The accessory gene regulator (agr) controls Staphylococcus aureus virulence in a murine intracranial abscesses model

The

Brazilian

Journal

of

INFECTIOUS

DISEASES

w w w . e l s e v i e r . c o m / l o c a t e / b j i d

Original

article

The

accessory

gene

regulator

(agr)

controls

Staphylococcus

aureus

virulence

in

a

murine

intracranial

abscesses

model

Jian

Gong

a,d

_,

_Dongzhi

_Li

b,d

_,

_Jun

_Yan

c,d

_,

_Yu

_Liu

c

_,

_Di

_Li

c

_,

_Jie

_Dong

c

_,

_Yaping

_Gao

c

_,

Tao

Sun

b,∗∗

_,

_Guang

_Yang

c,∗,∗∗

a_{DepartmentofNeurosurgery,BeijingTiantanHospital,CapitalMedicalUniversity,BeijingNeurosurgicalInstitute,Beijing,China} b_{NingxiaMedicalUniversity,IncubationBaseofNationalKeyLaboratoryforCerebrocranialDiseases,Yinchuan,China}

c_{BeijingInstituteofBasicMedicalSciences,Beijing,China}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received3January2014

Accepted18March2014

Availableonline13May2014

Keywords: S.aureus Agr

Intracranialabscessesmodel

a

b

s

t

r

a

c

t

Background:Intracranialabscessesareassociatedwithhighmortality.Staphylococcusaureus

isoneofthemainpathogensthatcauseintracranialinfection.Untilnow,thereisnoreport

toidentifythekeyeffectorsofS.aureusduringtheintracranialinfection.

Methods:ThemurineintracranialabscessesmodelinducedbyS.aureuswasconstructed.

Thevitalsignandsurvivalrateofmicewereobservedtoevaluatetheinfection.

Histolog-icalexaminationwasusedtodiagnosethepathologicalalterationsofmousetissues.The

sensitivityofS.aureustowholebloodwasevaluatedbywhole-bloodkillingassay.

Results:Inmurineintracranialabscessesmodel,itwasshownthatthemortalitycaused

bytheaccessorygeneregulator(agr)locusdeficientstrainwassignificantdecreased

com-paredwithitsparentstrain.Moreover,wefoundthatRNAIII,theeffectorofagrsystem,was

essentialfortheintracranialinfectioncausedbyS.aureus.Inthefurtherinvestigation,it

wasshownthatrestorationtheexpressionof␣-toxininagrdeficientstraincouldpartially

recoverthemortalityinthemurineintracranialabscessesmodel.

Conclusion: OurdatasuggestedthattheagrsystemofS.aureusisanimportantvirulence

determinantintheinductionandmortalityofintracranialabscessesinmice.

©2014ElsevierEditoraLtda.Allrightsreserved.

Introduction

Intracranialabscessesfrequentlyoccurasacomplicationof

sinusitis or neurosurgery, which are associated with high

∗ _{Correspondingauthorat:InstituteofBasicMedicalSciences,27TaipingRoad,Beijing100850,China.} ∗∗ _{Co-correspondingauthor.}

E-mailaddresses:[email protected](T.Sun),[email protected](G.Yang).

d _{Theseauthorscontributeequallytothiswork.}

mortality(19–43%).1_{Untilnow,theincidence,etiology,}

man-agementandmorbidityofintracranialabscessesarenotwell

understood.Staphylococcusaureusisoneofthemainpathogens

thatcauseintracranialabscesses.1–8

http://dx.doi.org/10.1016/j.bjid.2014.03.005

Table1–Bacterialstrainsandplasmids.

Strainorplasmid Comments Sourceorreference

Strain S.aureus

8325-4 Wild-type,rsbU− ₃₀

RN6390 Laboratoryvirulentstrainderivedfrom8325 31

RN6911 RN6390withanagrmutation,tetR ₃₁

RN6911-hla RN6911restoringHLAactivity,cmrR _Thisstudy

RNAIII Restriction-negativestrain,8325derivative,kanR ₃₂

RNAIII-R RNAIIIrestoringRNAIIIactivitycmrR ₃₂

E.coli

DH5␣ Ahoststrainforcloning Transgene

Theaccessorygeneregulator(agr)wasidentifiedasa

quo-rumsensingsysteminS.aureus.RNAIIIisthemajoreffector

ofagrsystem.ItactsasasmallRNAregulatingtheexpression

ofmanyvirulencefactors,includingmostofthoseencoding

cell-wall-associatedandextra-cellularproteins.9,10_Exotoxins

expressedbyS.aureusplayimportantrolesintheinfection.

