Structure of the type IV secretion system in different strains of Anaplasma phagocytophilum

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Structure of the type IV secretion system in different strains of Anaplasma phagocytophilum
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Al-Khedery et al. BMC Genomics 2012, 13:678 http://www.biomedcentral.com/1471-2164/13/678
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Barbet, Anthony
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Background: Anaplasma phagocytophilum is an intracellular organism in the Order Rickettsiales that infects diverse animal species and is causing an emerging disease in humans, dogs and horses. Different strains have very different cell tropisms and virulence. For example, in the U.S., strains have been described that infect ruminants but not dogs or rodents. An intriguing question is how the strains of A. phagocytophilum differ and what different genome loci are involved in cell tropisms and/or virulence. Type IV secretion systems (T4SS) are responsible for translocation of substrates across the cell membrane by mechanisms that require contact with the recipient cell. They are especially important in organisms such as the Rickettsiales which require T4SS to aid colonization and survival within both mammalian and tick vector cells. We determined the structure of the T4SS in 7 strains from the U.S. and Europe and revised the sequence of the repetitive virB6 locus of the human HZ strain. Results: Although in all strains the T4SS conforms to the previously described split loci for vir genes, there is great diversity within these loci among strains. This is particularly evident in the virB2 and virB6 which are postulated to encode the secretion channel and proteins exposed on the bacterial surface. VirB6-4 has an unusual highly repetitive structure and can have a molecular weight greater than 500,000. For many of the virs, phylogenetic trees position A. phagocytophilum strains infecting ruminants in the U.S. and Europe distant from strains infecting humans and dogs in the U.S. Conclusions: Our study reveals evidence of gene duplication and considerable diversity of T4SS components in strains infecting different animals. The diversity in virB2 is in both the total number of copies, which varied from 8 to 15 in the herein characterized strains, and in the sequence of each copy. The diversity in virB6 is in the sequence of each of the 4 copies in the single locus and the presence of varying numbers of repetitive units in virB6-3 and virB6-4. These data suggest that the T4SS should be investigated further for a potential role in strain virulence of A.phagocytophilum.
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RESEARCHARTICLEOpenAccessStructureofthetypeIVsecretionsystemin differentstrainsof AnaplasmaphagocytophilumBasimaAl-Khedery1,AnnaMLundgren1,SnorreStuen2,ErikGGranquist2,UlrikeGMunderloh3,CurtisMNelson3, ARickAlleman4,SumanMMahan5andAnthonyFBarbet1*AbstractBackground: Anaplasmaphagocytophilum isanintracellularorganismintheOrder Rickettsiales thatinfectsdiverse animalspeciesandiscausinganemergingdiseaseinhumans,dogsandhorses.Differentstrainshaveverydifferent celltropismsandvirulence.Forexample,intheU.S.,strainshavebeendescribedthatinfectruminantsbutnotdogs orrodents.Anintriguingquestionishowthestrainsof A phagocytophilum differandwhatdifferentgenomeloci areinvolvedincelltropismsand/orvirulence.TypeIVsecretionsystems(T4SS)areresponsiblefortranslocationof substratesacrossthecellmembranebymechanismsthatrequirecontactwiththerecipientcell.Theyareespecially importantinorganismssuchasthe Rickettsiales whichrequireT4SStoaidcolonizationandsurvivalwithinboth mammalianandtickvectorcells.WedeterminedthestructureoftheT4SSin7strainsfromtheU.S.andEurope andrevisedthesequenceoftherepetitive virB6 locusofthehumanHZstrain. Results: AlthoughinallstrainstheT4SSconformstothepreviouslydescribedsplitlocifor vir genes,thereisgreat diversitywithintheselociamongstrains.Thisisparticularlyevidentinthe virB2 and virB6 whicharepostulatedto encodethesecretionchannelandproteinsexposedonthebacterialsurface. VirB6 4 hasanunusualhighly repetitivestructureandcanhaveamolecularweightgreaterthan500,000.Formanyofthe virs ,phylogenetictrees position A phagocytophilum strainsinfectingruminantsintheU.S.andEuropedistantfromstrainsinfecting humansanddogsintheU.S. Conclusions: OurstudyrevealsevidenceofgeneduplicationandconsiderablediversityofT4SScomponentsin strainsinfectingdifferentanimals.Thediversityin virB2 isinboththetotalnumberofcopies,whichvariedfrom8 to15inthehereincharacterizedstrains,andinthesequenceofeachcopy.Thediversityin virB6 isinthesequence ofeachofthe4copiesinthesinglelocusandthepresenceofvaryingnumbersofrepetitiveunitsin virB6 3 and virB6 4 .ThesedatasuggestthattheT4SSshouldbeinvestigatedfurtherforapotentialroleinstrainvirulenceof A phagocytophilum Keywords: Anaplasma phagocytophilum Rickettsiales ,T4SS,ComparativegenomicsBackgroundAnaplasmaphagocytophilum isatick-bornepathogenin theOrder Rickettsiales thatisincreasinglyrecognizedas acauseofdiseaseinhumansandanimalsworld-wide [1,2].Itcausesthepotentiallyfataldiseaseofhuman granulocyticanaplasmosis,whichtypicallymanifestsasa flu-likeillnessaccompaniedbyleukopenia,thrombocytopeniaandanemia.Itwasinitiallyrecognizedinthe early1990'swhenpatientsfromWisconsinandMinnesota developedfebrileillnessfollowingatickbite[3].Sincethat timethenumberofhumancaseshasincreasedannually; between2000and2007thereportedincidenceintheU.S. increasedfrom1.4to3.0cases/millionpersons/year[4]. Thecasefatalityratewas0.6%andthehospitalization ratewas36%.InMassachusettsduringthe2009transmissionseasontherewere33confirmedcaseswith14 (42%)requiringhospitalization[5].Thehumandiseaseis alsopresentinEuropeandAsia[2].Arecentstudyof83 A phagocytophilum -infectedpatientsinChinareported amortalityrateinthiscohortof26.5%[6].IntheU.S., therehasbeenaparallelincreaseincasesofthedisease [7]andseroprevalence[8]indogsintheeasternand *Correspondence: barbet@ufl.edu1DepartmentofInfectiousDiseasesandPathology,CollegeofVeterinary Medicine,UniversityofFlorida,Gainesville,FL,USA Fulllistofauthorinformationisavailableattheendofthearticle 2012Al-Khederyetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsofthe CreativeCommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse, distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.Al-Khedery etal.BMCGenomics 2012, 13 :678 http://www.biomedcentral.com/1471-2164/13/678

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upperMidwesternstates.ThetickvectorsintheU.S. are Ixodesscapularis and Ixodespacificus andwild rodentsarethemainreservoirsofhumaninfections. A phagocytophilum alsoinfectsnumerousothermammalianspeciesincludingruminants,horses,cats,and bearsandthesymptomsareextremelyvariable,with somemammalianspeciesexhibitingacutediseaseand othersonlypersistentasymptomaticinfections[9,10]. Forexample, A phagocytophilum strainsisolatedfrom deerintheU.S.canhaveaslightlydifferent16S rRNAsequenceandbeuninfectivetomiceanditis thought,humans[11-13].InEurope,thisagenthasbeen knowntocausediseaseofruminantsfor>100years,yet therehavebeenfewhumaninfections[14].Thegenome sequenceisavailableforasinglestrainof A phagocytophilum derivedfromaninfectedhumanintheU.S.and itisapparentthat,althoughthisstrainlacksTypeII,III, VandVIsecretionsystems,aTypeIVsecretionsystem (T4SS)ispresent[15].Asinothermembersofthe Rickettsiales ,theT4SSof A phagocytophilum isorganized differentlyfrommostgram-negativebacteriawiththe component vir genesdistributedbetweenthreemajor genomelocations[16]. TheT4SStypicallyencodesamembrane-spanning multiproteincomplexthatformsatransmembrane channelthroughwhichsolutescanpassintohostcells. ItcanmediatetransferofDNAandproteinsinto eukaryotichostcells,interferewithhostsignaling,andis essentialforthesurvivalofintracellularbacteria[17].In A phagocytophilum ,whichpreferentiallycolonizeneutrophilicwhitebloodcells,itisthoughtthattheT4SS secretesvirulencefactorsthatareresponsibleforsubvertinginnateimmunityandinhibitinghostcellapoptosis[16].Interestingly,thereappearstobedifferential transcriptionoftheT4SSinticksandinthemammalian hostwith virB6 and virB9 upregulatedduringinfection ofhumanneutrophilsanddifferent virB2 paralogs expressedinmammalianandtickcells[18].ThereisevidencethatVirB2,VirB6andVirB9areexposedonthe outermembranesurfaceinthe Rickettsiales [18-20], whichhasstimulatedinterestintheirpotentialuseas vaccinecandidates.Thispossibilityhasbeeninvestigated moreextensivelyintherelatedorganism Anaplasma marginale [21-25].In A marginale ,unlikemanyother surface-exposedproteins,theT4SSproteinsareconservedbetweenstrains[26].Also,cattleimmunizedwith outermembranesandprotectedagainstchallengeinfectionrespondwithIgGandTcellstoVirproteins,notablyVirB2,VirB9andVirB10.Todate,onlytwoT4SS