericarditis, cellulitis, and brain abscess.1 _{Due to the}

rapidincreaseofdrug-resistantbacteria,thecurativeeffect of antibiotics is declining.2 _{To find new ways of treating}

pneumococcal infections, new virulence genes need to be identifiedandthemolecular mechanismsofpneumococcal virulenceanddrugresistanceshouldequallybethoroughly investigated.

OneoftheremarkablefeaturesofS.pneumoniaeistheir natural capability to undergo genetic transformation. This mechanismofDNAuptakemayresultinimportantchangesin thebacterialgeneticbackground,whichmayleadtothe emer-genceofbacterialsubpopulationscarryingdifferentgenetic traitsinrelationtotheparentalgeneration.3_WhenS.

pneumo-niaeenterinthecompetentstate,theautolysin—considered animportantS.pneumoniaevirulencefactor—isactivated.As consequence,S.pneumoniaereleasecell-wallfragmentsand hemolysin.4_{Anotherstudydemonstratedthatthevirulence}

ofS.pneumoniaemaysignificantlydecreaseaftermutationsin

someoftheirproteingenes.5_{Alltogether,thesedatasuggest}

thattransformationcandirectlyinfluencethevirulenceofS. pneumoniae.

Natural transformation in S. pneumoniae is a complex process that requires at least 23 genes. To integrate new DNAinto thegenome,abacterium mustenterinaspecial competent state for binding, taking up, and recombining exogenousDNA.InS.pneumoniae,competenceisinducedby DNA-damagingagentssuchasmitomycinC,topoisomerase inhibitors, and the fluoroquinolone antibiotics norfloxacin, levofloxacin, and moxifloxacin. Transformation protects S.

pneumoniaeagainstthebactericidaleffectofDNA-damaging

agents.6_{Additionally,theinductionofcompetenceinS.}

pneu-moniaewasassociatedwithincreasedresistancetooxidative

stressand upregulationofRecAprotein,whichplaysakey roleinremovingDNAdamage.7

InS.pneumonia,the ubiquitousrecombinases, RecA and

DprA are required for DNA integration into the bacterial genome.8 _{RecA plays important roles in the homologous}

recombinationpathway,catalyzingDNAstrandinvasionand homology search.4 _{DprA was reported to be involved in}

binding to the internalized single-stranded DNA (ssDNA) and promoting the loading ofRecA on ssDNA during nat-ural transformation.9 _{The inactivation of dprA results in}

>104-fold reduction in transformation.10 DNA internalized

indprA-deficientcompetent cellsappearstobecompletely

destroyed,10_{suggestingthatDprAplaysaprominentrolein}

protectingincomingssDNAfromnuclease(s).DprAcanalso represstheearlyexpressionofcompetencegenes—by inter-actingwithComEduringcompetenceregulation—butdoesnot affectproteolysisoflabileearlygeneproducts.11

Despite the knowledge about the involvement of DprA in the bacterial DNA uptake and integration, the specific

mechanism by which DprA influences the virulence of S.

pneumoniaeremainsunclear.Choline-bindingproteins(CBPs),

pneumolysin (Ply), capsular polysaccharide biosynthesis locus(Cps2A),andpneumococcalsurfaceproteinA(PsaA)are factorsassociatedwiththevirulenceofS.pneumonia.12,13_To

furtherexploretherole ofDprAinS.pneumoniaevirulence,

adprA-deficientmutantwasconstructed,andtheinfluence

of DprA on S. pneumoniae virulence was investigated by

assessingadherencerates,theexpressionlevelsofvirulence associatedgenes,andmicesurvivalafterinfectionswiththe isogenicdprA-deficientmutantandthewild-typestrainofS. pneumoniae.

Materials

and

methods

Bacterialstrainsandgrowthconditions

Thewild-typeS.pneumoniaestrainusedinthisstudywasD39, whichisavirulentencapsulatedtype2S.pneumoniaestrain obtainedfromtheNationalInstitutefortheControlof Phar-maceuticalandBiologicalProductsofChina.TheS.pneumoniae cellsweregrownat37◦Cin5%CO2inTHYmedium

contain-ing (perliter)36.4gofTodd-Hewittbrothand5.0gofyeast extract. Kanamycinwasadded tothe growthmediumata concentrationof350␮g/mLfortheselectionofpneumococcal transformants.

