MouR controls the expression of the Listeria monocytogenes Agr system and mediates virulence

15 Pages·2017·7.87 MB·English

9338–9352 NucleicAcidsResearch,2018,Vol.46,No.18 Publishedonline12July2018
doi:10.1093/nar/gky624
MouR controls the expression of the Listeria
monocytogenesAgr system and mediates virulence
Jorge Pinheiro1,2, Johnny Lisboa3, Rita Pombinho1,2, Filipe Carvalho1,2, Alexis Carreaux1,4,
Cla´udia Brito1,2, Anna Po¨ntinen5, Hannu Korkeala5, Nuno M. S. dos Santos3, Joa˜o
H. Morais-Cabral6, Sandra Sousa1,* and Didier Cabanes 1,*
1GroupofMolecularMicrobiology,IBMC–InstituteforMolecularandCellBiology;i3S–InstituteforResearchand
InnovationinHealth,Porto4200-135,Portugal,2ICBAS-InstitutodeCieˆnciasBiome´dicasAbelSalazar,Universityof
Porto,Porto4200-135,Portugal,3GroupofFishImmunology&Vaccinology,IBMC–InstituteforMolecularandCell D
o
Biology;i3S–InstituteforResearchandInnovationinHealth,Porto4200-135,Portugal,4SDV-UFRSciencesDu w
n
Vivant:Universite´ ParisDiderot-Paris7,Paris75013,France,5DepartmentofFoodHygieneandEnvironmental loa
d
Health,FacultyofVeterinaryMedicine,UniversityofHelsinki,Helsinki00014,Finlandand6GroupofStructural e
d
Biochemistry,IBMC–InstituteforMolecularandCellBiology;i3S–InstituteforResearchandInnovationinHealth, fro
m
Porto4200-135,Portugal h
ttp
s
ReceivedFebruary12,2018;RevisedJune25,2018;EditorialDecisionJune26,2018;AcceptedJune28,2018 ://a
c
a
d
e
m
ABSTRACT giventime,theirconstitutiveorover-expressionisenergeti- ic
.o
callywastefulandevenharmfultothepathogen(2,3). u
The foodborne pathogen Listeria monocytogenes Thus,thetightregulationofvirulencefactorsexpression p.c
o
(Lm) causes invasive infection in susceptible ani- becomesacrucialmechanismforpathogensurvivalandfit- m
mals and humans. To survive and proliferate within ness.Transcriptionalregulatorsofvirulencerespondtovar- /na
hcooosrtsd,intahtiessfathceuletaxtpivreesisnitornacoefllaulcaormpaptlhexogreengutliagthotrlyy icoourdsisniggnlyatlsr,igbgoetrhaesnwviitrcohnminetnhtealvairnudlehnocestf-adcetroivreedx,parnesdsiaocn- r/article
networkthatcontrolstheexpressionofvirulencefac- pattern(4,5). /4
6
tors. Here, we identified and characterized MouR, a Listeria monocytogenes (Lm) is a Gram-positive bac- /18
novel virulence regulator of Lm. Through RNA-seq terium and the causative agent of the systemic disease lis- /93
transcriptomic analysis, we determined the MouR teriosis. Although ubiquitous and commonly found grow- 38
regulonanddemonstratedhowMouRpositivelycon- ingindiverseenvironments,Lmswitchesfromasaprophyte /505
into a deadly pathogen (6,7), currently holding the high- 2
trolstheexpressionoftheAgrquorumsensingsys- 3
est mortality rate among foodborne pathogens in Europe 70
tem (agrBDCA) ofLm. The MouR three-dimensional (8).Lmiswellequippedtosurvivethehostileconditionsof by
structure revealed a dimeric DNA-binding transcrip- thehumandigestivetract,invadebothphagocyticandnon- gu
tion factor belonging to the VanR class of the GntR phagocyticcells,crossmajorbiologicalbarriers(intestinal, es
superfamily of regulatory proteins. We also showed blood-brainandplacentalbarriers),causesepticemiainim- t on
that by directly binding to the agrpromoter region, munocompromisedindividuals,infantsandtheelderlyand, 11
MouR ultimately modulates chitinase activity and inthecaseofpregnantwomen,severelyinfectthefoetus(9– Fe
b
biofilm formation. Importantly, we demonstrated by 11). ru
invitrocell invasion assays and invivomice infec- VirulenceregulationofLmgreatlydependsonthetran- ary
scription regulator PrfA, which controls the expression of 2
tionstheroleofMouRinLmvirulence. 0
a broad list of genes, including major virulence factors 23
and is thus regarded as the Lm master virulence regulator
INTRODUCTION
(12). Despite this, several other important regulators such
Pathogenicbacteriadependonanarsenalofgenesencod- as SigB (13), VirR (14), Hfq (15), MogR (16), DegU and
ingvirulencefactorstosuccessfullyinfecttheirhost.Dur- GmaR(17,18)contribute,toalesserextent,totheLmvir-
inginfection,byexertingspecificfunctions,eachvirulence ulenceregulatorynetwork.
factorplayscrucialrolesranging fromcellinvasiontonu- The GntR family of proteins is a large group of tran-
trientacquisitionandsubversionoftheimmunesystem(1). scription factors associated with the regulation of various
While the expression of such factors is fundamental at a
*Towhomcorrespondenceshouldbeaddressed.Tel:[phone];Email:[email protected]
CorrespondencemayalsobeaddressedtoSandraSousa.Tel:[phone];Email:[email protected]
(cid:2)C TheAuthor(s)2018.PublishedbyOxfordUniversityPressonbehalfofNucleicAcidsResearch.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionNon-CommercialLicense
(http://creativecommons.org/licenses/by-nc/4.0/),whichpermitsnon-commercialre-use,distribution,andreproductioninanymedium,providedtheoriginalwork
isproperlycited.Forcommercialre-use,[email protected]
NucleicAcidsResearch,2018,Vol.46,No.18 9339
biologicalprocessesinmanydiversebacteria(19,20).Mem- overnightcultureswerediluted1:100infreshBHIandab-
bersofthisgroupshareabasicstructureincludingahighly sorbance (OD ) was measured every 30 min. When
600nm
conservedDNA-bindingdomainattheNterminus,show- appropriate, antibiotics were added to the media: ampi-
ingacharacteristichelix-turn-helix(HTH)motif,andanef- cillin100(cid:2)g/ml,erythromycin5(cid:2)g/mlandkanamycin50
fectorbinding/oligomerization(E-O)domainattheCter- (cid:2)g/ml.
