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RESEARCHARTICLEOpenAccessIron-sparingResponseofMycobacteriumaviumsubsp.
paratuberculosisisstraindependentHarishKJanagama1,Senthilkumar1,6,JohnPBannantine3,AbiramiKugadas1,PratikJagtap4,LeeAnnHiggins5,BruceAWitthuhn5,SrinandSreevatsan1,2*AbstractBackground:TwogenotypicallyandmicrobiologicallydistinctstrainsofMycobacteriumaviumsubsp.
paratuberculosis(MAP)exist-SandCMAPstrainsthatprimarilyinfectsheepandcattle,respectively.
Concentrationofironinthecultivationmediumhasbeensuggestedasonecontributingfactorfortheobservedmicrobiologicdifferences.
WerecentlydemonstratedthatSstrainshavedefectiveironstoragesystems,leadingustoproposethatthesestrainsmightexperienceirontoxicitywhenexcessironisprovidedinthemedium.
Totestthishypothesis,wecarriedouttranscriptionalandproteomicprofilingoftheseMAPstrainsunderiron-repleteor-depleteconditions.
Results:WefirstcomplementedM.
smegmatisΔideRwithIdeRofCMAPorthatderivedfromSMAPandcomparedtheirtranscriptionprofilesusingM.
smegmatismc2155microarrays.
Inthepresenceofiron,sIdeRrepressedexpressionofbfrAandMAP2073c,aferritindomaincontainingproteinsuggestingthattranscriptionalcontrolofironstoragemaybedefectiveinSstrain.
WenextperformedtranscriptionalandproteomicprofilingofthetwostraintypesofMAPunderiron-depleteand-repleteconditions.
Underiron-repleteconditions,Cstrainupregulatedironstorage(BfrA),virulenceassociated(Esx-5andantigen85complex),andribosomalproteins.
Instrikingcontrast,Sstraindownregulatedtheseproteinsunderiron-repleteconditions.
iTRAQ(isobarictagforrelativeandabsolutequantitation)basedproteinquantitationresultedintheidentificationoffourunannotatedproteins.
TwoofthesewereupregulatedbyaCMAPstraininresponsetoironsupplementation.
Theiron-sparingresponsetoironlimitationwasuniquetotheCstrainasevidencedbyrepressionofnon-essentialironutilizationenzymes(aconitaseandsuccinatedehydrogenase)andupregulationofproteinsofessentialfunction(irontransport,[Fe-S]clusterbiogenesisandcelldivision).
Conclusions:Takentogether,ourstudyrevealedthatCandSstrainsofMAPutilizedivergentmetabolicpathwaystoaccommodateinvitroironstress.
Theknowledgeofthemetabolicpathwaysthesedivergentresponsesplayaroleinareimportantto1)advanceourabilitytoculturethetwodifferentstrainsofMAPefficiently,2)aidindiagnosisandcontrolofJohne'sdisease,and3)advanceourunderstandingofMAPvirulence.
BackgroundMycobacteriumaviumsubsp.
paratuberculosis(MAP),thecausativeagentofJohne'sdisease(JD)ofruminants,oftenrequireseighttosixteenweekstoseecoloniesinculture-amajorhurdleinthediagnosisandthereforeinimplementationofoptimalcontrolmeasures.
Unlikeothermycobacteria,whichmobilizeironviamycobac-tins,MAPisunabletoproducedetectablemycobactininvitroorinvivo[1-3].
Althoughthereasonsforthein:vitromycobactindependencyofMAParecurrentlyunknown,wehaverecentlyshownthatthemycobactin(mbt)operonpromoterisactiveandthatthemycobac-tingenesaretranscribedbyMAPinsidemacrophages[4]andintissuesofnaturallyinfectedanimals(acceptedforpublicationinBMCGenomics).
Pathogenicmycobacteriaencounterawidevarietyofstressorsinsidethehostcellsandtheirabilitytoover-comeirondeprivationandirontoxicityrepresentsamajorvirulencedeterminant[5].
TranscriptandproteinprofilingofMTBandotherpathogensinresponseto*Correspondence:sreev001@umn.
edu1DepartmentofVeterinaryPopulationMedicine,UniversityofMinnesota,SaintPaulMN,USAFulllistofauthorinformationisavailableattheendofthearticleJanagamaetal.
BMCMicrobiology2010,10:268http://www.
biomedcentral.
com/1471-2180/10/2682010Janagamaetal;licenseeBioMedCentralLtd.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(http://creativecommons.
org/licenses/by/2.
0),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
invitroironstressiswelldocumented[6-9].
WhileMAPtranscriptomeorproteomeprofilesinresponsetoheatshock,pH,oxidativestress,hypoxia,andnutrientstarvationhavebeendemonstrated[10-12],stressresponsestoironsupplementationorstarvationarelacking.
Irondependentregulator(IdeR)hasbeenverywellstudiedasaglobalregulatorinvolvedinmaintainingironhomeostasisinMycobacteriumtuberculosis(MTB)[13].
RecentlywehavedemonstratedthatIdeRofMAPinthepresenceofironrecognizesaconsensussequenceonthepromotercalled"ironbox"andregulatesexpres-sionofgenesinvolvedinironacquisition(mbt)andsto-rage(bfrA).
Moreinterestingly,wedemonstratedthatpolymorphismsinthepromoterofironstoragegene(bfrA)inSMAPstrainsrelativetoCMAPstrainsresultsinadifferentialgeneregulation[4].
IdeRdepen-dentrepressionofbfrAinthepresenceofironsuggestsvariationsinironstoragemechanismsand/orironrequirementsincattleandsheepMAPstrains.
Comparativegenomichybridizations,shortsequencerepeatanalysisandsinglenucleotidepolymorphismsofMAPisolatesobtainedfromdiversehostspecieshaveestablishedandindexedgenomicdifferencesbetweenCandSstrainsofMAP[14-19].