substrateshavebeenidentifiedandpartiallycharacterized in A phagocytophilum :theankyrinrepeatdomaincontainingprotein,AnkA,andthe Anaplasma translocatedsubstrate1,Ats-1.AnkAtranslocatestothehost nucleusandinteractswithDNA[27,28],whileAts-1is importedintothemitochondriawhereitisproposedto interferewiththeinductionofapoptosis[29]. Inthisstudy,wecomparedthestructureanddiversity oftheT4SSindifferentstrainsof A phagocytophilum infectinghumans,dogs,rodentsandruminants.Most diversitywasfoundintheproteinsthoughttobesurfaceexposed,whichmaybeassociatedwiththedifferent virulenceandcellinvasionpropertiesofthisspecies.ResultsanddiscussionThe vir lociweresequencedineightstrainsof A phagocytophilum ;sevenofthesewerestrainsforwhichpreviousstructuralinformationwasnotavailableand includedorganismsoriginallyisolatedfromU.S.dogs ( Ap Dog1, Ap Dog2),arodent( Ap JM),ahorse( Ap MRK), theruminant Ap variant1strain( Ap Var1)andtwo strainsfromNorwegiansheep( Ap NorV1, Ap NorV2). ThehumanHZstrainwasalsoresequenced,asoptical mappinghadsuggestedapossibleerrorinthepreviously sequenced virB6 4 locus.Thedataindicatedconsiderablediversityintheindividual vir locibetweenstrains thatwillbediscussedbelow.Inallstrains,however,as notedpreviously[20,30],the vir lociweredistributed mainlyinthreegeneclusterscomprising: virB8 1 virB9 1 virB10 virB11 and virD4 ; virB2 0 sand virB4 2 ;and virB3 virB4 1 ,andthefour virB6 paralogs(Figure1). Thesethreelocimayeachbetranscribedpolycistronically[31],althoughitisclearthatT4SSstructureinthe Rickettsiales isuniqueandmorecomplexthaninitially thought.Thenumberof virB2 paralogswasdifferentbetweenstrainswiththehumanHZstrainhavingtheleast (8totalparalogs)andtheruminantstrainshavingthe most(upto15totalparalogs).Thedescriptionofthe T4SScomponentspresentedherefollowsthefunctional classificationdescribedbyAlvarez-MartinezandChristie [20].Innermembranechannel/scaffoldsubunits:VirB3,VirB6, VirB8andVirB10ThemostconservedofthesesubunitsareVirB3,VirB8 andVirB10,withfewdifferencesbetweenstrains.VirB3 hasbeenlinkedin Agrobacteriumtumefaciens withpilus assemblyandsubstratetranslocation[32,33].Itisabsolutelyconservedbetweenstrainswithnoaminoacid changesandconformstothetypicalVirB3structure. Twoalpha-helicaldomainsforinsertionintothecytoplasmicmembranearestronglypredictedbyTMpred. VirB8,proposedtofunctionasanucleationfactorduringtheassemblyofT4SS[34,35],isalsowellconserved, particularlyVirB8-1inthepolycistronictranscription locus(oneaminoacidchangebetweenallstrains). VirB10,proposedtofunctionasascaffoldacrossthe entirecellenvelope[36],isalsogenerallywellconservedwiththeexceptionofoneruminantstrain,Al-Khedery etal.BMCGenomics 2012, 13 :678 Page2of15 http://www.biomedcentral.com/1471-2164/13/678

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Ap NorLamb-V1,whichhas31aminoacidsubstitutions withrespectto Ap HZ(datanotshown).However,all A phagocytophilum VirB100s,including Ap NorLamb-V1, havetwostronglypredictedtransmembranedomains, whichsupportstheirfunctionasmembranescaffolding subunitsintheseorganisms. Oftheseinnermembranechannelsubunits,thedata onVirB6arethemostinteresting.AllVirB6subunits thathavebeendescribedpossessahighlyhydrophobic membranedomainincludingfiveormorepredicted transmembranedomains[20].SomeVirB6proteinsalso haveanextendedC-terminalhydrophilicdomainthat hasbeenproposedtoprotrudethroughtheT4SSinto thetargetcell,ormaybeproteolyticallyreleasedfrom theN-terminaldomainandthentranslocatedintothe targetcell.Evidencehasbeenobtainedforsurface virB9 -2 v i rB 3 v i rB 4 1 virB6 -1 v i rB 6 -2 virB6 -3 v i rB 6 4 virB4 t v i rB8 -2 virB 7 virB4 -2 virB2 -8 virD4 v i rB9 -1 v i rB 8 1 v i rB 6-t virB4 t additionalvirB4-t in ApNorLamb-V1 and -V2: v i rB2 -7 v i rB 2 6 v i rB 2 5 v i rB 2 -4 v i rB2 -3 v i rB 2 2 v i rB 2 1 oriC v i rB11 v i rB10 B2-8B2-7B2-6B2-5 B2-9 B2-4B2-3B2-2B2-1ApHzApJMApDog1ApDog2ApMRKApNorLamb-V2ApVar-1ApNorLamb-V1 ? ? 1kb 1kb Figure1 Distributionandcontentof vir geneclustersineightdiverse A.phagocytophilum strains. Toppanel.Schematicrepresentationof all vir loci(coloredarrows)showingthethreeconservedgeneclusterislands(seetext). VirB 7 virB8 2 and virB9 2 arenotpartof vir geneclusters, buttheirlocationrelativetosurroundinggenesisalsohighlyconservedamongstrains.Asmallclustercomprisingtruncated(t) virB6 and virB4 genefragmentsispresentinallstrains,buttheNorwegianlambstrainshaveoneadditional virB4 t .Bottompanel.Magnificationofthe virB2 gene cluster.Numberingofparalogs1 – 8isbasedontheoriginal Ap HZannotatedgenome(GenBankCP000235).Artificialgaps(stippledlines)were introducedtoallowalignmentofthemorespatiallyconservedparalogs B2 1 2 – 2 and 2 – 3 atoneend,and B2 7 and 2 – 8 attheotherendofthe cluster.Withtheexceptionof virB2 9 ,lackingin Ap HZ,thenumberandarrangement(butnotnecessarilysequence)of virB2 genesishighly conservedinallbuttheUSruminant Ap Var-1and Ap NorLamb-V1,whichhaveseveraladditional virB2 genes.Inbothstrainsasub-clusterof6 distinctgeneswaspresent.Duetotherepetitivenatureofsequencesinthisregion,combinedwiththerelativelyshortlengthof454reads ( 550bp),theirplacementcouldnotbeconfidentlyascertained(highlightedbyarrowsand ‘ ? ’ ).Mapsaredrawntoscale.Doublelinesdesignate interruptioninsequences.Genesbelongingtothesamegroupinghavethesamecolor. oriC ;originofreplication. Figure2 PhylogenetictreetoshowtherelationshipofsyntenicVirB6proteinsfromdifferentstrainsof A phagocytophilum Ascalebar isshownunderneathrepresentingthenumberofaminoacidsubstitutions/site. Al-Khedery etal.BMCGenomics 2012, 13 :678 Page3of15 http://www.biomedcentral.com/1471-2164/13/678

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exposureofextendedVirB6insome Rickettsiales [37]. Ofallthemembranechannelsubunits,themostsequencediversitybetween A phagocytophilum strains wasinthefourVirB6paralogs(Figure1).Although therewerenoaminoacidchangesintheVirB6-1,VirB62andVirB6-3paralogsbetweenhuman,dogandrodent strains,theruminantandhorsestrainshadnumerous substitutionsthroughouteachmolecule,agreeingwith thecloserevolutionaryrelationshipbetweenstrains infectinghumansanddogsintheU.S.(Figure2).Furthermore,majordifferencesinrepeatnumberandsequencewerefoundintheC-terminalrepeatregionof VirB6-3(yellowboxesinFigure3AandAdditionalfile1: FigureS1)inruminantandhorsestrains,withthehorse strainshowingtheleastvariabilityfrom Ap HZ. Theonlyaminoaciddifferencesdetectedbetweenthe human,dogandrodentstrainswereintheVirB6-4subunit. VirB6 4 inthesestrainscontainsfourrepeat regions(R1-R4inFigure3A)andvariabilityinrepeat number,orderandsequencewerefoundmainlyinR3 andR4(Additionalfile2:FigureS2).WithinR1 (Figure3A),theonlydifferencedetectedwasin Ap Dog2 whichhad4and1partialof231bprepeatunits(data notshown),comparedto3and1partialrepeatsinthe Ap Dog1, Ap JMand Ap HZ virB6 4 R1.Opticalmapping oftheDog1genomeandcomparisonwith Ap HZsuggestedthatthesequenceobtainedpreviouslyforthe humanHZstrain virB6 4 wasincorrect(Figure3B).This wasconfirmedbyPCRandsequencing,andmapped specificallytothe30-mostR4region(Figure3C).Becauseofitssizeandunusualcompositionitwasonly possibletoresolvethissequenceusingthelongreadlengthPacificBiosciencestechnology(seeMethods). Thecorrected virB6 4 R4of Ap HZ,totaling6.89kb,differedfromtheoriginalby5.88kbofadditionalsequence composedexclusivelyof84bp[type1,aandb(T1a, T1b);light/darkblueboxes,respectively,inFigure3A] and162bp[type2,aandb(T2a,T2b);light/darkorangeboxes,respectively,inFigure3A]repeatunits,givingacomplexrepeatstructurecontaining53and1 4 6 B r i v 3 6 B r i v 2.7kb 1.15kb virB6-4 inCP000235 R1 R2 4 R 3 R AB1393 AB1395 AB1466 AB1466Bam HI: 1kbCP000235 CP000235 corrected Dog1 optical map Dog1genome sequence 1kb kb 12 10 9 8 7 6 5 kb 48.5 24.5 17.0 13.8 12.1 10.2Ap Hz Ap Dog1 Ap Dog2 Ap JM Ap MRK Ap Var-1 Ap NorLamb -V2virB6-4 2.32 4.81 4.30 3.61 3.19 2.23 2.11 3.40 4.22 4.36 2.46 4.90 16.637.2516.687.38 4 11 Ap Hz Ap Dog1 Ap JM AB1395/1466:R4 AB1393/1466:R3 R4JMgenome sequence B A C R4:811p R4:531p R4:81p + + + + Figure3 The30endof A.phagocytophilumvirB6 4 genesiscomposedofanunusuallylargetandemrepeatregion,whichexhibits dramaticvariabilityamongstrains.A .MapofthehumanHZstrain virB6 3 and virB6 4 genes,highlightingthelocationandstructureofseveral repeatregions(R1-R4).ThemostvariabilityoccurredinR4;thisregionis5.88kblargerthanpreviouslyreportedforthe Ap HZgenome (CP000235).Theoriginalsequenceisdiagrammedabovethemap,withthedashedlinerepresentingthesegmentmissinginCP000235.Larger repeatedR4segmentsof2.7kband1.15kbareindicatedabove.Verticalblackbarswithineachgenedesignatesegmentsencodingpredicted transmembranedomains.BamHIsites,ofwhichthereisoneinallR4type2repeats(seeFigureS2B),areindicated.Alsoshownarethepositions ofPCRprimersusedinC. B .BamHIgenomicmapsdepictingthe virB6 4 locus(blackarrows).ThesegmentencompassingR4ishighlightedbelow eachrespectivemap.Intheregionsoutsidethe virB6 4 locus,correspondingBamHIfragmentsareshowninthesamecolor.Overall,theoptical mapsizeswereingoodagreementwiththeactualsizes,exceptwithinR4.Thisisattributedtothelimitationofopticalmappinginresolving fragments<2kb.Despitethesediscrepancies,thecumulativesizeofthegenomicregionencompassing virB6 4 intheopticalmapisinclose agreementwiththatinthe Ap Dog1genomesequence. C .ThevariabilityinsizeofPCRproductsspanning virB6 4 repeatregionsR4andR3/R4in diverse A phagocytophilum strains. Al-Khedery etal.BMCGenomics 2012, 13 :678 Page4of15 http://www.biomedcentral.com/1471-2164/13/678