minus (21). Due to its higher diversity, the C terminus is
used to categorize the diverse GntR sub-families (FadR,
Mutantconstructionandstraincomplementation
HutC,MocR,YtrA,AraRandPlmA)(19).FadRfromEs-
cherichiacoliisoneofthebestcharacterizedGntRregula- ThedeletionofmouR(lmo0651)fromtheLmEGD-eWT
tors.FadRdimersbindtospecificDNAsequencesthrough strainwasachievedbydoublehomologousrecombination
theHTHmotif,whileacyl-CoAbindingtotheE-Odomain using the suicide plasmid pMAD (36). The detailed pro-
causes dramatic conformational changes impairing DNA cedure was previously described (34). The primers used
binding (22). FadR regulates fatty acid biosynthesis and are listed in Supplementary Table S2. Complementation
degradation through activation and repression of several of the (cid:2)mouR mutant strain was performed by genomic Do
w
genes(23). reintroductionofthegeneintrans,asdescribedbefore(7). n
Theaccessorygeneregulator(agr)locusencodesabac- Complementation was mediated by the Lm specific inte- loa
d
terialcommunicationsystemconsistingofaquorumsens- grative plasmid pIMK (37), through the construction of ed
ing module paired with a classical two-component system pIMK(mouR)(SupplementaryTableS1). fro
m
(24).FirstdescribedinStaphylococcusaureus,theAgrsys-
h
temisfoundinmanyotherGram-positivebacteria(25).In Plasmidforproteinoverexpression ttp
Lm, it has been associated with survival and competitive The coding region of mouR was C-terminally fused to a s://a
advantageinsoil,adhesiontosurfacesandbiofilmforma- c
tion,invasionofmammaliancells,infectivityinthemouse 6-His tag by in-frame restriction enzyme cloning into the ad
e
pET28aexpressionvector,usingtheprimerslistedinSup- m
modelandglobalchangesingeneexpression(26–30).Inter-
estingly,despitebeingtheonlyquorumsensingsystemde- plementaryTableS2,tocreatepET28a(mouR-6His). ic.o
u
scribedinLm,itappearsthatmonitoringpopulationden- p.c
sityisnotitsmainfunction(31)andtheroleofitsquorum Site-directedmutagenesis om
sensingpropertiesismostlyunknown(32).Also,noregula- /n
To create the MouR amino acid substitutions (R44A, a
toroftheAgrsystemhasbeendescribedsofarforLm,its R48A, Y80F, H133F andH134F), site-directedmutations r/a
apuotsoedre(g2u6l,a2t8io).n being the only regulatory mechanism pro- 6wHeries)puesrifnogrmtheedQounikpCIMhaKng(meoIIuRX)LaSnidte/-oDripreEcTte2d8aM(mutoaugRe-- rticle/4
We previously provided the first comprehensive view nesisKit(AgilentTechnologies)accordingtothemanufac- 6/1
of the genome expression of Lm directly in deep organs turer’srecommendationsandwithprimerslistedinSupple- 8/9
(spleen) of infected mice (7). This enabled us to identify mentaryTableS2. 338
andcharacterizenovelvirulencefactors,otherwisedifficult /5
0
to predict (33–35). It also showed that the in vivo differ- 5
ential expression of the Lm genome is coordinated by a Bioinformaticanalyses 23
7
0
complexregulatorynetwork,inparticularthroughtheup- GenesequenceswereobtainedfromtheGenbankdatabase b
regulationofthetwomajorvirulenceregulators,PrfAand (38) and homologue searches were performed with the y g
u
VirR,andtheirdownstreameffectors(7).Interestingly,dur- BLAST tool (39). Search of conserved protein domains e
s
ing mouse infection, Lm appears also to overexpress sev- andpredictionofproteinfunctionwereperformedwiththe t o
n
eralnewpotentialvirulenceregulators.Here,weidentified web-basedPROSITE(40)andNCBI’sConservedDomain 1
1
a novel transcription factor––MouR––upregulated during Database (41) tools. Comparative analyses of protein se- F
infectionandinvolvedintheorchestrationofLmvirulence quenceswasconductedinMEGA7:MolecularEvolution- eb
regulation. Notably, we demonstrated that mouR encodes aryGeneticsAnalysisversion7.0(42). rua
atranscriptional activatoroftheAgr system andisneces- ry 2
saryforfullvirulence.WecharacterizedMouRatthestruc- RNAisolationandRT-qPCR 023
tural level, identified its regulon and demonstrated how it
controlsbiofilmformationandLmchitinase-dependentim- Lm cultures were grown in BHI to exponential phase
muneevasion,aswellasrevealeditsroleinLmvirulence. (OD = 1.0) and total RNA isolated by the phenol-
600nm
chloroform method described elsewhere (43), with modifi-
cationsasdescribednext.Afterlysis,RNApurificationwas
MATERIALSANDMETHODS performedusingtheTripleXtractorreagent(Grisp,Porto)
followingthemanufacturer’srecommendations.DNAwas
Bacterialstrainsandgrowthconditions
eliminated by DNase treatment (Turbo DNA-free, Am-
Strains used are detailed in Supplementary Table S1. Lm bion) and RNA purity and integrity was verified by 1%
EGD-e(ATCC-BAA-679)(WT)andE.coliwereroutinely (w/v)agarosegelelectrophoresisandExperionAutomated
culturedinBrainHeartInfusion(BHI)andLysogenyBroth ElectrophoresisSystem(Bio-RadLaboratories).One(cid:2)gof
(LB)(Difco),respectively,at37◦Caerobicallywithshaking. RNA was reverse-transcribed into cDNA using a random
BHI-agarandLB-agar(Difco)plateswereusedforgrowth hexamercocktail-basedkit(iScriptKit,Bio-RadLaborato-
on solid media. To draw growth curves of Lm strains, ries).qPCRwasperformedonone(cid:2)gofcDNAina20(cid:2)l
9340 NucleicAcidsResearch,2018,Vol.46,No.18
reactionvolumeusingtheiTaq™UniversalSYBR®Green and lysed with a French Pressure Cell Press (Thermo) at
Supermix(Bio-RadLaboratories)andareal-timePCRde- 12 000 psi. Proteins were trapped in a nickel-based ma-
tectionsystem(iQ5,Bio-RadLaboratories)withthefollow- trix(Ni-NTA Agarose, Qiagen) and elutedwithimidazole
ing cycling protocol: 1 cycle at 95◦C (3 min); 40 cycles at inalow-pressureliquidchromatographysystem(BioLogic
95◦C(10s),56◦C(20s)and72◦C(20s).Primersarelisted DuoFlowQuadTec10System,Bio-Rad).Lastly,imidazole
inSupplementaryTableS2.Eachgroupcomprisesthreebi- was removed by membrane dialysis (Spectra/Por Dialysis
ologicalreplicateseachwiththreetechnicalreplicates.Data Membrane, SpectrumLabs) and protein was concentrated
were normalized to that of a reference housekeeping gene byVivaspin(SartoriusStedimBiotech)columncentrifuga-
(16S rRNA) and analyzed by the comparative threshold tion. For expression and purification of SeMet-MouR, E.
