FrontiersinCellularandInfectionMicrobiology|www.frontiersin.org 1 September2018|Volume8|Article313 ORIGINALRESEARCH published:06September2018doi :10.3389/fcimb.2018.00313
Editedby:F rancoisVandenesch,Unive rsitédeLyon,France Reviewedby:N ickWheelhouse,EdinburghN apierUniversity, UnitedKingdom StefanoGiulieri, UniversitédeLausanne,Switzerland
*Correspondence:B enedyktWladykabenedykt.wl adyka@uj.edu.pl
†Theseauthorshavecontributed equallytothiswork
Specialtysection:Th isarticlewassubmittedtoMolecular BacterialPathogenesis, asectionofthejournalFr ontiersinCellularandInfection Microbiology Received:10May2018 Accepted:16August2018 Published:06September2018 Citation:
BonarEA,BukowskiM,HydzikM,Jank owskaU,Kedracka- KrokS,GroborzM,DubinG,Akkerboo mV,MiedzobrodzkiJ,SabatAJ,Friedri chAWandWladykaB(2018) JointGenomicandProteomicAnalysisId entifiesMeta-TraitCharacteristicsof VirulentandNon- virulentStaphylococcusaureusStrai ns.Front.Cell.Infect.Microbiol.8:313 .doi:10.3389/fcimb.2018.00313
JointGenomicandProteomicA nalysisIdentifiesMeta-
TraitCharacteristicsofVirulent
and Non-virulentStaphylococcusaureus Strains
EmiliaA.Bonar1†,
MichalBukowski1†,
MarcinHydzik1,
UrszulaJankowska2, SylwiaKedracka-
Krok3,
MagdalenaGroborz1,
GrzegorzDubin2,4,
ViktoriaAkkerboom5,
JacekMiedzobrodzki4, Artu rJ.Sabat5,
AlexanderW.Friedrich5a
ndBenedyktWladyka1*
1D
epartmentofAnalyticalBiochemistry,FacultyofBiochemistry,BiophysicsandBiotechnology,JagiellonianUniversity,Krakow,Po land,2M
alopolskaCentreofBiotechnology,JagiellonianUniversity,Krakow,Poland,3D
epartmentofPhysicalBiochemistry,Facultyo fBiochemistry,BiophysicsandBiotechnology,JagiellonianUniversity,Krakow,Poland,4D
epartmentofMicrobiology,FacultyofBioc hemistry,BiophysicsandBiotechnology,JagiellonianUniversity,Krakow,Poland,
5D
epartmentofMedicalMicrobiology,UniversityMedicalCenterGroningen,UniversityofGroningen,Groningen,Netherlands
Staphylococcusa ureusi sa nopportunisticpathogenofhumansa ndwarm- bloodedanimalsandpresentsagrowingthreatintermsofmulti-
drugresistance.Despitenumerousstudies,t hebasisofstaphylococcalv irulencea ndswitchin gbetweencommensalandpathogenicphenotypesisnotfullyunderstood.Usinggenomics,we showheret hatS.a ureusstrainse xhibitingv irulent( VIR)a ndnon-
virulent( NVIR)phenotypesinachickenembryoinfectionmodelgeneticallyfallintotwoseparate groups,witht heVIRgroupbeingmuchm orec ohesivethant heNVIRgroup.Significantly,t heg enese ncodingknownstaphylococcalvirulencefactors,suchasc lumpingfactors,areeitherfo undindifferentallelicvariantsinthegenomesofNVIRstrains(comparedtoVIRstrains)ora rei n activepseudogenes.M oreover,t hepyruvatec arboxylasea ndgamma-
aminobutyratepermeasegenes,whichwerepreviouslyl i nkedwithv irulence,arepseudogeni zedi nNVIRstrainc h22.Further,weusec omprehensiveproteomicstoolstocharacterizestrain sthatshowopposingphenotypesinachickenembryovirulencem odel.VIRstrainCH21hada ne levatedl evelofdiapolycopeneoxygenaseinvolvedinstaphyloxanthinproduction(protectiona gainstfreeradicals)andexpressedahigherlevelofimmunoglobulin-
bindingproteinSbionitssurfacecomparedtoNVIRstrainch22.Furthermore,jointgenomican dproteomicapproacheslinkedtheelevatedproductionofsuperoxidedismutaseandDNA-
FrontiersinCellularandInfectionMicrobiology|www.frontiersin.org 2 September2018|Volume8|Article313
bindingproteinbyNVIRstra inch22withgeneduplicatio ns.
Keywords:genome,genomics,patho gen,proteome,proteomics,Staphylo coccusaureus,v irulence
GenomicsandProteomicsinStudiesofS.aureusVirulence Bonaretal.
INTRODUCTION
Thegeneticdeterminantsandproteineffectorsthatareresponsiblefo rt hev irulenceofStaphylococcusaureusescapeourfullunderstandin gdespiteanumberofcomprehensivestudies.Inhomeostasis,S.aur euscoexistswithitshostwithoutdistinguishedadverseeffects.Howe ver,inanimbalancedstate,thenatureofwhichispoorlyunderstood,t hisopportunisticpathogenmaycauseinfectionandposeasignificant healththreat.Thus,theJanus-
facebacteriaconstantlybalancescommensalandvirulentphenotype s,copingwithdifferentlevelsofhostdefenses(RasigadeandVanden esch,2014).Indeed,itwasrecentlydemonstratedt hatwithint hesa mec lonalc omplex,p h enotypicdifferencesmaybel inkedwitht hes everityofi nfections.Moreover,factorscorrelatedwithhighpathogeni cityinthegroupofgeneticallyrelatedS.aureushadlittleeffectonthem ortalityratesassociatedwithi nfectionsc ausedbyb acteriafromother clonalcomplexes(Reckeretal.,2017).Thisfindingindicatesboththeg enetica ndp h enotypicb asisofs taphylococcalv irulence.Asidefro mmaintainingh ost/pathogenb alancei na s inglehosts pecies,s tap hylococcihavebeendemonstratedt os witchbetweenanimalandhu manhosts.Suchswitchingisassociatedwiththeexchangeofhost- specificvirulencefactorsthatareresponsibleforc olonizationa nds pr ead(Lowdere ta l.,2 0 0 9).Thisp l asticitys ignificantlyc omplicates s tudiesonv irulencedeterminants,especiallyintermsoflikelyhuman specificfactorsthatcanbeexperimentallytestedexclusivelyinanima lmodels.Geneticmethodshavebeensuccessfullyusedtopredictanti bioticresistancewithhighcredibilityandtherecentadventofmassive parallelsequencingpromisesclinicalutility(Aanensenetal.,2016).H owever,onlyafewgeneticmarkers,whosemechanismofactionhasb eendeterminedatthemolecularlevel,havebeenconvincinglyl inked withs uccessfulc olonizationa ndv irulence[e.g.,argininec atabolis mm obilee lement,ACME(Diepe ta l.,2008;Thurlowe ta l.,2 0 1 3),e xfoliativet oxins(Bukowskie ta l.,2010)].O thergeneticmarkers,b as edons tatisticala nalysisofwholegenomes equencinga ndD NAmic roarrayassaysd ata,werep ointedt op ositivelyc orrelatewiths everit yofi nfections,includingbacteremiaandinfectiveendocarditis(Gillet al.,2011;Calderwoodetal.,2014;Bouchiatetal.,2015).Neverthele ss,apaletteofgeneticp redictorsofs taphylococcalv irulencei ss tillli mited.Anumberofstudieshaveusedproteomicsapproachestotrytoi dentifytheproteineffectorsofvirulentphenotypes(Bonaretal.,2015).
Earlier,wecomparedexoproteomesinasetofstrainsexhibitinghigha ndlowvirulenceinachickenembryoinfectionmodel.Despitethehigh heterogeneityoftheanalyzedproteomes,wewereneverthelessable toidentifyalpha-
hemolysinandbifunctionala utolysinasi ndicatorsofv irulence,wher easglutamylendopeptidaseproductionwascharacteristicofnon- virulents trains(Bonare ta l.,2 0 1 6).Thisp riors tudy,h owever,didn ottakeintoaccountsurface-
attachedproteins,whichmaycontaina dditionalv irulencef actors.Th ei ntracellularp roteomewasnott akeni ntoc onsideratione ither,buti tmayp otentiallycontainregulatoryp roteins.
However,anothersourceofinformationonstaphylococcalviru lencefactorstraditionallycomesfromtheinvestigationofknock- outandknock-instrainsdevoidoforsupplementedwith
testedf actorsa nds ubsequentlychallengedi na nimalmodels(Kim etal.,2014b).Unfortunately,alackofappropriatemodelshasdetrim entallyaffectedtheresults(Polakowskaetal.,2012).Moreover,thisa pproachdoesnotallowtodistinguish“true”virulencefactorsfromthos eaffectingtheoverallfitnessofbacteria.