Escherichia coliDH5˛cells (storedinourlab) weregrown

onLuria-Bertani(LB)agarorbroth.pGEM-Teasyvectorbased plasmid were introduced into E. coli by transformation as previously described by Hanahan.14 _{For the selection of}

E.colitransformants,ampicillin(100␮g/mL)wasaddedtothe growthmedium.

Forthedeterminationofgrowthcurves,S.pneumoniaecells weregrowninTHYmediumtoanOD620of0.4,andtheculture

fluidwassubsequentlydilutedtoanOD620of0.01.TheOD620

valuewasdeterminedeach30min,andagrowthcurvewas plotted.

Constructionofapartialdeleted-dprAmutant

TocreateamutationinthedprAgeneinS.pneumoniae,a 1351-bpJuatcassettewasamplifiedwithJuat1(5 -ATTGGCGCGCCA-CCGTTTGATTTTTAATGGATAATG-3)andJuat2(5

-ATTGGCC-GGCCCCTTTCCTTATGCTTTTGGAC-3) from

kanamycin-resistantE.colichromosomalDNAandusedtodisruptdprA.A 1111-bpfragment(dprAup)containingpartofdprAwas ampli-fied with dap1 (5 -CGCGAGCTCGAAAAATCCTCTACTCTAC-TAATCCAC-3)anddap3(5 -ATTGGCCGGCCAGATTCCAACAT-CTGACAAAATAG-3) from S. pneumoniae DNA. An 872-bp fragment (dprA down) containing part of both the down-stream dprA sequence and licC was amplified with dap2 (5-ATTGGCGCGCCGCTTTCACACGAATCTCCTTTATT-3) and dap4 (5-TCCCCGCGGTTTTCTGGGGCTTGGGTCTA-3) from

S. pneumoniaeDNA.The resultingthree PCRproducts were

digestedusingrestrictionenzymes(FseI,AscI)knowntocutat thecorrespondingsiteswithintheprimers,andthedigested productswereligatedtogetherintoapGEM-Tvector(Promega, USA).ThepGEM-Ttargetvectorwassubsequentlyintroduced

intoS.pneumoniaeD39bytransformation,andrecipient

intotheirchromosomesbyhomologousrecombinationwere selectedbytheirresistancetokanamycin.Primersdap1and dap4were usedtoscreenfortheappropriatedeletion,and theresultingPCRproductswereconfirmedbysequencing.S.

pneumoniaecontainingthecorrectdeletionwithindprAwas

namedD39andwasusedforfurtherstudies.

Adherenceofthewild-typeanddprA-deficientS. pneumoniaestrainstoPEC-A549airwaycells

ThecapabilityofthedprA-deficientmutanttoadheretothe humanpulmonary epithelial cell line, PEC-A549(Shanghai Institute of Cell Biology, China) was assessed in 12-well tissue culture plates. ThePEC-A549 cells were cultured in DMEM/Ham’sF-12medium(pH7.5)with5%fetalcalfserum for2–3days.ThePEC-A549cellmonolayerswerethenrinsed twicewithphosphate-bufferedsaline(PBS), andS. pneumo-niaecellssuspendedinDMEM-Ham’sF-12medium(2.0×107

CFU/mL;1mL/well)wereaddedtothecellson12-wellplates. After1h-incubationat37◦C,thePEC-A549cellswerewashed fivetimeswithPBS (pH7.5)toremoveanyremaining non-adherentbacteria.Themixture(epithelialandbacterialcells) wastreatedwith0.25%trypsinwith0.02%EDTA(200␮l/well) at37◦Cfor5min.After,400␮lof0.025%coldTritonX-100was addedandthe mixturewasrepeatedlypipettedtolysethe epithelialcells.ThemixturewasdilutedwithPBS,and100␮l oftrypticasesoyagarplatescontaining5%defibrinatedsheep serumwereinoculatedintoeachwell.Thebacterialcellswere culturedfor16handthenumberofbacterialcoloniescounted. Adherencewasdeterminedbydividingthenumberof adher-entbacteriaper100epithelialcells.Thevaluesreflectaverages offourwellsfromindependentexperiments.