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partialrepeatunitscomparedto8and1partialinthe originalsequence.Further,the50-and30-most2.7kbof thiscomplexstructureareidenticalinsequence,andthe 30-most1.15kbofeachofthesesegmentsisrepeated againinthecenterofR4(Figure3AandAdditionalfile 2:FigureS2).Althoughthepossibilityexiststhatthe Ap HZpopulationfromwhichweisolatedgDNAdiffers withinthe virB6 4 R3/R4repeatregionsfromthepopulationusedtogenerateCP000235,thefactthatall strainsinvestigatedhereinpresentedexpansiveR3/R4 regions(Figure3C)wouldcontradictthat.Instead,itis moreplausiblethattheexistenceof2.7kbofidentical repeatsattheendsofthe Ap HZR4mayhaveleadto theexcisionofmostofitssequenceduringconstruction/propagationofthoselibraries.Interestingly, virB6 4 R3andR4wereidenticalbothinsizeandsequencein theDog1androdentstrainsdespitedifferingmarkedly fromtheHZandDog2strainregions(Additionalfile2: FigureS2A).WithinR3,thesestrainshad2additional 405bprepeatscomparedto Ap HZandonemorecomparedtotheDog2strain.However,differencesbetween strainsweremostdramaticwithinR4.Notonlywasthis regionin Ap Dog1/ Ap JM2.87kblargerthanin Ap HZ bringingthetotalnumberofrepeatsto81and1partial, butintriguingly,therepeatpatternwascompletelyunrelatedtothatintheHZstrain.Also,theDog1androdent strainR4lackedT1brepeatunits,whilehavingathird type2repeatvariant,namelyT2c,whichdifferedfrom T2bby1SNPanda12bpdeletion(Additionalfile2: FigureS2).Partialanalysisofthe Ap Dog2454reads spanningR4(estimatedat~8kbbyPCR;Figure3C) showedthattheorderofthe50-and30-mostthreerepeat unitsdifferedfromeithertheHZorDog1/rodentstrain R4repeatpatterns(Additionalfile2:FigureS2A).Notably,ourpreliminaryanalysesofthehorseandruminant 454readssuggesttheabsenceofdistinctR3andR4 regionsin virB6 4 inthesestrains.Rather,thefewrepeat unitsidentifiedtodateappeartobeacombinationofR3 andR4repeats(datanotshown).Itisalsounclearifthe ~17kband~25kbPCRproductsgeneratedwithprimersAB1393/1466in Ap Var-1and Ap NorLamb-V2,respectively(Figure3C),arecomposedmainlyofrepeats, oralternativelyifafifth virB 6 geneparalogexistsin thesestrains.Takentogether,thedatapresentedhere clearlydemonstratetheextremevariabilityoftheT4SS VirB6-4subunitamong A phagocytophilum strains.Althoughthedifferencesbetweenthemorecloselyrelated human,dogandrodentUSstrainsweremainlywithin repeat-ladenregions,thefactthatanextensive,distinct repeatpatternwasmaintainedintwostrainswould speakagainstthepossibilitythatthevariabilitymaybe attributedsolelytothehighlyrecombinogenicnatureof suchstructures.Worthnoting,CampRipley,wherethe infectedjumpingmousewascaptured(2001)isonly~20 milesawayfromthecityofBaxter,MN,whereDog1 resides.Althoughtherearenorecordsofwherethisdog mayhaveactuallyacquiredtheinfection,itpresented withsevereclinicaldiseasein2007. Theunusualstructureandlikelyantigenicityofthe C-terminalregionofthe A phagocytophilum VirB6-40sis apparentinhydrophobicityplots(Figure4).Whatspecificpropertiesthesedistinctrepeatpatternsmayconfer ontoeachstrainawaitsfunctionalanalysisoftheseproteinsin A phagocytophilum .ThecorrectedVirB6-4 translatedproteinhadapredictedmolecularweightof 470,695Dacontaining4,322aminoacidresiduescomparedtomolecularweightsof90,742,103,204and 158,321DafortheHZstrainVirB6-1,VirB6-2and VirB6-3,respectively.Interestingly,thepredictedacidity oftheVirB60salsoincreasedfromVirB6-1toVirB6-4 (pI ’ sof8.4,6.8,5.1and4.0forthe Ap HZVirB6-1,VirB62,VirB6-3andVirB6-4,respectively).The Ap Dog1/ Ap JM VirB6-4polypeptideshadapredictedmolecularweight of603,529Dacontaining5,550aminoacids,andapIof 3.96.Despitethesedissimilarities,atleasteighttransmembranesegmentswerepredictedforallVirB6 paralogs.Periplasmic/outermembranechannelsubunits:VirB2, VirB7andVirB9SeveralotherT4SSsubunitscontributetothesecretion channelacrosstheperiplasmandoutermembrane. VirB7subunitsaretypicallysmalllipoproteinsthatmay stabilizeVirB9[38,39].In A phagocytophilum strainsa putativeVirB7isabsolutelyconservedbetweenstrains andmaybelipidmodifiedthroughanN-terminalcysteineonthematuremolecule.VirB9ishydrophilicand alsolocalizestotheperiplasmandoutermembrane.In A tumefaciens theC-terminalregionofVirB9ispartof theoutermembraneproteinchannelandissurfaceaccessible[40].Thereisalsoevidenceforsurfaceexposure ofVirB9in Ehrlichiachaffeensis and A phagocytophilum [18,19,41].VirB9-1,whichisencodedonthepolycistronic virB8 1 virD4 transcript[31],hasastronglypredictedsignalpeptideandtwotransmembranehelices.Of allthepotentiallyexposedcomponentsoftheT4SS, VirB9of A phagocytophilum appearstobetheleastdiverseamongstrains.Therearesomeaminoacidsubstitutionsinruminantandhorsestrains(2 – 6total comparedto Ap HZ)butintheotherstrainsVirB90sare unchanged(datanotshown). UnlikeVirB90s,VirB20sarethemostdiverseofall T4SSsubunitsin A phagocytophilum ,intermsofboth copynumberandsequence.VirB2proteinsaretypically constituentsofpiliandofthesecretionchanneland theirdiversityin Anaplasma suggeststhepossibilityof exposed,antigenicallyvariablestructures.In A marginale ,VirB2isexpressedtogetherwiththemajorouterAl-Khedery etal.BMCGenomics 2012, 13 :678 Page5of15 http://www.biomedcentral.com/1471-2164/13/678

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membraneproteinMSP3onasequence-variablepolycistronictranscript[25,42].Themechanismofexpressionin A phagocytophilum isnotknown.VirB20sof othergeneraaretypicallysmallhydrophobicproteins withalongsignalpeptidesequenceandtwohydrophobicalphahelicesforintegrationintothecytoplasmic membrane.Thisalsoappearstobethecasefor A phagocytophilum .TheVirB2paralogsinthedifferent strainsarepredictedtohavetwohydrophobicalphahelicesoflengths22+/ 3and20+/ 0.2aminoacidsand signalpeptidesoflength27+/ 2aminoacids.Thisis truedespitetheirsequencediversity(Figure5).Aswith manyotherT4SScomponents,theruminantandhorse strainsaremoredistanttaxonomicallyinVirB2 Figure4 HydrophobicityplotsofVirB6-4proteinsfrom A.phagocytophilum HZ(top)orDog1(bottom)strains. Al-Khedery etal.BMCGenomics 2012, 13 :678 Page6of15 http://www.biomedcentral.com/1471-2164/13/678

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sequencecomparedtoVirB20sofhumananddog strains.AlignmentofallVirB2paralogsandorthologs showsthatsequencediversityisprimarilylocalizedto twohypervariableregionseitherprecedinganNterminalcysteineorclosetotheC-terminus(Figure6). Thisissimilartothehypervariableregionsfoundamong VirB2paralogsof A marginale [25].Energeticsubunits:VirB4andVirB11ATPasesaretypicallyusedinT4SStoenergizesubstrate transferandhavebeenfoundineveryT4SSdescribed. Ingram-negativebacteriathesearetypicallyintegral membraneproteinsencodedbygenesresidingupstream of virB2 (encodingpilin).Thisistrueforallstrainsof A phagocytophilum andithasbeensuggestedthatthisarrangementofmultiple virB2 paralogsand virB4 2 may allowassemblyofanantigenicallyvariablesurfaceorganelle[20].Theenergeticsubunititself,VirB4-2,is however,wellconservedbetweenstrains.Themostdistanttaxonomicrelationshipwasfoundbetweenhuman andruminantstrains(29totalaminoacidsubstitutions in Ap NorLamb-V1comparedto Ap HZ,Figure7).The otherenergeticsubunit,VirB11,wasalsowell-conserved betweenstrains(6aminoacidsubstitutionsbetween Ap NorLamb-V1and Ap HZ;datanotshown).Type4couplingprotein:VirD4Type4couplingproteinssuchasVirD4areATPases thatfunctioninsubstraterecognitionandtranslocation usingtheT4SS.Theyareassociatedwithmosteffector translocatorsystems.Theytypicallypossessaminimum oftwoN-terminaltransmembranedomains.Oftenmost heterogeneityexistsintheseN-terminalregions[20]. The A phagocytophilum VirD40sconformsomewhatto thisstereotypewiththreestronglypredictedN-terminal transmembranesegments.AswiththeotherATPasesof the A phagocytophilum T4SS,thereislittlevariationin VirD4,atotalof17aminoacidsubstitutionsofwhich4 areN-terminalbutmore(12)areC-terminal.Again,the evolutionaryrelationshipsamongVirD4sequencespositiontheruminantandhorsestrainsmoredistantlyto theU.S.dog,humanandrodentstrains(Figure8).ConclusionsA phagocytophilum representsarecentreclassificationof intracellularorganismsinfectingdifferentanimalspecies Figure5 PhylogenetictreestoshowtherelationshipofsyntenicVirB2proteinsfromdifferentstrainsof A.phagocytophilum Al-Khedery etal.BMCGenomics 2012, 13 :678 Page7of15 http://www.biomedcentral.com/1471-2164/13/678