Givent hel imitationsoft hesec urrenta pproaches,h ere,wedesig nedandappliedanew,combinedworkflowtosuccessfullyidentifyvir ulence-andcommensalism-relatedmeta-
traitswithinthegenomesa ndp roteomesofv irulent( VIR)a ndnon- virulent(NVIR)S.aureusstrains.Twobelongingtothesamesequenc etypewild-typestrainsthathavebeenwell-
characterizedintermsofvirulenceinaninvivomodelwerecompared andcontrastedusingacombinedgenomicandproteomicmethodolo gy.Weshowthatthenon-
virulentstrainch22ischaracterizedbyamorecomplexexoproteomet hani tsv irulentc ounterpartCH21.Thisfindingi sassociatedwitht he s mallergenomeofCH21t hanch22.Interestingly,CH21isnotcharac terizedbytheproductionofanyclassicalvirulencefactorscomparedt och22.Itisratherthecombineddifferentialexpressionofmultiplefact orsthatdeterminesthevirulenceofCH21;therationalebehindthiscon clusionisdiscussedinourcommunication.
MATERIALSANDMETHODS
BacterialStrainsandGrowthConditionsP
oultry- isolatedS.aureuss trainsexhibitinge itherh igh( CH3,CH5,CH9,CH 21,a ndCH23)orl ow( ch22,ch24,p a 3 ,a ndph2)v irulence( VIRa n dNVIR,respectively)i na chickenembryoexperimentali nfectionmo delwereusedi nt hes tudy(SupplementaryTable1).Strainorigina n dgeneralgeneticandp h enotypiccharacteristics,i ncludingb asicp h ylogeneticrelationshipsandv irulence,weredescribedpreviously(L owderetal.,2 0 0 9 ;P olakowskae ta l.,2 0 1 2 ;Bonare ta l.,2 0 1 6).Th ebacteriawereculturedintrypticsoybroth(TSB)for16hat37◦C withvigorousshakingunlessindicatedotherwise.
GenomeSequencingandAssembly
WholeGenomeSequencing
GenomicDNAwasisolatedusingaDNeasyBloodandTissueKit(Qiag en)fromanovernightculturederivedfromasinglecolony.PurifiedD N AwasquantifiedwithaQubit2 .0Fluorometer(LifeTechnologies).Wh olegenomesequencingwasperformedusinga nI lluminaM i Seqs ystemwithD NAfragmentl ibrariespreparedusinga NexteraXTv 3k i t(Illumina)accordingt ot hemanufacturer’sprotocol.Thesampleswe resequencedtoobtainaminimumof100-
foldcoverage.Readsweredenovoassembledintoc ontigsusingCLC G enomicsWorkbench( version8 .5.1).Contigswereorderedona t e mplateoft heS.aureusED98completechromosomesequence(Gen BankCP001781.1)usingself-
developedPythonscripts,whichutilizednucleotideBLAST fromtheNCBIBLAST+toolkit[version2.3.0(Camacho eta l.,2 0 0 9)].Thec ompletegenomics equencesoft heCH21 andch22strainswereobtainedbyclosingtheremaininggapsusin gPCRamplificationandSangersequencing.Automatedgenom eannotationwasperformedusingtheNCBIProkaryoticGenomeA nnotationP ipeline( http://www.ncbi.nlm.nih.gov/
genome/annotation_prok/).ThesequencesweredepositedinGenB ankwitht heaccessionnumbers:CH3,M O YG00000000;CH5,MSG Q00000000;CH9,MOYH00000000;CH21,CP017804,CP017804, CP017806;ch22,CP017807,CP017808,CP017809;ch23,M O YI0 0000000;ch24,M O YJ00000000;p a3,
MOXP00000000;ph2,MOYK00000000.Detailedinformationmay befoundintheSupplementaryTable1.
IdentificationofMobileGeneticElements(MGEs)Thecontigsthat didnotmatchthechromosomesequencewereexaminedforp ossibl ep l asmidoriginbyevaluatingtheirsimilaritytoexistingplasmidsorfra gmentsthereof.Wholegenomics equenceswereexaminedfork now nstaphylococcalp hagesa ndp a thogenicityi slandsusinga nexhau stivesetofreferencesequencesobtainedfromGenBank(Suppleme ntaryTable2).Shortsequencefragmentsofatleast1kbshowingsimi larity(butnotidentical)toknownphagesandpathogenicityislandsw ereclassifiedasputativelynovel.
ConstructionofPhylogeneticTrees
Phylogenetictreesoftheidentifiedprophagesandpathogenicityislan dswerec onstructedusingCLCMainWorkbench( version7.7.2)andt hek -
merbasedt reec onstructionmethodwiththeNeighborJoinalgorithm (k-mersize1 5;distancemeasure:fractionalc ommonk -
merc ount).SNPa nalysiswasp e rformedusingtheCSIPhylogeny1.
4server(Kaasetal.,2014).Asthechromosomesequencesweresu bmittedtotheserverminimumdeptha tSNPp ositions,minimumrelat ivedeptha tSNPpositions,minimumdistancebetweenSNPsandmin imumSNPqualityasinputp arametersweredisabledduringanalysis.
Thereadmappingqualitywassettominimum25andthez-scoreto 1.96.ThemaximumlikelihoodtreeproducedbyCSIPhylogeny 1.4s erverwasv isualizedi nM EGA6s oftware(Tamurae ta l.,2013) .
InsilicoM LSTandST5GroupPhylogeneticAnalysisAp i pelinewa sdevelopedi nP ython3 fori nsilicoM L STa ndphylogeneticanalysis ofstrainsbelongingtoST5group.All8,688S.aureusgenomes,compl etegenomicsequencesaswellasshotgunsequencingresults,weref etchfromNCBIGenBankdatabase( ftp://ftp.ncbi.nlm.nih.gov/geno mes/genbank/bacteria/,accessedon20 18 -07-
06).28 9 genomeswhichdidnotp rovideac ross-
referencet oNCBIBiosampled atabasewereomitted.Locip rofilesfr omBISGsd atabasewereusedt oc lassifyt hegenomesaccordingto theirsequencetype(https://pubmlst.org/saureus/,accessedon2 0 1 8 -07-
06(Jolleya ndMaiden,2010)].L ocis equencesusedforS.aureusM L STt ypingwereobtainedfromUSA300straingenomicsequence(acc essionversionCP014420.1)andusedasqueriesfornucleotideBLAS TsearchoftheremaininggenomeswithE-valuethreshold0.0001 andrequiredquerycoverage100%(ver.2.7.1+(Camacho etal.,2009).For2,129assemblies,classifiedasST5,cross-
referencestoNCBIBiosampledatabase(ftp://ftp.ncbi.nlm.nih.go v/biosample/,accessedon2018-07-
06)wereusedtoobtaininformationonthehost,yearofisolationand countryoforigin(attributesof“harmonized_name”propertyequal to“host,”“ geo_loc_name,”a nd“ collection_date,”respectively).
1,635assembliesc ontaineds uchi nformation.Thei nformationonth ereferencestrainsED98andN315,missinginNCBIBiosampledatab ase,wasaddedmanually.Thesegenomesweresearchwithnucleotid eBLASTwithE -
valuet hreshold0 .0001andminimalqueryc overage9 5 %forl ocis e quencesobtainedfromN315straingenome(accessionversionBA00 0018.3),whichwereutilizedbeforeforST5phylogeneticanalysisbyN ubeleta l.
( 2008).H owever,t oavoidf alsep ositiveh its,onlyl ociofl engthequ alorgreaterthan4 00 bpweres elected(intotal97of126,Suppleme ntaryTable10).Forasignificantover-
representationofstrainsisolatedfromhumanhostandoriginatingfro mtheUSA(1,248of1,474,85%),arandomsampleofsize50wasobta inedforthisgroup.Selectedinsuchaway279strainstogetherwithoth ersixbeinganalyzedinthisresearchandbelongingtoST5group(Sup plementaryTable11)weres ubjectedt op h ylogenetica nalysis.Fo reverys traint hesequencescorrespondingtotheaforementionedN3 15strainlociwereordered,c oncatenateda nda lignedusingClustalO megawithdefaultparameters[ver.1.2.1(SieversandHiggins,2014)]
.Subsequentlyeverycolumncontainingagapwasremovedfromt he a lignment,whichshortenedi tfrom4 4 ,295t o4 3 ,872bp.Thea lignm entwasc onvertedt oP HYLIPformat)a ndusedtocreateaphylogen etictreewithRAxML[ver.8.2.9(Stamatakis,2014)]basedonGTRG AMMAImodel[generaltime-
reversible,GTR(Tavaré,1986)].Thetreewassubsequentlyimported t oCLCMainWorkbench(Qiagen,CLCBio)t ogetherwithdataobtain edfromBiosampledatabase,asmetadata,andvisualized.
AnalysisofCodingSequencesandPutativePr omoters
Self-
developedP ythonscriptsutilizingnucleotidea ndp roteinBLASTfro mNCBIBLAST+toolkit( version2 .3.0)a ndmuscle[version3 .8.31(
Edgar,2 0 0 4)]wereusedforc reatingmultiplesequencealignmentst oanalyzecodingsequencesandputativepromoterregions.Annotat edc odings equenceswereextracted
fromtheanalyzedgenomestogetherwith200bpupstreamfragments containingtheputativepromoters.Translationsofthecodingsequen ceswereclusteredwithasimilaritythresholdof9 0 %.A lli denticalp r oteins equenceswithina c lusterweredesignatedasasingleallele.T hemultiplesequencealignmentsofdifferentallelesandpromoterseq uenceswithineachclusterwerescannedfordifferentiatingcolumns.