PhylogeneticanalysisofsequenceshasidentifiedCandSstrainsasseparatepathogenicclonesthatshareacommonancestor[20-23].
Furthermore,cellularinfectionstudiesshowdis-tinctivephenotypesbetweenthetwoMAPstraintypes[24,25].
WealsorecentlydemonstratedthatSstrainshavedefectiveironstoragesystems,leadingustopro-posethatthesestrainsmightexperienceirontoxicitywhenexcessironisprovidedinthemedium[4].
Takentogether,theliteraturesuggeststhatMAPstrainsvaryintheirirondependentgeneregulation.
Totestthisfurther,weprofiledtheirtranscriptomesandproteomesinresponsetoironanddemonstratedthatironinducedmetabolicpathwaysaresignificantlydiverse.
MethodsBacterialstrains,DNAmanipulationsandmediaMycobacteriumaviumsubsp.
paratuberculosisstrainsMAP1018(CMAP)andMAP7565(SMAP)weregrowninMiddlebrook7H9supplementedwithOADCenrichmentmediumandmycobactinJ(2mg/mL;AlliedMonitor,Fayette,MO).
TotestthehypothesisthatgeneregulationmaybedependentonironavailabilityMAPstrainsweregrowninMiddlebrook7H9mediumwithoutmycobactinJorSautonmedium(0.
5gKH2PO4,0.
5gMgSO4,4.
0gL-asparagine,60mlglycerol,0.
05gferricammoniumcitrate,2.
0gcitricacid,0.
1ml1%(w/v)ZnSO4and2.
5ml20%Tween80in1liter).
GrowthofMAPstrainsintheabsenceofmycobactinJtookover6monthstoprovidesufficientmaterialforproteomicsandtranscrip-tionalprofiling.
Forironrestriction,2,2'-dipyridyl(SigmaAldrich,St.
Louis,MO)wasaddedataconcentrationof200μM.
MAP7565andMAP1018havebeengenotypedbySSRaswellascomparativegenomicsusingoligoarrays.
Theyrepresentthetypicalgenomotypesofsheepandcattlestrains,respectively[18]andshowdistinctphenotypesinbothhumanandbovinemacrophages[24,25].
M.
smegmatis(mc2155)andE.
coliTOP10F(Invitro-genCorporation,Carlsbad,CA)competentcellsweregrowninLuriaBertani(LB)mediumandantibiotics(kanamycin(20μg/ml)orhygromycin(100μg/ml))wereaddedwhennecessary.
TheopenreadingframesofideR(MAP2827)derivedfromCorSMAPstrainswereclonedintopSM417andM.
smegmatisΔideR(SM3)wascomplementedaspreviouslyreported[4].
Briefly,MAP2827fromMAP1018(cideR)orMAP7565(sideR)wasamplifiedviaPCRusingprimersthatcarriedrestric-tionsitesforBamHIandHindIII.
AmplifiedproductsweredoubledigestedwithBamHIandHindIIIandligatedintoapredigested(BamHIandHindIII)expres-sionplasmidpSM417.
AccuracyoftheligationandorientationofMAP2827inpSM417wasverifiedbysequencing.
SM3wastransformedwithpSM417carry-ingMAP2827fromCorSMAPstrains.
Aseedstockfromlogarithmicallygrown(OD600=1.
0)culturesweredilutedtofreshmediumtoyieldanOD600=0.
1.
Theseweregrowninvariousaliquotsunderconstantshaking(120rpm)at37°C.
Thesecul-turesweremonitoredfortheirgrowthatweeklyinter-valsbymeasuringtheirabsorbanceat600nmwavelengthusingSpectraMaxM2(MolecularDevices,Sun-nyvale,CA)untiltheyreachedanabsorbanceof1.
0(Additionalfile1,FigureS1).
Atthispoint,thecultureswerethenpelleted,washedinicecold1XPBSandre-suspendedinfreshculturemedium(withorwithouttheadditionof2,2'-dipyridyl(SigmaAldrich,St.
Louis,MO)).
Dipyridylwasaddedataconcentrationof200μM.
Followingthreehoursofincubationat37°Cunderconstantshaking,cellswerepelletedandwashedwithicecold1XPBSandeitherusedinmicroarraysoriTRAQ.
ThedetailedexperimentaldesignisprovidedasAdditionalfile1,FigureS2.
NucleicacidandproteinextractionLogphaseMAPorM.
smegmatiscultureswerepelleted,washedandre-suspendedinfreshculturemediumwithorwithout200μMof2,2'-dipyridyl.
Thecultureswereincubatedat37°Cwithshakingfor3hr.
immediatelypriortoRNAandproteinextraction.
ForRNA,cellswerehomogenizedinMinibead-beaterfor4min.
byadding0.
3mlof0.
1mmsterileRNase-freezirconiumbeadsfollowedbyextractionusingTrizolJanagamaetal.
BMCMicrobiology2010,10:268http://www.
biomedcentral.
com/1471-2180/10/268Page2of11(Invitrogen,Carlsbad,CA).
AllsamplesweretreatedwithRNase-freeDNaseI(Ambion,Inc.
,Austin,TX)toeliminategenomicDNAcontamination.
ThepurityandyieldoftotalRNAsampleswasconfirmedusingAgilent2100EBioanalyzer(AgilentTechnologies,Inc.
,SantaClara,CA).
RNAwasstoredat-80untilusedinmicro-arraysandrealtimeRT-PCRassays.
Forprotein,cellswerere-suspendedinminimalquan-tity(250μL)ofiTRAQdissolutionbuffer(0.
5MTEABpH8.
5)and0.
1%SDS.
Thesolutionwastransferredtoa2mlscrewcaptubecontaining0.
1mmzirconiumbeads(Biospec)anddisruptedinminibeadbeater(Bios-pec)for4*1minutepulseswithsampleskeptonicebetweenpulses.