␣-Toxinisoneofexotoxins,whichisregulatedbyRNAIII.As

aporeformingtoxin,␣-toxincancausehemolysisandtissue

damage.Recently,ithasbeenfoundthat␣-toxincanalso

inter-actwithitsreceptorADAM10toaggravateS.aureusinfection.11

Herein we describe an animal model of intracranial

abscessescausedbyS.aureusthatwehaveconstructed.The

pathogenicroleofagrsystemisinvestigatedinthismodel.

Materials

and

methods

Mice

AdultfemaleC57BL/6J mice(6–8weeksold),obtainedfrom

VitalRiverLaboratories,wereusedforallexperiments.Mice

werehousedingroupcages,maintainedona12:12hour

light-dark cycle, in a controlled environment (25◦C) and given

unrestricted access to food and water. All animal

experi-mental protocols ofthe study are in accordance with the

nationalguidelinesfortheuseofanimalsinscientificresearch

“Regulations for the Administration of Affairs Concerning

ExperimentalAnimals”.ItisalsoapprovedbyAnimalCareand

UseCommitteeofBeijingInstituteofBasicMedicalSciences

withtheapprovalnumberBMS-130112.

Staphylococcusaureusstrainsandcultureconditions

S. aureuscells were grown in5mLof brain heart infusion

(BHI),andEscherichiacolicellsweregrowninLuria-Bertani(LB)

medium.Bacteriawereculturedat37◦Cfor12hwithshaking

at200rpmina25-mLtesttube.Cellsweretransferredfrom

1mLofpre-cultureto100mLofBHIorLBmediumina500-mL

flask.S.aureuscellswereroutinelygrowninBHIandE.colicells

weregrowninLBmediumeitherwithnoantibiotics,orwith

40␮g/mLChloramphenicol,50␮g/mLtetracycline,100␮g/mL

ampicillin,and50␮g/mLkanamycin.

ThestrainsusedinthisstudyarelistedinTable1.

Mousebraininfectionmodel

Mice were anesthetized with 1% pentobarbital sodium

(3–5mg/100gbw i.p.). While under anesthesia, mice were

preparedforinjection bysecuringthe headina

stereotac-tic frame and quickly locating appropriate coordinates in

the frontalcortexregionontheskull.Thegenerallocation

for injection is1mm anterior tobregma and 1mm lateral

to the sagittal suture on the right side.12 _{Parting the fur}

withaneedlepointalongbothaxesisappropriatefor

injec-tion.Tomakeaninjection,orienttheneedle perpendicular

to the skull and press through the calvarium to

approxi-mately 2mm depth. Inject 5␮L bead suspension (106_CFU)

into the brain by pressing the syringe button down and

waiting2sforthebacteriatoenterthebrain.Controlmice

receivedinjectionsofsterilesaline.Carefullyremovethe

nee-dle and place the mouse ina clean, warm cage. All mice

survivedtheinjectionprocedure.Afterrecoveryfrom

anesthe-sia,micewerekeptatroomtemperature(25◦C)andprovided

with standard chow and watered ad libitum.48-hour

mor-tality, average time of death and rectal temperature were

monitored.

Histologicalexaminationofmousetissues

Mice were killed 12h after injection and brains were

removedimmediately.Mousebrainwasfixedin10%

neutral-buffered formalin for48h. Thefixed tissuewas processed

through graded series of ethanol, followed byxylene, and

embedded in paraffin. Tissue blocks through the entire

abscess were sectioned at 5␮m and slides stained with

hematoxylin–eosin.

Whole-bloodkillingassays

Bacteria cultures were washed twice inPBS, dilutedto an

inoculum of105 _{CFUin25␮LPBS,andmixedwith75␮Lof}

freshlydrawnmouseblood inheparinizedtubes.Thetubes

wereincubatedat37◦Cfor4hwithagitation,atwhichtime

dilutionswereplatedonBHIagarforenumerationofsurviving

Rectal temper ature (ºC) Rectal temper ature (ºC) Rectal temper ature (ºC) Rectal temper ature (ºC) Rectal temper ature (ºC) Rectal temper ature (ºC) P ercent sur viv al