VisualGenomeComparisonWithBRIG
Theninegenomes,p ublishedh ere,a ndt hegenomeofN315strainas a h umanreferenceofST5werec omparedt oED98genomeusingB RIGtool(Alikhanetal.,2011).Theidentityt hresholdsweres ett o9 8 a nd9 5 %a ndED98mobilegeneticelements,prophagesϕAv1,ϕAv β,thepathogenicityislandSaPIAv,andplasmidspAvX,pT181,and pAvYwereannotated.
WholeGenomeGeneralComparisonofCH21andch 22Strains
Continuoussimilarsequencesegmentswereidentifiedusingnucleoti deBLAST.Mostsegmentswereseparatedbyshort
insertionsorduplications.Similarsequencesegmentsandtheireq uivalencebetweenstrainswerevisualizedusingCircos[version0.69- 3(Krzywinskietal.,2009)].
ProteomicA nalysis
ExoproteomeAnalysis
Forc omparativea nalysisoft heexoproteomesoft heCH21andch 22s trains,t riplicatesofc learedc ulturef l uidswerehandledasd escribedpreviously(Bonaretal.,2016).Toassurenocontaminatio nwithintracellularproteins,thesupernatantswerepassedthroug ha0.22-
µmPVDFfilter.Then,theproteinswereprecipitatedwithanequal volumeof20%
(w/v)trichloroaceticacidinacetoneandrecoveredbycentrifugatio n.Thepelletwaswashedwithacetoneandairdried.Thesamplesw eredissolvedi nl ysisbuffer( 30mMT risHClp H
8.5c ontaining7 Murea,2 Mt hioureaa nd4 %CHAPS).Theproteins ampleswerelabeledwithspectrallyresolvablefluorescentG- dyes(NHDyeAGNOSTICSGmbH)andsubjectedtotwo- dimensionaldifferencegelelectrophoresis[2DDIGE;
(Albane ta l.,2 0 0 3 ;Timmsa ndCramer,2 0 0 8 ;M i ndene ta l.,200 9)].Isoelectrofocusing(IEF)wasperformedusing17-
cmimmobilizedpH(3–10)gradientstripsandProteanIEFCell(Bio- Rad).P roteinsweres eparatedi nt hes econddimensionusingan1 2%acrylamidegelaccordingtotheLaemmlimethod
(Laemmli,1970).ThegelswerescannedusingTyphoonTrio+
(GE),andimageswereanalyzedusingImageQuantv.7.0and DeCyder2Dsoftwarev.7.2(GE).Proteinspotswereconsideredasdif ferentiatingift hestandardizedaveragespotv olumeratioexceeds1.
5-foldatt he95 %confidencelevel(Student’st-testp -
value<0.05).Subsequently,t hegelsweres ilvers tained(Shevchenk oetal.,1996).Thedifferentiatingspotswereexcisedanddestainedby severalsubsequentwashesin25%and50%
(v/v)acetonitrile( ACN)i n2 5 mMa mmoniumbicarbonatebuffer(NH
4HCO3),pH8.0at37◦C.ThegelfragmentsweredehydratedinACN,dr iedusingavacuumconcentrator,andrehydratedusingt rypsins oluti on( 10ng/µli n2 5 mMNH4HCO3,p H 8.0),anddigestionwascarried outovernightat37◦C.Peptideswereextractedbysonication,dehydr atedinACNanddriedusingavacuumconcentrator.Samplesweresu spendedin2%(v/v)ACNi nwaterc ontaining0 .05%
( v/v)t rifluoroaceticacid(TFA)andseparatedusinganUltiMate300 0RSLCnanoSystem(Dionex).P eptideswerea nalyzedona c ouple dM i crOTOF-
QIImasss pectrometer( Bruker)e quippedwitha nA polloSourceE SInanosprayerwithlow-flownebulizer.Rawdatawerepre- processedwithD ataA nalysis4 .0s oftware( Bruker,G ermany)into MascotG enericformat.TheSwissProtnon-
redundantproteind atabaset axonomicallyrestrictedt oFirmicutes(
gram-
positivebacteria)wasqueriedwiththeobtainedpeaklistsusingani n- houseMascots erver.A dditionally,a ni n-
housep repareddatabaseconstructedbasedonthefullgenomeseq uencesofCH21andch22obtainedinthisstudywasused.Onlyidentifi cationswithascorevalueover100wereconsideredrelevantforfurthe ranalysis.Ifmorethanasingleproteinwasidentifiedinparticularspot ,onlyhitsscoringover50%oft heh ighestscoringp roteinwerec onsid eredi nfurtheranalysis.
AnalysisofIntracellularProteome
ForcomparativeanalysisoftheintracellularproteomesofstrainsC H21andch22,3mlofovernightcultures(OD600∼12)wasusedintr iplicate.Thecellswerecollectedbycentrifugation,washedthreeti meswith10mMTrisHClpH8.0andsuspendedin1 mlofT riR e age nt( SigmaA ldrich),t ransferredt oL ysis
MatrixTubes(MPBiomedicals)anddisruptedwithPrecellys24H omogenizer(BertinInstruments).Lysateswereclarifiedbycentrif ugation,andtheproteinphasewasisolatedaccordingtotheTriR eagentprotocol.Theobtainedproteinpelletwassuspendedinlysi sbufferandanalyzedby2DDIGEandMSasdescribedforexoprotei ns.
SurfaceProteomeAnalysis
Proteolytic“shaving”wasusedtocomparesurfaceproteomes(sur facomes)ofs trainsCH21a ndch22.A llsampleswereanalyzedi n biologicalt riplicates.Bacteriawerec ulturedovernighti nTSBa t3 7◦Cwithagitation.Thefreshmediumwasinoculatedwiththeovern ightcultureat1:100dilutionandcultureduntilOD600reached1.The culturesweredividedintotwosamples,experimentalandcontrol,b asedonthemethodofSolisetal.
(2010)tocontrolforcontaminationbycytoplasmicproteinsresulti ngfromunspecificc elll ysis.Bacteriawerecollectedbycentrifuga tion,washedwithphosphatebufferedsaline(PBS)andsuspendedi n30%sucroseinPBS.Experimentalsampleswereincubatedwithtry psin[1µg/µl;“Gold,MSGrade”(Promega)]for30minat37◦Cwithg entleagitation,clarifiedbycentrifugationandfilteredthrougha0.
22-
µmPVDFfilter.Thecontrolsamplewashandledasabovesavetha ttrypsinwasnotaddedduringtheinitialincubation.Thecontrolsam plewastreatedwitht rypsin( 1µg/µl)onlyafterf i ltering( condition sasabove).AllsampleswerereducedwithDTTandalkylatedwithi doacetamide,digestedovernightwitht rypsin0 .2µg/µl(Biocentru m)at37◦C,supplementedwith0.5%TFAand5%ACN( finalc on centration),c leanedusingP ierceC18Spincolumns(ThermoFis herScientific)andvacuumdried.SampleswereanalyzedbyMSas describedforexoproteins,savescorevaluesover50wereconsidere drelevant.
RESULTSANDDISCUSSION
OverallGenomicCharacteristicsofVirulenta ndNon-virulentStrainsofS.Aureus
Inap riors tudy,weevaluatedthev irulenceinachicken
embryoinfectionmodelofa numberofp oultryoriginatingstrainsofSta phylococcusaureusonabackgroundoftheirgeneticrelationships.T odeterminet hegenomicb asisofp h enotypicdifferencesbetweent heselectedstrains,inthisstudy,weobtainedthegenomicsequences offivehighlyvirulentstrains(CH3,CH5,CH9,CH21,andCH23)andfo urstrainscharacterizedbylowvirulence(ch22,ch24,pa3,andph2).
Twogenomes(CH21andch22)wereobtainedi nt heirc ompleteform ,whereast heremainingsevengenomeswereobtainedintheformofo rderedcontigs.Theoverallcharacteristicsoftheobtainedgenomesar esummarizedinSupplementaryTable1.Genomesizedoesnotcorr elatewithvirulentphenotypessinceallgenomes,savethatofstrainch 22(consideredinmoredetailinthenextsection),have
FIGURE1|
ComparisonofproteinclustersamongnineS.aureusstrains.Clusterswereobtainedforannotatedcodingsequencesatthresholdof90%similarity.Withinaclusteridenticalseque ncesaredenotedbythesamecolor.Whitedenotestheabsenceofanortholog.Longwhitegapsattheendoftheplotcorrespondtotheabsenceofdifferentmobilegeneticelementssu chasplasmids.TheclusterorderisrelatedtotheorderofcodingsequenceoccurrenceinthegenomeofED98.
asimilarsizeofca.2.8Mbp.Furthermore,theoverallnumberofgen
esandcodingsequencesdoesnotcorrelatewithvirulent TABLE1|Numberofclustersgatheringopenreadingframesexhibitingover90%proteinsequence similarityinthestudiedgroupofS.aureusstrains.