((cid:2)(cid:2)Ct)method. coliB834(DE3)harboringpET28a(mouR-6His)wasgrown
in40mlofLBovernightat37◦C,collectedandwashedthree
timeswithsteriledeionizedwaterandfinallyusedtoinoc-
RNA-seq
ulate1lofSelenoMethionineMedium(MolecularDimen-
D
DNA-free total RNAs were depleted for rRNA species sions).Purificationwasperformedfollowingtheprocedure o
w
by processing with MICROBExpress Bacterial mRNA describedforthenativeprotein. n
lo
Enrichment Kit (Ambion) following the manufacturer’s a
d
irniostnruAcutitoonms.atEefdficEielencttreonprhicohrmeseisntSywsatesmve(rBifiioe-dRabdy LEaxbpoe-- CrystallizationandstructuredeterminationofMouR ed fro
m
ratories) virtual gel analysis and Qubit 3.0 Fluorometer Both native and SeMet-substituted MouR proteins were
h
(Thermo Fisher Scientific) analysis. Sequencing of mR- stored in 50 mM Tris–HCl pH 8.0, 300 mM NaCl. Na- ttp
N(TAhesrwmaos pFeisrhfoerrmSecdieunstiinfigc)thaendIoqnuTaolittyalcRoNntAro-lSefoqrKRitNvA2 toifveac1r0ysmtagl/smwleprerogtreoinwnsoalutt2i0o◦nCwfirtohmaacr0y.s1t:a0l.l1iza(cid:2)tliomnixsotulure- s://ac
a
fragmentation and library construction was assessed by tion composed of 1.5 M lithium sulfate, 100 mM sodium de
m
Qubit3.0Fluorometer(ThermoFisherScientific)and2200 Hepes pH 7.5. SeMet-MouR crystals were grown from a ic
TapeStation (Agilent) analyses. Template preparation was mixtureof6mg/mlproteinsolutionwithacrystallization .o
u
achieved with an Ion Chef system (Thermo Fisher Scien- solution composed of 3 M NaCl, 100 mM Tris pH 8.5. p.c
tific) and sample sequencing was done in triplicates with Crystals were flash-frozen directly in liquid nitrogen. Na- om
an Ion Proton System (Thermo Fisher Scientific). Gen- tive and SeMet diffraction data were recorded on beam- /n
a
eration of sequence reads and read trimming and filter- linesProxima2A(Soleil,Gif-sur-Yvette,France)andID29 r/a
ing was done with Torrent Suite v4.4 software with a (ESRF, Grenoble, France). The structure was determined rtic
FileExporter v4.4 plugin for generation of FASTQ/BAM by the SAD (Single wavelength Anomalous Dispersion) le/4
files. To assess differentially expressed genes between Lm method using the anomalous signal from the selenium el- 6/1
WT and (cid:2)mouR mutant strain, sequencing reads were ement.DatawereprocessedwithXDS(48)inspacegroup 8/9
aligned to the reference genome sequence of Listeria P41,withtwocopiesperasymmetricunit.AllexpectedSe 33
8
monocytogenes EGD-e (RefSeq: GCF 000196035.1, Gen- sites(eightbymonomer)werefoundwithSHELXDusing /5
Bank: GCA 000196035.1, assembly ASM19603v1 -http:// reflectionsinthe50–3.5A˚ resolutionrange(49).Refinement 05
2
www.ncbi.nlm.nih.gov/assembly/GCF 000196035.1) using ofSeatompositions,phasinganddensitymodificationwere 3
7
0
thealignerTopHat2(44).Aftertranscriptassembly,therel- performedwithPhenix-EP.Thehighqualityoftheexperi- b
y
ative abundance of each transcript was estimated by cal- mentalphasesallowedautomaticbuilding(Autobuildpro- g
u
culationofthemetricfragmentsperkilobaseoftranscript gram)ofmostoftheproteinmodel.Themodelwasrefined e
permillionmappedreads(FPKM)(45)usingCufflinksand againstthe2.2A˚ nativedatasetusingPHENIX(50).The st o
n
Cuffdifftools(46).Statisticalsignificancewasattributedto finalmodelcontainsresidues3–217frombothmonomers. 1
1
transcriptswithfoldchangeofexpressionhigherthan2or In addition, 85 water molecules, 10 sulfate ions, one 4- F
lowerthan0.5,aP-valuebelow0.5andanFDR-adjusted (2-hydroxyethyl)-1-piperazineethanesulfonic acid and six eb
P-value below 0.1. Sequencing results are available on the ethylene-glycolcouldbemodelledintotheelectrondensity rua
GEO database (https://www.ncbi.nlm.nih.gov/geo/) under maps.Statisticsfordatacollectionandrefinementaresum- ry 2
theaccessionnumberGSE106833. marized in Supplementary Table S3. Data of the reported 02
3
crystal structure was deposited in the Protein Data Bank
(PDB)(https://www.rcsb.org)undertheID:6EP3.
Proteinpurification
Purificationof6xHis-taggedMouRandMouR(R44/48A)
Electrophoreticmobilityshiftassays
(Supplementary Table S1) proteins was performed by E.
coli heterologous overexpression and chromatography as Protein-DNAbindingwassetupin20(cid:2)lreactionscontain-
described elsewhere (47) with some modifications. Briefly, ing 240 ng of DNA synthesized with primers described in
the growth of E. coli BL21(DE3), harbouring either Supplementary Table S2, binding buffer (50 mM HEPES
pET28a(mouR-6His) or pET28a(mouR(R44/48A)), for 3 pH 7.5, 500 mM NaCl, 1mM EDTA, 0.1 mM DTT, 5%
h at 37◦C with agitation in LB supplemented with 0.5 M glycerol,0.01mg/mlBSA)andincreasingamountsofpuri-
IPTG was determined as a favorable condition for high fiedproteinaspreviouslydescribed(35).DNAwasfirstin-
protein expression. Cultures were inoculated at 1:50 with cubatedinbindingbufferfor5minfollowedbygentlemix-
anover-nightculturepelletedbycentrifugationat200rpm ingoftheproteinand20minincubationatroomtempera-
and re-suspended in LB. One litre of culture was grown ture.Thetotalreactionwasloadedintoa10%acrylamide
NucleicAcidsResearch,2018,Vol.46,No.18 9341
native gel and ran in TAE buffer. The gel was stained for dilutedandplatedonBHI-agarplatesforCFUdetermina-
10 min in a 0.01% GreenSafe Premium (NZYTech) TAE tion.
buffersolutionandimagedinaGelDocXR+System(Bio-
RadLaboratories).