Thelysatewasthencentrifugedat12,000*gfor10minutesat4°C.
SupernatantwastransferredtoafreshtubewithoutdisturbingthepelletandusediniTRAQlabelingfordetectionofproteome(Additionalfile1,FigureS3).
MicroarrayexperimentsGeneexpressionprofilingofS(1018)andC(7565)MAPstrainswasperformedusingMAPK-10microar-raysobtainedfromDr.
MichaelPaustian,NADC,IA.
ExpressionprofilingofM.
smegmatisΔideRcomplemen-tedwithcorsideRwascarriedoutusingM.
smegmatismc2155arraysprovidedviaPathogenFunctionalGeno-micsResourceCenter(PFGRC)atJ.
CraigVenterInsti-tute(JCVI).
ArrayhybridizationsandanalyseswereperformedasdescribedpreviouslyandaccordingtotheprotocolsestablishedatPFGRCwithminormodifica-tions[26]andaccordingtoMIAME2.
0guidelines.
Briefly,synthesisoffluorescentlylabeledcDNA(Cya-nine-3orCyanine-5)fromtotalRNAandhybridizationsoflabeledcDNAtoMAPK-10ormc2155oligoarraywasperformed.
Microarrayhybridizationswereper-formedfromcDNAisolatedfromtwoindependentexperiments.
Oneachindependentoccasion,bacterialculturesgrowingunderiron-repleteoriron-limitingmediumwereusedforRNAextractions,cDNAlabelingandarrayhybridizations.
EachslidewascompetitivelyhybridizedwithcDNAobtainedfromiron-replete(labeledwithcy3orcy5)andiron-limitinggrowthmed-ium(counterlabeledwithcy5orcy3)torevealrelativeexpressionaldifferences.
About4μg(2μgeachfromironlimitationorsufficient)ofcDNAwasusedtohybri-dizeontothearray.
However,ifthecDNAyieldislowforasampletheRNAfromthesamesamplewasusedtosynthesizemorecDNA,pooledandlabeledontothearrays.
HybridizedslideswerescannedusingHPScanarray5000(PerkinElmerInc.
,Waltham,MA).
Theimageswereprocessedandnumericaldatawasextractedusingthemicroarrayimageanalysissoftware,BlueFuse(BlueGnomeLtd,Cambridge)andTM4micro-arraysuiteavailablethroughJCVI.
Genesdifferentiallyregulatedatafoldchangeof1.
5orgreaterwereidenti-fiedatafalsediscoveryrateof1%byStatisticalAnalysisofMicroarrays(SAM)program[26].
Genesthatshowedafoldchange1.
5orgreaterinallthereplicatearrayswereretainedandreportedasbeingup-ordownregu-latedinthepresenceofiron.
RealtimeRT-PCRRNAisolatedfromMAPstrainsgrownunderiron-repleteoriron-limitinggrowthmediumwasusedinrealtimeRT-PCRassays.
Geneswereselectedbasedontheirdiverserolesandmicroarrayexpressionpattern.
Selectedgenesincludedsiderophoretransport(MAP2413c,MAP2414c),esx-3secretionsystem(MAP3783,MAP3784),aconitase(MAP1201c),fattyacidmetabo-lism(MAP0150c)andvirulence(MAP0216,MAP3531c,MAP1122andMAP0475).
RNAwastreatedwithDNa-seI(Ambion,Austin,TX)andonestepQ-RTPCRwasperformedusingQuantiFastSYBRGreenmix(Qiagen,Valencia,CA)andgenespecificprimers(Additionalfile1,TableS1)inaLightcycler480(Roche,Indianapolis,IN).
iTRAQexperimentsProteinextractedfromthetwoMAPstrainsgrowniniron-repleteoriron-limitingmediumwasusediniTRAQanalysis(Additionalfile1,FigureS3).
iTRAQlabelingandproteinidentificationwascarriedoutasdescribedpreviouslywithminormodifications[27].
Briefly,celllysatewasquantifiedusingthebicinchoni-nicacid(BCA)proteinassay(Pierce,Rockford,IL)priortotrypsindigestion.
PeptideswerelabeledwithiTRAQreagents(114and115forMAP1018growniniron-repleteandiron-limitingmediumrespectively;116and117forMAP7565growniniron-repleteandiron-limit-ingmediumrespectively)atlysineandarginineaminoterminalgroups.
Thelabeledpeptideswerepooled,driedandre-suspendedin0.
2%formicacid.
There-sus-pendedpeptideswerepassedthroughOasisMCX3CC(60mg)extractioncartridgespermanufacturerrecom-mendations(WatersCorporation,Milford,MA)fordesaltingpriortostrongcationexchange(SCX)fractionation.
ElutedpeptidesweredriedanddissolvedinSCXbuf-ferA(20%v/vACNand5mMKH2PO4pH3.
2,withphosphoricacid)andfractionatedusingapolysulfoethylAcolumn(150mmlength*1.
0mmID,5μmparticles,300poresize)(PolyLCInc.
,Columbia,MD)onamagic2002HPLCsystem(MichromBioResources,Inc.
,Auburn,CA).
Peptideswereelutedbyrunninga0-20%bufferBgradientforgreaterthan55min.
and20%-100%bufferB(20%v/vACN,5mMKH2PO4pH3.
2,500mMKCL)for20min.
atacolumnflowrateof50μl/min.
SeveralfractionswerecollectedatfrequentJanagamaetal.
BMCMicrobiology2010,10:268http://www.
biomedcentral.
com/1471-2180/10/268Page3of11intervalsandsevenfractionsthatshowedmAU280>2wereanalyzedbyLC-MS/MSaspreviouslydescribed[28].
Fractionswerereconstitutedinreversed-phaseloadbuffer(10mMphosphatebuffer)andanalyzedina4800MALDITOF/TOFinstrument(ABSciex,FosterCity,CA).
ProteinpilotSoftware3.
0.