phenotypesandiscomparableamongtheanalyzedstrainssaveforch 22.Significantly,t hev irulentphenotypecorrelateswiththep resence ofp hageϕ AvB( 46,768bp;6 2 O RFs)a ndp AvYplasmid(1,442bp,1 ORF).AllVIRstrainscarrytheabovegenetic
Totalnumberofi dentifiedcluster
s
Number(percent )ofidenticalclust
ers
elements,whereasallNVIRstrains,withtheexceptionofch22,arede voidofbothoft hesee lements.A partfromt heabovemobilegenetice lements,weadditionallyidentifiedanumberofnovelp rophagesa nd p a thogenicityi slandsa nds everalk nownplasmids,neitherofwhich differentiatedtheVIRandNVIRgroups(SupplementaryTables3,4;
SupplementaryFigures1,2).Anothercleardistinctiondifferentiatin gtheVIRandNVIRgroupsbecomesa pparentwhenO RFsl ongert h an1 6 5 nucleotidesareextractedandclusteredaccordingtoproteins equencesimilaritiesabove90%
(Figure1).Whenthecoregenomeofallninestrainsisconsidered,only approximatelyone-
quarterofgenesfallwithinsuchdefinedclusters,whichclearlydemon stratess ignificantgeneticdifferencesa mongs trains.Interestingly,t hepercentageofgenesclusteredwiththeabovecriteriaamongt hec oregenomesoft heVIRa ndNVIRgroupsismuchhigherat72and42
%,respectively,demonstratingthecloserrelatednessofs trainswithi nbothgroupst hanbetweengroups.Sucha nalysisa lsoshowst hatt h eVIRgroupi smorehomogenoust hant heNVIRgroup( Table1).Whe nc omparedtohuman-
originstrainsofsequencetype5(ST5),theST5poultry-
origins trainsc omprisea c learevolutionarys ubgroup.Thisphylog eneticrelationshipbasedonmoreindepthanalysisofgenome- extracted97differentloci(Nubeletal.,2008)placespoultry- originST5strainsonaseparatebranchofthetreeregardlesst ogeogr aphicalorigin.I nterestingly,b asedont hisanalysisCH21a ndch22s trainsa res tillgeneticallyi dentical(Figure2).Markedly,thewholege nomeanalysisconfirmedtheirhighgeneticresemblancebyrevealing only85singlenucleotidep olymorphisms( SNPs).ThemaximalSNP numberamongt hewholep oultryST5s ubgroupi s3 5 5 ,whereast h edistancetothetypestrainN315ofhumanoriginishigher(from663to 823SNPs).Non-
ST5poultrystrainsanalyzedinthisstudy,allbeingnon-
virulent,areclearlymoredistantfromtheaforementionedST5subgro upbyhavingfrom19,384to21,127SNPs.TheST692strainspa3and ph2,although
Pan-genome 3,307 1,277(38.62)
Core-genome 2,317 586(25.29)
VIRstrainspan-genome 2,949 2,513(85.22)
VIRstrainscore-genome 2,527 1,826(72.26)
NVIRstrainspan-genome 3,009 1,862(61.88)
NVIRstrainscore-genome 2,471 1,050(42.25)
isolatedfromdifferentp oultryh osts,respectivelyp a rtridgeandp h e asant,a regeneticallyh ighlys imilart oe achotherbydisplayingonly1 6 5 SNPs.TheST1s trainch24i sh ighlydistantfromt herestoft hea nalyzeds trainsbyhavingfrom19,384to20,875SNPs(Figure3;Sup plementaryTable12).Allthesegeneticsimilaritiesanddifferencesar ealsovisiblewhent hea nalyzedgenomesa rev isuallyc omparedt o ED98genomeasareference(Lowderetal.,2009).Notably,noneoft henon-
ST5s trainsc ontainmobilegenetice lements uchasprophagesϕAv 1,ϕAvβandthepathogenicityislandSaPIAv,whichs eemt obechara cteristict ot heST5p oultrys ubgroup(Figure4).
GeneticDifferencesBetweenV irulent(CH21)andNon -
virulent(ch22)StrainsC
H21a ndch22showoppositep h enoty pesi na chickenembryoi nfectionmodel( VIRvs.NVIR,respectively) buta reindistinguishablebyc ommont ypingt echniques.Weassum edthattheirdifferentphenotypesmaybeexplainedbydeepgenomese quencing.Suchanapproachisgenerallyappliedtoelucidatethegenet icbasesofthesuddenacquisitionofantibioticresistance(Sabatetal., 2015).However,itwaslikelythattheswitchbetweentheVIRandNVI Rph enotypesinCH21vs.ch22wasalsoduetomutationsorthelossor acquisitionofgeneticmaterial,whichwouldbetraceablebyNGS.Ther efore,weobtainedthecompletegenomicsequencesofbothstrains.T heFIGURE2|
PhylogeneticrelatednessofanalyzedpoultrystrainstoarepresentativegroupofotherstrainsbelongingtoST5groupdeterminedbytheanalysisof97differentloci,44kbofsequen ceintotal.Thepoultry-
associatedstrainscompriseadistinctivephylogeneticgroup.Markedly,CH21andch22strainsareindistinguishable.FragmentsofthetreecontainingreferencestrainsED98and N315forpoultry-andhuman-originstrains,respectively,aremagnified.
FIGURE3|
PhylogeneticrelationshipofstrainsmappedagainstreferencetypestrainED98.PhylogeneticmaximumlikelihoodtreeconstructedonthebasisofSNPswasobtainedbyCSIphylog eny1.4.Aconfidencescorerangingfrom0to1(×100duringvisualizationinMEGA6software)wascalculatedforrobustnessevaluationofthenodes.Thescalebarindicatestheevol utionarydistancebetweenthesequencesdeterminedby0.05substitutionspernucleotideatthevariablepositions.
firststrikingdifferencebetweenthestrainswasthetotallengthoftheir genomes,whichwas2.8and3.1Mbp,respectively,forthevirulentan dnon-
virulentstrains.Theadditionalgeneticpoolofover2 5 0 kbpwithint he
genomeofch22a rosemainlyfromthreeduplications.Thelongestone(
CP0178071552041..
1722216and1777324..1947499)encompassed170kbpandenc oded230proteins.Twoshorterones(CP0178071496929..1551606an d1722212..1776889,54kbp;andCP017807
1978955..1990796and2244863..2256780,12kbp)encoded 70a nd1 6 p roteins,respectively( SupplementaryTables5–9).
FIGURE4|Visualcomparisonofgenomesofpoultry-
originS.aureusstrainsagainstareferencestrainED98.Thegroupofvirulentstrainsisgeneticallymorehomogenouswhencomparedwiththemorediversegroupofnon-
virulentstrains.ParticularlyregardingindistinguishableCH21andch22strains.Noticeably,mobilegeneticelementssuchasprophagesϕAv1,ϕAvβandpathogenicityislandSaPIA vseemtobecharacteristicfortheST5poultrysubgroup.Human-originMRSAstrainN315belongingtoST5wasalsoincluded.
Interestingly,themajorduplicationsresidedwithinthecorepartoft he b acterialchromosome,whereast hep oolofmobilegeneticelements ,encompassingplasmidspAvXandpAvY,twoprophagesandpatho genicityislandSaPIAv,remainedidenticalinbothstrains(Figure5;S upplementaryTable3).Therefore,thefirstc onclusioni st hatl ossof t hev irulentp h enotypebych22wasnotrelatedtolosingasubstantial partofitsgeneticmaterial,whichcouldhavebeenresponsibleforvirule nce.Ontheopposite,pathogenicb acteriawereshownt ohavegenera llys mallergenomesthantheirclosenon-
virulentrelatives(GeorgiadesandRaoult,2 0 1 1).Therefore,onec a ni maginet hatt heburdenofdispensablegeneticmaterialc ouldi mp airt herateofgrowthandt hust hev irulenceofch22.H owever,t hegr owthratei nrichTSBmediumaswellasinminimalM9-
CAAmediumwerecomparablebetweenCH21andch22(Suppleme ntaryFigure6).Moreover,apossiblerelationbetweengenomesizea ndvirulenceisnots upportedbya nalysisoft hegenomesoft heremain ingstrains,whichhavesimilarsizeswithinboththeVIRandNVIRgrou ps.
Adetailedcomparativeanalysisrevealedgeneticdifferencesthat possiblyaffectthevirulencestatusofCH21andch22.Thefirstnotable differencebetweenCH21andch22followstheoveralldistinctionchar acteristicfortheanalyzedVIRandNVIRstrainsandencompasses10 ORFs(Table2)withestablishedrolesinstaphylococcalcolonizationa ndvirulence(Fosteretal.,2014).