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andhumansandcausingdiversediseasesymptomatology [43].Thesebacteriawerepreviouslyknownas Ehrlichia phagocytophila Ehrlichiaequi ,andtheagentofhuman granulocyticehrlichiosis.Despitethedifferenceswithin thisspecies,theoverallgenomestructureandsyntenyof theT4SSismaintained.However,genestructuralanalysis revealsevidenceofgeneduplicationandconsiderablediversityofT4SScomponentsinstrainsinfectingdifferent animals.Taxonomictreessuggestacloseevolutionaryrelationshipof A phagocytophilum strainsinfectingU.S. humans,miceanddogsandamoredistantrelationship withruminantandhorsestrains.Thisrelationshipisnot uniquetotheT4SSbutisalsosupportedbysimilartaxonomictreesofother A phagocytophilum proteinsofconservedmetabolicfunction(Figure9).WithintheT4SS multicomponentmembranecomplex,theenergeticand internalscaffoldingproteincomponentsarethemostconserved.Incontrast,componentsthatformtheproposed exposedstructuresoftheT4SS,suchasVirB2andVirB6, aremorevariable.T4SSareimportantvirulence determinantsofbacteria,thereforethesedifferencesmay resultinthedifferentinfectivityandvirulenceprofiles observedwithdifferentstrains.ItwillbeofinteresttodeterminethemoleculararchitectureofVirB6paralogsin differentstrains,includinginteractionswithotherT4SS componentsandeffectors.Oftheknownsurfaceexposed componentsoftheT4SS,VirB9isthemostconserved. Thisproteinhasbeenproposedasavaccinecomponent against A marginale andmayalsobesuitableagainst A phagocytophilum .MethodsA.phagocytophilum strains,cellculture,andexperimental infectionThe A phagocytophilum U.S.strainsHZ(human-origin, NY),MRK(horse-origin,CA),JM(rodent-origin,MN) andDog1(dog-origin,MN)werepropagatedinHL-60 cellsinRPMI-1640medium(ThermoFisherScientific, Inc.,Waltham,MA)supplementedwithfinal10%heatinactivatedfetalbovineserum(ThermoScientific)and ApDog1-VirB2-1 M-----------------M S N L T G F V A VL S V IMMF ------G V A G A ---IDAC-GVEPTAEKDHTVA V PI K -----GDV A V K SVSG V LQT V R R FC L P V M I G V VS G AV I ITV F G R S A W F A I A ML I VF SC IF L G GS EF I Q K F T E G V G D ----S A G T K H S R V I ASRL-----ApHz-VirB2-1 M-----------------M S N L T G F V A VL S V IMMF ------G V A G A ---IDAC-GVEPTAEKDHTVA V PI K -----GDV A V K SVSG V LQT V R R FC L P V M I G V VS G AV I ITV F G R S A W F A I A ML I VF SC IF L G GS EF I Q K F T E G V G D ----S A G T K H S R V I ASRL-----ApDog2-VirB2-1 M-----------------M S N L T G F V A VL S V IMMF ------G V A G A ---IDAC-GVEPTAEKDHTVA V PI K -----GDV A V K SVSG V LQT V R R FC L P V M I G V VS G AV I ITV F G R S A W F A I A ML I VF SC IF L G GS EF I Q K F T E G V G D ----S A G T K H S R V I ASRL-----ApJM-VirB2-1 M-----------------M S N L T G F V A VL S V IMMF ------G V A G A ---IDAC-GVEPTAEKDHTVA V PI K -----GDV A V K SVSG V LQT V R R FC L P V M I G V VS G AV I ITV F G R S A W F A I A ML I VF SC IF L G GS EF I Q K F T E G V G D ----S A G T K H S R V I ASRL-----ApVar1-VirB2-1 M-----------------M S N L T G F V A VL S V IMMF ------G V A G A ---IDAC-GVEPTAEKDHTVA V PI K -----GDV A V K SVSG V LQT V R R FC L P V M I G V VS G AV I ITV F G R S A W F A I A ML I VF SC IF L G GS EF I Q K F T E G V G D ----S A G T R H S R V I ASRL-----ApMRK-VirB2-1 I-----------------M S N L T G F V A VL S V IMMF ------G V A G A ---IDAC-GVEPTAEKDHTVA V PI K -----GDV A V K SVSG V LQT V R R FC L P V M I G V VS G AV I ITV F G R S A W F A I A ML I VF SC IF L G GS EF I Q K F T E G V G D ----S A G T R H S R V I ASRL-----A pNorV1-VirB2-1 M-----------------M S N L T G F V A VL S V IIMF ------G V A G A ---IDAC-GVEPTAEKDHTVA V PI K -----GDV A V K SVSG V LQT V R R FC L P V M I G V VS G AV I ITV F G R S A W F A I A ML I VF SC IF L G GS EF I Q K F T E G V G D ----S A G T R H S R V I ASRL-----A pNorV2-VirB2-1 M-----------------M S N L T G F V A VL S V IMMF ------G V A G A ---IDAC-GVEPTAEKDHTVA V PI K -----GDV A V K SVSG V LET V R R FC L P V M I G V VS G AV I ITV F G R S A W F A I A ML I VF SC IF L G GS EF I Q K F T E G V G D -----P A G T R H S R V I ASRL-----ApDog1-VirB2-2 ------------------M FG L T R F M A VL A L VVA L V G F GT S A F A S ---------------------T TG S -----DDV A A K VIC N VV V F V Q R L G L P IM T G VIL G A S I M A IF G K L A W A A I V ML V VF T A IFF G AG K L I Q K F A A G V G S DIIG K A D S F E C K GNGATTLS---ApJM-VirB2-2 ------------------M FG L T R F M A VL A L VVA L V G F GT S A F A S ---------------------T TG S -----DDV A A K VIC N VV V F V Q R L G L P IM T G VIL G A S I M A IF G K L A W A A I V ML V VF T A IFF G AG K L I Q K F A A G V G S DIIG K A D S F E C K GNGATTLS---ApHz-VirB2-2 ------------------M FG L T R F M A VL A L VVA L V G F GT S A F A S ---------------------T TG S -----DDV A A K VIC N VV V F V Q R L G L P IM T G VIL G A S I M A IF G K L A W A A I V ML V VF T A IFF G AG K L I Q K F A A G V G S DIIG G N A E S F E C K GNGATTLSS--ApDog2-VirB2-2 ------------------M FG L T R F M A VL A L VVA L V G F GT S A F A S ---------------------T TG S -----DDV A A K VIC N VV V F V Q R L G L P IM T G VIL G A S I M A IF G K L A W A A I V ML V VF T A IFF G AG K L I Q K F A A G V G S DIIG G N A E S F E C K GNGATTLSS--ApVar1-VirB2-2 ------------------M FG L T R F M A VL A L VVA L V G F GT S A F A N A ---STA--------------S AG S -----DDV A A K VIC N VV V F V Q R L G L P IM T G VIL G A S I M A IF G K L A W A A I V ML V VF T A IFF G AG K L I Q K F A A G V G S DIVG D TN S F E C K G G GGTVLK---ApMRK-VirB2-2 ------------------M F S L T R F M A VL A L VVA L V G VGT SD F A S A ---SAP--------------A TG S -----DDV A A K VIC N VV V F V Q R L G L P IM T G VIL G A S I M A IF G K L A W A A I V ML V VF T A IFF G AG K L I Q K F A A G V G S DIIG N TD S F E C K G G GQTVLGK--ApNorV2-VirB2-2 ------------------M FG L T R F M A VL A L VVA L V G F GT S A F A T------A---------------QYA S -----DDV A A K VIC N VV V F V Q R L G L P IM T G VIL G A S I M A IF G K L A W A A I V ML V VF T A IFF G AG K L I Q K F A A G V G S DIIG D A N S F E C R GNGETKLGG-SK ApNorV1-VirB2-2 ------------------M FG L T R F M A VL A L VVA L V G F GT S A F A T T ---GST---------------QYA S -----DDV A A K VIC N VV V F V Q R L G L P IM T G VIL G A S I M A IF G K L A W A A I V ML V VF T A IFF G AG K L I Q K F A A G V G S DIIG D A N S F E C R GNGETKLGGVSK ApDog1-VirB2-4 ------------------M A K I V R F F T ST A G MFLL L L L CS Q G V A A G ---AS----AN---------D EH K -----KEE T S K VIC N VV L F A Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I S K F A N G V G E ---I K A N D F D C K E V AEK------ApHz-VirB2-4 ------------------M A K I V R F F T ST A G MFLL L L L CS Q G V A A G ---AS----AN---------D EH K -----KEE T S K VIC N VV L F A Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I S K F A N G V G E ---I K A N D F D C K E V AEK------ApDog2-VirB2-4 ------------------M A K I V R F F T ST A G MFLL L L L CS Q G V A A G ---AS----AN---------D EH K -----KEE T S K VIC N VV L F A Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I S K F A N G V G E ---I K A N D F D C K E V AEK------ApJM-VirB2-4 ------------------M A K I V R F F T ST A G MFLL L L L CS Q G V A A G ---AS----AN---------D EH K -----KEE T S K VIC N VV L F A Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I S K F A N G V G E ---I K A N D F D C K E V AEK------ApDog1-VirB2-5 ------------------M A K I V R F F T ST A G MFLL L L L CS Q G V A A D ---TA----TN----------EEH K -----KEE T S K VIC N VV L F A Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I G K F A N G V G D ---L K A TE F D C K E V TK-------ApJM-VirB2-5 ------------------M A K I V R F F T ST A G MFLL L L L CS Q G V A A D ---TA----TN----------EEH K -----KEE T S K VIC N VV L F A Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I G K F A N G V G D ---L K A TE F D C K E V TK-------ApHz-VirB2-5 ------------------M A K I V R F F T ST A G MFLL L L L CS Q G V A A G ---AS----AN---------D EH K -----KEE T S K VIC N VV L F A Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I G K F A N G V G D ---L K A TE F D C K E V TK-------ApDog2-VirB2-5 ------------------M A K I V R F F T ST A G MFLL L L L CS Q G V A A G ---AS----AN---------D EH K -----KEE T S K VIC N VV L F A Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I G K F A N G V G D ---L K A TE F D C K E V TK-------ApMRK-VirB2-5 ------------------M A K I V R F F T ST A G MFLL L L L CS Q G V A A G ---AS----AN---------D EH K -----KEE T S K VIC N VV L F A Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I G K F A N G V G D ---L K A TE F D C K E V TK-------ApNorV2-VirB2-5 ------------------M A K V V R F F T ST A G MFLL L L L CS Q G V A A G ---AS----AN---------D DH K -----KEE T S K VIC N VV L F A Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I G K F A K G V G D ---L N A N D F D C K T V TG-------ApDog1-VirB2-9 ------------------M A K I V R F F T ST A G MFLL L L L CS Q G V A A G ---AS----AN---------D EH K -----KEE T S K VIC N VV L F A Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I G K F A K G V G E ---L N A N D F D C K T