1(ABSciex,Fos-tercity,CA)whichutilizestheparagonscoringalgo-rithm[29]wasusedtoidentifyandquantifytherelativeabundanceofthelabeledpeptides.
Relativeabundanceofproteins(iron-repletev/siron-limitation)foreachMAPstrainwasdeterminedbycomparingthereporterionratios(114/115forCand116/117forSMAP).
iTRAQexperimentswererepeatedontwoindependentexperimentsforeachtreatmentofeachstrain.
WesearchedagainsttheMAPK-10,nonredundant(nr)mycobacteriaproteinsandentirenrproteindatabasedepositedintheNCBIalongwiththecontaminantstoidentifyMAPspecificpeptidesatafalsediscoveryrateof1%.
ResultsTranscriptionalprofilingofMAPIdeRWerecentlycharacterizedMAPIdeRandcomputation-allypredictedthatIdeRinthepresenceofironregulatesexpressionof24genes[4].
Inthecurrentstudy,weidentifiedthat20ofthe24previouslypredictedgenesweredifferentiallyexpressedinresponsetoironbyMAPmicroarrays.
Mycobactinsynthesis,transportandfattyacidbiosynthesisgeneswererepressedinthepre-senceofironbybothcattleandsheepMAPstrains(Additionalfile1,TableS2).
HoweverironstorageandoxidoreductasegeneswereupregulatedinthepresenceofirononlyinCMAP(Additionalfile1,FigureS4).
Wefirstconfirmedifthesedifferencesareduetoreg-ulationviaIdeR.
IdeRisessentialinMAPandattemptstodeletethisgenefailed[26].
WecomplementedM.
smegmatisΔideR(SM3)withCorSstrainideRandcomparedregulationaldifferencesinthepresenceorabsenceofiron.
Genesthatshowedalog2foldchangeof1.
0inSM3orSM3complementedwithemptyplas-mid(negativecontrols)inthepresenceorabsenceofironwhilehavingafoldchange>±1.
5inthecomple-mentedstrains(test)andplasmidcarryingM.
smegmatisideRandmc2155(wildtype)(positivecontrol)werecon-sideredasbeingregulatedbyMAPIdeR.
Fourteenofthe20geneswereregulatedbyIdeRsofbothMAPstrainsinM.
smegmatis.
Furthermore,ourresultssug-gestedthatsIdeRfunctionsbyprimarilyrepressinggenesinthepresenceofironwhereascIdeRfunctionsbothbyrepressingmycobactinsynthesisandde-repres-singironstoragegenesinthepresenceofiron(Addi-tionalfile1,TableS3).
ThesewerefurthervalidatedbyrealtimeRT-PCRinbothM.
smegmatistransformantscarryingMAPideRsandMAPgeneticbackground.
ThedataispresentedonlyforMAP(Additionalfile1,TableS4).
WenextcomparedthetranscriptomeandproteomesofCandSMAPstrainsunderiron-repleteandiron-limitingconditions.
Transcriptomeanalysisundertwoindependentobservationsidentifiedthat94and57geneswereconsistentlyexpressedbytheCandSMAPstrainrespectively.
Similarly,proteomedatarevealedaconsistentexpressionof64and60proteinsbythecattleandsheepMAPstrainsrespectively.
Acomparisonoftheseconsistentlydetectedtranscriptsandproteinsrevealedthat,inthepresenceofiron,onethirdofthedifferentiallyregulatedgenes(P95%confidencewereusedtoquantifytherelativeabundance(iron-repletev/siron-limitation)ofeachprotein.
ApeptidewithnohitsontheMAPgenomebutwithidentitieswithothermycobacterialproteinswasconsideredasunannotatedMAPprotein.
Proteinexpressionunderiron-limitingconditionsConsistentwiththetranscriptionprofile,theCstrainofMAPupregulatedproteinsbelongingtoSUFoperoninvolvedinFe-Sclusterassembly,fattyacidmetabolismandapyruvatedehydrogenase(MAP2307c).
Transporterproteins,twocomponentsystems,andcelldivisionasso-ciatedproteins(MAP1906c,MAP0448andMAP2997c)werealsoupregulatedbytheCstrain(Table1andAdditionalfile1,TableS8).
Thesheepstrainalsoupre-gulatedtransporterproteins,fattyacidbiosynthesis,DNAreplicationprotein(MAP3433),andstressresponseproteins(MAP3831c,MAP2764)(Table2,Additionalfile1,TableS9andFigureS3).
Figure1Transcriptomeandproteomecomparisons:Venndiagramshowingthecomparisonoftranscriptsandproteinsthatweredifferentiallyexpressedatafoldchangeof1.
5orgreaterincattleorsheepMAPstrainsinresponsetoiron.
Onethirdofthegenesdifferentiallyexpressedinresponsetoironwererepresentedinboththetranscriptomeandtheproteome.
Table1TranscriptandproteinexpressionincattleMAPunderiron-limiting(LI)conditionsMAPORFIDPredictedfunctionaFoldchangeProteinTranscriptMetabolismMAP1587calphaamylase2.
03±0.
22.
87±0.
7MAP1554cFadE33_2(acyl-coAsynthase)1.
79±0.
51.
88±0.
8MAP2307cpdhCalpha-ketoaciddehydrogenase1.
68±0.
32.
52±0.
4MAP3189FadE23(acyl-CoAdehydrogenase)2.
41±0.
23.
51±1.
0MAP3694cFadE5(acyl-CoAdehydrogenase)1.
87±0.
83.
15±0.
2CellularprocessesMAP3701cheatshockprotein2.
18±0.
62.
48±0.
3MAP1188FeSassemblyproteinSufD2.
23±1.
02.
73±0.
2MAP1189FeSassemblyATPaseSufC1.
78±0.
52.
03±0.
1MAP4059heatshockproteinHtpX1.
48±0.
11.
66±0.
5PoorlycharacterizedpathwaysMAP1012cpatatin-likephospholipase1.