Amongothers,a deletiona nda ni nternals topc odonwithintherep eat-
containingfragmentsofclfAandclfBresultinthetruncationofprot einsequencesby113and254 aminoacidresidues,respectively ,inCH22.InthecaseofsdrD,eightsensemutationsandapointmuta tionupstreamofthecodingsequencearedifferentinthestrains.CH 21andch22areindistinguishablewithp opulart ypingmethods,i n cludingM LVF(Sabate ta l.,2003).Significantly,MLVFisbasedo nvirulence-
relatedgenesandthusallowedustoclearlydistinguishvirulent(CH 3,CH5,CH9,CH21,a ndCH23)fromnon-
virulent( ch24,p a 2 ,a ndph2)strains(Polakowskaetal.,2012).T hisstudyshowsthatCH21andch22indeedcontaindifferentallele softhreegenes(clfA,clfB,andsdrD)amongthesevenusedfortypin g;however,thedifferenceswereonlydetectableusingdeepseque ncing.Thisf i ndingc orroboratesmultiplep reviousreportsont h einvolvementoftheseproteinsinstaphylococcalvirulence,where clumpingfactorsa ndotherSdrp roteinsf acilitates uccessfulcolo nization-
mediatinginteractionswithhostproteins,suchasfibrinogen,desm oglein-
1a ndc ytokeratin1 0 (O’Briene ta l.,2002;Walshe ta l.,2 0 0 8 ;W ertheime ta l.,2 0 0 8 ;Askarianeta l.,20 16).Asa na dditionalexa mpledifferentiatingCH21andch22,thegenesencodingpyruvatec arboxylase(pycA)andexotoxinSeN(seN)arepseudogenizedinth eNVIRch22strain(Table2).Interestingly,seNistotallyabsentinal
lotherNVIRstrains.PycAc atalyzest heATP- dependentc arboxylationof
FIGURE5|
ComparisonofwholegenomesequencesoftheCH21andch22strains.Colorbandsr epresentcontinuoussimilarsequencesegmentscharacterizingthetwostrains.Parti cularsegmentsareseparatedbyshortinsertionsorduplicationsunlessindicatedoth erwise.Threelongerduplicationsinthech22genome(170,54,and11kbp)areadditio nallymarkedbyoutwardribbons.Lociofintrachromosomalmobilegeneticelements, suchasprophageϕAvβ,putativeprophageϕch21andpathogenicityislandSaPIAv,a remarked.Thelocidifferentiatingthetwostrainsareidentifiedwithacronymsandcolor edasfollows.Genomics:codingsequencesinblueandpromotersingreen(pseudog enesinsquarebrackets).Proteomics:intracellularinred;extracellularinorange;ands urfaceinmagenta(upregulatedproteinsonly).
pyruvatetooxaloacetate,animportantanapleroticreactionthatpr ovidesintermediatesforthetricarboxylicacidcycle(SauerandEik manns,2005).TheactivityofPycArequiresC-terminaldomain- driventetramerization(XiangandTong,2008).Inch22,ani nternals topc odont runcatesP ycAby1 1 0 a minoacidresidues,mostlikel ydisturbingtetramerizationandthusproteinfunction.Bentonandc olleagues(Bentonetal.,2004)previouslydemonstratedtheimport anceofPycAinvirulencebyshowingthatthepycAmutantwasamo ngthemostseverelyattenuatedmutantsinamurinemodelofsyste micinfection.Theavirulentphenotypeofch22isinlinewiththosest udies.
Thes econddistinctione ncompasses1 2 differencesfoundonly betweenCH21a ndch22,butt hesedifferencesa renotcharacteristic oftheVIRorNVIRgroupsaltogether.Inthisgroup,gamma-
aminobutyratepermease(gabP)isapseudogeneinch22butnotinCH 21,whereaspeptideABCtransporterATP-
bindingprotein(nikE)isapseudogeneinCH21butnotinch22.Allrem ainingdifferencesares ensemutationst hataffecttheaminoacidseq uenceoftheirrespectiveproteins(Table3).Interestingly,amongthe attenuatedmutantsstudiedbyBentonetal.,onemutantcarriedamuta tioningabP,whichcodesfora t ransmembranep roteinc atalyzingt he t ranslocationof4 -aminobutyrate[ GABA;(Marchler-
Bauere ta l.,2 0 1 7)].I nch22
aninternalstopc odontruncatest herespectiveproteinby206amino acidresidues.Thus,togetherwithpycA,t wogenesindependentlyde monstratedasimportanttothevirulenceofS.aureusareswitchedoffi nch22.NikE,pseudogenizedinCH21butnotinch22,i sencodedbyth enikBCDEoperon,whichconstitutesa nickelt ransports ystem.Ther efore,Ni2+uptakeislikelydisturbedinCH21,butnickelionsarerequire dforureaseactivity.A ni ncreasei ne nvironmentalp H duet ot hepr oductionofammoniabyureasewasshowntobeasignificantfactori ns taphylococcalurinaryt racti nfections(GatermannandMarre,1 9 8 9
;G atermanne ta l.,1 9 8 9 ;H irone ta l.,2 0 1 0).Moreover,anincrea seinskinpHduetotheactivityofargininedeiminaseencodedinACM E(Diepetal.,2008)ofMRSAUSA300strainswaslinkedtosuccessf ulcolonizationandconsideredtobeahuman-
hostadaptation(Thurlowetal.,2013).Onecouldt husexpectt hatt he p s eudogenizationofnikEi nCH21shouldattenuatevirulence,which ishowevernotthecase.Instead,weattributenikEpseudogenizationt oanadaptationtoapoultryhost.Itwasshownthatthehuman-to- poultryhostjumpwasassociatedwiththelossofcertaingenesdispen sableforvirulenceinanewhost(suchasspa,encodingIgG- bindingproteinA ) a ndt heacquisitionofothergenes(Lowdere ta l., 2009).SincethepHofchickenskin(pH6.6–
7.2)issignificantlyhighert hant hatofh umans kin( pH5 .5),i ti sreas onablet ospeculatethatanacidicpHisnotasignificantbarrierforpoult rycolonizationbystaphylococci.Hence,itsincreasebyureasewould bedispensable,andassuch,thepseudogenizationofnikEwouldnotm anifestasa na ttenuationoft hev irulenceoft heCH21straininachick enembryoinfectionmodelasfoundinourexperiments.
Thelastgroup,differentiatingCH21andch22,encompasses1 9differencesinthepromoterregions,whichmayinfluenceexpres sionofthedownstreamgenes/proteins.Thedifferencesrangefro msinglenucleotidechanges[e.g.,fibrinogen-
bindingprotein(FnBP),thiaminase(TenA)andSdrD]toentirelydi fferentsequenceswithinthepromoterregionasexemplifiedbycyst eineproteasestaphopainA(ScpA;Table4).
DifferencesinProteomesofCH21andch 22Strains
Genomicc omparisonoft heCH21a ndch22s trainsp rovided certainc luesregardingt hel ikelygeneticdeterminantsofdifferences inthevirulentphenotype.Nevertheless,theinfluenceofgeneticalterat ionsontheoverallproteomicspectrumremainshardtoreliablypredict andthuswastestedexperimentally.Wehavep reviouslyc omparedt heexoproteomesofa numberofVIRandNVIRS.aureusstrainstode monstratetheextracellularfingerprintsofe achp h enotype(Bonare t a l.,2 0 1 6).H ere,wefocusedoncomparingthecellular,cellwall/me mbraneassociatedandsecretedproteomesoftwogeneticallyrelate dstrainsCH21andch22t op i npointt hecharacteristicsdeterminingt heirdifferentvirulencep henotypes.A mongt hec ellularproteins,12 differentiatings potswerei dentifiedascharacteristicofCH21compa redt och22,wherease ights potswerecharacteristicofch22.M S a n alysisi dentified1 0 a ndt hreeuniquep roteinsi n
TABLE2|DifferencesingenesbetweenVIRandNVIRstrainsexemplifiedbydifferencesidentifiedintheVIRCH21andNVIRch22strains.
No. Cl.* Product Acronym CH21loci ch22loci Proteinlengths Differences
1. 188 Hypotheticalprotein(associated BJL64_01345 BJL65_01345 166 161 Mergebetweentwo
withtypeVIIsecretionsystemop eron)
BJL64_01350 —————— 161 —— codingsequencesof
CH21.
2. 438 Serine-aspartaterepeatproteinD SdrD BJL64_02690 BJL65_02680 1,385 1,385 Eightsensemutations,o nepointmutationupstre amofCDS.
3. 670 ClumpingfactorA ClfA BJL64_03875 BJL65_03870 875 762 113aadeletionwithinth
erepeat-
containingfragment.
4. 891 Hypotheticalprotein(associated withbacteriocino peron)
BJL64_05095 [BJL65_05090] 654 —— Internalstopcodoninch2
2.
5. 938 Hypotheticalprotein(lipoprotein) BJL64_05335 [BJL65_05325] 69 —— Internalstopcodoninch2
2.
6. 975 Pyruvatecarboxylase PycA BJL64_05525 [BJL65_05515] 1,150 —— Internalstopcodoninch2
2.
7. 1017 Hypotheticalp rotein BJL64_05735 [BJL65_05725] 55 —— Internalstopcodoninch2
2.
8. 1702 Exotoxin SeN BJL64_09410 [BJL65_10575] 251 —— Internalstopcodoninch2
2.
9. 1767 Celldivisionprotein FtsK BJL64_08675 BJL65_09840 453 453 Fivesensemutationand
13pointmutationsupstr eamofCDS.
10. 2529 ClumpingfactorB ClfB BJL64_13955 [BJL65_15235] 865 —— Internalstopcodoninch2
2.
*Cluster’snumber,ford etailedp r o teins equencesandalignments ee“https://mol058.mol.uj.edu.pl/extra/clusters.htm.”