V TG-------ApJM-VirB2-9 ------------------M A K I V R F F T ST A G MFLL L L L CS Q G V A A G ---AS----AN---------D EH K -----KEE T S K VIC N VV L F A Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I G K F A K G V G E ---L N A N D F D C K T V TG-------ApDog2-VirB2-9 ------------------M A K I V R F F T ST A G MFLL L L L CS H G V A A G ---AS----AN---------D EH K -----KEE T S K VIC N VV L F A Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I G K F A K G V G E ---L N A N D F D C K T V TG-------ApMRK-VirB2-9 ------------------M A K I V R F F T ST A G MFLL L L L CS Q G V A A G ---AS----AN---------A EH K -----KEE T S K VIC N VV L F A Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I G K F A K G V G E ---L N A N D F D C K T V TG-------ApMRK-VirB2-4 ------------------M A K I V R F F T ST A G MFLL L L L CS Q G V A A D ---TA----TN---------A EH K -----KEE T S K VIC N VV L F A Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I G K F A K G V G E ---L N A N D F D C K E V AEK------ApNorV2-VirB2-4 ------------------M A K V V R F F T ST A G MFLL L L L CS Q G V A A G ---ASAG-TAN----------EEH K -----KEE T S K VIC N VV L F A Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I G K F A K G V G E ---L N A N D F D C K S V SDK------ApNorV2-VirB2-9 ------------------M E K I V R F F T NT A G MFLL L L L CS Q G I A V G ---VAADQKAN----------EEH K -----KEE T S K VIC N A V S F A Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I G K F A K G V G D ---L N A N D F D C K E V AEK------ApVar1-VirB2-novel2 ------------------M V R I V R F F T ST A S MFLL L L L CS Q G V A A G ---VSAG-SPS---------V DH K -----NED T S K VIC N VV T F A Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I G K F A N G V G D ---L N A K D F D C K S V SNGK-----ApNorV1-VirB2-novel1 ------------------M A R I V K F L T HT T G MFLL L L L CS Q G V A A G ---ASTG-AQS---------A EH K -----NED T S K VIC N VV S F A Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I S K F A N G V G G---V N A D G F D C K D V ATKP-----ApNorV1-VirB2-novel5 ------------------M A R I V K F L T HT T G MFLL L L L CS Q G V A A G ---ASTG-AQS---------A EH K -----KED T S K VIC N VV M F A Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I G K F A E G V G G---V K GN D F D C K S V SDVK-----ApNorV1-VirB2-4 ------------------M V R I V R F F T ST A G MFLL L L L CS Q G V S A G ---ASAG-SLD---------D GH K -----NED T S K VIC N VV T F V Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I S K F A N G V G E ---I K A D G F D C K K V TG-------ApVar1-VirB2-novel6-partial ------------------M V R I V R F L T RT T G MFLL L L L CS Q G V A A G ---ASAD-KAN---------T DH K -----KED T S K VIC N VV T F A Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I ---------------------------------ApVar1-VirB2-4 ------------------M V R T V R F W T RI T G MFLL L L L CS Q G V A A G --------------------A EE H -----KGD T S K VIC N VV E F V Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I S K F A N G V G D ---L N A DK F D C K D V KGEGQHN--ApVar1-VirB2-novel4 ------------------M V K V V R F F T ST A G MFLL L L L CS Q G V A A ----DGA---------------KTD H -----DGA T S R VIC N VV E F V Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I G K F A D G V G N ---L D A K G F D C K N V KGDN-----ApVar1-VirB2-novel7-partial -----------------------------------------------------------------------------------------------P IM T G EF L G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I G K F A D G V G N ---L D A K G F D C K N V KGDN-----ApNorV1-VirB2-novel2 ------------------M V R I V R F L A RT T G MFLL L L L CN Q G I A S ----AVS--------------A DD H -----KGD T S R VIC N VV E F V Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I G K F A D G V G N ---L N A K G F D C K T V KGDN-----ApNorV1-VirB2-novel4 ------------------M A R I V K F L T RT T G MFLL L L L CN Q G I A V G --------------------A EE H -----KED T S K VIC N VV G F V Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I G K F A D G V G N ---L N A K G F D C K D V KGDK-----ApNorV1-VirB2-novel6 ------------------M A R I V K F L T RT T G MFLL L L L CN Q G I A V G --------------------A EE H -----KED T S K VIC N VV G F V Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I G K F A D G V G N ---L N A K G F D C K T V KGDN-----ApNorV1-VirB3-novel3 ------------------M A K V V R F F T ST T W MFLL L L L CN Q G I A G A VSPAVG---------------QGD H -----KGD T S K VIC N VV E F V Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I G K F A D G V G N ---L N A K G F D C K T V KGDN-----ApVar1-VirB2-5 ------------------M E K I V R F L MRT T G M C FL L L L CS Q G V A G A --GAAS--------------T DD H -----KGD T S K VIC N VV M F V Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I S K F A N G V G D ---L K A N D F D C K K V TT-------ApNorV1-VirB2-6 ------------------M E K I V R F L MRT T G M C FL L L L CS Q G V A V A --GAAL--------------A DD H -----KGD T S K VIC N VV L F V Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I S K F A N G V G D ---L K A N D F D C K K V TS-------A pVar1-VirB2-novel5 ------------------M V R I V R F L A RT T G M C FL L L L CS Q G V A E P --GTVS--------------A GD H -----KGD T S K VIC N VV L F A Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I S K F A N G V G D ---L K A D N F D C K T V QGDK-----ApVar1-VirB2-novel3 ------------------M V R I V R F L T RT T G MFLL L L L CS Q G V A V A SLGGTS--------------T DD H -----KGD T S K VIC N VV L F V Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I S K F A N G V G D ---L N A EK F D C K D V TKQ------ApNorV1-VirB2-5 ------------------M E K I I R F L MRT T G M C F V L L L CS H G I V A S --AAAA--------------A TD H -----NGA T S K VIC N VV E F V Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I S K F A K G V G G---L D A D N F D C S K V KDDASGSSSP ApVar1-VirB2-novel1 ------------------M V R I V R F L MRT T G M C F V L L L CS H G I A S A ---AAE--------------A TK H -----DGA T S K VIC N VV L F A Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I G K F A K G V G E ---L E A D N F D C S K V KDDASGSSSL ApDog1-VirB2-6 ------------------M A K I V R F F T ST V G MFLL L L L CS H G I A S A ---AAA--------------G TD H -----NGV T A K VIC N VV L F V Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I G K F A K G I G E ---L D A D N F D C S K V QAEESSSV-ApHz-VirB2-6 ------------------M A K I V R F F T ST V G MFLL L L L CS H G I A S A ---AAA--------------G TD H -----NGV T A K VIC N VV L F V Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I G K F A K G I G E ---L D A D N F D C S K V QAEESSSV-ApDog2-VirB2-6 ------------------M A K I V R F F T ST V G MFLL L L L CS H G I A S A ---AAA--------------G TD H -----NGV T A K VIC N VV L F V Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I G K F A K G I G E ---L D A D N F D C S K V QAEESSSV-ApJM-VirB2-6 ------------------M A K I V R F F T ST V G MFLL L L L CS H G I A S A ---AAA--------------G TD H -----NGV T A K VIC N VV L F V Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I G K F A K G I G E ---L D A D N F D C S K V QAEESSSV-ApMRK-VirB2-6 ------------------M A K V V R F F T ST V G MFLL L L L CS N G I A S A ---AAA--------------A TK H -----DGA T S K VIC N VV L F A Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I G K F A K G I G E ---L D A D N F D C S K V KAEESNSV-ApNorV2-VirB2-6 ------------------M A K V V R F F T ST V G MFLL L L L CS H G I A S A ---AAAV-------------A TK H -----DGA T S K VIC N VV L F A Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I G K F A K G I G E ---L D A D N F D C S K V KAEESNSVLP ApVar1-VirB2-6 ------------------M S K I V R F F T HT T C MFLL L F L CN Q G I A A A ---AEA----------------TK H -----DGA T S K VIC N VV L F A Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I G K F A K G I G E ---L D A D N F D C S K V KAEESNSV-ApDog1-VirB2-7 ------------------M A K V V R F F T ST V G MFLL L L L CS H G I A S A ---AAA--------------G TD H -----NGV T A K VIC N VV L F V Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I G K F A Q G V G E ---W E A EK F D C K D I KAG------ApJM-VirB2-7 ------------------M A K V V R F F T ST V G MFLL L L L CS H G I A S A ---AAA--------------G TD H -----NGV T A K VIC N VV L F V Q K L G L P IM T G VIL G SS V M A IF G R L A W P A I A ML I VF T A IFF G SS K I I G K F A Q G V G E ---W E A EK F D C K D I KAG------ApHz-VirB2-7 ------------------M A K V V R F F T ST V G MFLL L L L CS H G I A S A ---AAA--------------G TD H -----NGV T A K VIC N VV L F V Q K L G L P IM T G VIL G SS V M A IF G R L A W P S I A ML I VF T A IFF G SS K I I G K F A Q G V G E ---W E A EK F D C K D I KAG------ApDog2-VirB2-7 ------------------M A K V V R F F T ST V G MFLL L L L CS H G I A S A ---AAA--------------G TD H -----NGV T A K VIC N VV L F V Q K L G L P IM T G VIL G SS V M A IF G R L A W P S I A ML I VF T A IFF G SS K I I G K F A Q G V G E ---W E A EK F D C K D I KAG------A pMRK-VirB2-7 ------------------M A K V V R