Genome-wide RNAi selection identifies a regulator of transmission stage-enriched gene families

25 Pages·2017·2.55 MB·English

RESEARCHARTICLE
Genome-wide RNAi selection identifies a
regulator of transmission stage-enriched
gene families and cell-type differentiation in
Trypanosoma brucei
EvaRico1,AlasdairIvens1,LucyGlover2¤,DavidHorn2,KeithR.Matthews1*
1 CentreforImmunity,InfectionandEvolution,InstituteforImmunologyandInfectionResearch,Schoolof
BiologicalSciences,UniversityofEdinburgh,Edinburgh,Scotland,UnitedKingdom,2 TheWellcomeTrust
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CentreforAnti-InfectivesResearch,SchoolofLifeSciences,UniversityofDundee,Dundee,Scotland,United
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Kingdom
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a1111111111 ¤ Currentaddress:DepartmentofParasitesandInsectVectors,InstitutPasteur,Paris,France
a1111111111 *[email protected]
Abstract
OPENACCESS Trypanosomabrucei,causingAfricansleeping-sickness,exploitsquorum-sensing(QS)to
Citation:RicoE,IvensA,GloverL,HornD, generatethe‘stumpyforms’necessaryfortheparasite’stransmissiontotsetse-flies.These
MatthewsKR(2017)Genome-wideRNAiselection quiescentcellsaregeneratedbydifferentiationinthebloodstreamfromproliferativeslender
identifiesaregulatoroftransmissionstage-
forms.Usinggenome-wideRNAiselectionwescreenedforrepressorsoftransmission
enrichedgenefamiliesandcell-typedifferentiation
inTrypanosomabrucei.PLoSPathog13(3): stage-enrichedmRNAsinslenderforms,usingthestumpy-elevatedESAG9transcriptasa
e1006279.https://doi.org/10.1371/journal. model.ThisidentifiedREG9.1,whoseRNAi-silencingalleviatedESAG9repressioninslen-
ppat.1006279
derformsandtsetse-midgutprocyclicforms.Interestingly,trypanosomesurfaceprotein
Editor:ChristianTschudi,YaleSchoolofPublic Family5andFamily7mRNAswerealsoelevated,which,likeESAG9,areT.bruceispecific
Health,UNITEDSTATES
andstumpy-enriched.Wesuggestthesecontributetothedistincttransmissionbiologyand
Received:January10,2017 vectortropismofT.bruceifromotherAfricantrypanosomespecies.Aswellassurfacefam-
Accepted:March8,2017 ilyregulation,REG9.1-depletiongeneratedQS-independentdevelopmenttostumpyforms
invivo,whereasREG9.1overexpressioninbloodstreamformspotentiatedspontaneousdif-
Published:March23,2017
ferentiationtoprocyclicformsintheabsenceofanexternalsignal.Combined,thisidentifies
Copyright:©2017Ricoetal.Thisisanopen
REG9.1asaregulatorofdevelopmentalcellfate,controllingtheexpressionofTrypano-
accessarticledistributedunderthetermsofthe
CreativeCommonsAttributionLicense,which somabrucei-specificmoleculeselevatedduringtransmission.
permitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginal
authorandsourcearecredited.
DataAvailabilityStatement:RNA-seqandIon Authorsummary
torrentdataareavailableat:http://www.ncbi.nlm.