Hours after injection

42

a

c

b

d

e

10h 5h 0h P=0.9648 P=1.0000 12.5h P=0.8633 0h 4h 8h 15h P=0.7569 P=0.0770 7.5h P=0.8597 40 38 36 34 32 42 40 38 36 34 32 42 40 38 36 34 32 42 40 38 36 34 32 42 40 38 36 34 32 42 40 38 36 34 32 Saline 83 25-4 Saline 83 25-4 Salin e 83 25-4 Saline 83 25-4 Salin e 83 25-4 Saline 83 25-4 Salin e 83 25-4 Salin e 83 25-4 Saline 83 25-4 Saline 83 25-4 Saline 83 25-4 Saline 83 25-4 100 80 40 60 20 0 100 80 40 60 neutrophilic g ran ulocyte (%) neutrophilic g ran ulocyte (%) neutrophilic g ran ulocyte (%) 20 0 100 80 40 60 20 0 100 80 40 60 20 0 0 12 24 36 48 8325-4 Saline 8325-4 Saline P=0.08254 P<0.0001 8h P=0.7856 P=0.9259 4h P=0.2282 P=0.8778 25 20 15 10 5 0 25 20 15 10 5 0 25 20 15 10 WBC(10 9/L) WBC(10 9/L) WBC(10 9/L) 5 0 0h 400um 400um 25um 25um

Fig.1–ConstructionofmurineintracranialabscessesmodelcausedbyS.aureus.(A)Measurementofrectaltemperatureof

miceatdifferenttimepointsafterchallengewithS.aureus8325-4(0h,5h,7.5h,10h,12.5h,15h)(n=9ineachgroup).(B)

Measurementofsurvivalcurveofmiceinthetwogroups(n=52miceineachgroup).MicewerechallengedwithS.aureus

8325-4orsaline.Thenumberofdeadmicewasrecorded.Thesurvivalratesofthetwogroupswerecalculatedindividually,

p<0.0001.(C)Histologicalexaminationofmousebraintissue.Braintissuesfromthetwogroupswerecollected8h

post-challengewithS.aureus8325-4andfixed.ThefixedtissueswerestainedbyH&E.(D)Whitebloodcellcountofthetwo

groupsatdifferenttimepoints(0h,4hand8h)post-challengewithS.aureus(n=8ineachgroup).(E)Percentageof

neutrophilsinthetwogroupsatdifferenttimepoints(0h,4hand8h)post-challengewithS.aureus(n=8ineachgroup).

RestoringHLAactivityinRN6911

ThewholeHLAcodingregionwasamplifiedbyPCRandcloned

intopOS1vector.Theresultingplasmid(pOS1-hla)was

trans-formed into E. coli DH5␣. Positive clones were selected on

LBplatescontaining100␮g/mLampicillin.Therecombinant

plasmidwasisolatedfrompositiveclonesandtransformedto

S.aureusRN4220cells.Transformantswereselectedon

tryp-ticsoyagar plates containing40␮g/mLchloramphenicol at

37◦C.ThepositiveclonewasselectedbycolonyPCR.Thenthe

plasmidwasisolatedandtransformedtoRN6911.The

trans-formantswereselectedontrypticsoyagarplatescontaining

40␮g/mLchloramphenicolat37◦Cfor20h.Plasmidsof

posi-tiverestoringcolonieswereextractedandconfirmedbyPCR.

Statisticalanalysis

Dataare expressedasmean±SEM. Thesignificanceof

dif-ferences between experimentalgroups was determined by

two-tailedt-testassuminga95%confidenceinterval.

Results

Constructionofmurineintracranialabscessesmodel causedbyS.aureus

InjectionofS.aureus8325-4intomurinefrontalcortexcould

produceafocalbrainabscesswithaclinicalcoursesimilar

tothat seeninclinical patientswithintracranial infection.

Within48hpostbacterialchallenge,micedemonstrated

clin-icalsigns ofillness including hunchedposture,ruffled fur,

lethargy, spasm, and finallyexpired. In contrast,any overt

changes in posture, grooming, or physical activity ofmice

injectedwithsterilesalinewerenotobserved.However,

chal-lengewithS.aureuscouldnotinducesignificantchangesof

rectaltemperature(Fig.1A).Morethan50%ofmiceweredead

after48h(Fig.1B).Brainswerecollected8hafterinjectionof

Bacteria.Awell-circumscribedabscesscontainingneutrophil

and macrophageinfiltrate,withsignificantmasseffectwas

observedinhistologicalexaminationofbrainsinjectedwith

400um 400um 25um 25um

b

RN6390B RN6911 P ercent sur viv al

Hours after injection

a

100 80 40 60 20 0 0 12 24 36 48 RN6390B RN6911 Saline P ercent sur viv al

Hours after injection

d

e

100 80 40 60 20 0 0 Sur viv al percentage 100 80 40 60 20 0 12 24 36 48 8325-4 ΔRNAIII ΔRNAIII-R RN6390B RN6911 Saline Neutrophilic g ran ulocyte (%)

c

0h 4h 8h P=0.7653 P=0.1541 P=0.1981 P=0.9837 P=0.8668 0 20 40 60 80 100 Salin e RN6390 B RN691 1 Neutrophilic g ran ulocyte (%) 0 20 40 60 80 100 Saline RN6390 B RN69 11 P< 0.0001 P=0.5702 Neutrophilic g ran ulocyte (%) 0 20 40 60 80 100 Salin e RN6390 B RN69 11