67±0.
31.
56±0.
3MAP1944cironsuphurclusterbiosynthesis1.
56±0.
91.
66±0.
2MAP2482Glyoxalase/Bleomycinresistance1.
84±0.
32.
19±0.
8MAP3838cRESdomaincontainingprotein1.
50±0.
72.
40±0.
2aMAPoligoarraywasusedtomeasuregeneexpressionwhereasiTRAQwasusedtoquantitateproteinexpressionintheculturesofcattleMAPstraingrowniniron-replete(HI)oriron-limiting(LI)medium.
Foldchangeforeachtargetwascalculatedandrepresentedasalog2ratioofLI/HI.
ShownaretheMAPgenesthatdemonstratedthepresenceof1.
5timesormoreoftranscriptsandproteinsinLIcomparedtoHI.
GenesareannotatedbasedonthemotifsearchesinKEGGdatabase.
Janagamaetal.
BMCMicrobiology2010,10:268http://www.
biomedcentral.
com/1471-2180/10/268Page5of11Theiron-sparingresponsetoironstarvationoccurswhennon-essentialironutilizationproteinssuchasaco-nitaseandsuccinatedehydrogenasesarerepressedandintracellularironisusedtomaintainessentialcellularfunctions[34,35].
Interestingly,duringironlimitation,thecattlestrainbutnotsheepMAPdownregulatedexpressionofaconitase(MAP1201c)andsuccinatedehydrogenases(MAP3697c,MAP3698c)(Figure2).
Repressionofaconitaseinresponsetoironstarvationispost-transcriptionallymediatedviasmallRNAs[36].
Consistentwiththisfinding,ourresultsrevealanupre-gulationofaconitasetranscripts(bothbymicroarrayandQ-RTPCR)withaconcomitantdownregulationattheproteinlevelintheCMAPaloneunderiron-limit-ingconditions.
Proteinexpressionunderiron-repleteconditionsThesheepstrainupregulatedasmanyas13uniquepep-tides(>95%confidence)thatweremappedtoMAP2121c.
ArepresentativepeptidemapisshowninFigure3A.
Interestingly,noneoftheseweredifferentiallyregulatedinresponsetoironbyCstrainofMAP.
MAP2121cwasoriginallydescribedas35-kDaantigenandisanimmune-dominantproteininvolvedinMAPentryintobovineepithelialcells[37,38].
Althoughstatis-ticallynotsignificant,furthermicroarrayanalysisrevealedatwo-foldincreaseofMAP2121cinbothcattleandsheepstrainsunderiron-repleteconditions(datanotshown)suggestingapossibleposttranscriptionalrepressionofMAP2121cbythecattlestrainofMAP.
Asexpected,transcriptsidentifiedasupregulatedunderiron-repleteconditionsinCMAPstrainwerealsoupregulatedintheproteome(Table3,Additionalfile1,TableS10).
Therewasincreasedexpressionoffiveribosomalproteinsandaribosomereleasingfactor(MAP2945c)bycattleMAPunderiron-repletecondi-tions.
Aspreviouslyreported,BfrAwasupregulatedincattleMAP(Figure3B).
Antigen85AandMAP0467c(mycobacterialheme,utilizationanddegrader)werealsoupregulated.
However,MAP0467candotherstressresponseproteinsweredownregulatedintheSMAPstrain(Figure4).
IdentificationofunannotatedMAPproteinsWeidentifiedtwouniquepeptides(SSHTPDSPGQQPPKPTPAGKandTPAPAKE-PAIGFTR)thatoriginatedfromtheunannotatedMAPgenelocatedbetweenMAP0270(fadE36)andMAP0271Table2TranscriptandproteinexpressioninsheepMAPunderiron-limiting(LI)conditionsMAPORFIDPredictedfunctionaFoldchangeProteinTranscriptMetabolismMAP3564methyltransferase1.
54±0.
11.
58±0.
6MAP1942cCbhK,ribokinase1.
74±0.
32.
05±1.
0MAP2286cthioredoxindomaincontainingprotein1.
82±0.
12.
04±0.
3MAP1997acylcarrierprotein1.
90±0.
51.
68±0.
5CellularprocessesMAP4340TrxC,thioredoxin1.
50±0.
42.
29±0.
3MAP3840DnaKmolecularchaperone1.
63±0.
63.
52±0.
5InformationstorageandprocessingMAP4142FusA,elongationfactorG1.
52±0.
22.
58±0.
7MAP4268ctranscriptionalregulatoryprotein1.
52±0.
31.
50±0.
1MAP4233DNA-directedRNApolymerasealphasubunit1.
56±0.
11.
83±0.
3MAP3024cDNAbindingprotein,HU1.
60±0.
61.
81±0.
5MAP418430SribosomalproteinS51.
75±0.
11.
55±0.
3MAP3389cresponseregulator1.
94±0.
31.
59±0.
2MAP4111transcriptionantiterminationprotein,NusG1.
98±0.
31.
82±0.
5MAP4143elongationfactorTu2.
08±0.
42.
16±0.
1PoorlycharacterizedpathwaysMAP2844conservedalanineandargininerichprotein1.
54±0.
22.
27±0.
5MAP3433initiationofDNAreplication1.
63±0.
11.
91±0.
2MAP0126transcriptionalregulatorlikeprotein1.
75±0.
61.
50±0.
2MAP1065pyridoxoxidase1.
83±1.
01.
52±0.
5aMAPoligoarraywasusedtomeasuregeneexpressionwhereasiTRAQwasusedtoquantitateproteinexpressionintheculturesofsheepMAPstraingrowniniron-replete(HI)oriron-limiting(LI)medium.
Foldchangeforeachtargetwascalculatedandrepresentedasalog2ratioofLI/HI.
ShownaretheMAPgenesthatdemonstratedthepresenceof1.
5timesormoreoftranscriptsandproteinsinLIcomparedtoHI.