nih.gov/geo/query/acc.cgi?token= AfricantrypanosomescauseimportantdiseaseofhumansandlivestockinsubSaharan
ovahieycdlahlyh&acc=GSE81765 Africaandaretransmittedbytsetseflies.Inpreparationfortransmission,Trypanosoma
Funding:ThisworkwassupportedbyaWellcome bruceiusesquorumsensingtogenerate‘stumpyforms’thatarearrestedandexpressadis-
Trust(https://wellcome.ac.uk)SeniorInvestigator tinctsubsetofgenestothe‘slenderforms’thatproliferatetoestablishtheparasitaemiain
awardtoKRM[grantnumber103740/Z/14/Z],a thebloodstream.Thisnecessitatesthatstumpy-enrichedtranscriptsarerepressedinslen-
RoyalSociety(https://royalsociety.org)Research
derforms,althoughthemolecularcontrolofthisisunknown.Here,wehavedevelopeda
meritaward[grantnumberWM140045]toKRM,a
genome-wideselectionalstrategytoisolaterepressorsofstumpy-enrichedgenes,and
WellcomeTrust(https://wellcome.ac.uk)Strategic
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Trypanosomedevelopmentalcontrol
AwardtotheCentreforImmunity,Infectionand
Evolution[grantnumber095831],andaWellcome successfullyidentifiedanovelregulatorymolecule,termedREG9.1.SilencingofREG9.1
Trust(https://wellcome.ac.uk)SeniorInvestigator alleviatestherepressionofthepreviouslycharacterisedstumpy-enrichedESAG9gene
AwardtoDH[grantnumber100320/Z/12/Z].The
family,andalsotwonovelpredictedsurfaceproteinfamiliesthatarespecifictoTrypan-
fundershadnoroleinstudydesign,datacollection
somabruceibutabsentfromotherAfricantrypanosomespecies.REG9.1silencingalso
andanalysis,decisiontopublish,orpreparationof
drivesdensity-independentdifferentiationtostumpyforms,whereasitsectopicexpres-
themanuscript.
sioninbloodstreamformspotentiatesdifferentiationtotsetsemidgutprocyclicformsin
Competinginterests:Theauthorshavedeclared
theabsenceofanexternalsignal.REG9.1isthereforeidentifiedasanoveldevelopmental
thatnocompetinginterestsexist.
regulatorwhoseactionmaycontributetothespecies-specifictransmissionbiologyofTry-
panosomabrucei,whichdiffersfromthatofeitherTrypanosomacongolenseorTrypano-
somavivax.
Introduction
Africantrypanosomescompriseacollectionofdifferentspecies(Trypanosomabruceispp.,Try-
panosomacongolense,Trypanosomavivax)thatdifferintheirhostrange,pathology,morphol-
ogy,motilityandlife-cycledevelopment[1,2].Theseunicellularparasites,responsiblefor
HumanandAnimalAfricanTrypanosomiasis(HAT,AAT),arehighlyunusualintheeukar-
yotainthattheirgenomeisorganisedintopolycistronicgeneclustersinwhichdistinctgenes
canshowdifferentialregulationeitherattheleveloftranscriptabundance,translationorpro-
teinturnover[3].
Regulationoftrypanosomegeneexpressionisparticularlyimportantastheseparasites
progressthroughtheircomplexlifecycle[4].ForT.congolense,developmentislimitedtothe
tsetsemidgutandmouthparts,whereasforT.vivaxtheparasitesarerestrictedtothemouth-
partsalone.InT.brucei,incontrast,thereareseveraldevelopmentalformsbothinthe
bloodstreamoftheirmammalianhostandindifferentcompartmentsofthetsetsefly[5,6].
Specifically,theparasitesproliferateinthetsetseflymidgutasearlyandlatestageprocyclic
formsbeforemigrationtothesalivaryglandswheretheyestablishasepimastigotesbefore
developingthemetacyclicformsthataremammalinfective[7].Progressionthroughthese
developmentalformscanbedrivenbytheoverexpressionofjustasinglepredictedRNAbind-
ingprotein,RBP6,thatallowstheproductionofinfectivemetacyclicformsfromprocyclic
formswithoutanexternalstimulus[8].Inthebloodstream,T.bruceiestablishtheparasitaemia
asproliferativeslenderforms,whichevadethehostimmuneresponsethroughtheirexpression
andabilitytochangetheexpressionofadensevariant-specificsurfaceglycoprotein(VSG)
coat[9–11].Asbloodstreamparasitedensityincreases,thetrypanosomesexhibitaquorum-
sensinglikeresponsewhereupontheyarrestinG1asaquiescentandmorphological‘stumpy
form’[12].Thesearecompetentfortransmissiontothetsetseflyupontheiruptakeinablood-
mealwheretheydifferentiatetoprocyclicforms,whereasthebloodstreamslenderformsare
killed[13].Hence,whilstsharingthesamebloodstreamenvironmentasslenderforms,the
stumpyformrepresentsadistinctdevelopmentalstage,whichalsoexhibitsadistinctdevelop-
mentalgeneexpressionprofile[14–17].Thisrequiresregulatorsthateitheractivelymaintain
theslendercellform,orinhibitorpromotethedifferentiationtostumpyforms.