TABLE3|IdentifieddifferencesingenesexclusivelyfoundbetweentheVIRCH21andNVIRch22strains.
No. Cl.* Product Acronym CH21loci ch22loci Proteinlengths Differences
1. 89 Hypotheticalp rotein BJL64_00820 BJL65_00820 199 199 Sevensensemutationsinahi
ghlyvariableregion.
2. 385 Zincmetalloprotease FtsH BJL64_02360 BJL65_02355 710 697 Onesensemutation.OneSNPres
ultinginaframeshiftattheendofth egene.36terminalaachangedto 23differentaa.
3. 475 NAD(P)H-
dependentoxidoredu ctase
WrbA BJL64_02885 BJL65_02880 178 178 Onesensemutation.
4. 1221 Cholinetransporter BetT BJL64_06800 BJL65_06835 548 548 Onesensemutation.
5. 1368 Hypotheticalp rotein (lipoprotein)
BJL64_07580 BJL65_07610 230 308 Ahighlyvariableregionshorten edby78aainCH21.
6. 1573 Gamma-
aminobutyratepermeas e
GabP BJL64_08625 [BJL65_09790] 453 —— Internalstopcodoninch22.
7. 1700 Hypotheticalp rotein BJL64_09400 BJL65_10565 258 258 Onesensemutation.
8. 1766 AAAfamilyATPase(FtsKo peronassociated)
VirB4 [BJL64_08695] BJL65_09860 —— 832 InternalstopcodoninCH21.
9. 2127 Malonatetransporter YfdV BJL64_11910 BJL65_13180 302 302 Twosensemutations.
10. 2185 Conjugaltransferprotein TpcC BJL64_00290 BJL65_00290 353 353 Foursensemutations.
(FtsKoperonassociated) BJL64_08710 BJL65_09875 358 358 Avariable17aaregioncontaining
15sensemutations.
11. 2356 PeptideABCtransporterA TP-bindingp rotein
NikE [BJL64_13030] BJL65_14305 —— 249 InternalstopcodoninCH21.
12. 2902 Hypotheticalp rotein BJL64_11025 BJL65_12290 30 30 Identicalproteinsequences.
—————— BJL65_12295 —— 30 Tandemduplication.Acopywithon
epointmutation.
*Cluster’snumber,ford etailedp r o teins equencesandalignments ee“https://mol058.mol.uj.edu.pl/extra/clusters.htm.”
September2018|Volume8|Article313
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FrontiersinCellularandInfectionMicrobiology|www.frontiersin.org
TABLE4|IdentifieddifferencesinputativepromoterregionsbetweentheVIRCH21andNVIRch22strains.
No.
Downstreamcodings equence
Acronym CH21loci ch22loci Proteinlengths Differences
1. Ring-cleavingdioxygenase GloA BJL64_01535 BJL65_01530 308 308 OnepointmutationupstreamofCDS.
2. Hypotheticalp rotein BJL64_01915 BJL65_01910 227 227 Completelydifferentsequencesfrom113
bpupstreamofCDS.
3. Serine-
aspartaterepeatprotein D
SdrD BJL64_02690 BJL65_02680 1,385 1,385 OnepointmutationupstreamofCDS.
4. Hypotheticalp rotein BJL64_04510 BJL65_04505 37 37 Completelydifferentsequencesfrom145
bpupstreamofCDS.
5. Hypotheticalp rotein BJL64_05100 BJL65_05095 106 106 Completelydifferentsequencesfrom54
bpupstreamofCDS.Inch22,thepreced ingCDSispseudogenized.
6. Fibrinogen-bindingp rotein FnBP BJL64_05765 BJL65_05755 116 116 Onesinglenucleotidedeletionu pstreamofCDS.
7. Hypotheticalp rotein BJL64_07020 BJL65_07055 55 55 FivepointmutationsupstreamofC
DS.
8. DNA-
bindingresponseregula tor
OmpR BJL64_08605 BJL65_09770 234 234 ThreepointmutationsupstreamofC
DS.
9. Hypotheticalp rotein BJL64_08650 BJL65_09815 120 120 TwopointmutationsupstreamofC
DS.
10. Hypotheticalprotein(FtsKoper onassociated)
BJL64_08655 BJL65_09820 197 197 TwopointmutationsupstreamofC DS.
11. Hypotheticalprotein(FtsKoper onassociated)
BJL64_08670 BJL65_09835 77 77 19pointmutationsandonesinglenucleo tidedeletionupstreamofCDS.
12. Celldivisionprotein FtsK BJL64_08675 BJL65_09840 453 453 13pointmutationsupstreamofCDS.
13. Hypotheticalprotein(FtsKoper onassociated)
BJL64_08680 BJL65_09845 110 110 OnepointmutationupstreamofCDS.
14. Exotoxin SeO BJL64_09435 BJL65_10600 254 254 Fourone-
nucleotidedeletionsupstreamo fCDS.
15. Cysteineprotease (staphopainA)
ScpA BJL64_10140 BJL65_11405 388 388 Completelydifferentsequencesstartin
gfromthefirstbpupstreamofCDSbeca useoftranslocation.
16. Hypotheticalp rotein BJL64_11025 BJL65_12290 30 30 Geneduplication.fourpoint
BJL65_12295 30 mutationsintheregionupto100bpupst
reamofCDSandsubstantialdifference sintheregionfrom101to200bpupstrea mofCDS.
17. ThiaminaseI I TenA BJL64_11050 BJL65_12320 229 229 OnepointmutationupstreamofCDS.
18. Hypotheticalp rotein BJL64_11905 BJL65_13175 43 43 OnepointmutationupstreamofCDS.
19. Hypotheticalp rotein BJL64_13105 BJL65_14385 140 140 Deletionof46bprightupstreamofCD
SinCH21.
therespectivegroupsofdifferentiatingspots(Table5;Figure6;Supp lementaryTable13).
Althoughi ntracellularp roteinsdonotdirectlyi nteractwiththehos t,theysignificantlycontributetowardmaintainingthevirulentphenot ypebymaintainingmetabolisminstressconditions.Proteinscharact erizedbymoreabundantexpressioni nv irulents trainCH21c ompare dt och22i ncludeddiapolycopeneoxygenase( CrtP),whichi si nvolv edi nt hebiosynthesisofstaphyloxanthin.Interestingly,howeverboth s trainsexhibitedt hesamel evelofp i gmentation(SupplementaryF igure5).Thisgoldenpigmenthasbeenlinkedwithstaphylococcalviru lenceasitshieldsthemicrobefromoxidation-
basedclearance,whichistheinnatehostimmuneresponset oi nfecti on(Clauditze ta l.,2 0 0 6).I nterestingly,
ithasbeenshownthatstrainswithgeneknock-
outsinthestaphyloxanthinbiosynthesispathwayexhibitattenuatedv irulence(Liue ta l.,2 0 0 5).Nevertheless,s taphyloxanthinoverex pressiondoesnotdirectlycorrelatewithvirulence.Ithasbeenshownt hatstrainswithelevatedp igmentation(associatedwithmutationsi n genesresponsibleforoxidativephosphorylationandpurinebiosynth esis)werelessvirulentinamurineabscessmodelofinfection.Somest rainswereevenunablet oexhibitl ong-
termc olonization(Lane ta l.,2010).Furthermore,supplementation ofS.argenteuswithagenec lusterresponsiblefors taphyloxanthinp r oductionl edtoi ncreaseds usceptibilityt ot heh ostdefensep e ptide sL L-37andh NP-
1i nvitroa ndreducedv irulencei na nexperimentalrabbite ndocardit ismodel(Xionge ta l.,2 0 1 5).Therefore,CrtP
TABLE5|ListofproteinsdifferentiallyexpressedbyVIRCH21andNVIRch22strainsasidentifiedbyproteomics.