Intracellularmultiplication
Chitinhydrolysisassay Assays of intracellular multiplication in mouse
macrophages were performed as described (33). RAW
Evaluationofchitinaseactivitywasperformedasdescribed 264.7 cells (ATCC TIB-71, ∼2 × 105/well) were propa-
(51).Fivegramofshrimpshellchitin(Sigma-Aldrich)were
gated in DMEM (Lonza) supplemented with 10% FBS
pre-treated with 50 ml of HCl (37%) overnight. Treated
(Biowest), infected as described above with exponential
chitinwasadjustedtopH8withNaOHandpelletedat8300
phasebacteriaatamultiplicityofinfectionof10for30min
g for 5 min, washed with ultrapure water seven times and andtreatedafterwardswith20(cid:2)g/mlgentamicin.At,2,5,
usedtosupplementLB-agarplates(6mg/ml).10(cid:2)lofLm
7and20hpost-infectioncellswerewashedwithPBS,lysed D
oatve3r0n◦iCghfotrcu6ltduaryess.wCehrietisnphoyttderdoolynsitshweapslaetveaslaunatdeidnbcuybmateead- in cold 0.2% Triton X-100 and bacterial suspensions were own
serially diluted and plated on BHI-agar plates for CFU lo
suringtheradiusofthedegradationhalo. a
determination. d
e
d
Biofilmformation from
Animalinfections h
Lm ability to form biofilm was evaluated by crystal vio- ttp
Animal infections were performed with 6–9-week-old spe- s
ldeitluttuerdbi1d:i1m00etrinyBasHsaIya(n5d2)1.0O0v(cid:2)erln(iogchttapculilctautreess)owfeLremtrwanerse- cific pathogen-free female BALB/c mice (Charles River ://ac
ferredtosterile96-wellPVCplates(Corning)andincubated Laboratories)maintainedattheIBMCanimalfacilities,in ade
at30◦Cfor20h.WellsfilledwithsterileBHIservedascon- high efficiency particulate air (HEPA) filter-bearing cages m
trol. Media was removed and loose bacteria were washed under 12 h light cycles and in an ad libitum regiment of ic.o
tfihllreedetwimithes1w5i0th(cid:2)dliostfil0le.5d%wactreyrs.tAalirvdiorlieetd(wBeDllsH()30fomri4n5)wmeinre. swteerreilepecrhfoowrmaenddbayutinooclcauvleadtiownatoefr.1I0n4trCavFeUnosutshrionufegchtiotanisl up.com
Excessstainwaswashedthreetimeswithdistilledwater,the vein injection as described (56). For oral infections mice /n
prel-astueswpaensdaeirddirnie2d0a0n(cid:2)dlboifo9fi9lm%-aetshsoacnioalt.e1d0c0ry(cid:2)sltaolfveiaoclehtwwealsl wlaeterde wstiatrhve2d×fo1r098–C1F2Uhsb(ienfoPrBeSthweitphro1c5e0dmurge/manldCainCoOcu3)- ar/artic
wogsurvaorBesewridntonrifiiagnalnhmtast3fcfep0ourl◦rralCtemtudeoarrettneoisoagwdnaleaewnrrseesa(ws(cid:2)dcoaiQlplvusluetaoreatsadenlsit1aps,:ensB1sd0(si2oe0t2dthienea×ksf)Or.d2eD2essh5cc9mrB5inb)HmesIduwab(an5ms3de)mw.rgLeeearmde- bhewmayapecsnoghhtaseeatvdd-naiingmwtefwe.iatciMlhctewio1icewn0er0ieatwhna(cid:2)edsgEree/tpMhmtseiacEllcaiMgrvlelieyfin,rc,triaeensdmmpcluyobebecvyanientgdeae.(dnnLPdeioenryPnaeezlEryaa’Mse)nrpfa’EsoaeMrtpscta1hhtsecehushs,piewaapsenl7ored2ef- le/46/18/9338/5
washedtwicewithEMEMandoncewithPBS.Organswere 0
in a six-well plate. Three days post-incubation, the cover- 5
slipswerewashedthreetimeswithdistilledwater,fixedwith homogenized in PBS and homogenates were serial diluted 23
7
andplatedonBHI-agarplatesforliversandspleensoron 0
4% glutaraldehyde during 2 h and stained with 0.1% acri- b
Listeria Selective Agar (Oxoid) plates supplemented with y
dineorangefor15min.Coverslipsweremountedontomi- g
croscopeslideswithAqua-Poly/Mount(Polysciences)and Listeria Selective Supplement (Oxoid) for Peyer’s patches. ue
examinedunderaconfocallaser-scanningmicroscope(Le- Animal procedures were in agreement with the guidelines st o
icaSP5II).Sectionsweretakenevery0.5(cid:2)mandprocessed oanfitmheaElsu(rdoipreecatnivCeo2m01m0/is6s3io/EnUfo)r,tthheehPaonrdtulignugeosefllaebgoisrlaattoiorny n 11
usingFiji-ImageJ(54,55).Biofilmthicknesswascalculated F
usingLeicaSP5IIsoftware. for the use of animals for scientific purposes (Decreto-Lei eb
113/2013),andwereapprovedbytheIBMCAnimalEthics ru
a
CommitteeaswellasbytheDirecc¸a˜oGeraldeAlimentac¸a˜o ry
Cellinvasionassays e Veterina´ria, the Portuguese authority for animal protec- 20
2
tion,underlicense015302. 3
Human cell lines invasion assays were performed as de-
scribed(33).Briefly,cellsweregrowntoconfluencyinEa-
gle’smediumwithL-glutamine(EMEM)(Lonza),supple-
Statisticalanalyses
mented with nonessential amino acids (Lonza), sodium
pyruvate(Lonza)and20%fetalbovineserum(FBS,Biow- StatisticalanalyseswereperformedwiththesoftwarePrism
est) (Caco-2, ATCC HTB-37) or in Dulbecco’s modified 7 (GraphPad Software). Unpaired two-tailed Student’s t-
Eagle’s medium (DMEM) with glucose (4.5 g/l) and L- testwasusedtocomparethemeansoftwogroups;one-way
glutamine (Lonza), supplemented with 10% FBS (HeLa, ANOVA was used with Tukey’s post-hoc test for pairwise
ATCCCCL-2andJeg-3,ATCCHTB-36).Lmwasgrownto comparisonofmeansfrommorethantwogroups,orwith
exponentialphase,washedandinoculatedatamultiplicity Dunnett’spost-hoctestforcomparisonofmeansrelativeto
ofinfectionof75for1h.Cellswereincubatedwithmedium themeanofacontrolgroup.Differenceswithacalculated
supplementedwith20(cid:2)g/mlgentamycin(Lonza)for1h30 P-valueabove0.05wereconsiderednon-significantandsta-
min to eliminate extracellular bacteria, washed and lyzed tisticallysignificantdifferenceswerenotedasfollows:*P<
with0.2%TritonX100.Bacterialsuspensionswereserially 0.05;**P<0.01;***P<0.001.