Amongthemostpredominantgenefamiliesup-regulatedinstumpyformsistheESAG9
genefamily[14,18].ESAGs(ExpressionSiteAssociatedGenes)arecommonlyfoundinthe
polycistronicexpressionsitelocusresponsibleforVSGgenetranscription.However,ESAG9
genesareonlyoccasionalcomponentsoftheVSGgeneexpressionsitesandseveralcopies
ofthehighlydiversegenefamilycanbesimultaneouslyexpressedupondifferentiationto
stumpyforms[18–20].AnanalysisofthepredictedcellsurfacephylomeofdifferentAfrican
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trypanosomespeciesdeterminedthatESAG9genes(includedincellsurfacephylomeFamily
2)werespecifictoT.bruceiandmissingintherelatedparasitesT.congolenseandT.vivax[21].
Thiswasalsothecaseforeightothercellsurfacephylomefamilies,whichmayhavefunctions
relatedtotheparticularbiologyofT.bruceiintheirmammalianhostortsetseflyvector.
Thestumpy-enrichedexpressionoftheESAG9genefamilyisregulatedthroughtheir
3’UTR[22].Exploitingthis,herewereportagenome-wideRNAiselectionalregimendesigned
toisolaterepressorsofstumpy-enrichedgeneexpressionoperativeinproliferativeblood-
streamslenderforms.Thissuccessfullyidentifiedseveralnegativeregulators,includingone
(REG9.1;REGulatorofESAG9)whoseindependentdepletionconfirmeditsfunctionasagen-
eralregulatorofESAG9familygeneexpression.Strikingly,depletionofthismoleculealso
resultedinpremature,density-independent,differentiationtostumpyformsinvivoandthe
strongupregulationofmembersoftheT.bruceicellsurfaceproteinfamilies5and7,inaddi-
tiontoESAG9.EachofthesegenefamiliesisabsentinT.congolenseandT.vivax.Interestingly,
ectopicexpressionofREG9.1attheelevatedlevelsnormallyobservedinprocyclicforms,also
causedbloodstreamformstoelevateprocyclicformsurfaceproteinmRNAsandpotentiated
differentiationtothatform.ThisidentifiesREG9.1asanoveldifferentiationregulatorand
controllerofthesurfacemoleculesassociatedwiththespecies-specificdevelopmentalbiology
ofT.brucei.
Results
AgenomewideRNAiscreenforrepressorsofmoleculesexpressedin
theT.bruceitransmissionstage
Usinggenome-wideRNAiselectionwesoughttoidentifymoleculesthatactinT.bruceislen-
derformstorepressstumpyformenrichedmRNAs.Ourstrategywastogenerateacellline
thatallowedustoselectforincreasedresistancetoG418resultingfromthelossofarepressor
thatactedonaNeoRgeneregulatedbytheexpressioncontrolsignalsofastumpy-enriched
transcript,ESAG9(Fig1A).Genome-widescreensrequireuseofthes427monomorphic
bloodstreamformcellline,competentforefficienttransfectionwithatetracycline-inducible
RNAilibrary[23–26].Thesecells,likeslenderformsinnatural(pleomorphic)bloodstream
infections,expresslittleESAG9mRNAduetoposttranscriptionalrepression[18,22].Tocarry
outthescreen,weinitiallytransfectedthes427monomorphicparentalcelllinewithacon-
structcomprisingtheneomycinresistance(NeoR)geneflankeddownstreambya1057ntfrag-
mentcontainingthepreviouslycharacterised3’UTRcontrolsignalsandintergenicsequence
oftheESAG9geneTb927.5.4620[22](‘ESAG9-EQlong3’UTR’inFig1A).Whentheresulting
celllinewasassayedwithatitrationofG418,cellstransfectedwiththeNeoRgenecoupledto
theESAG9flankingsequencewerekilledat(cid:21)10μg/mlG418,whereascellstransfectedwiththe
sameconstructbutwheretheNeoRgenewasflankedbytheconstitutively-expressedaldolase
gene3’UTRwereviableat>750μg/mlG418(Fig1B).Thisconfirmedourpreviousanalyses
demonstratingthattheESAG9downstreamsequencesrepressgeneexpressioninproliferative
slenderforms[22].