Real-timeRT-PCR

ToinvestigateifdprAplaysaroleintheexpressionofsome importantvirulencegenes duringbacterial transformation, both the wild-type (D39) strain and dprA-deficient (D39) pneumococcalmutant(grownatapHof8.0)wereinducedfor competencebyaddingcompetence-stimulatingpeptide(CSP; 20ng/mL)attheoptimalbacterialcelldensity(OD550of0.1)

toinducecompetence.Forthereal-timequantitativereverse transcription-PCR(realtimeRT-qPCR)analysis, thebacteria werecollectedat0and20minintervalsaftertheadditionof CSPtotheculture.

TotalRNAwasobtainedusingthehotacidphenolmethod aspreviouslydescribed.15_{LevelsofmRNAforpneumolysin(ply),}

psaA,cbpA,andcps2Ageneswerequantifiedbyone-step

real-time RT-PCRwith the Takara SYBR RT-PCRsystem (Takara Bio). The specific primers used for the real time qRT-PCR assayshavebeenpreviouslydescribedelsewhere16_andwere

usedatafinalconcentrationof100nMperreaction.Specific primersforthe16SrRNAwereincludedasaninternalcontrol. Thereal-timeqPCRwasperformedinanABI7500Real-Time cycler(AppliedBiosystems,USA).TheRT-PCRcycling condi-tionsweresetasinstructedbythemanualfortheTakaraSYBR RT-PCRsystem.Amplificationdatawereacquiredatthe exten-sionstepandanalyzedusingtheABISoftwareversion2.0with comparativecriticalthresholdvalues.TheCtcalculation fortherelativequantificationoftarget wasusedasfollows

Ct=(Ct,targetgene–Ct,16SrRNA)␹–(Ct,targetgene–Ct, 16SrRNA)y,where␹=S.pneumoniaestrainD39withand

with-outCSPandy=S.pneumoniaestrainD39withandwithoutCSP.

Aftervalidationofthemethod,resultsforeachsamplewere expressedinN-foldchangesin␹targetgenecopies, normal-izedto16SrRNArelativetothecopynumberofthetargetgene, according to the following equation: amount of target= 2-Ct.17_{Allexperimentswerecarriedoutinquadruplicate.}

Analysisofcholine-bindingproteins(CBPs)expression

For CBPs analysis experiments, wild-type (D39) and dprA-deficient(D39)S.pneumoniaeisogenic cellswere grownin HYT medium to an OD620 of 0.6, centrifuged at 5000rpm

for20min,andwashedtwicewithPBS.Thecellswerethen harvested, resuspended in a PBS solution containing 2% cholinechlorideat1010_{cells/mLandequilibratedfor20min}

atroomtemperature.Thecellsweresubsequentlycentrifuged at5000rpmfor20min,and resuspendedin 100mLofTBS. The cell suspension was subjected to ultrasonication with 155-secondpulseswith10-secondintervalsbetweenpulses at 400W (Misonix XL2020, Farmingdale, NY) on ice. SDS-PAGE(12%polyacrylamidegel)wascarriedoutaspreviously describedbyLaemmli,18_{andtheproteinsinthegelwere}

visu-alizedbyperformingCoomassiebrilliantbluestaining.

Virulencestudies

Intraperitoneal challengewithahighlyvirulent strainofS.

pneumoniae(D39)oritsisogenicdprA-deficientmutant(D39)

was performed to evaluate the role ofdprA on S. pneumo-niaevirulence.Male6-to8-week-oldBALB/cmiceweighing 18–22gwereusedinthisexperiment.Inoculationofthemice

withpneumococciwasconductedaccordingtothepreviously

describedprocedure.13_{Thewild-typestrainanddprAmutant}

were tested intwo separateexperiments, involving each a totalof21mice.ToassessS.pneumoniaevirulenceinblood, theBALB/cmicewereintraperitoneallyinjectedwith0.1mL of5×106_{CFU/mLofasuspensionofpneumococcalcells.The}

numbersofsurvivinganimalswerethenrecordeddailyfor14 days.Allexperimentalprocedureswereconformedwiththe guidelinesforthecareanduseoflaboratoryanimalsofPLA GeneralHospital,Beijing,China(NoPLAGH2014078).