9342 NucleicAcidsResearch,2018,Vol.46,No.18
RESULTS teria grown to exponential phase. RT-qPCR results and
RNA-seqdataexhibitedaverystrongPearsoncorrelation
lmo0651encodesaputativetranscriptionregulator
coefficient(R2=0.93)(Figure2B),thereforevalidatingthe
In a previous study, we reported the transcriptome profil- differentialexpressionlevelsdetectedbytranscriptomics.
ing of Lm while infecting the mouse spleen and identified Together, these results pointed Lmo0651, renamed
the gene lmo0651 as highly expressed during spleen infec- MouR,asthetranscriptionalactivatoroftheAgrsystem.
∼
tionwhencomparedtogrowthinBHImedium(=100-fold)
(7). In addition, lmo0651 was also previously shown to be
MouRisadimericGntRprotein
upregulated when Lm was infecting the mouse intestinal
lumen or incubated in human blood (57). Through bioin- To further explore the function of MouR, we purified
formaticsanddatabaseanalysis,theLmo0651proteinwas MouRandresolveditsthree-dimensionalstructure(Figure
predicted as a transcriptional regulator containing a pu- 3). The crystal structure of MouR was determined by the
tative typical GntR winged HTH DNA-binding domain SADmethodusingSeMet-substitutedprotein.Theatomic
(19)(Figure1A).Proteinsequencealignmentsshowedthat modelwasrefinedagainstanativedatasetto2.2A˚ resolu- Do
w
Lmo0651appearstobeconservedacrosstheListeriagenus, tion (Supplementary Table S3). The final model of MouR n
lo
found in the genome of almost all pathogenic and non- showsahomodimerthatincludesresiduesAsn3toArg217 a
d
pathogenic species and serogroups (Figure 1B and S3A). forbothsubunitsandthreehistidinesfromtheHis-Tagfor ed
However, it has no close relatives outside the genus, the oneofthesubunits(Figure3A).Thesubunitsinthehomod- fro
closesthitbeingaproteinfromIsobaculummelisannotated imerdisplayaRMSDof1.5A˚ (http://zhanglab.ccmb.med. m
h
asa‘DNA-bindingtranscriptionalregulator,GntRfamily’ umich.edu/TM-align/)(65).Theproteinpresentsthefoldof ttp
s
(Figure1B).Althoughflankedbytwopredictedtranscrip- theGntRsuperfamilyofdimerictranscriptionfactorswith ://a
tionterminators,lmo0651appearstobepartofanoperon each subunit composed by two domains (Figure 3B). The c
a
(lmo0648 to lmo0651) (57) comprising genes encoding po- N-terminal domain of MouR (Asn3–Cys73) is a winged- de
m
tentialmembraneproteins(lmo0648andlmo0650)orsimi- helix dsDNA-binding domain, characteristic of the GntR
ic
lartotranscriptionregulators(lmo0649andlmo0651)(Fig- family,andincludesthecanonicalHTHDNA-bindingmo- .o
u
ure1C). tif((cid:3)1–(cid:3)2–(cid:3)3)followedbya(cid:4)-hairpin((cid:4)1–(cid:4)2).TheFadR p.c
C-terminalputativeregulatorydomain(FCDdomain),en- om
compassingresiduesVal75–Arg217,containsanantiparal- /n
MouRisthetranscriptionalactivatoroftheAgrsystem a
lel array of six (cid:3)-helices ((cid:3)4–(cid:3)5–(cid:3)6–(cid:3)7–(cid:3)8–(cid:3)9) that form r/a
To study the role of Lmo0651 as a regulator and identify a barrel-like structure. The presence of only six (cid:3)-helices rtic
genes under its control, we designed an RNA-seq-based within the FCD domain classifies MouR as a member of le/4
experiment to assess the genome-wide expression of both theVanRsubclassoftheFadRfamilyofGntRregulators. 6/1
Lm EGD-e WT and the (cid:2)lmo0651 deletion mutant in ex- The dimer interface is mediated exclusively by helix (cid:3)4 of 8/9
ponential growth phase in BHI at 37◦C. When compared the FCD domain (Figure 3A and C), burying a surface 33
to the WT strain, six transcripts (agrD, agrB, agrC, agrA, of∼1150A˚2 permonomer(calculatedwith‘Proteininter- 8/5
fruA, lmo0278) appeared less abundant in the (cid:2)lmo0651 facesandassemblies’servicePISAattheEuropeanBioin- 05
2
mutant,whileonlyone(lhrA)wasmoreabundant(Figure formatics Institute; http://www.ebi.ac.uk/msd-srv/prot int/ 3
7
0
2A), suggesting that Lmo0651 acts mainly as a transcrip- pistart.html) (66). Residues that contribute to the dimer- b
tionalactivator.Fourofthesegenes(agrD,agrB,agrCand izationarelistedinSupplementaryTableS4,togetherwith y g
u
agrA)areclusteredinanoperonlocated639kbawayfrom anestimateoftheaccessibilityofindividualresiduesinthe e
s
lmo0651 and encoding the components of the conserved dimer versus the monomer. Interface residues are located t o
AgrquorumsensingsystemofLm(32,58)(Supplementary mainlyon(cid:3)4andtheloopbetween(cid:3)6and(cid:3)7(Figure3C). n 1
1
FigureS3B–E),whichisinvolvedinsurvivalintheenviron- The interface involves apolar contacts mediated by Ile89, F
ment, biofilm formation and virulence (26–28,30). lhrA is Phe90,Ala93andIle152,anetworkofhydrogenbondsand eb
anon-codingshortRNA(ncRNA)negativelyregulatedby electrostatic interactions involving Gln82, Glu85, Thr86, rua
Agr(51)andknowntoregulateseveralgenes,includingthe Lys97, Glu145 and Gln149 (Figure 3C). It is known that ry 2
chitinase-codinggenechiAthroughmRNAbindingandin- the regulatory domains of the FadR family bind small or- 02
3
terference with ribosome recruitment (59,60). In addition ganic ligands leading to conformational changes that re-
to Agr system related genes, fruA and lmo0278 were also orient their winged-helix domains and affect DNA bind-
less expressed in absence of Lmo0651 (Figure 2A). These ing (67,68). Analysis of the electron density map revealed
genesarerelatedtosugaruptake,fruAencodesoneofthe an internal polar cavity within the FCD domain, suggest-
componentsofafructose-specificphosphotransferasesys- ingaputativeligand-bindingsite(Figure3AandD).FCD
tem and lmo0278 encodes a maltose uptake ABC trans- domainscommonlydisplaythreeconservedhistidinesthat
porter(61,62).Bothgenesseemtobeessentiallyrelatedto interact and bind a metal ion in the cavity (68). The den-