ThecelllinewiththeconstructcomprisingtheNeoRgeneflankedbytheESAG9intergenic
sequencewasthenstablytransfectedwiththetetracyclineinduciblegenome-wideRNAi
library[25].Thereafter,uninducedandinducedpopulationswereselectedwith10μg/mlG418
(Fig1C);eventualoutgrowthofsurvivingcellsoccurredat10μg/mlG418withRNAiinduction
butnotat25μg/mlG418.Incontrast,intheabsenceofG418ortetracycline,growthwasmain-
tainedunimpededthroughthedurationoftheexperiment(Fig1C).AftergenomicDNAisola-
tionandanalysisoftheRNAiinsertampliconsfromparasitesthroughouttheselectionperiod,
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Fig1.Selectionofnegativeregulatorsofstumpyenrichedtranscriptexpression.A.Schematicrepresentationoftheselectionschematoisolate
negativeregulatorsofESAG9expressioninslenderforms;NRdenotesanegativeregulator.TheselectionconstructusedtotransfecttheRNAilibraryis
shownbeneath.TheNeoRgeneisflankedbythe5’UTRofthetrypanosomealdolasegene,whilstthe3’UTRisderivedfrom1057bpdownstreamofthe
ESAG9-EQstopcodon(longformreporter,blue).B.TitrationofthesensitivityoftrypanosomelineswithNeoRflankedbyeithertheESAG9-EQ3’UTRor
aldolase3’UTR.Errorbarssymbolizestandarddeviationsofbiologicaltriplicates.C.RNAilibraryselectionofparasitescontainingNeoRflankedbythe
ESAG93’UTR(diamonds).TherighthandpanelshowsthelibraryinsertampliconprofilefromcellswithNeoRflankedbytheESAG93’UTRatdifferent
stagesofselectioninG418whenRNAiwaseitherinduced,ornot.ThegelshowsgDNAisolatedfromuntreated(notetracycline,noG418)parasitesat
days2and3(asacontrol),andtreatedparasitesatdays11and14afterexposureto1μg/mloftetracyclineand10μg/mlG418,whenparasiteswere
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outgrowingasaresistantpopulation.D.Genome-widedistributionofselectedRNAiinsertsdeterminedbyiontorrentsequencing,verticaldashes
representingtheboundariesofeachchromosome(withChromosome1attheleft).Targetshighlightedcomprisethosewithahighenrichmentinthe
selectedpopulationandapredictedRNAbindingdomain.ThefulldatasetisavailableinS1Table.
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asignificantchangeintheampliconprofilewasobservedwithstrongenrichmentofdiscrete
bandsafterselectionfor11and14daysinG418(Fig1C,righthandpanel).
Toanalysetheselectedmaterialindetail,iontorrentdeepsequencing[12]wascarriedout
onreplicatesamplesoftheday14selectedpopulations.Theresultingreadsweremapped
againsttheT.bruceiv9genomeassembly(Fig1D;S1Table)andthetop30enrichedlociana-
lysedforalignmenttogeneswithapredictedroleinmRNAregulation.Thisidentifiedthree
candidategenes,ofwhichtwowerealreadycharacterisedtosomeextent(Tb927.6.3480,
DRBD5,[27];Tb927.3.720,TbZFP3;[28,29])andanovelgeneencodingahypotheticalprotein
(REGulatorofESAG9-1,REG9.1;Tb927.11.14220)(Fig1D).Toverifythescreenoutputs,inde-
pendentRNAiconstructsderivedfromeachcandidategenewerethentransfectedintothe
monomorphicreportercelllinecontainingtheNeoRgeneflankedbytheESAG9intergenic
region.NeoRproteinexpressionwasincreaseduponsilencingofeachpotentialregulator,vali-
datingtheselectionofeachcandidate,butRNAitargetingDRBD5andZFP3didnotelevate
endogenousESAG9transcript(S1Fig).InthecaseofDRDB5,thiswasbecausethe5’UTRof
ESAG9contributestoESAG9repressioninadditiontothe3’UTR(RicoandMatthews,per-
sonalobservations).Incontrast,REG9.1knockdown(Fig2A)resultedinenhancedG418
resistance(Fig2B),elevationofNeoRmRNA(Fig2C)andprotein(Fig2D),andelevationof
endogenousESAG9-EQtranscripts(Fig2E)inmonomorphiccells.