Statistics

StatisticalanalyseswereperformedusingunpairedStudent’s

t-tests.Dataarepresentedasmeans±thestandarddeviation

ofthemeanforfourindependentexperiments.Differencesin themeansurvivaltimebetweengroupswereanalyzedusing two-tailedMann–WhitneyUtest.

Results

ConstructionandgrowthrateofthedprAmutantstrain (D39)

For the construction of the dprA mutant, a DNA fragment containing a Juat cassette insertion was amplified by PCR

3287bp

FseI

1351bp 312bp

AscI

DprA Juat DprA

Fig.1–MutatedregionofdprA.AschematicofthedprA

mutationthatwascreatedbytheinsertionofa1351-bpJuat

cassette.

andincorporatedintothechromosomebytransformationas described in Materialsand Methods (Fig. 1). The construc-tionofthemutantwasconfirmedbyPCRdemonstratingthe absenceofthedprAsegment.ThegrowthrateofdprAmutant at37◦CwassimilartothatoftheparentalstrainD39,witha doublingtimeof55min,comparedwithabout54minforD39 (Fig.2).Thus,dprAdoesnotseemtobeessentialforthegrowth

ofS.pneumoniaeat37◦C.

EffectofadprAmutationontheS.pneumoniaeadherence rate

TheeffectofthedprAgeneontheadherencerateofS. pneumo-niaestrainD39wasdeterminedbycomparingthecapabilityof thewild-typestrainD39andtheisogenicdprAmutant(D39) toadheretopulmonaryepithelialcelllinePEC-A549invitro.In comparisonwithD39,theadherencerateofD39toPEC-A549 cellswassignificantlylower(p<0.01).Specifically,the adher-encerateofD39wasonly30%oftheadherencerateachieved bytheparentstrain,D39(Fig.3).ThisresultsuggeststhatdprA playsanimportantroleintheadherenceofS.pneumoniaeto hostcells.

EffectofdprAmutationontheexpressionof choline-bindingproteins(CBPs)

CBPsaremajorS.pneumoniaevirulencefactorsthatexistonthe bacterialsurface.ChangesinCBPsexpressioncaninfluence theadherencerateandvirulenceofS.pneumoniae.Unlikethe observedforthewild-typestrain,12,13 _{wedidnotdetectthe}

0.7 0.6 0.5 0.4 0.3 V alue of OD260 0.2 0.1 0 0 1 2 3 Hours 4 5 6 7 D39 ΔD39

Fig.2–GrowthcurvesofS.pneumoniaestrainsD39and D39.GrowthcurvesofS.pneumoniaewild-type(D39) strainanddprA-insertional(D39)mutant.Dataare representativeoffourindependentexperiments.

120 P<.01

∗∗

100 80 60 P ergent adherence 40 20 0 D39 _ΔD39

Fig.3–AdherenceofS.pneumoniaestrainsD39andD39 toPEC-A549cells.Adherencewasexpressedasthenumber ofpneumococciattachedper100PEC-A549cells.

100-KDCBPinthedprAmutant(Fig.4).Thisresultsuggests that theexpressionofCBPsseems tobedependent onthe expressionofthedprAgene.

Modulationoftheexpressionofvirulence-associated factorsbyCSP-inducedcompetence

Competence, induced byCSP, can affect the expressionof virulencefactors.19_{Thus,themodulation ofvirulencegene}

expressionwasexaminedatthemRNAlevelinthewild-type

S.pneumoniaestrainandinthedprA-mutant.ThemRNAlevels

ofply,cbpA,psaA,andthecapsulesynthesisgenecps2Awere

determinedbyreal-timeRT-PCR(Fig.5).