metabolicpathwaysand,furthermore,theyappeartohave sity map of MouR structure revealed that only two his-
acomplexdynamictypeofregulationsincetheyseemprone tidines(His133andHis134)areconservedwhereasatyro-
tobesimilarlyregulatedincomparativegenome-widetran- sine(Tyr80)ispresentinplaceofthethirdhistidine(Figure
scriptomes(7,57,63,64). 3D).Inaddition,thispocketislarge,withavolumeof∼170
WefurtherconfirmedourRNA-seqresultsbyRT-qPCR. A˚3andtheelectron-densitymapshowsunexplaineddensity
Weselectedasubsetofup-anddown-regulatedgenesand in the cavity where a molecule of ethylene glycol, the cry-
performedqPCRoncDNAfromWTand(cid:2)lmo0651bac- oprotectantusedtofreezethecrystal,wasmodelled.Alto-
NucleicAcidsResearch,2018,Vol.46,No.18 9343
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Figure1. lmo0651encodesanovelGntRtranscriptionfactor.(A)Bioinformaticanalysispredictsthelocuslmo0651ofLmtoencodeatranscription
factorwithatypicalN-terminalDNA-bindingwingedhelix-turn-helixGntRdomainandaC-terminalFadRdomain.(B)Alignmentofproteinsequence
ofLmo0651withorthologsfromotherLmstrainsandListeriaspecies,withconservedaminoacidresidueshighlightedinred.Aminoacidsconserved
betweenLmEGD-eLmo0651andtheputativeGntRtranscriptionalregulatorinIsobaculummelis(theclosestrelativeoutsideoftheListeriagenusfound
byBLASTanalysis)aremarkedinyellow.CriticalDNA-bindingargininesarehighlightedwithblackboxes.(C)GenomicorganizationofthemouRregion
inLmEGD-e.
9344 NucleicAcidsResearch,2018,Vol.46,No.18
the binding to DNA. Among the other E. coli FadR (ID:
IHW2)residues(Arg35,His65andLys67)involvedinDNA
contact, only the histidine (His63) is conserved in MouR,
Arg35beingreplacedbytheLys35.However,inourmodel
these residues do not contact with DNA (Supplementary
Figure S1), probably because the MouR protein will un-
dergostructuraladjustmentswhenboundtoDNA,accom-
modatingtheinteractionofthoseaminoacidresidueswith
theDNAstrand.Ontheotherhand,thismodelshowsthat
MouRLys49isalsoclosetothemajorgrooveoftheDNA
andmayalsobeinvolvedintheinteraction(Figure4B).Al-
though all known structures of winged-helix domain are
very similar, their mode of interaction with dsDNA can
D
varygreatly.Whilethemajorityoftheseinteractionsinvolve o
w
the(cid:3)3–helixbindinginthemajorgrooveoftheDNA(71), n
lo
ourmodeloftheMouR–dsDNAcomplex(Figure4A)sug- a
d
gests that only thetop of this helix is implicatedin the in- ed
teractionwithdsDNA(Figure4B). fro
m
h
MouRpositivelyregulatestheexpressionoftheagrlocusby ttps
bindingtotheoperonpromoter ://a
c
a
Based on MouR structure analysis, we hypothesized that de
the (cid:3)3-helix and in particular the conserved Arg44 and m
ic
Arg48 residues would be essential for its ability to bind .o
u
target DNA and, thus, crucial for its activity as a tran- p.c
scriptional regulator. To test this, we engineered an Lm om
straintoexpress,ona(cid:2)mouRbackground,amutatedver- /n
a
sionofMouRinwhichArg44andArg48werereplacedby r/a
alanine residues (Lm (cid:2)mouR+mouR(R44/48A)). We con- rtic
firmed that mouR(R44/48A) is expressed in this strain at le/4
levels comparable to mouR in the WT strain (Supplemen- 6/1
Figure2. Genesdifferentlyregulatedin(cid:2)mouRinvitro.(A)Foldchangeof traegryulaFtiegdurgeenSe2s),inantdheanWalTy,ze(cid:2)dmtohueRe,x(cid:2)prmesosuioRn+mofouMRouaRnd- 8/933
tiadhsaeLteiooxgpn2roeosfsfRiofNonlAdof-csgheeqannetgrsaernebsgecutrlwiapteteeodnmbiiyncsMvditoaruotaRgbeaynseRshTeox-wqpPrneCsbsRyio.RnDNoaAfta-Lsremeqp.Wr(eBsTe)nVatnaeldd- (cid:2)(cid:2)mmoouuRR+smtroauinR(tRhe44e/x4p8rAes)ssiotrnaionfs.tWheeofobusreravgedr tgheanteisnwthaes 8/5052
(cid:2)mouRinexponentialgrowthinBHIat37◦CasmeasuredbyRNA-seq highly repressed while lhrA was upregulated (Figure 5A), 37
andRT-qPCR. as detected by RNA-seq. In the (cid:2)mouR+mouR comple- 0 b
y
mented strain the expression of all five genes was re- g
u
getherthissuggeststhatthepocketbindsasmall-molecule storedbacktolevelscomparabletothoseoftheWT(Fig- e
s
insteadofametal. ure 5A), definitively demonstrating the role of MouR in t o
n
the expression of the agr locus and lhrA. Inversely, the 1
MouR-dsDNAcomplexmodel e(cid:2)lsmoofuaRg+rmgeonueRs(sRim44il/a4r8tAo)thstorsaeinobexsehrivbeitdedforex(cid:2)pmreossuiRon(Fleigv-- 1 Feb
The GntR superfamily of dimeric transcription factors is ure 5A). This strongly suggests that these conserved argi- rua
characterized by the presence of an N-terminal winged- nineresiduesattheNterminusofMouRarecrucialforthe ry 2
helix DNA-binding domain. Three structures of FadR- transcriptionalactivationoftheagrgenes. 02
3
dsDNAcomplexareavailableonPDB:twofromE.coli(ID: To investigate whether MouR controls the expression
1HW2and1H9T)(22,69)andonefromVibriocholera(ID: of the agr locus by direct binding of the operon pro-
4P9U) (70). These FadR proteins in complex with DNA moter, we performed electrophoretic mobility shift assays
presentanidenticalstructuralarrangementoftheirwinged- totestprotein-DNAinteractions.Weobservedthatincreas-
helixdomainsandconservedDNA-interactingaminoacid ingamountsofMouRwereabletodelaythemigrationof
residues. The superimposition of MouR winged-helix do- thebandcorrespondingtotheagrpromoter(Pagr),0.2(cid:2)M
main onto FadR-dsDNA complexes (ID: 1HW2, 1H9T of MouR being sufficient to delay the agr promoter mo-
and4P9U)showsthatithasasimilarstructure.Therefore, bility(Figure5B).Furthermore,increasingamountsofthe
wemodelledtheMouR-dsDNAcomplexusingtheFadR- mutatedMouR(R44/48A)variantfailedtodelayPagrmi-
dsDNA complex from E. coli (ID: 1HW2) (Figure 4A). gration,clearlyindicatingthatthearginineresiduesarees-
AnalysisofthemodelstronglyshowsthatArg44andArg48 sentialforthebindingofMouRtotheagrlocuspromoter.