Location Locustag CH21
Locustag ch22
Protein Acronym Clasteroforthologou
sgroups(functionalc ategory)
Elevatedin/
shavedfro mstrain
in BJL64_13565 BJL65_14845 Diapolycopeneoxygenase CrtP 1233(Q) CH21
in BJL64_11045 BJL65_12315 Bifunctionalhydroxymethylpyrimidine kinase/phosphomethylpyrimidinekin ase
ThiD/J 0351(H) CH21
in BJL64_01840 BJL65_01835 NAD(P)-
dependentoxidore ductase
3β-HSD 0702(R) CH21
in BJL64_14305 BJL65_15585 ArylamineN-acetyltransferase NhoA 2162(Q) CH21
in BJL64_11040 BJL65_12310 Hydroxyethylthiazolekinase ThiM 2145(H) CH21
in BJL64_09170 BJL65_10335 Translaldolase TalA 0176(G) CH21
in BJL64_09115 BJL65_10280 Riboflavinsynthasesubunitalpha RibE 0307(H) CH21
in BJL64_00690 BJL65_00690 Hypotheticalp rotein Hyp n/a CH21
in BJL64_01760 BJL65_01755 Peroxiredoxin AhpC 0450(V) CH21
in BJL64_11525 BJL65_12795 Alkalineshockp rotein Asp23 1302(S) CH21
in BJL64_00965 BJL65_00965 FormateC-acetyltransferase PflD 1882(C) ch22
in BJL64_08575 BJL65_09740 TypeI
glyceraldehyde-3- phosphatedehydrogenase
G3p2 0057(G) ch22
in BJL64_12445 BJL65_13715 HeminABCtransporterA
TP-bindingp rotein
HrtA 1136(M) ch22
out BJL64_09350 BJL65_10515 DUF4888d omain-
containingprotein
DUF4888 n/a CH21
out BJL64_02695 BJL65_02685 MSCRAMMfamilyadhesion
SdrE
SdrE 4932(S) ch22
out BJL64_07905 BJL65_09090 Superoxided ismutase SodM1 0605(P) ch22
out BJL64_01760 BJL65_01755 Peroxiredoxin AhpC 0450(V) ch22
out BJL64_03485 BJL65_03480 Glycerolp hosphatelipoteichoica cidsynthase
LtaS 1368(M) ch22
out BJL64_01445 BJL65_01440 Lipase2 Lip2 1075(I) ch22
out BJL64_11645 BJL65_12915 Toxin n/a ch22
out BJL64_14165 BJL65_15445 Lipase1 Lip1 1075(I) ch22
surf BJL64_02580 BJL65_02570 50Sr ibosomalproteinL7/L12 RL7/12 0222(J) CH21
surf BJL64_12760 BJL65_14035 Immunoglobulin-
bindingproteinSbi
Sbi n/a CH21
surf BJL64_14035 BJL65_15315 N-acetylmuramoyl-L-
alanineamidase
Y2979 1705(MN) CH21
surf BJL64_03805 BJL65_03800 Phosphopyruvateh ydratase Eno 0148(G) ch22
surf BJL64_07465 BJL65_08690 DNA-bindingp rotein HU 0776(L) ch22
surf BJL64_11780 BJL65_13050 30SribosomalproteinS5 RS5 0098(J) ch22
surf BJL64_13785 BJL65_15065 Fructosebisphosphatealdolase Alf2 3588(G) ch22
surf BJL64_14045 BJL65_15325 Adhesin(surfaceproteinF) SasF n/a ch22
overexpressioni nCH21maynotnecessarilyrelatet ot hevirulenceoft hisstrain.
Peroxiredoxin,a nothere nzymep rotectingt heseb acteriaag ainstoxidativehostattackbyscavenginghydrogenperoxide,was a lsoupregulatedi nCH21c omparedt och22.Thisf i ndingdemon stratest hegenerali mportanceofa ntioxidativemechanismsi ns t aphylococcalv irulence.Thelevelofa lkalineshockprotein( Asp2 3),whichwaslinkedwithc ellh omeostasisa ndp rotectionoft hec ellenvelopeinnon-
growingcells(Mülleretal.,2014),wasalsoe levatedi nCH21c om paredt och22.I na ddition,
enzymesi mplicatedi nt hes ynthesisofv itamins[ thiamine(hydr oxymethylpyrimidine/phosphomethylpyrimidinek inaseandh yr oxyethylthiazolek inase),a ndriboflavin( riboflavinsynthasesubu nitalpha)]andanenzymeinvolvedinthemetabolismofbileacid[ N AD(P)-
dependentoxidoreductase]werealsoelevatedinthevirulentCH2 1straincomparedtoch22;however,thesignificanceofthisfactinvir ulenceremainselusive.
Proteinscharacterizedbyh igherexpressioni nnon-
virulentstrainch22comparedtovirulentstrainCH21includedtypeIgl yceraldehyde-3-
phosphatedehydrogenase2 ( G3P2),a nisoenzymeofG 3PD- 1i nvolvedi nglycolysis,a ndformate
FIGURE6|2D-
DIGEofcellularproteinsisolatedfromCH21andch22strains.Proteinspotspositively differentiatinginVIRCH21andNVIRch22aremoreintensiveinredandgreencanal,re spectively.Proteinspotswithequalexpressioninbothstrainsareyellow.Identifieddiff erentiatingproteinsaremarkedwithacronyms.
C-
acetyltransferase,whichi si mplicatedi na naerobicglucosemetab olism.H eminA BCt ransporterATP-
bindingp rotein(HrtA)wasalsoelevatedinch22(Figure6).HrtAisres ponsibleforc opingwithh emes tressa ndwasa lreadydemonstratedt onegativelycorrelatewithstaphylococcalvirulence(HammerandSk aar,2011).Thetransporterpreventsheme-
mediatedtoxicity.Ithasbeendemonstratedt hata ni nabilityt oc ope withh emestressparadoxicallyyieldsahypervirulentphenotype(H ammerandSkaar,2011).InhrtAmutants,suchaphenotypewasass ociatedwitht hel ossofmembranei ntegritya ndi ncreasedsecretion ofimmunomodulatoryproteins(Attiaetal.,2010).Theabovef i nding sp ointt ot hel ikelys ignificanceoft heinabilitytoproduceHrtAbyCH2 1inmaintainingthestrainvirulence.
Inconclusion,althoughtheexpressionofonly13intracellularpro teinsiss ignificantlydifferentbetweent heVIRandNVIRstrains,th epatternofexpressionisstrikinglyconsistent.VIRstrainCH21ov er-
expressesproteinsinvolvedincopingwithoxidativestress,which characterizesthehostresponseagainstinvadingpathogens.Inco ntrast,NVIRstrainch22overproducesenzymesa daptingt hec el lt oa naerobicc onditions,whichcharacterizei tsnichesofc omm ensalc oexistencewitht hehost.
TheexoproteomesofstrainsCH21andch22aredominatedbys ta phopainC,a p l asmid-encodedc ysteinep rotease
overproducedbya rangeofp oultry-
derivedS.aureuss trainsthatwaspreviouslydemonstratedtobeafact orunrelatedtovirulence(Bonare ta l.,2 0 1 6).O nlyt hreedifferentia tings potswereidentifiedasoverexpressedwithintheexoproteomeof virulentstrainCH21comparedtoch22,andallthreespotscontainedD UF4888domain-
containingprotein(Table5;SupplementaryTable13).Thisproteino f193residueshasnok nownfunction(Marchler-
Bauere ta l.,2 0 1 7).H omologesareprimarilyfoundinvariousspec iesofstaphylococci,suggestingaspecies-
specificrole,theelucidationofwhichisofsignificanti nteresti nt hel ig htoft hiss tudy.I nterestingly,t heexoproteomeoft hev irulents train CH21i snotcharacterizedbytheoverexpressionofanyknownvirulen cefactorscomparedtononvirulentstrainch22.Evenalpha-
hemolysin,whichwasp reviouslyc onsistentlyfoundt obeoverexpre ssedi nt heexoproteomesofv irulents trainsisolatedfromp oultry,i s notoverproducedbyCH21c omparedt och22(Bonare ta l.,2016).
WithintheexoproteomeofNVIRstrainch22,sevenproteinswer efoundtobeoverexpressedcomparedtoCH21(Table5;Supple mentaryTable13).Themostpronounceddifferencesinexpressi onwerecharacterizedbylipase1(Lip1,foundin15differentiating p roteins pots)a ndl ipoteichoicacids ynthase(LtaS,foundin10di fferentiatingspots).Further,peroxiredoxin(AhpC)wasidentifiedi nthreedifferentiatingspots,whereassuperoxidedismutase1(So dM1)andlipase2(Lip2)werefoundintwodifferentiatingspots.Addi tionalproteinswereidentifiedassingledifferentiatingspots.
Theh igherc omplexityoft heexoproteomeofnonvirulentstrain ch22comparedtovirulentstrainCH21corroboratestheresultsofo urp reviouss tudy,whichdocumenteda generalregularityinthem orecomplexedexoproteomesofNVIRstrainsrelativetoVIRstrain s(Bonaretal.,2016).Moreover,ClustersofOrthologousGroups(
COGs)analysisconfirmedthatproteinsdifferentiatingbothexo- andintracellularproteomesofCH21andch22wereassignedtodiff erentfunctionalcategories(Figure7;SupplementaryFigure4).
Gelproteomicsperformspoorlyincomparingcellmembrane /
c ellwallassociatedp roteomes.Therefore,t oc omparet hesesubset softheproteomesofCH21andch22,weusedthec ell“shaving”appro ach(Solisetal.,2010)coupledtodirect,LC-
MSbasedidentification.Thisapproachenableddirectcomparisons, butunlikesemiquantitativegelproteomics,thedirectapproachisqua litativeonlyandsuffersfroml argevariabilityamongpoolsidentifiedin differentbiologicalrepeats.Assuch,onlyidentificationspresentintw oormoresampleswereconsideredfurther( Table5;S upplementar yTable13).P roteinsi dentifiedexclusivelyont hes urfaceofv irulents trainCH21i ncludeimmunoglobulin-bindingprotein(Sbi)andN- acetylmuramoyl-L-
alanineamidase(Y2979;BJL64_14035).Sbiandstaphylococcalprot einA(SpA)areIgGbinderswithademonstratedroleins taphylococca lv irulence(Gonzaleze tal.,20 15).WhileSpAbindsFcγofI gGs,Sbih ast woI g-
bindingdomainsa ndt wodomainst hatbindt oc omplementc ompon entC3(Zhaoe ta l.,2016).Apriorstudydemonstratedthepseudogen izationofspa,agenee ncodinga majorS.aureusI gG-
bindingp rotein,asa specificadaptationtoapoultryhostsinceSpAisu nabletobind
FIGURE7|
DistributionoffunctionalcategoriesofClustersofOrthologousGroups(COGs)withindifferentiatingproteinsidentifiedduringanalysisofintracellular(in),extracellular(out)andcell membrane/wall-associated(surf)proteome.OnelettercodeoffunctionalcategoriescorrespondstothecodeinCOGsdatabase[https://www.ncbi.nlm.nih.gov/COG/;
(Galperinetal.,2015)].
chickenimmunoglobulins(Lowderetal.,2009).Itistemptingtosp eculatethatSbimayreplaceSpAinpoultrystrains.Sbihasabroad erabilitytobindmammalianIgGthanSpA,butitsabilitytobindavian Igsremainstobetested(Atkinsetal.,2008).AmidaseY2979isconsi stentlyfoundintheexoproteomesofdifferentvirulentS.aureusstr ainsisolatedfrompoultry(Bonaretal.,2016),butitsroleinstaphyl ococcalvirulenceremainsunknown.