Thebaseline expressionlevels ofcbpA,cps2A,and psaA were lower inD39than inD39 (p<0.01), but thebaseline

M 130 KD 96 KD 72 KD 56 KD 36 KD 28 KD 17 KD 1 2

Fig.4–Detectionofcholine-bindingproteins(CBPs)inS.

pneumoniaestrainsD39andD39.Arepresentative

SDS-PAGEgelofthecorrespondingcelllysisproductsis shown.Lefttoright:slotM,proteinmolecularweight marker;slot1,D39;slot2,D39.Arrowindicatesthe choline-bindingproteins(CBPs)band.

8

A

B

C

D

1.2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 1 0.8 0.6 0.4 0.2 0 7 ∗∗ ∗∗ ∗∗ ∗∗ ∗∗ ∗∗ 6 5 4 3 2 CbpA/ 16S rRNA Ply/ 16S rRNA Cps2A/ 16S rRNA PsaA/ 16S rRNA 1 0 7 6 5 4 3 2 1 0 CSP– NS NS CSP+ CSP– CSP+ CSP– CSP+ CSP– CSP+ D39 ΔD39

Fig.5–RelativequantityofcbpA,cps2A,ply,andpsaAmRNAinS.pneumoniaestrainsD39andD39withandwithoutCSP. Real-timeqRT-PCRwasperformedtoassesstherelativemRNAconcentrationsofcbpA(A),cps2A(B),ply(C),andpsaA(D)in

S.pneumoniaestrainsD39andD39intheabsenceorpresenceofCSP.ThelevelsofindividualmRNAspecieswere

normalizedusingasreferencetheinternalcontrol(16SrRNA).**p<0.01,NS=nosignificance.

expressionlevelsofplywerenotstatisticallydifferentbetween thewild-type and the isogenicdprA mutant.Following the addition of CSP, the relative cbpA mRNA level increased 7.15-fold (p<0.01) in D39, but the level of cbpA expres-sion only increased 2.1-fold (p<0.01) in the dprA mutant (Fig.5A).Additionally,themRNAlevelsofplywereincreased 5.83-and5.4-foldinD39andthedprAmutant,respectively, after CSP was added (both p<0.01) (Fig. 5C). Furthermore, expression of cps2A in the dprA mutant was significantly decreased following CSP addition (p<0.01) (Fig. 5B). The expression of psaA was significantly increased (p<0.01) in D39 after CSP was added. In contrast, CSP treatment decreased the mRNA level of psaAby 1.8-fold in the dprA mutant(p<0.01)(Fig.5D).Together,ourresultssuggestthat the dprAmutationmay negatively affect the expressionof psaA.

EffectofadprAmutationonS.pneumoniaevirulence

TofurtherinvestigatetheeffectofthedprAmutationonthe virulenceof S. pneumoniae, the survival time ofmice after intraperitonealinfectionwithapproximately105_CFUofD39

orD39pneumococciwasmeasured.Themediansurvivaltime

(36h)formiceinfectedwiththeparentalstrain(D39)was sig-nificantly lower than that(44h)formice infectedwiththe dprAmutant(Fig.6),aswastheoverallsurvivalrate(p<0.01). ThisresultindicatesthatdprAfunctioniscriticalforvirulence factorexpressioninS.pneumoniae.

Discussion

CPBsare proteinsthat werepreviouslyassociatedwithcell lysis20_{andcontainanumberofshortcholine-bindingrepeats}

that anchor them non-covalently to teichoic and lipotei-choicacids inthepneumococcal cellwall.21 _Wefoundthat

S. pneumoniae cells with mutated dprA lack 100-KD CBPs,

suggesting that dprA plays a key role in CBPs expression and thus might influence the CBPs competence-induced cell lysis. However,furtherstudies are neededto precisely determine the protein(s) affected and the correspondent mechanisms.

Our results also demonstrated that the adherence to PEC-A549cellsofpneumococcalstrainD39wasreducedby mutationindprA.BecausePspA,PspC,CbpAareassociated withbacterialadherencetohostcells,22–24_{itseemslogicalto}

examinetherelationshipbetweendprAandtheseproteins. Here,weassessedtheexpressionsofcbpA,cps2A,andpsaAin

theS.pneumoniaeD39strainandintheisogenicdprAmutant

byreal-timeqRT-PCR.OurdatademonstratethatthedprA haslowerlevelsofcbpA,cps2A,andpsaAexpressioncompared withtheparentalwild-typestrain.