onthe(cid:3)3-helixappearreadytointeractwithDNA(Figure As controls, we used Pagr with a purified unrelated regu-
4B).ThesetwoarginineresiduesareconservedintheGntR lator (Lmo0443), or mixed MouR with its own promoter
superfamily (Figure 1B), supporting their involvement in region.Inneithercondition,wewereabletodetectaband
NucleicAcidsResearch,2018,Vol.46,No.18 9345
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Figure3. MouRstructureanddimerizationinterface.(A)RibbonrepresentationofMouRstructure.Dimerizationinterfaceandligandbindingcavities gu
indicatedbydashed-linedboxes.(B)Top:ribbonrepresentationoftheMouRsubunitstructure.TheNandCterminiandsecondarystructureelementsare es
indicated.Colourcode:blue,wingedHTHdomain;green,FCDdomain.Bottom:linearrepresentationofMouRstructuraldomainsaccordingtoPfam t o
n
(colorcodeasthetop).(C)Thedimerizationinterfaceisenlargedtoshowsomeoftheresiduesinvolvedintheinteraction(representedasmagentasticks). 1
Hydrogenbondsarerepresentedbydashedlines,anddistancesbetweenresiduesareindicatedinA˚.(D)Highlightoftheputativeligand-bindingcavityof 1
F
MouR(graysurface)withtheconservedresiduespotentiallyrelevantforligandbindingshownasorangesticks. e
b
ru
a
shift(Figure5B),demonstratingthatMouRactsasaDNA- bydecreasingthepolarcharacterofthecavityandreducing ry
2
bindingtranscriptionfactorwithspecificaffinitytothepro- the availability of hydrogen-bond partners. We confirmed 0
2
3
moteroftheagroperon. thatmouRisexpressedinthesestrainsatlevelscomparable
Above, our analysis of the MouR structure revealed an to the WT (Supplementary Figure S2). The expression of
internalpolarcavitywithintheFCDdomain,suggestinga agrB was analyzed by RT-qPCR in these strains as com-
putative ligand-binding site with two conserved histidines pared to the WT. Our data showed that agrB expression
(His133 and His134) and a tyrosine (Tyr80) susceptible to in the mutant strains was not significantly different from
interact with a potential ligand in the cavity (Figure 3D). the WT strain, indicating that none of the mutations im-
To evaluate if the residues oriented towards the putative pairedtheabilityofMouRtoactivateagrBexpression(Fig-
C-terminalligand-bindingcavityplayaroleintheMouR- ure5C).
dependent transcriptional activation of agr genes, we en-
gineered Lm (cid:2)mouR strains to express MouR with the
residue substitutions: Y80F (Lm (cid:2)mouR+mouR(Y80F)), MouRregulateschitinaseactivityandbiofilmformation
H133F (Lm (cid:2)mouR+mouR(H133F)) and H134F (Lm WhiletheAgrsystemhasnotbeendeeplycharacterizedin
(cid:2)mouR+mouR(H134F)). These substitutions alter the Lm,itwasconsistentlyassociatedwithLmchitinaseactiv-
physical-chemicalpropertiesoftheresiduesliningthecavity ity and capacity to develop a biofilm (28,51). We thus an-
9346 NucleicAcidsResearch,2018,Vol.46,No.18
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Figure4. ModelofMouR-dsDNAcomplex.(A)RibbonrepresentationofthemodelledMouR–DNAcomplex.TheDNAismodeledbasedonthe ttp
s
sduopmearpinosoiftiMonooufRth(sehFoawdnRin–DblNueA),cwoimthppleuxta(tPivDeBDINDA:-1bHinWdi2n)gornetsoidtuheeswshinogwend(-rHepTrHesednotmedaiansostficMkso)u.TRh.e(Bh)elTixh(e(cid:3)o3v)eirnavlollavrecdhiitnetchtuerientoefrathcteiowninwgietdh-DHNTHA ://ac
iscoloredinred. ad
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Figure5. MouRpositivelyregulatestheexpressionoftheagrlocusbydirectlybindingtotheoperonpromoter.(A)ExpressionofagrgenesandthencRNA
lhrAdeterminedbyRT-qPCRperformedwithtotalRNAisolatedfromLmWT,(cid:2)mouR,(cid:2)mouR+mouRor(cid:2)mouR+mouR(R44/48A)culturesinexpo-
nentialgrowthinBHImediumat37◦C.ExpressionlevelsarerepresentedrelativetoWT.Valuesaremean±SDofthreeindependentexperiments.**P≤
0.01;***P≤0.001.(B)ElectrophoreticmobilityshiftassaysofincreasingamountsofpurifiedMouR,MouR(R44/48A)oranunrelatedputativeregulator
(Lmo0443)withaDNAfragmentcontainingthepromoterregionofagrormouR.Resultsarerepresentativeofatleastthreeindependentexperiments.
(C)ExpressionofagrBdeterminedbyRT-qPCRperformedwithtotalRNAisolatedfromLmWT,(cid:2)mouR+mouR(Y80F),(cid:2)mouR+mouR(H133F)or
(cid:2)mouR+mouR(H134F)culturesinexponentialgrowthinBHImediumat37◦C.ExpressionlevelsarerepresentedrelativetoWT.Valuesaremean±SD
ofthreeindependentexperiments.