GenesareannotatedbasedonthemotifsearchesinKEGGdatabase.
Janagamaetal.
BMCMicrobiology2010,10:268http://www.
biomedcentral.
com/1471-2180/10/268Page6of11(ABCtypetransporter).
Wealsoidentifiedtwopeptides(DAVELPFLHKandEYALRPPK)thatdidnotmaptoanyoftheannotatedMAPproteinsbuttotheaminoacidsequenceofMAV_2400.
FurtherexaminationoftheMAPgenomerevealedthatthepeptidesmaptothereversedaminoacidsequenceofMAP1839.
Thesetwouniqueproteinswerenotdifferentiallyregulatedinresponsetoiron.
However,twomoreuniquepeptidesthatweretranslatedfromotherunannotatedMAPgeneswereupregulated(>1.
5fold)underiron-repleteconditionsinCMAPstrain(Figure3B).
DiscussionJohne'sdiseaseisamajoranimalhealthproblemofruminantspeciesworldwideandimposessignificanteconomiclossestotheindustry.
Ourabilitytoculturethecausativeagent–Mycobacteriumaviumsubsp.
para-tuberculosis(MAP)–andthereforeitsrapiddiagnosisandourunderstandingofitsvirulenceislimited.
MAPisdifficulttoculturebecauseofitsunusuallystrictironrequirements.
Foroptimalgrowthinlaboratorymedia,MAPrequiresasiderophore(mycobactin)supplementa-tionthatmakesMAPfastidious[39].
,oftenrequiringeighttosixteenweekstoproducecoloniesinculture-amajorhurdleinthediagnosisandthereforeimplementa-tionofoptimalcontrolmeasures.
Understandingironregulatorynetworksinthepathogeninvitroisthereforeofgreatimportance.
AtaleoftwostraintypesofMAP-AcasetostudyironregulationSeveralmicrobiologicalandgenotypingstudiesandclin-icalobservationssuggestthatJohne'sincertainhostssuchassheep,goats,deer,andbisoniscausedbyadis-tinctsetofstrainsthatshowarelativelyhighdegreeofhostpreference[18,40].
AtleasttwomicrobiologicallydistincttypesofMAPhavebeenrecognized.
Alessreadilycultivabletypeisthecommon,butnotinvari-able,causeofparatuberculosisinsheep(SMAP)[39,41,42],whileanotherreadilycultivabletypeisthemostcommoncauseofthediseaseincattle(CMAP).
CellinfectionstudieshavealsorevealeddistinctivehostresponsephenotypesbetweencattleandsheepMAPstrains-theformerelicitprimarilyapro-inflammatoryTable3TranscriptandproteinexpressionincattleMAPunderiron-replete(HI)conditionsMAPORFIDPredictedfunctionaFoldchangeProteinTranscriptMetabolismMAP0150cFadE25_2(acyl-coAdehydrogenase)1.
72±0.
11.
88±0.
2MAP0789acetyl-CoAacetyltransferase1.
73±0.
31.
56±0.
1MAP1846cATPphosphoribosyltransferase1.
69±0.
23.
68±0.
3MAP2332cFas(fattyacidsynthase)1.
61±0.
52.
28±0.
4MAP3404AccA3(acetyl-/propionyl-coenzymeA)1.
45±0.
12.
18±0.
2MAP3698csuccinatedehydrogenase1.
89±0.
34.
57±0.
5CellularprocessesMAP1339ironregulatedconservedprotein1.
62±0.
20.
78±0.
3MAP1653thiolperoxidase1.
79±0.
52.
29±0.
2InformationstorageandprocessingMAP2907ctranslationinitiationfactorIF-21.
57±0.
21.
89±0.
2MAP2945cribosomereleasingfactor1.
66±0.
32.
11±0.
5MAP411350SribosomalproteinL11.
61±0.
11.
57±0.
2MAP4125rplJ50SribosomalproteinL101.
52±0.
11.
66±0.
5MAP4142fusAelongationfactorG2.
13±0.
43.
05±0.
3MAP4160rpsJ30SribosomalproteinS101.
68±0.
32.
87±0.
4MAP4181rpsH30SribosomalproteinS81.
79±0.
52.
42±0.
1MAP4233rpoADNA-directedRNApolymerase1.
56±0.
11.
65±0.
4PoorlycharacterizedpathwaysMAP0216FbpAantigen85-A1.
87±0.
22.
16±0.
3MAP1122mycobacterialintegrationhostfactor1.
73±0.
32.
00±0.
5aMAPoligoarraywasusedtomeasuregeneexpressionwhereasiTRAQwasusedtoquantitateproteinexpressionintheculturesofcattleMAPstraingrowniniron-replete(HI)oriron-limiting(LI)medium.
Foldchangeforeachtargetwascalculatedandrepresentedasalog2ratioofHI/LI.
ShownaretheMAPgenesthatdemonstratedthepresenceof1.
5timesormoreoftranscriptsandproteinsinHIcomparedtoLI.
GenesareannotatedbasedonthemotifsearchesinKEGGdatabase.
Figure2Repressionofnon-essentialironusingproteinsunderiron-limitingconditionsbycattleMAPstrain:Reporterionregions(114-117m/z)ofpeptidetandemmassspectrumfromiTRAQlabeledpeptidesfromMAP3698c,MAP3697candMAP1201careshown.
Quantitationofpeptidesandinferredproteinsaremadefromrelativepeakareasofreporterions.
PeptidesobtainedfromcattleMAPculturesgrowniniron-repleteandiron-limitingmediumwerelabeledwith114and115reporterions,respectively.
.
PeptidesobtainedfromsheepMAPculturesgrowniniron-repleteandiron-limitingmediumwerelabeledwith116and117reporterions,respectively.
Thepeptidesequencesandshownintheparenthesisandthereddashedlineillustratesthereporterionrelativepeakintensities.