Fig.2–Theroleofagrsysteminmurineintracranialabscessesmodel.(A)Survivecurveofmicechallengedwithdifferent

strains(n=16miceineachgroup).MicewerechallengedwithRN6390B,RN6911,andsaline,respectively.Thenumberof

deadmicewasrecorded.Thesurvivalrateofthetwogroupswascalculatedindividually.(B)Histologicalexaminationof

mousebraintissue.BraintissuesfromRN6390BandRN6911groupswerecollectedat12hpost-challengewithbacteriaand

fixed.ThefixedtissueswerestainedbyH&E.(C)Percentageofneutrophilsinthethreegroups(RN6390B,RN6911andsaline)

atdifferenttimepoints(0h,4hand8h)post-challengewithS.aureus(n=10ineachgroup).(D)Survivecurveofmice

challengedwithdifferentstrains(n=22miceineachgroup).MicewerechallengedwithS.aureus8325-4,RNAIII,

RNAIII-R,andsaline,respectively.Thenumberofdeadmicewasrecorded.Thesurvivalrateofthetwogroupswas

calculatedindividually.(E)Survivalpercentageofthedifferentstrains(RN6390BandRN6911)inmurinewholeblood.

Bacteria(1×105_{CFU)wereincubatedwithmurinewholebloodfor4h.SurvivalrateofbacteriaweredeterminedatBHIplate.}

ofmiceat4hand8hafterinfection.Thenumber ofwhite

bloodcellswasnotsignificantlyaltered(Fig.1D),butthe

per-centageofneutrophilwassignificantlyincreasedatdifferent

timepointsinthismodel(Fig.1E).

Theroleofagrsysteminmurineintracranialabscesses model

Given that agr system can regulate most of exotoxins

expressedbyS.aureus,wetriedtodeterminewhetheragr

sys-temwasinvolvedinintracranialinfection.Micewereinjected

withS.aureusRN6390BorRN6911(agr-nullstrain).Injection

withRN6911couldnotinducedeathofmice(Fig.2A).

How-ever,histologicalexaminationshowedthatRN6911induced

similarhistologicalalterationofbraincomparedtoRN6390B

(Fig. 2B). The percentage of neutrophil was significantly

increasedinRN6390BandRN6911groups,comparedto

ster-ile saline (Fig. 2C). Itis well known that RNAIII isthe key

effectorofagrsystem.Furtherinvestigating,wedetermined

whetherRNAIIIdeletionaswellasagr-nullstrainwas

asso-ciatedwithdecreasedmortalityofmice.TheRNAIIIdeletion

strains(RNAIII)andtherestoredstrain(RNAIII-R)were

pre-viouslyconstructed.Micewerechallengedwithwild-typeS.

aureus 8325-4, RNAIII and RNAIII-R, respectively. In line

withexpectation,RNAIIIcouldnotinducethedeathofmice

andchallengewiththerestorationRNAIIIstrainrecovered

vir-ulence and the consequent mortality ofmice(Fig. 2D). All

theseresultssuggestedthatagr systemwasessentialforS.

aureusinducedmortalityinthemurineintracranialabscesses model.

As the alteration ofhemogram induced byRN6911 was

notdifferent fromthat ofRN6390B, wewonderedifa

defi-cientagr systemcouldmakethebacteriamoresensitiveto

wholebloodkilling.RN6911andRN6390Bwereincubatedwith

murinewholebloodrespectively.Thebacterialsurvivalrate

P

ercent sur

viv

al

Hours after injection 100 80 40 60 20 0 0 12 24 36 48 Saline RN6390B RN6911 RN6911-hla

Fig.3–Determinationoftheroleof␣-toxininmurine

intracranialabscessesmodel.Survivecurveofmice

challengedwithdifferentstrains(n=12miceineach

group).MicewerechallengedwithRN6390B,RN6911,

RN6911-hla,andsaline,respectively.Thenumberofdead

micewasrecorded.Thesurvivalrateofthetwogroupswas

calculatedindividually.

bacteria was not significantly different when compared to

RN6390Bbacteria(Fig.2E).Thisresultsuggeststhatthe

mor-talityalterationcausedbyagrnullstrainwasnotrelatedwith

thesensitivitytowholebloodkilling.