F F T ST V G MFLL L L L CS N G I A S A ---AAA--------------G TD H -----NGV T A K VIC N VV L F V Q K L G L P IM T G VIL G SS V M A IF G R L A W P S I A ML I VF T A IFF G SS K I I G K F A Q G V G E ---W E A EK F D C K D I KAG------ApVar1-VirB2-7 ------------------M A K V V R F L T GT V G MFLL L L L CS H G I A S A ---AAA--------------G TD H -----NGV T A K VIC N VV I F I Q K L G L P IM T G VI M G SS I M A IF G R L A W P S I A ML I VF T A IFF G SS K I I G K F A Q G V G E ---W E A EK F D C K D I KAG------ApNorV2-VirB2-7 ------------------M G R I V R F L T RT V G MFLL L L L CS Q G I A S A ---AAA--------------D TD H -----SGV T A K VIC N VV L F V Q K L G L P IM T G VIL G SS V M A IF G R L A W P S I A ML I VF T A IFF G SS K I I G K F A Q G V G E ---W E A EK F D C K D I KAG------ApNorV1-VirB2-7 ------------------M A K I V K F F MHT T C MFLL L F F CN Q G I A A A ---AT----------------HIEP-----KDPI S R VIC N VV I F I Q K L G L P IM T G VI M G SS I M A IF G R L A W HT I A T L V VF T A IFF G AG K I I S K I A S G I G G---L N A EK F D C K P G KAKKQEIWII ApDog1-VirB2-8 M-----------------F T N I L R S C V ISI I FFIF L I L PA V S V S A A ---PVAH-------------A AG D -----GEVI S K VIC N VV V F V Q R L G L P IM T G VIL G SS I M A V F G R L A W P A I V ML V VF T A IFF G AG K L I S K F A G G I S E ---L G A E D F D C R V L AGKNI----ApDog2-VirB2-8 M-----------------F T N I L R S C V ISI I FFIF L I L PA V S V S A A ---PVAH-------------A AG D -----GEVI S K VIC N VV V F V Q R L G L P IM T G VIL G SS I M A V F G R L A W P A I V ML V VF T A IFF G AG K L I S K F A G G I S E ---L G A E D F D C R V L AGKNI----ApJM-VirB2-8 M-----------------F T N I L R S C V ISI I FFIF L I L PA V S V S A A ---PVAH-------------A AG D -----GEVI S K VIC N VV V F V Q R L G L P IM T G VIL G SS I M A V F G R L A W P A I V ML V VF T A IFF G AG K L I S K F A G G I S E ---L G A E D F D C R V L AGKNI----ApMRK-VirB2-8 M-----------------F T N I L R S C V ISI I FFIF L I L PA V S V S A A ---PVAH-------------A AG D -----GEVI S K VIC N VV V F V Q R L G L P IM T G VIL G SS I M A V F G R L A W P A I V ML V VF T A IFF G AG K L I S K F A G G I S E ---L G A E D F D C R V L AGKNI----ApNorV2-VirB2-8 M-----------------F T N I L R S C V ISI I FFIF L I L PA V S V S A A ---PVAH-------------A AG D -----GEVI S K VIC N VV V F V Q R L G L P IM T G VIL G SS I M A V F G R L A W P A I V ML V VF T A IFF G AG K L I S K F A G G I S E ---L G A E D F D C R V L AGKNI----ApNorV1-VirB2-8 M-----------------F T N I L R S C V ISI I FFIF L I L PA V S V S A A ---PVAH-------------A AG D -----GEVI S K VIC N VV V F V Q K L G L P IM T G VIL G SS I M A V F G R L A W P A I V ML V VF T A IFF G AG K L I S K F A G G I S E ---L G A E D F D C R V L AGKNI----ApVar1-VirB2-8 M-----------------F T N I L R S C V ISM I FFIF L I L PA V S V S A A ---PVAH-------------A AG D -----GEVI S K VIC N VV V F V Q K L G L P IM T G VIL G SS I M A V F G R L A W P A I V ML V VF T A IFF G AG K L I S K F A G G I S E ---L G A E D F D C R V L AGKNI----ApHz-VirB2-8 M-----------------F T N I L R S C V ISI I FFIF L I L PA V S V S A A ---PVTH-------------A AG D -----GEVI S K VIC N VV V F V Q R L G L P IM T G VIL G SS I M A V F G R L A W P A I V ML V VF T A IFF G AG K L I S K F A G G I S E ---L G A E D F D C R V L AGKNI----ApDog1-VirB2-3 MDTQGRAIAEDRRSFARTF F N K K V F F L IIQ G S LFF V L L L IL D E A Y A G V ---AESN-LFPAVAQHG---S AT N -----EDV T S K VIC N VV K F V R G I G L P IM T G VIL G SS V M A IF G R L A W P A I A A L V I F T A V FF G A E K V I S K F T D G I S V---M Q TG N C D T I-----------ApHz-VirB2-3 MDTQGRAIAEDRRSFARTF F N K K V F F L IIQ G S LFF V L L L IL D E A Y A G V ---AESN-LFPAVAQHG---S AT N -----EDV T S K VIC N VV K F V R G I G L P IM T G VIL G SS V M A IF G R L A W P A I A A L V I F T A V FF G A E K V I S K F T D G I S V---M Q TG N C D T I-----------ApDog2-VirB2-3 MDTQGRAIAEDRRSFARTF F N K K V F F L IIQ G S LFF V L L L IL D E A Y A G V ---AESN-LFPAVAQHG---S AT N -----EDV T S K VIC N VV K F V R G I G L P IM T G VIL G SS V M A IF G R L A W P A I A A L V I F T A V FF G A E K V I S K F T D G I S V---M Q TG N C D T I-----------ApJM-VirB2-3 MDTQGRAIAEDRRSFARTF F N K K V F F L IIQ G S LFF V L L L IL D E A Y A G V ---AESN-LFPAVAQHG---S AT N -----EDV T S K VIC N VV K F V R G I G L P IM T G VIL G SS V M A IF G R L A W P A I A A L V I F T A V FF G A E K V I S K F T D G I S V---M Q TG N C D T I-----------ApMRK-VirB2-3 MDTQGRAIAEDRRSFARTF F N K K V F F L IIQ G S LFF V L L L IL D E A Y A G V ---AESN-LFPAVAQHG---S AT N -----EDV T S K VIC N VV K F V R G I G L P IM T G VIL G SS V M A IF G R L A W P A I A A L V I F T A V FF G A E K V I S K F T D G I S G---M Q TG N C D T I-----------ApNorV2-VirB2-3 MDTQGRAMAEDRRSFARTF F N K K V F F L IIQ G S LFF V L L L IL D E A Y A G V ---AESN-LFPAVAQHG---S AT N -----EDV T S K VIC N VV K F V R G I G L P IM T G VIL G SS V M A IF G R L A W P A I A A L V I F T A V FF G A E K V I S K F T D G I S G---M Q TG N C D T I-----------ApNorV1-VirB2-3 MDTQGRAMAEDRRSFARTF F N K K V F F L IIQ G S LFF V L L L IL D E A H A G V ---AESN-LFPAVAQHG---S AA N -----EDV T S K VIC N VV K F V R S I G L P IM T G VIL G SS V M A IF G R L A W P A I A A L V I F T A V FF G A E K V I S K F T D G I S G---M Q TG N C D T I-----------ApVar1-VirB2-3 MNTQGRAMAEDRRSFARTF F N K K V F F L IIQ G S LFF V L L L IL D E A H A G V ---SESN-LFPAVAQHG---S AA N EDVTSEDV T S K VIC N VV K F V R S I G L P IM T G VIL G SS V M A IF G R L A W P A I A A L V I F T A V FF G A E K V I S K F T D G I S G---M Q TG N C D T I-----------Consensus/80% ...................bsp lsbFhs..shbbbblhb..s.thAts.....................s..p........stKVICNVV.Fsp+lGLPIMTGVILGSSlMAIFGRLAWsAIAMLlVFTAIFFGttKlI.KFs.Gltp. ...hpA.sb-sp.h.......... Figure6 MultiplesequencealignmentofVirB2aminoacidsequencesfromdifferentstrainsof A.phagocytophilum Al-Khedery etal.BMCGenomics 2012, 13 :678 Page8of15 http://www.biomedcentral.com/1471-2164/13/678

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4mML-glutamine(Lonza,Rockland,ME),andinthe absenceofantibiotics. Ap HZand Ap MRKhavebeen describedpreviously[15,44].The Ap JMstrain(CR011258)originatedfromameadowjumpingmouse( Zapus hudsonius )trappedatCampRipley,MN[45].The Ap Dog1strainoriginatedfromthebloodofadogfrom Baxter,MNnaturallyinfectedwith A phagocytophilum asevidencedbythedetectionofdistinctivemorulaeina diagnosticbloodsample,andsequencingoftheExpressionSite-linked msp2 / p44 gene.Briefly,wholebloodwas collectedfromtheanimalwithEDTAasananticoagulant.Thebuffycoatlayerwascollectedafterlow-speed centrifugationofthewhole-blood,washedin1xphosphatebufferedsaline(PBS,Hyclone,cat.no.SH30256.01), thenaddedtoacultureofuninfectedHL-60cells.The culturewasleftundisturbedfor3days,afterwhichmorulaebegantoappear.The Ap Dog2strainalsooriginated fromaMNdogandwaspassagedtoandmaintainedin the Ixodesscapularis ISE6tickcelllineasdescribed[46]. TheApvariant1CRT35strain(tick-origin,MN), maintainedinISE6cells,hasbeendescribed[47].For DNAisolation,culturesweremaintaineduntil90-100% ofcellswereinfectedwithmaturemorulae.Cellswere pelletedbycentrifugationat2500xgfor20minat4C. Pelletsweregentlyresuspendedin1.5mlcoldPBS,transferredtoscrew-capmicrofugetubes,andcentrifugedat Figure7 PhylogenetictreestoshowtherelationshipofsyntenicVirB4proteinsfromdifferentstrainsof A.phagocytophilum Figure8 Phylogenetictreetoshowtherelationshipofsyntenic VirD4proteinsfromdifferentstrainsof A.phagocytophilum Al-Khedery etal.BMCGenomics 2012, 13 :678 Page9of15 http://www.biomedcentral.com/1471-2164/13/678

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1500xgfor20minat4C.Supernatantswereremoved andthecellpelletsstoredat 80Cuntilfurtheruse. TwonaturallyoccurringNorwegianlamb A phagocytophilum strainsdifferinginthe 16S rRNAgeneanddegreeofvirulencewereusedtoexperimentallyinfect lambsraisedinanindoorenvironmentwithbarriers againsttickentryandtickinfestation.Lamb00186was infectedwiththemorevirulentvariant1(identicalto GenBankM73220)andlamb0054withvariant2(identicaltoGenBankAF336220)[48],tobereferredtoas Ap NorLamb-V1and-V2fromhereon.Infectionswere monitoredbymicroscopyandbloodwasharvestedat maximumparasitemia.Topurifybuffycoatscontaining theinfectedneutrophils,approximately2.5lofNacitratedbloodwascollectedfromeachanimal.The bloodwastransferredto1lcentrifugebottlesandcentrifugedat2,500-3,000xginaswing-outbucketrotor for30minat4C.Afterremovingmostoftheplasma layer,thebuffycoatlayerwascollectedwithminimal contaminationofredbloodcells.Thecellswerediluted 1:3withPBS,mixedgentlyandcentrifugedat1,500xg for20minat4C.FollowingthreePBSwashes,supernatantswereremovedandthecellpelletsstoredat 80C. Theexperimentalstudyinsheepwasapprovedbythe NorwegianAnimalResearchAuthority.Purificationofhostcell-free A phagocytophilum and genomicDNA(gDNA)isolationFortheHZ,JM,Dog1,MRKandNorLamb-V1and-V2 strains,intact,hostcell-freeorganismswithminimal Figure9 Phylogenetictreetoshowtherelationshipofotherconservedproteinsfromdifferentstrainsof A.phagocytophilum. These proteinsare:PolA,DNApolymeraseI;LeuS,leucyl-tRNAsynthetase;AtpA,ATPsynthaseF1,alphasubunit;ValS,valyl-tRNAsynthetase;RecG, ATP-dependentDNAhelicase;LigA,NAD-dependentDNAligase. Al-Khedery etal.BMCGenomics 2012, 13 :678 Page10of15 http://www.biomedcentral.com/1471-2164/13/678