ToexploretheregulationoftheESAG9-EQmRNAfurther,REG9.1RNAiwascarriedout
inreporterlinesharbouringESAG9-EQdownstreamsequencesextendingonly400ntfrom
thestopcodon(ESAG9-‘shortform’),orlackingapreviouslymappedregulatoryelementcon-
tributingtostumpy-enrichedexpression(ESAG9ΔE)(S2AFig).TheESAG9-shortform3’UTR
waslesssensitivetoG418thanthecontrolreporterwith1057ntofdownstreamsequence,
consistentwiththedeletionofrepressiveelementsfromthe3’UTR(S2BFig).Nonetheless
uponREG9.1depletion,boththeshortform3’UTRandtheΔEmutantexhibitedincreased
NeoRmRNA,demonstratingtheretentionofREG9.1-dependentregulatorymotifswithin
each3’UTR(S2CandS2DFig).Thus,REG9.1negativelyregulatesESAG9throughdown-
streamsequences,butthiswasnotsimplyrestrictedtodiscreteproximalordistalsequencesin
theESAG9intergenicregion,norasmallregionofthe3’UTR.
IndependentRNAilinesfor10potentialfurthertargetsidentifiedinthelibraryscreenwere
alsogeneratedbuttheseeitherexhibitednoeffectiveRNAknockdown,didnotenhanceG418
resistanceuponRNAiorgeneratedastronggrowthphenotypeprecludinginformativefollow-
upstudies(S1Table).
REG9.1isdifferentiallyexpressed
REG9.1ispredictedtoencodea54.4kDaproteinwithanacidicPI(pH5.5)andapredicted
RNA-bindingmotif(SuperfamilySSF54928domain)(Fig3A).InapreviousmRNAtethering
screen,directbindinginparasitesofaREG9.1-lambdaNfusionproteintoareporterconstruct
mRNAwithBoxBbindingsitesrepressedexpression,supportingapotentialregulatoryfunc-
tionforREG9.1[27].
AnantibodywasgeneratedagainsttheREG9.1proteinandthedevelopmentalexpression
profileoftheproteinassessedinpleomorphicslenderandstumpyforms,andculturedinsect
procyclicforms(Fig3B).Inadditiontoabandat54kDa,whichwasinconsistentlydetected,
twobandsweredetectedat57kDaand59kDapotentiallyrepresentingdifferentpost-
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Fig2.ValidationofRNAilinessilencingREG9.1selectedfromtheRNAilibrary.A.Northernblot
demonstratingtheinduciblegenesilencingofREG9.1.TwoindependentRNAicloneswereanalysed
(REG9.1,clone3and4).);rRNAwasusedasaloadingcontrol.Smearingiscausedbythetranscriptbeing
largerthan6kb.B.G418resistance(at10μg/ml)oftherespectiveRNAicloneswhenRNAiwasinducedor
not.RNAiresultedindecreasedsensitivitytoG418.Errorbars(obscuredbythesymbols)symbolizestandard
deviationsofthetriplicateassaysofeachclone±tetracycline.C.NorthernblotofNeoRtranscriptlevelswhen
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eachREG9.1RNAilinewasinducedornot;rRNAwasusedasaloadingcontrol.D.WesternblotofNeoR
proteinwheneachREG9.1RNAilinewasinducedornot;EF1αwasusedasaloadingcontrol.E.Northern
blotofESAG9-EQtranscriptlevelswheneachREG9.1RNAilinewasinducedornot;rRNAwasusedasa
loadingcontrol.