CattlestrainofMAPshowsanironsparingresponsebydownregulatingexpressionofironusingproteins.
Janagamaetal.
BMCMicrobiology2010,10:268http://www.
biomedcentral.
com/1471-2180/10/268Page7of11responsewhilelatterstrainssuppressinflammationandupregulateanti-apoptoticpathways[24,25].
Inaddition,sinceMAPgenomesequencewaspublishedin2005,verylittleresearchhasfocusedonironphysiologyanditscontributiontometabolicnetworksofthisfastidiousorganism.
Basedontheseclassicalmicrobiologic,genotypic,andclinicalobservations,weaddressedthehypothesisthattheirondependentgeneregulationisdifferentbetweencattleandsheepMAPstrainsusingasystemsapproach.
Iron-sparingresponsetoiron-limitationisuniquetocattleMAPstrainIronisacriticalcomponentofseveralmetabolicenzymes[43].
Mostbacteriarespondtoironstarvationwithauniqueregulatorymechanismcalledtheiron-sparingresponse[35].
Iron-sparingisaphysiologicalphenomenonusedbycellstoincreasetheintracellularironpoolbypost-transcriptionallyrepressingthesynth-esisofnon-essentialironusingproteinsandsparingironforessentialcellularfunctions[44].
Therefore,theparadigmistotranscriptionallyupregulateallironuptakesystemswhilerepressingnon-essentialenzymesviapost-transcriptionalregulatorymechanismstosur-viveiron-limitingconditions.
BothMAPstrainsupregu-latedgenesinvolvedinsiderophorebiosynthesis(mbt),abilitytoacquireironfromsynthesizedsiderophores(esx-3),andtotransportironboundsiderophores(irtAB)intothebacterium(Additionalfile1,TableS2).
Furthermore,cattleMAPstrainunderiron-limitingcon-ditionsupregulatedtranscriptionofaconitase(Addi-tionalfile1,TableS4)whiledownregulatingitsproteinexpression(Figure2).
Itislikelythattargetsforpost-transcriptionalrepressionofthesenon-essentialironusingproteinsaremediatedviasmallRNAs[34].
Stu-diestotestthishypothesisinthetwoMAPstraintypesareunderway.
DifferentialmetabolicresponsesofcattleandsheepMAPstrainstoiron-limitationUnderiron-limitingconditionsmostotherbacteriaincludingM.
tuberculosis(MTB)upregulateSUFoperon[26,45].
SUFsynthesizes[Fe-S]clustersandtransportsthemtoiron-sulfurcontainingproteinsinvolvedindiversecellularfunctionssuchasredoxbalanceandgeneregulation[46].
ThisiscriticalbecauseunlikeE.
coli,MTBandMAPgenomesencodeforonlyonesuchsystemtosynthesizeallthe[Fe-S]neededbythecellandfreeironandsulfideatomsaretoxictocells[47].
Ourdatarevealedthatcattlestrain,butnotSstrainFigure3PeptidequantitationofproteinsexpressedbyCandSMAPstrainsunderiron-repleteconditions:Reporterionregions(114-117m/z)ofpeptidetandemmassspectrumfromiTRAQlabeledpeptidesfromthe(A)35-kDamajormembraneprotein(MAP2121c)and(B)BfrA,andtheintergenicregionsofMAP1508-1509andMAP2566-2567c.
Quantitationofpeptidesandinferredproteinsaremadefromrelativepeakareasofreporterions.
Severaluniquepeptides(>95%confidence)weremappedtoeachprotein.
However,onlyonerepresentativepeptideisshownforeachprotein.
PeptidesobtainedfromcattleMAPculturesgrowniniron-repleteandiron-limitingmediumwerelabeledwith114and115reporterions,respectively.
PeptidesobtainedfromsheepMAPculturesgrowniniron-repleteandiron-limitingmediumwerelabeledwith116and117reporterions,respectively.
Thepeptidesequencesandshownintheparenthesisandthereddashedlineillustratesthereporterionrelativepeakintensities.
MAP2121calonewasupregulatedinthesheepMAPstrainunderiron-repleteconditions.
Figure4ProteinsexpressedbytypeIIMAPunderiron-repleteconditions:ProteinsupregulatedincattleMAPstrainwhereasdownregulatedinsheepstraininthepresenceofiron.
Foldchangeforeachtargetiscalculatedandrepresentedasaratioofiron-replete/iron-limitation.
Anegativefoldchangerepresentsrepressionandapositivefoldchangeindicatesde-repressionofthatparticulartargetgeneinthepresenceofiron.
MhuD=mycobacterialhemeutilization,degrader;USP=universalstressprotein;CHP=conservedhypotheticalprotein;MIHF=mycobacterialintegrationhostfactor;CsbD=generalstressresponseproteinJanagamaetal.
BMCMicrobiology2010,10:268http://www.
biomedcentral.
com/1471-2180/10/268Page8of11upregulatedSUFoperonatthetranscriptandproteinlevelunderiron-limitingconditions(Table1).
CattleMAPstrainupregulatedpyruvatedehydrogen-aseoperoninvolvedincatabolismofpropionateakeycomponentoflipidbiosynthesisunderlimitingironcon-ditions[48].
Incontrast,sheepstrainupregulatedisopre-noidsynthesisgenesinvolvedincellwallbiogenesis[49].
Thesheepisolatealsoupregulatedoxidoreductaseandstressresponsesinitstranscriptomeandproteomeduringiron-limitation(Table2).
CarDandtoxin-anti-toxinsystemsprimarilyfunctionduringunfavorableconditionssuchasstarvationoroxidativestressbyarrestingcellgrowth[50,51].
Sheepstrainupregulatedtranscriptsoftoxin-antitoxinsysteminvolvedinarrest-ingcellgrowth,suggestingatrendtowardstringencyresponse(Additionalfile1,TableS6).