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hostcellgDNA/RNAcontaminationwerepurifiedfrom frozenPBSpelletsofinfectedcellspreparedasabove. Samplesandreagentsweremaintainedonicethroughouttheentireprocedure,andallcentrifugationsperformedat4C.Followingaquickthaw,hostcellswere disruptedbyvigorousvortexingfor5min.AnequalvolumeofPBSwasaddedandvortexingcontinuedfor 3min.Cellulardebriswasremovedbycentrifugationat 200xgfor15min.Afterremovingmostofthesupernatantstofreshtubes,thesewerepassedseveraltimes througha31Gneedleandsavedonice.Pelletswere resuspendedwellinfinal500 lPBSthenpassedserially through22G,25G,28Gand,whenpossible,31Gneedlesattachedtoa1mlsyringe.3 – 5volumesPBSwere addedandmixedbyvortexing.Debriswasremovedby centrifugationat200xgfor10min.Supernatantswere pooledtothosefromthepreviouscentrifugationstep. RNaseAwasaddedtoafinal250 – 300 g/mlandthe samplesincubated45 – 60minat37C.Sampleswere centrifugedat21,000xgfor30minandthesupernatantsremovedcompletely.Pelletswereresuspendedin 50 – 100 lPBSeachandtransferredtofreshtubes.To ensurehomogeneityofthesuspension,initiallyadrawnout10 lpipettetipwasusedtodisruptthepelletby swirlingfollowedbyup/downpipettingandgentlevortexing,beforeswitchingtoalargertip.Thesamplewas furtherhomogenizedbyseveralpassesthrougha2831Gneedle.PBSwasaddedtofinal500 – 700 land DNaseItofinal250 g/ml.Following45 – 60minincubationat37Cthesampleswerecentrifugedat21,000xg for30min.Pelletswerehomogenizedasaboveandthe DNaseItreatmentrepeated.EDTA(pH8.0)wasadded tofinal25mMandthesamplescentrifugedasabove. TubeswerewashedtwicewithPBSwithoutdisturbing thepelletsandresidualPBSwasremovedafter3min centrifugationat21,000xg.Pelletswerehomogenized asabovein600 – 800 lRPMIculturemedium(containing10%fetalbovineserum)addedincrementallyand transferredtoa50mltube.Culturemediumwasadded toafinalvolumeof6mlbeforepassagethroughaprewet,2 mpore-size,25mm,GMF-150glassmicrofiber syringefilter(Puradisc25GD;WhatmanInc.,Florham Park,NJ).Thefilterwaswashed3-4xwithculture medium.Washeswerepooledtothefiltrateandcentrifugedat22,000xgfor30min.Thepellets,comprisedof free,non-viableorganismsandhostcellmitochondria, wereresuspendedinPBS,transferredtomicrofugetubes andre-pelletedat21,000xgfor30min.Supernatants wereremovedcompletelyandthepelletswereprocessed immediatelyorstoredat 20C.Forevery108hostcells usedat90-100%infectivity,enoughorganismswere recoveredtoyieldonaverage1 – 1.5 ghigh-quality DNAusingeithertheGentraPuregeneYeast/Bact.kit (QiagenInc.,Valencia,CA)ortheQIAGENBlood& CellCultureDNAminikitfollowingthemanufacturer ’ s protocols. FortheDog2andApvariant1strains,organismswere culturedandisolatedfromISE6tickcellsasdescribed [49].Hostcell-freebacteriawerepreparedfromtwoculturesin25cm2flasks,collectedbycentrifugationfor 10minat11,000xgat4C,andlysedinGentraPuregenelysisbuffer(Qiagen)at80Cfor5min.Sincethese DNAsamplesalsocontainedaconsiderableamountof small(<500bp)DNAspeciesnaturallyassociatedwith theISE6hostcellline,the A phagocytophilum gDNA wasfurtherpurifiedbyelectroelutionfromagarosegels, followedbyphenol/chloroformextractionandEtOH precipitationusingconventionalprotocols.Preparationofhostcell-free A phagocytophilum agarose plugsforopticalmappingAp Dog1wasinitiallyselectedforcompletegenomesequencingtocomparewiththepublishedHZstrain. Whenadraftgenomewasassembledfor Ap Dog1itwas largelysyntenicwithHZexceptforthe vir B6locus,indicatingapossibleerrorinthesequenceofoneorbothof thestrains.Accordingly,the Ap Dog1draftgenomesequencewasverifiedbyOpticalMapping.Inpreparation forOpticalMapping(performedbyOpGenInc., Gaithersburg,MD),hostcell-freeorganismswereembeddedin0.5%low-meltingpointagaroseplugsand subsequentlylysed,allowingaccesstotheintact, ~1.48Mbcircular A phagocytophilum chromosome.A procedurerecommendedbyOpGenwasfollowed.All solutionsweremadefreshusingOpGensuggested reagents.Intact Ap Dog1organismswerepurifiedas above,exceptthatthepelletoffreeorganismsobtained followingcentrifugationofthefiltratewasresupended andwashedincellsuspensionbuffer[200mMNaCl, 100mMEDTA-Na2(pH8.0),10mMTris(pH7.2)]. Plugsweremadeimmediatelyoncompletionoftheisolationprocedure.Briefly,followingthefinalcentrifugationofthepurifiedorganisms,thepelletwas resuspendedincellsuspensionbufferusing40 – 50 lfor every108hostcellsusedat>95%infectivity.Thesample waspassed2xthrougha31Gneedle(3/10mlcapacity InsulinSyringewithfused8mmlongneedle,BD #328438;Becton,Dickinson&Co.,FranklinLakes,NJ) toensurehomogeneityofthethicksuspension,andan equalvolumeof1%lowmeltingpointSeaPlaqueGTG agarose[(Lonza#50111)dissolvedinDEPC-treated water(Invitrogen#750023;Carlsbad,CA)andheldat55C] wasimmediatelyadded.Followingmixing,100 laliquots weredispensedintoplugmolds(Bio-Rad#170-3713; Hercules,CA)andallowedtosetfor1hrat4Cpriorto transferintoa50mltubecontaining5 – 10ml,50CNDSK solution[filtersterileNDSsolu tion(1%N-lauroylsarcosine (Sigma#L5000;St.Louis,MO)in0.5MEDTA-Na2Al-Khedery etal.BMCGenomics 2012, 13 :678 Page11of15 http://www.biomedcentral.com/1471-2164/13/678

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(pH9.5),supplementedwithfinal2mg/mlproteinase K(Pierce#17916;Rockford,IL)immediatelypriorto use].Thetubewasincubateduprightat50Cwith mildshaking(40rpm)for8 – 24hrs,untiltheplugs turnedclearandcolorless.Plugsweregentlywashed 3xin5ml0.5MEDTA-Na2(pH9.5),thentransferred toafreshtubeandstoredinEDTAat4C.Optical MappingdatageneratedfromtheBamHI-digested Ap Dog1chromosomewasanalyzedusingtheOpGen MapSolversoftware.454GenomesequencingandbioinformaticsIsolatedDNAwasprovidedtotheInterdisciplinaryCenterforBiotechnologyResearch(ICBR)corefacilities, UniversityofFloridaforlibraryconstructionandpyrosequencingontheRoche/454GenomeSequenceraccordingtostandardmanufacturerprotocols.Regularread librariesweregeneratedforallstrains.Additionally,3kb pairedendlibrariesweremadefor Ap HZ, Ap Dog1and Ap MRK.Genomecoveragerangewas31.3xto72.1x. Foreachstrain,theSFFformatflowfiles,returnedby ICBRforbioinformaticsanalysis,werefirstcombined andconvertedto.fastaand.qualfiles(orthetwocombinedin.fastqformat)usingRoche/454GenomeSequencerFLXSystemsoftware.Genomedraftswere assembledusingtheCLCGenomicsWorkbenchsoftwaresuite(version4.0-4.9)bymappingreadsinitially againstthefullyannotated,Sangersequenced Ap HZ genome(GenBankCP000235),thenagainstthecompleted Ap Dog1genome.Defaultparameterswereused: lengthfraction,0.5;similarity,0.8;andforpairedend reads,minimumdistance,1500/maximumdistance, 4500.Toobtainthe vir loci,theresultingconsensussequenceandunderlyingalignedreadswereinspectedfor conflictsandmismatchedpairedendssuggestingthe presenceofinsertionsand/ordeletionsnotmirroredin theconsensus.Theseweremanuallycorrected.Gaps werealsomanuallyclosedwherepossible.Briefly,overlappingreadscoveringatleast2kbofsequenceonboth sidesofagapandextendingintoitwereindividually extractedfromthealignment.Anewconsensusforeach sidewasobtainedbyassemblingthereadsagainsteach other,and250N ’ swereaddedtoitsends.Thesewere individuallyusedasthereferencesequenceagainst whichallthe454readswerere-mappedtopullout novelreadsextendingintotheunknownregion.The processwasrepeatedmultipletimes,allowingfortheincrementalfillingofthegap.PCR,followedbysequencingwasperformedwhensequencesextrapolatedinthis fashionspannedcomplextandemrepeatregionssuchas repeatregions1and3(R1andR3inFigure3A)ofthe virB6 4 gene,orwhengapclosurecouldnotbecompletedduetosuchstructures,aswasthecasewiththe extremelylong virB6 4 R4(Figure3A)region. AminoacidsequenceswerealignedwithMAFFT[50] anddisplayedwithCHROMA[51].Taxonomicrelationshipsusedaneighbor-joiningtreeandtheITTsubstitutionmodel[52]andweredisplayedusingArchaeopteryx (http://www.phylosoft.org/archaeopteryx).Hydrophobicity analyseswereconductedusingthemethodofHoppand Woods[53,54]atweb.expasy.organdtransmembranesegmentswerepredictedwithTMpredathttp://www.ch. embnet.org/software/TMPRED_form.html.PCRamplificationofvirB6-4generepeatregions,cloning, andPacificBiosciencessequencingDuetodifficultiesinamplifyi ngtandemrepeat-containing DNA,allPCRreactionsspanningthe virB6 4 generepeat regionswereperformedinthepresenceof1.5-1.7M Betaine(Sigma).The8.36kbPCRproductspanningR3 andR4inthe Ap HZstrain(Figure3A,3C,andAdditional file2:FigureS2A)wasamplifiedusingtheiProof High-FidelityDNAPolymerasesystemwithGCbuffer (Bio-Rad).Reactionstotaled50 lwith5ngpurified A phagocytophilum gDNA,1.0Upolymerase,1.5mMMgCl2, 200 MeachdNTP,and250nMeachprimer(AB1393: 50-CGGGATCTAAGACAGATGATGATTC-30,forward; AB1466:50-CTCATCCTGATGCGTCTCCTTAG-30,reverse;Figure3A).35cyclesof30secdenaturingat98C, 