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translationallymodifiedformsoftheprotein(Fig3B).Theupperbandwassomewhatless
abundantinstumpyformscomparedtoslenderandprocyclicforms,andinprocyclicforms
theoverallabundanceofboth57and59kDabandswasgreaterthanineitherbloodstream
form.Consistentwiththis,quantitativeproteomicanalysishaspreviouslyindicatedthat
REG9.1is2-[17]to3-fold[30]moreabundantinprocyclicformsvs.stumpyforms.
TofurtherexplorethefunctionofREG9.1,pleomorphiccelllinesweregeneratedthateither
silencedthisgenebyRNAiorinduciblyoverexpressedtheproteinusingthepLEWectopic
expressionvector[31].Pleomorphiccellsareconsiderablymoredifficulttoanalyseandgeneti-
callymanipulatethanlaboratoryadaptedmonomorphiclinesbutarenecessaryfordevelop-
mentalstudiessincetheyundergonaturalprogressiontostumpyforms,whichisassociated
withphysiologicalESAG9regulation.Fig3Cshowsnorthernanalysisoftwoindependentpleo-
morphicRNAireplicatesdemonstratingeffectivedepletionofREG9.1.Matchingtheoriginal
selectioninmonomorphiccells,inbothcases,REG9.1depletioncausedupregulationof
ESAG9-EQmRNAinvitro.Inseparatesamples,theproteinlevelsofREG9.1werealsocorre-
spondinglyreduceduponRNAi(Fig3D).
ToassessthephenotyperesultingfromREG9.1depletioninvivo,thepleomorphicRNAi
linewasinoculatedintomice,withgenesilencingbeinginducedbyprovisionofdoxycycline
tothedrinkingwaterofreplicateinfectedanimals1-daypostinfection.Strikingly,thisresulted
inarapidcessationoftheincreasingparasitaemiasuchthatinducedparasitesremainedbelow
5x107parasites/mlcomparedtouninducedparasitesthatreached2.5x108parasites/ml(Fig
4A).Despitetheirlowparasitaemia,themorphologyoftheinducedparasitesresembledinter-
mediate/stumpyformsgeneratedbyuninducedparasitesathighparasitaemiaafter4daysof
infection(Fig4B;S3AFig).Todeterminewhetherthesehadtheexpectedphysiologicalcharac-
teristicsofstumpyforms,wetestedtheircellcyclearrestinG0/G1,theirexpressionofthe
stumpyspecificmarkerproteinPAD1[32]andtheircapacityfordifferentiationtoprocyclic
formsuponharvestandexposureincultureto6mMcisaconitate.Fig4Cshowsthatthe
inducedparasitesaccumulatedwitha1kinetoplast1nucleusconfiguration(consistentwith
G1/G0arrest)andexpressedPAD1asassessedbybothflowcytometryandwesternblotting
(Fig4D),despitebeingat5-10-foldlowercelldensitythanuninducedparasites.Further,the
isolatedparasiteswereabletodifferentiatesynchronouslytoprocyclicforms,expressingthe
procyclicformsurfaceproteinEPprocyclinon>60%ofcells4hafterexposureto6mMcis
aconitate(Fig4E).Thecellsalsore-enteredaproliferativecellcycleafter24hinthesedifferen-
tiationconditions(S3BFig).AlloftheseassaysdemonstratedthatREG9.1depletiondrives
cellstogeneratephysiologicallyvalidstumpyformsatmuchlowerparasitaemiathanwhen
uninduced,overridingnormalquorumsensingmechanisms.
REG9.1regulatesmRNAsfordevelopmentallyregulatedcellsurface
proteins
Thewiderconsequencesfortheparasites’transcriptomeofsilencingREG9.1werethenexam-
ined.Thus,invitrogrownbiologicalreplicatesofthepleomorphicRNAiline,inducedornot
for48h,wereanalysedbyRNA-seq(Fig5;S2Table).Fig5Aand5Bdemonstratethatnotonly
ESAG9-EQshowedelevatedexpressionbutalsomanyothermembersoftheESAG9family
(lilacsquaresinFig5A;GreenbarsinFig5B),includingthosewithonlydistantrelationshipto
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Fig3.REG9.1developmentalexpressionprofileandREG9.1RNAiinpleomorphicparasites.A.