Takentogether,ourdatasuggeststhatcattlestrainisabletoefficientlymodulateitsmetabolismduringiron-limitation-prob-ablyasurvivaladvantage.
MAP2325,ahypotheticalproteindeletedinthesheepstrainwasfoundtobeupregulatedunderiron-limitingconditionsbytheCstrain(Additionalfile1,TableS5).
ThisisinterestingbecauseanorthologofMAP2325inMTBcalledenhancedintracellularsurvival(eis)interactswithhostTcells.
StimulationofrecombinantEisfromMTBresultsinincreasedproductionofIL-10anddecreasedproductionofTNF-athuscontributingtomycobacterialsurvivalinsidemacrophages[52].
WehavealsodemonstratedasimilarresultinbovineorhumanmacrophagesstimulatedwithdiverseMAPstrains.
CattlestrainsproducedrelativelymoreIL-10andlessTNF-aandpersistedforlongerperiodsoftimeinsidemacrophages[24,25].
ThereisincreasedproteinsynthesisandturnoverinresponsetoironinMTB[31].
Similarly,weobservedanincreasedexpressionofribosomalproteinsinthetran-scriptomeandproteomeinCMAPunderiron-repleteconditions.
Instrikingcontrast,iron-limitationinducedasimilarthemeinsheepstrain.
Hemedegradationisasignificantphysiologicalphenomenonwhereinpatho-gensrecycleironandgainasurvivaladvantageinsidethehost[53].
RecentlythecrystalstructureofRv3592ofMTBwassolvedanddemonstrateditsabilityashemedegrader[54].
WeobservedanupregulationofMAP0467cprotein(orthologofRv3592)underiron-repleteconditionsinCMAPwhileitwasdownregulatedinthesheepstrain(Figure4).
Similartoourpreviousreports,ironstorageprotein,BfrAwasupregulatedonlyFigure5IrondependentmetabolicprogrammingincattleandsheepMAP:Underiron-repleteconditions,thereisupregulationofribosomalproteins,bacterioferritin,mycobacterialheme,utilizationanddegraderproteinsincattlestrainalone.
Underironlimitingconditions,siderophoresynthesisandtransportgenesareupregulatedinbothcattleIandsheepMAPstrains.
However,underironlimitationthereisdownregulationofaconitase,succinatedehydrogenasesandsuperoxidedismutaseincattleMAPstrainalone.
Thissuggestsaniron-sparingresponseexclusivelyincattlebutnotsheepstrain.
Janagamaetal.
BMCMicrobiology2010,10:268http://www.
biomedcentral.
com/1471-2180/10/268Page9of11byCMAPunderiron-repletion(Figure3)[4].
AlthoughthereasonsfordifferentialironstoragemechanismsinsheepcomparedtocattlestrainsofMAParecurrentlyunknown,differentialroleofferritinsinbacterialpatho-gensisnotuncommon[55].
ConclusionsOurdatarevealedstrikingdifferencesinmetabolicpath-waysusedbycattleandsheepstrainsofMAPtoadapttoironstarvation(Figure5).
WehaveidentifiedandcharacterizedkeyirondependentpathwaysofMAP.
Sinceironmetabolismiscriticalfortheinvivoandinvi-trosurvivalofthebacterium,ourcurrentstudiesareexpectedtoimproveourabilitytoprovidebetterinvitroculturemethodsforMAPandprovideanunderstandingofironregulationasakeyvirulencedeterminantofMAP.
AdditionalmaterialAdditionalfile1:Descriptiveandpathwayanalysisoftranscriptomeandproteomedata.
Thisfilecontainstheexperimentaldesign,additionalmicroarray,proteomicandQ-RTPCRdataalongwithpathwayanalysisofironstressresponseproteinsofCandSMAPstrains.
AcknowledgementsThisworkwassupportedinpartbyaUSDA-NRIgrant(2005-35204-16106)andJohne'sdiseaseIntegratedProgram(USDA-CSREES2008-55620-18710)awardedtoSS.
WewouldliketothankMicrobialandPlantGenomicsInstitute,BiomedicalGenomicsCenterandComputationalGeneticsLaboratoryattheUniversityofMinnesotaforprovidingresourcesandservicestoperformthesestudies.
WewouldalsoliketothankJCVIforprovidingM.
smegmatismicroarrays.
Authordetails1DepartmentofVeterinaryPopulationMedicine,UniversityofMinnesota,SaintPaulMN,USA.
2DepartmentofVeterinaryandBiomedicalSciences,UniversityofMinnesota,SaintPaulMN,USA.
3NationalAnimalDiseaseCenter,AgriculturalResearchService,UnitedStatesDepartmentofAgriculture,Ames,IA,USA.
4MinnesotaSupercomputingInstituteforAdvancedComputationalResearch,UniversityofMinnesota,MinneapolisMN,USA.
5DepartmentofBiochemistry,MolecularBiologyandBiophysics,UniversityofMinnesota,MinneapolisMN,USA.
6DepartmentofAnimalBiotechnology,MadrasVeterinaryCollege,Chennai,India.
Authors'contributionsSSdesignedthestudy.
HKJparticipatedintheexperimentaldesignwithSSandperformedmostoftheexperiments.
SKandAKhelpedinsomeexperiments.
JBPcontributedtonewreagents.
BAWperformedmassspectrometry.
PJandLAHhelpediniTRAQdataanalysis.
HKJandSSanalyzedthedataandwrotethemanuscript.
Allauthorsreadandapprovedthemanuscript.
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paratuberculosisisstraindependent.
BMCMicrobiology201010:268.
SubmityournextmanuscripttoBioMedCentralandtakefulladvantageof:ConvenientonlinesubmissionThoroughpeerreviewNospaceconstraintsorcolorgurechargesImmediatepublicationonacceptanceInclusioninPubMed,CAS,ScopusandGoogleScholarResearchwhichisfreelyavailableforredistributionSubmityourmanuscriptatwww.
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