SasFa dhesina ndD NA-
bindingp rotein( HU)a redistinguisheda mongp roteinsi dentifiedex clusivelyont hesurfaceofnon-
virulents trainch22.SasFwasi nitiallyidentifiedasa c ellwalla ttache da dhesionp rotein(Rochee ta l.,2 0 0 3),butlater,itwasdemonstrat edthatitprovidesresistancetounsaturatedfreef attyacids,s uchasl i noleicacid(Kennyeta l.,2 0 0 9).I ti st husl ikelyt hatS asFhasa p rot ectiverole,compensatingtheincreasedlipasecontentintheexoprot eomeofch22.Highlipasewouldresultinhighproductionoffreefattyaci ds,whichinturn,couldadverselyaffecttheintegrityofthebacterialce llmembrane(Cadieuxetal.,2014).SasFwouldcounteractsuchadv erseeffects.HUproteinisoneofthemajornucleoid-
associatedproteinsinvolvedinDNAbendingandthusthedeterminat ionofprokaryoticchromosomestructure(Kimetal.,2014a).Italsoact sasatranscriptionalregulatorofgenes,respondingtoanaerobiosis,a cidstress,highosmolarityandSOSinduction(Obertoe ta l.,2 0 0 9).
Abundanti ntracellularp roteinsareoftenconcomitantlypresentatth esurfaceofthesebacteria,andt heyusuallyhavemoonlightingfunctio ns.ExtracellularDNAi sa ni mportantc omponentofb acterialbiofilms ,a ndDNA-
bindingp roteinsothert hanH Ua reassociatedwithbiofilmformation (JooandOtto,2012),suggestingthatHUmayhaveasimilarfunction.
AdvantageousInfluenceof“Individualized”
GenomicsinProteomicIdentifications
Thenumberofs tudiesc ombininggenomica ndp roteomic approachesi srelativelyl imited,butt hec omplexityofh ost- pathogeninteractionscallsforsuchaholisticapproach.Byapplyi ngtheadvantageofthegrowingavailabilityofNGS,t hiss tudy,fort hef i rstt ime,b asedp roteomicidentificationson“individualized”in formationretrievedfromt hegenomesoft hes tudieds trains.M or ei mportantly,however,suchanapproachenabledthecorrelation ofgenomicandp roteomicd ata,p rovidingrationalexplanationsfo rt hevariedexpressionofspecificproteinsdifferentiatingthevirule ntandnonvirulentstrains.Bifunctionalhydroxymethylpyrimidine kinase/phosphomethylpyrimidinekinase(BJL64_11045)andh y droxyethylthiazolek inase( BJL64_11040),p roteinsupregulate di nCH21c omparedt och22,a ree ncodedi na singleoperon(Mül leretal.,2009)togetherwiththethiaminasegene(BJL64_11050,T enA).Genomicsdemonstratesasinglenucleotidep olymorphismi nt hep utativep romoterregionoftheoperoninwhichapolymorphi smmayberesponsiblefordifferentialexpression.Evenmorei nter estingly,i nt hec aseofsuperoxidedismutase(BJL65_09090)and DNA-
bindingprotein(BJL65_08690;HU),whichwereupregulatedinch 22comparedtoCH21,additionalcopies(BJL65_07935andBJL6 5_07500,respectively)oftheencodinggeneswerefoundin1 7 0 a nd5 4 kbpduplications,respectively,explainingt heobservedincre asedexpression.Nevertheless,ofthe28proteinsdifferentiallyexpre ssedinCH21andch22,differencesintheexpressionofonlyt heab ovefourp roteinsc ouldhavebeenexplainedongeneticbasis.This resultdemonstratesthatglobalexpressionregulatorsinfluenceth eproteomemoresignificantly
thana lterationsi np a rticularp romoterregions.Superoxidedismuta ses(SODs)givebacteriaadefensemechanismagainstprofessional phagocytes.Mn-
dependentSOD(SodM1)isspecificforS.aureusandnotfoundincoag ulase-
negativestaphylococci,agroupofstaphylococcithatisgenerallyless pathogenicthantheformerspecies(WrightValderasetal.,2002).Sod M1isgenerallyacytosolicprotein,howeverextracellularSodM1was previouslyidentifiedandimplicatedinbiofilmformation(Atshanetal., 2015).AlthoughCH21andch22areweakbiofilmproducers,thelatteri ndeedformsaslightlyhigherbiofilmwhentestedinvitrothantheforme r(SupplementaryFigure3).Intheabovecontext,itisinterestingtono tethatanotherprimarilyintracellularDNA-
bindingprotein(HU),identifiedintheexoproteomeofch22,maybei nv olvedi nbiofilmformation.ExtracellularD NAi sa nimportantcompon entofbacterialbiofilms,andDNA-
bindingproteinshavebeenassociatedwithbiofilmformation(Jooa nd Otto,2012).Theimportanceofsuchmoonlightingproteinsisshown byt hef actt hats pecificmonoclonala ntibodiesa reablet odisrupta nestablishedbiofilma ndrestoret hea ntibioticsensitivityofreleasedb acteria(Estellésetal.,2016).Nonetheless,itremainstobedetermined whethertheoverexpressionofHUbych22trulyinfluencesbiofilmfor mationandvirulence.
Inconclusion,staphylococcalvirulencehasbeeninvestigatedusi nggenetic,proteomic,biochemicalandmolecularapproaches(Heck ere ta l.,2 0 1 8).I ti sc urrentlywell-
establishedt hatt hevirulentphenotypereliesonmultiplefactorsthata resubjecttof i ne-
tunedregulationa ndacti na h ighlyorchestratedmanner(Thomeret al.,2016).Nevertheless,thecorrespondinginterconnectionsandgu idingprinciplesremainelusive.Here,wedemonstratedt hatt hel evel ofv irulenceofwild-
typeS.aureusstrainsmaybes ignificantlyi nfluencedbyminordiffere ncesi ntheirgeneticmaterial.Performedproteomicsindicatethatcopi ngwithoxidativestressiscrucialforvirulentstrainCH21,whereasbas icmetabolismandnutrientacquisitionarefi ne-tunedinnon- virulentch22.H owever,wea rea lsoa waret hatt hes tudywasperfor medonalimitednumberofstrainsandchangesinp roteomeswereass essedusingi nvitrob acterialc ultures
whichnotoptimallyreflectsinvivoconditions.Nevertheless,wep oint edmutationsi nwild-
typeS.aureuswhichhavebeenpreviouslyshowedt obel inkedwiths t aphylococcalv irulenceusingrecombinantstrains(Bentonetal.,2004 ),whichstrengthenaccuracyofourfindings.Otherresultsarecandidat esforfurtherdetailedstudies.
AUTHORCONTRIBUTIONS
EB,MB,andBWdesignedthestudy.EB,MB,MH,UJ,SK- K,VA,andASperformedtheexperiments.EB,MB,SK-
K,MG,GD,JM,AS,AF,andBWanalyzedandinterpreteddata.EB,MB, GD,andBWwrotet hemanuscript.A lla uthorsrevisedt hemanuscrip tandagreedtobeaccountableforallaspectsoftheworkherein.
FUNDING
Thisresearchwass upportedbyfundsgrantedbytheNationalScienc eCentre(NCN,Poland)onthebasisofdecisionno.DEC-
2012/07/D/NZ2/04282( toBW).P roteomicss tudieswerecarriedout withequipmentpurchasedthroughEuropeanUnionstructuralfunds, grantPOIG.02.01.00-12-
167/08(MalopolskaCentreofBiotechnology).
ACKNOWLEDGMENTS
Wea ret hankfult oI wonaWojcikforh elpi np roteomics.TheFacultyo fBiochemistry,Biophysicsa ndBiotechnologyofJagiellonianUniver sityisapartneroftheLeadingNationalResearchCenter( KNOW),wh ichi ss upportedbyt heM i nistryofScienceandHigherEducation,Wa rsaw,Poland.
SUPPLEMENTARYM ATERIAL
TheSupplementaryMaterialforthisarticlecanbefoundonlinea t:h ttps://www.frontiersin.org/articles/10.3389/fcimb.2018.00313/f ull#supplementary-material
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