AfterCSPwasaddedtoinducecompetence,the expres-sion level of cps2A, which is the first gene in the capsule biosynthesislocus,wasreducedinboththewild-typestrain and the dprA mutant, what might result in a potentially lower resistance to the host immune system.25 _However,

16.0 P<.01 ∗∗ 14.0 12.0 10.0 8.0 Sur viv al time (da ys) 6.0 4.0 2.0 0 D39 ΔD39

Fig.6–IntraperitonealchallengewithS.pneumoniae

strainsD39andD39.GroupsofBALB/cmicewere

intraperitoneallyinjectedwith5×100CFUofS.pneumoniae

strainsD39andD39.Thesurvivaltimeofeachofthemice isindicated(pvalue<0.01).Transverselinesdenotemedian survivaltimesforeachofthegroups.

thanthatofwild-typestrain,whichmayprobablyhassome consequenceforthebacterialvirulence.In thepresenceof CSP,theexpressionlevelofcbpAincreasedinbothwild-type anddprAmutant,buttheexpressionlevelofcbpAinthedprA mutantwasmuchlowercomparedwiththewildtype.This resultseemstoindicatethatdprAupregulatescbpAmainly duringthecompetencestate.

Notably,theexpressionofpsaAwasincreasedintheD39 followingtheadditionofCSP,butitwasdecreasedinthedprA mutant,suggestingthatdprAplaysanimportantroleinthe regulationofpsaAexpression.Therefore,virulencegene reg-ulationmightbemodulatedbyDprA.Previousstudieshave shownthatthecombinationoflowvirulencewithhigh resis-tancetohostimmunecellsmayleadtotheestablishmentof chronicbacteremia,inwhichthebacteriaareabletoevadethe hostimmunesystemandsurviveinthehostbutareunable to cause fulminant disease, a phenomenon that has been previously demonstrated in a pneumolysin-negative mutant

ofS.pneumoniaestrain R6.4,5 _{Althoughwedidnottestthat}

hypothesis,itispossiblethatthedprAmutantmightexhibita wild-typelevelofresistancetohostmacrophagesuponstress challenge,eventhoughtheoverallvirulencewasdecreased.

Infact, themouse infectionmodel indicatesthat muta-tion in dprA can significantly attenuate the virulence of a pneumococcal strain. This seems to suggest that factors relatedwithcompetenceandtransformationmechanismsare possiblylinkedwithpathogenicprocessofS.pneumoniae. How-ever,thisconnectionalsoappeartorely,atleastinpart,on

the geneticbackgroundofthe bacterium.For example,the virulence of another pneumococcal strain was reduced by approximately8000-foldinasystemicinfectionmodelwith acomD genemutantcomparedwiththe isogenicwild-type strain.5_{Whetherornotthesefactorsarecategoricallyinvolved}

incompetenceandtransformationisamatterthatrequires furtherinvestigation.

Insummary,DprAisanessentialrecombination-mediated proteinthat isinvolvedinthe naturaltransformationofS.

pneumoniae. This protein mainly protects the intracellular

plasmidorDNAthatweretakenupbythebacteriafrom degra-dationbyDNase.Here,wereportthatadprAmutationleadsto adecreasedvirulenceofS.pneumoniaethatwasparalleledby theabsenceofCBPs.Becausenaturaltransformationisclosely correlated withthe emergenceofdrugresistance,thefinds thatdprAparticipatesinbothtransformationandvirulence suggeststhatDprAmaybeaneffectivetargetforinhibiting drugresistanceandreducingS.pneumoniaevirulence.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

ThisworkwassupportedbytheNationalBasicResearch Pro-gram of China (973 Program, No. 2014CB744400), National MajorScientificandTechnologicalSpecialProjectfor “Signif-icant New Drugs Development”(grantno. 2015ZX09J15102-003),NationalNaturalScienceFoundationofChina(grantno. 81402471)andBasicResearchProgramofGeneralHospitalof ChinesePeople’sArmedPoliceForces(grantno.WZ2014026).

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