˛-ToxinwasinvolvedinthemortalitycausedbyS.aureus inmurineintracranialabscessesmodel

Exotoxins expressed by S. aureus are important for the

infection.10,14,15 _{Some exotoxins are regulated by the agr}

system10,16 _{and ␣-toxinis oneofthe major exotoxins}

reg-ulated by agr system. RN6911 do not express ␣-toxin. In

the following study, we investigated whether ␣-toxin was

involvedinintracranialabscesses.Theexpressionof␣-toxin

wasrecoveredinRN6911bytheplasmidpOS1-HLAto

gen-eratestrain(RN6911-HLA).Micewerechallengedwiththree

differentstrains(RN6390B,RN6911andRN6911-HLA),

respec-tively. Thesurvivalrateofmicefrom different groupswas

assessed at the indicated time points. It was shown that

restorationof␣-toxincouldpartiallyrecoverlethality(Fig.3).

Theseresultssuggestedthat␣-toxinplayedanimportantrole

intheintracranialinfectioncausingabscesses.

Discussion

Intracranialabscessescausedbybacteriaareusually

associ-atedwithhighmortalityrate.S.aureusisacommoncauseof

variouskindsofinfections,includingintracranialabscesses.

However,thereisnoreportaboutanimalmodelof

intracra-nialabscessescausedbyS.aureus.Moreover,theeffectofa

knownregulatorinS.aureusinintracranialinfectionhasnot

beendetermined.Inourstudy,weconstructedamurinemodel

ofintracranialabscessescausedbyS.aureus.Wealso

demon-stratedthatagr,thequorumsensingsystem,inS.aureuswas

essentialforcausingintracranialinfection.Furthermore,our

resultsrevealedthat␣-toxinregulatedbyagrsystemplayed

animportantroleintheprocessofintracranialabscesses.

There are some constructed S. aureus infection models

includingarthritis,pneumonia,acuteperitonitis,

endocardi-tis,andskininfection.17–26 _{Untilnow,therearefewreports}

about intracranial infection model caused by S. aureus. In

this study,we constructed amurine model by injecting S.

aureus8325-4intothefrontalcortex.MicechallengedwithS. aureusshowedsomeclinicalsignsofillness.Thenumberof

whitebloodcellsandtherectaltemperaturewerenot

signif-icantalteredafterinjectingS.aureussuggestingthatthesign

ofmurineintracranial abscesswasnotexactlythesameas

humanintracranialabscess.Thismaybeduetodifferencein

resistancetobacteriabetweenmiceand humans.However,

thepercentageofneutrophilinmicechallengedwithS.aureus

wassignificantlyhigherthanthatofcontrolgroup.Thisresult

isinaccordancewiththeclinicalsignseeninpatients.

AgrsystemiswellstudiedinS.aureusandisconsidered

essential forinfection.10,16,27 _{In line with expectations, we}

foundthatthemortalityassociatedwithagr-nullstrainwas

significantlydecreasedwhencomparedtothewildtypeon

intracranial abscessesmodel.Moreover,wealsofoundthat

deletionofRNAIIIsuppressedvirulenceofS.aureusinfection.

S.aureuscanexpressvariouskindsofexotoxinswhichare

necessaryforcausinginfection.␣-Toxinisthemajorexotoxin

ofS.aureus,regulatedbyRNAIII.10,16,28,29_{Ourresultsshowed}

thatrestorationofthelevelof␣-toxininagr-nullstrain

par-tiallyrecoveredthemortalityassociatedwithofS.aureuson

theintracranialabscessesmodel.Thisresultsuggestedthat

␣-toxinshouldplayanimportantroleinintracranialinfection.

Moreover,someothermoleculesregulatedbytheagrsystem

mightalsobeinvolvedinintracranialinfection.Theroleof

␣-toxinandothermoleculesexpressedbyS.aureusin

intracra-nialinfectionshouldbefurtherinvestigatedinthefuture.

Ourstudypresentsamurineintracranialabscessesmodel

that canbe usedtoinvestigate S.aureusinfection inbrain

tissue. Furtherinvestigation on therole of agr systemwill

increase our understanding of the intracranial abscesses

causedbyS.aureus.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

This research was supported by National Natural

Sci-ence Funding (31271119, 81072074), Beijing Educational

CommitteeKeyFunding(KZ201110025033)andBeijing

Orga-nizationDepartmentofMunicipalCommitteeTalentFunding

(3034000004).

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