NucleicAcidsResearch,2018,Vol.46,No.18 9347
alyzed the potential connection between MouR and these Lm was previously shown to preferentially invade intesti-
specificprocesses.Wefirstconfirmedthattheinvitrogrowth nalPeyer’spatchesfollowingoralinoculationinthemouse
of the WT, (cid:2)mouR and (cid:2)mouR+mouR strains was simi- model used in our study (73,74). We thus assessed num-
lar (Figure 6A). To test Lm chitinase activity, we spotted bersofLmpresentinmousePeyer’spatches72hafteroral
overnightculturesofthedifferentstrainsonLBagarplates inoculation. The results showed that invasion of intestinal
supplemented with chitin. The formation of a translucent Peyer’s patches was less efficient in the absence of MouR
halo on the opaque medium directly correlates with the (Figure7E).
bacterial chitinase activity (51). Our data showed that the Altogether,ourdataindicatethatMouRisnecessaryfor
mutant (cid:2)mouR had a striking reduction in chitin hydrol- Lmvirulenceinamouseoralinoculationmodel,thoughnot
ysis when compared to both the WT and (cid:2)mouR+mouR inanIVinoculationmurinemodel.
strainswhich,inturn,werecomparabletoeachother(Fig-
ure 6B). A deletion mutant for agrC ((cid:2)agrC), the sensor
DISCUSSION
kinase component of the Agr system (72), showed unde-
D
tectablechitinhydrolysis(Figure6B).Wealsoinvestigated Virulenceregulatorsarecrucialelementsofthegenomeof o
w
theroleofMouRinbiofilmformationonPVCmicroplates pathogenic bacteria (10). They coordinate the transcrip- n
lo
bythecrystalvioletturbidimetrymethod(52).Weobserved tionalshiftthatmediatestheswitchfromasaprophyteinto a
d
that the (cid:2)mouR mutant developed a significantly reduced a pathogen, granting the adaptability necessary to infect ed
biofilm relative to the WT strain, and that this defect was hostorganisms(6,7).Ithasbeenpredictedthatatleast209 fro
m
successfullyrevertedbygenecomplementation(Figure6C). transcriptionregulator-codinggenesexistinthegenomeof
h
Again,thebehaviourof(cid:2)agrCfurthershowedthatthelack LmEGD-e(75).WhereasPrfAoccupiesthehighestseatin ttp
of a functional Agr system renders Lm unable to form a thevirulenceregulationofLm,controllingtheexpressionof s://a
WT-likebiofilm,thusconfirmingtherelationshipbetween mostofthemainvirulencefactors(12),theidentificationof c
a
MouR and biofilm formation. 3-D analysis of 3-day-old novelregulatorsiskeytounderstandhowLmfine-tunesthe de
biofilms grown at 30◦C on a glass surface confirmed that expressionofvirulencedeterminantsinordertooptimally m
ic
themouRmutantformedamuchthinner(maximaldepth: promote infection dissemination (12). In addition, despite .o
u
WT=14±2(cid:2)m;(cid:2)mouR=4±1(cid:2)m)andlessdensebiofilm thehighabundanceoftranscriptionfactorsencodedinbac- p.c
thandidtheWTstrain(Figure6D). terial genomes, proper characterization of their 3D struc- om
Altogether, these results demonstrate how, by directly tures is limited (76,77). Here, we report the discovery and /n
a
regulatingtheAgrsystem,MouRcontrolsLmchitinaseac- molecular characterization of a novel virulence regulator, r/a
tivityandbiofilmformation. MouR,alongwiththedemonstrationofitsrolesintheLm rtic
virulenceregulatorynetworkasthetranscriptionalactiva- le/4
MouRisrequiredforcellinvasionandvirulence toroftheAgrsystem. 6/1
To our knowledge, this is the first regulator of the Lm 8/9
TakingintoaccounttheupregulationofmouRduringLm Agrsystemidentified.Atleastadozenregulatorshavebeen 33
8
infectioninmiceanditsroleinchitinaseactivityandbiofilm linked to the activation and repression of agr in S. aureus /5
0
formation, we hypothesized that MouR could be impor- (78).Althoughwedonotruleouttheexistenceofotherreg- 5
2
tant for the invasion of eukaryotic cells and establishment ulatorscontrollingAgrinLmandalsotheexistenceofan 3
7
0
of systemic infection. To test this hypothesis, we infected autoregulationmechanismassuggestedbefore(26,28),our b
thehumancelllinesCaco-2,Jeg-3andHeLawithLmWT datastronglyindicatethatMouRhasanimportantroleas y g
and(cid:2)mouRstrainsandassessedthenumberofintracellu- activatoroftheAgrsystem. ue
s
larbacteria.AscomparedtotheWTstrain,the(cid:2)mouRmu- By resolving the structure of MouR we opened ways to t o
n
tantshowedaslightdefectintheinvasionofallcelllines,es- theclassification and deeper characterization of this novel 1
1
peciallyinthecaseofJeg-3andHeLacells(Figure7A),sug- regulator.Asinitiallypredictedbybioinformatics,thecrys- F
gestingaroleforMouRincellinvasion.Wethenanalyzed tal structure of MouR revealed that a monomer is com- eb
the potential role of MouR in intracellular multiplication posed of a typically conserved GntR N terminus with a rua
by following the behavior of the WT and (cid:2)mouR strains winged-HTH DNA-binding domain and a variable C ter- ry 2
afterinternalizationinRAWmurinemacrophage-likecells. minus with an E-O domain. Deeper analysis of the C- 02
3
Bothstrainsgrewwithsimilarmultiplicationratesafterup- terminalstructureledustoclassifyMouRasamemberof
take,indicatingthatmouRdeletionhasnoconsequenceon theFadRsub-family,thelargestsub-groupoftheGntRsu-
intracellularmultiplication(Figure7B). perfamily(19).Thepresenceofexactlysix(cid:3)-heliceswithin
To investigate the role of MouR in vivo, mice were the C terminus allowed us to further classify it as a mem-
intravenously or orally infected with WT, (cid:2)mouR or beroftheVanRsub-class(79).Despitethelargesizeofthe
(cid:2)mouR+mouRstrainsandthebacterialloadperliverand FadR sub-family, currently making up close to 50% of all
spleenwasassessed72hpost-infection(Figure7CandD). GntRproteinssequencesonPfam,onlyfiveVanRregula-
Whilenosignificantdifferencesweredetectedinthebacte- torsarepresentlyavailableinthePDBdatabase(67,68,79).
rialburdenofintravenouslyinfectedanimals (Figure7D), To our knowledge, this is the second GntR member (80)
a significant reduction in the number of (cid:2)mouR bacteria andthefirstVanRtyperegulatordescribedinListeria.Our
was observed in the liver of orally infected mice (Figure structuraldatashowthatMouRisorganizedasadimeric
7C). Furthermore, mice infected with the (cid:2)mouR+mouR unit.GntRtranscriptionfactorspredominantlybindtheir
complemented strain showed bacterial levels comparable DNA–targets as dimers (79,81), however, the quaternary
to the WT, revealing a restored phenotype (Figure 7C). organization of these proteins and their mode of interac-