20secannealingat67C,and5minextensionat72Cwere performed.PCRproductsspanningR4in Ap JMand Ap Dog1(both~10.3kb;Figure3C)werederivedusing Takara ’ sPrimeSTARGXLDNAPolymerasesystem(ClontechLaboratories,MountainView,CA).Reactionscontained5nggDNA,1.25Upolymerase,1.0mMMgCl2, 200 MeachdNTP,and200nMeachprimer(AB1395:50CACCAGAGGATGCAGCATTAG-30,forward;AB1466, reverse;Figure3A)intotal50 l.Followingthemanufacturer ’ srecommendations,2-stepPCRwasperformedwith 30cyclesof10secdenaturingat98Cand10minannealing/extensionat68C.PCRproductswereanalyzedon 0.5%agarosegelsalongsidethe1kbPlus(Invitrogen)and theGeneRulerHighRange(Fe rmentas,Inc.,GlenBurnie, MD)DNAladders.InordertoTA-clonetheamplicons,Aoverhangswereaddedtotheendsusing0.5-1.0units AmpliTaqDNApolymerase(AppliedBiosystems,Foster City,CA)ina10 – 15minreactionat72C.Productspurifiedfromagarosegels(beforeorafterA-overhangaddition) wereclonedintothepCR-XL-TOPOvector(Invitrogen) andtransformedinto E coli Stbl2(Invitrogen),whichis morepermissivetorepeat-ladenforeignDNA.Recombinantscontainingthecorrectsizeinsertwereendsequenced toverifytheiridentity. Inpreparationforsequencingwiththelong-read lengthPacificBiosciences(PacBio)next-generationsequencingRSinstrument,constructswerelinearizedwith restrictionenzymeswhichcutthevectoronly,buton oppositesidesoftheinsertwithintheMultipleCloningAl-Khedery etal.BMCGenomics 2012, 13 :678 Page12of15 http://www.biomedcentral.com/1471-2164/13/678

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Site.For Ap HZ,equimolaramountsoftheTAclone werecutwitheitherHindIIIorEcoRV.FollowingpoolingandEtOHprecipitation,thelinearizedDNAmix wassubmittedtoICBR/UFforSMRTbelllibraryconstructionandsequencing.Librarieswereconstructed usingacommercialstrobelibrarypreparationkit (#001-326-530;PacificBiosciences,MenloPark,CA) followingstandardmanufacturerprotocols.Tofurther increasethelikelihoodoffullcoverage,thestrobesequencingrunwasperformedusingtwodifferent conditions:I)45minlightperiod(continuouscollectiontime);andII)(5minlightperiod,10mindark period),followedby(45minlightperiod,10mindark period).The Ap JMand Ap Dog1constructswere double-digestedwithHindIII/XbaItoexcisethe~10.3kb inserts.Followingseparationon0.5%agarosegels,the insertswererecoveredfromagaroseslicesbyelectroelutionandfurtherpurifiedandconcentratedbypassage overQIAquickspincolumnsfollowingthePCRPurificationkitprotocol(Qiagen).SMRTbelllibrariesweremade asabovethensequencedusingasingle75minmovie timerun. Duetotherepetitivenatureoftheclonedgenefragments,combinedwiththerelativelyhigherror-rateof thePacBiosystem,allattemptstoassemblethereads de novo failedtoyieldasequenceoftheexpectedsize. Therefore,foreachconstruct,reads>3kbwereselected fromthemulti-fastafilesusingtheGalaxysuite[55], andimportedintotheCLCGenomicsWorkbenchfor assemblyandfurtheranalysis.Thesewereassembledat lowstringencyinitiallyagainstaconsensussequence representinganentire(vectorandinsertsequence)linearconstructtowhichsufficientN ’ swereaddedbased ontheestimatedgap-size.Startingwithreadsinitiating outsidetherepeatregion,thelongestoftheassembled readswerevisuallyinspectedforthepresenceof virB6 4 R4repeatsignature-sequences(Additionalfile2:Figure S2)andtheirsequencemanuallycorrectedwherenecessary.TheextendedsequenceswereusedtoreplaceN ’ s intheconsensusandtheprocessrepeatedseveraltimes untilsufficientreadswith>2kbsequenceoverlapwere recoveredspanningtheentireinsertregion.Forverification,thecompletedsequenceforeachstrainwasusedas thereferencetore-mapalltherespective>3kbPacBio readsandtheRoche/454readsathigherstringency.GenBankAccessionNumbers:foreachisolate,the vir genesarelistedinorderThesequencesof vir lociarecompleteforstrains Ap Dog1and Ap JM.Thesequenceoftherepetitive virB6 4 locuswasincomplete( Ap Dog2)ornotdeterminedfortheotherstrainsexcept Ap Hz.Weprovidea revisedsequenceof virB6 4 forthepreviouslysequenced [15] Ap HZstrain. Ap Dog1:JX415845-JX415868 B2-1B2-2,B2-3,B2-4,B2-5,B2-6,B2-7,B2-8,B2-9, B3,B4-1,B4-t1,B4-2,B6-1,B6-2,B6-3,B6-4,B8-1,B82,B9-1,B9-2,B10,B11,D4 Ap JM:JX415869-JX415892 B2-1,B2-2,B2-3,B2-4,B2-5,B2-6,B2-7,B2-8,B2-9, B3,B4-1,B4-t1,B4-2,B6-1,B6-2,B6-3,B6-4,B8-1,B82,B9-1,B9-2,B10,B11,D4 Ap Dog2:JX415893-JX415915( virB6-4 submitted separatelyasgapped) B2-1,B2-2,B2-3,B2-4,B2-5,B2-6,B2-7,B2-8,B2-9, B3,B4-1,B4-t1,B4-2,B6-1,B6-2,B6-3,B8-1,B8-2,B91,B9-2,B10,B11,D4 Ap NorLambV2:JX415916-JX415938 B2-1,B2-2,B2-3,B2-4,B2-5,B2-6,B2-7,B2-8,B2-9, B3,B4-1,B4-t1,B4-2,B6-1,B6-2,B6-3,B8-1,B8-2,B91,B9-2,B10,B11,D4 Ap NorLambV1:JX415939-JX415966 B2-1,B2-2,B2-3,B2-4,B2-5,B2-6,B2-7,B2-8,B2novel1,B2-novel2,B2-novel3,B2-novel4,B2-novel5, B2-novel6,B3,B4-1,B4-t1,B4-2,B6-1,B6-2,B6-3,B81,B8-2,B9-1,B9-2,B10,B11,D4 Ap HZ virB6-4 :JX415967 Ap Var1:JX415968-JX415996 B2-1,B2-2,B2-3,B2-4,B2-5,B2-6,B2-7,B2-8,B2novel1,B2-novel2,B2-novel3,B2-novel4,B2-novel5, B2-novel6,B2-novel7,B3,B4-1,B4-t1,B4-2,B6-1, B6-2,B6-3,B8-1,B8-2,B9-1,B9-2,B10,B11,D4 Ap MRK:JX415997-JX416019 B2-1,B2-2,B2-3,B2-4,B2-5,B2-6,B2-7,B2-8,B2-9, B3,B4-1,B4-t1,B4-2,B6-1,B6-2,B6-3,B8-1,B8-2,B91,B9-2,B10,B11,D4 ApDog2 virB6-4 Gapped:JX416020.AdditionalfilesAdditionalfile1:FigureS1. Multiplesequencealignmentof VirB6-3aminoacidsequencesfromdifferentstrainsofA. phagocytophilum.ArrowsindicatethelocationsofC-terminal41-mer repeats. Additionalfile2:FigureS2. StructureofthevirB6-4repeatregionsR3 andR4infourUSA.phagocytophilumstrains.A.Comparativemapsof AB1393/AB1466PCRproductsdetailingtherepeatunitcontentofR3and R4inthehuman,rodentanddogstrains. Ap JMand Ap Dog1have identical virB6 4 genesandare,therefore,representedbyonemap. ModeratevariabilityinthenumberandsequenceoftheR3405bp repeatunits(lightbluearrows)isapparent.Thesmallbarattheendof R3correspondstothe30-mostpartialrepeatunitpresentinallstrains.Al-Khedery etal.BMCGenomics 2012, 13 :678 Page13of15 http://www.biomedcentral.com/1471-2164/13/678

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ThecoloredarrowswithinR4representthefiverepeattypesT1a(yellow), T1b(green),T2a,(red),T2b(darkblue)andT2c(grey).Therepeatpattern in Ap HZshowsnorelationshiptothatof Ap JM/ Ap Dog1,whichisalso 2.87kblarger,totaling9.76kb.Thisregionwasnotfullycharacterizedin Ap Dog2asindicatedbyabrokenline,buttherepeatpatternofthe50and30-mostrepeatsisclearlydifferentfromthatoftheotherstrains.The smallbardownstreamofthesecondrepeatunitrepresentsapartially characterizedtype2repeatunit.Linesaboveandbelowthe Ap HZand Ap JM/ Ap Dog1mapsdelineatesegmentsofsequenceidentitywithinthe respectiveR4regions.Theirsizesarespecified. B .Alignmentofthe nucleicacidsequenceofall virB6 4 R4repeatunittypesidentifiedto date.Type1repeatsareshowninblack,type2inblue.Differences betweensub-typesarehighlighted.AsingleBamHIsitepresentinall type2repeatsisunderlined.Withtheexceptionofonlyafew nucleotidesateachend,type1andtype2repeatunitsdonotshareany sequences. C .Alignmentoftheaminoacidsequencesoftherepeatunits showninB.Thesinglenucleotidedifferencesbetweensub-typesdonot leadtochangesinaminoacidsequence. Competinginterests Theauthorsdeclarethattheyhavenocompetinginterests. Authors ’ contributions BAKandAFBconceivedthestudy,performedbioinformaticsanalysesand draftedthemanuscript.BAKgrewinfectedHL-60cellcultures,purified organisms,isolatedgDNA,designedandsupervisedPCRandsubmitted sequencestoGenBank.AMLperformedPCRanalysesandcloningand superviseddatatransferbetweenunits.SSandEGGisolatedtheEuropean sheepstrains,infectedandmonitoredsheep,andpreparedorganismsat maximalparasitemia.UGMandCMNisolatedandcultured invitro theJM, MRK,Dog2andApvariant1strains,andpreparedDog2andApvariant1 straingDNA.ARAandSMMestablishedtheDog1strain.Allauthorsreadand approvedthefinalmanuscript. Acknowledgements TheresearchdescribedherereceivedsupportfromgrantsRO1GM081714 andGM081714-03S1andfromPfizerAnimalHealth.WethankDr.Roberta Veluci-Marlow,SusanBendaandAdamWebsterforhelpwithculturingcells infectedwith A phagocytophilum ,andDr.SavitaShankerforhighthroughputDNAsequencing. Authordetails1DepartmentofInfectiousDiseasesandPathology,CollegeofVeterinary Medicine,UniversityofFlorida,Gainesville,FL,USA.2Departmentof ProductionAnimalSciences,SectionofSmallRuminantResearch,Norwegian SchoolofVeterinaryScience,Sandnes,Norway.3DepartmentofEntomology, UniversityofMinnesota,StPaul,MN,USA.4PhysiologicalSciences,Collegeof VeterinaryMedicine,UniversityofFlorida,Gainesville,FL,USA.5PfizerAnimal Health,Kalamazoo,MI,USA. 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Submit your next manuscript to BioMed Central and take full advantage of: € Convenient online submission € Thorough peer review € No space constraints or color “gure charges € Immediate publication on acceptance € Inclusion in PubMed, CAS, Scopus and Google Scholar € Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Al-Khedery etal.BMCGenomics 2012, 13 :678 Page15of15 http://www.biomedcentral.com/1471-2164/13/678