SchematicrepresentationofREG9.1showingthepositionofitssinglepredictedRNAbindingdomain(RBD).
B.ProteinexpressionofREG9.1.Theantibodydetectsthreebands,thelowerbandbeingdetected
inconsistentlybetweenblots.Theabundanceofthetwoupperbandsdifferindifferentlifecyclestages,with
theuppermostbandbeinglessabundantinstumpyforms.ProcyclicformsexpressmoreREG9.1than
bloodstreamforms.C.NorthernblotforREG9.1andESAG9-EQintheparentalline(T.bruceiAnTat90:13)or
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tworeplicatesofthepleomorphicRNAilinesinducedornottosilenceREG9.1.Smearingiscausedbythe
REG9.1transcriptbeinglargerthan6kb.rRNAprovidestheloadingcontrol.ESAG9-EQmRNAiselevated
uponREG9.1depletionineachreplicate.D.WesternblotofREG9.1expressioninpleomorphiccellsinduced
todepleteexpressionviaRNAi.TheloadingcontrolwasanantibodydetectingEF1α.
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ESAG9-EQ,demonstratingco-regulationofthefamily.Interestingly,membersofothergene
familieswerealsoelevatedandcoregulatedwithESAG9uponREG9.1depletion.Thisincluded
membersofthecellsurfacephylomeFamily5(redsquares,Fig5A)andFamily7(bluesquares,
Fig5A)bothofwhich,likeESAG9,arerestrictedtoT.brucei,beingabsentfromthegenomesof
T.congolenseorT.vivax[21].Family5has8members,ofwhich5wereelevateduponREG9.1
depletion(Fig5B,redbars);Family7comprises9membersofwhich6were>log2FCelevated
intheabsenceofREG9.1(Fig5B,bluebars).Northernanalysisusingriboprobessimultaneously
detectingseveralmembersofthesesurfaceproteinfamilymRNAsdemonstratedtheirelevated
expressioninstumpyforms,matchingtheexpressionprofileofESAG9(Fig5C).HenceREG9.1
actsasanegativeregulatorofatleastthreeT.bruceispecificsurfaceproteinfamilieseachof
whichisexpressedintheparasite’stransmissionstage.Apartfromthesefamilies,otherupregu-
latedtranscriptsalsoencodedpredictedsurfaceproteins(e.g.MSP-A,Gp63;Fig5AandFig
5C),whereasdown-regulatedtranscriptswererelatedtocellproliferationandstructure(e.g.
histones,flagellarandparaflagellarrodproteins)(Fig5A).TranscriptsforthePADproteins,
diagnosticforstumpyforms,werenotsignificantlyregulated(S2Table).
Toinvestigatewhetherthetranscriptregulationobservedsimplyrepresentedaconse-
quenceoftheREG9.1RNAicellsdevelopingtowardastumpyphenotypeandtherebyupregu-
latingstumpy-enrichedtranscripts,weanalysedREG9.1-depletioninprocyclicforms,which
donotnormallyexpressESAG9transcripts.Thus,theuninducedpleomorphicbloodstream
formREG9.1RNAilinewasdifferentiatedwith6mMcisaconitateand,oncestablyestablished
asprocyclicforms,REG9.1depletionwasinducedwithtetracycline.Theinducedcellsmain-
tainedgrowth,albeitatareducedlevelcomparedtouninducedcells(Fig6A),andthecells
appearedelongatedandthepopulationcontainedmanyanucleatecytoplasts(zoids;[33])(Fig
6B).WesternblottingconfirmedthattheREG9.1proteinwaseffectivelydepletedinthe
inducedpopulation(Fig6C),wheremanyofthecellsalsoshowedseverelyreducedmotility,
accumulatingatthebottomofthecultureflask,contrastingwiththeuninducedpopulation
thatremaineddistributedthroughoutthemedium(S1andS2Supplementarydata).Nonethe-
less,whenRNAwasanalysedintheinducedpopulations,ESAG9-EQtranscriptswerefound
tobeupregulated(andtoalimitedextentinuninducedcellswhereREG9.1RNAiwassome-
whatleaky),consistentwiththeobservationsinbloodstreamforms.Thiseliminatedtheregu-
lationofthesemRNAsinbloodstreamformsbeingasimpleindirectconsequenceofinduced
stumpyformationandfurthersupportedafunctionforREG9.1intherepressionofstumpy-
enrichedmRNAsregardlessoflifecyclestage.
EctopicoverexpressionofREG9.1potentiatesdifferentiationtoprocyclic
forms
ToassesswhetheranelevationofREG9.1levelsgeneratedconverseregulatoryeffectstothatof
RNAi,andsodelayedstumpyformation,pleomorphicbloodstreamcellsweregeneratedthat
wereabletooverexpressREG9.1.SupportingthephysiologicalrelevanceoftheREG9.1ectopic
expressiongenerated,thelevelofREG9.1proteininbloodstreamformsgeneratedusingthe
pLEWexpressionvectorwasequivalenttothelevelnormallyseeninprocyclicforms(Fig7A).
Theinducedbloodstreamformparasitesremainedviableandcontinuedtoproliferate,albeit
ataslightlyreducedlevel(Fig7B).However,analysisoftheglobaltranscriptomedatasetfor
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Fig4.PhenotypeofREG9.1genesilencinginvivo.A.GrowthofREG9.1pleomorphicRNAilinesinvivo,withRNAiinducedornotbydoxycycline.
TheinsetshowstherespectiveabundanceofREG9.1transcriptineachcaseaftercellswereharvestedonday4postinfectionandRNAprepared.
Twomicereplicates(R1andR2)wereusedforeachcondition(–DOXand+DOX).B.Morphologyofcellsonday4ofinfection.Twocellsareshownfor
theuninducedpopulation,bothbeing‘intermediate’inmorphology,althoughtheuppercellissomewhatmorestumpywiththenucleusmoreproximal
tothekinetoplast;intheinducedpopulationastumpycellisshown,withthenucleusmovedtowardthecellposterior.Hencealthoughatamuchlower
parasitaemia(seepanelA),thestumpymorphologyofinducedcellswasequivalentto,orgreaterthan,uninducedcells.TheDAPIpanelattheleft
handsideshowsthepositionsofthenucleus(N)andkinetoplast(K)ineachcaseBar=10μm.C.CellcycleprofileofpleomorphicRNAilines(R1,R2)
inducedornottosilenceREG9.1expression.Thekinetoplast/nuclearconfigurationwasassessedonday3andday4ofinfection,withtheassociated
parasitaemiashownaboveeachbar.Atleast250cellswerecountedforeachcondition/replicate/day.D.PAD1expressionprofiledeterminedbyflow
cytometryintheREG9.1RNAilines(R1,R2)inducedornot,withslenderorstumpyT.bruceiAnTat1.1cellsascontrols.Theparasitaemiaofeach
populationatharvestisshown.PADpositivitywasdeterminedbygatingwithrespecttotheSlender(negative)andStumpy(positive)controlsamples.
BelowthechartisthePAD1expressionprofiledeterminedbywesternblottingintheREG9.1RNAilines(R1,R2)inducedornot,whenharvested,with
stumpyAnTat1.1cellsascontrol(‘ST’).EF1αabundancewasusedasaloadingcontrol.E.FlowcytometryoftheexpressionofEPprocyclinduring
differentiationtoprocyclicformsofeachREG9.1RNAiline(R1,R2)0h,4hand24hafterexposureto6mMcisaconitate.EPprocyclinexpressionon
slender,stumpyandprocyclicformsprovidecontrols.AlowlevelofEPprocyclinexpressionisdetectedonstumpycells,thisbeinginducedduring
isolationandcellpreparationprocedures.Althoughatlowerparasitaemia(panelA)theinducedcellsdifferentiateaseffectivelyasuninducedcells
demonstratingthephysiologicalrelevanceofthestumpyformsgenerated.
https://doi.org/10.1371/journal.ppat.1006279.g004
PLOSPathogens|https://doi.org/10.1371/journal.ppat.1006279 March23,2017 10/25