Characterization of the small RNA content of Trypanosoma cruzi extracellular vesicles

Molecular&BiochemicalParasitology193(2014)71–74

ContentslistsavailableatScienceDirect

Molecular

&

Biochemical

Parasitology

Short

communication

Characterization

of

the

small

RNA

content

of

Trypanosoma

cruzi

extracellular

vesicles

Ethel

Bayer-Santos

,

Fábio

Mitsuo

Lima

,

Jeronimo

Conceic¸

ão

Ruiz

,

Igor

C.

Almeida

,

José

Franco

da

Silveira

a_{DepartamentodeMicrobiologia,ImunologiaeParasitologia,UniversidadeFederaldeSãoPaulo,SãoPauloSP04023-062,Brazil} b_{BorderBiomedicalResearchCenter,DepartmentofBiologicalSciences,UniversityofTexasatElPaso,ElPaso,TX79968,USA} c_{CentrodePesquisasReneRachou,Fundac¸ãoOswaldoCruz,MinasGerais,MG30190-002,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received2December2013

Receivedinrevisedform11February2014

Accepted15February2014

Availableonline25February2014

Keywords: Trypanosomacruzi

Extracellularvesicles

SmallnoncodingRNA

High-throughputdataanalysis

Comparativetranscriptome

a

b

s

t

r

a

c

t

Agrowingbodyofevidenceinmammaliancellsindicatesthatsecretedvesiclescanbeusedtomediate intercellularcommunicationprocessesbytransferringvariousbioactivemolecules,includingmRNAsand microRNAs.Basedonthesefindings,wedecidedtoanalyzewhetherTrypanosomacruzi-derived extracel-lularvesiclescontainRNAmoleculesandperformedadeepsequencingandgenome-wideanalysisofa size-fractionedcDNAlibrary(16–40nt)fromextracellularvesiclessecretedbynoninfectiveepimastigote andinfectivemetacyclictrypomastigoteforms.OurdatashowthatthesmallRNAscontainedinthese extracellularvesiclesoriginatefrommultiplesources,includingtRNAs.Inaddition,ourresultsreveal thatthevarietyandexpressionofsmallRNAsaredifferentbetweenparasitestages,suggestingdiverse functions.Takentogether,theseobservationscallattentiontothepotentialregulatoryfunctionsthat theseRNAsmightplayoncetransferredbetweenparasitesand/ortomammalianhostcells.

©2014TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/3.0/).

Recentreportshavedescribedthattheintercellularexchange ofgeneticmaterialalsooccurineukaryotesandmulticellular orga-nismsthroughseveraldistinctpathwayssuchasthosedependent onnanotubes,extracellularvesicles(EVs),andnucleicacid-binding proteins[1].ThephenomenonofmicroRNA(miRNA)secretionand itsrole in intercellular crosstalk hasrecentlygained increasing attentionassomestudieshaveprovidedevidencethat membrane-boundvesiclesreleasedbyseveralcelltypescantransfermiRNAs toneighboringordistantcells,playingapreviouslyunrecognized roleinmodulatingcellfunctions.Severalmechanismshavebeen hypothesized describing the interactions of membrane-bound vesiclesand recipientcells,theseincludereceptor–ligand inter-actions,fusionofvesicleswithtarget-cellmembraneandvesicle endocytosis[2].

TherearecurrentlythreetypesofEVsthatarenamedaccording tothemechanismsbywhichtheyarereleasedintotheextracellular environment:(i)exosomes,whicharesmallcup-shapedvesicles rangingaround20–100␩mindiameterreleasedbytheexocytic

fusion of multivesicular bodies (MVBs); (ii) ectosomes, which

∗Correspondingauthor.Tel.:+19157476086.

∗∗Correspondingauthor.Tel.:+551155711095.

E-mailaddresses:[email protected](I.C.Almeida),[email protected],

[email protected](J.F.daSilveira).

arelargeheterogeneousvesiclesrangingaround100–1000␩min

diameterreleasedbythebuddingoftheplasmamembrane;and (iii)apoptoticbodies,whicharelargervesicles(>1␮min

diame-ter)thatoriginatefromapoptoticcells[3].Inapreviousreport,we demonstratedthatTrypanosomacruzireleasesatleasttwotypes ofmembranevesicles(i.e.,ectosomesandexosomes),generated bydistinctpathways. We alsoshowedthat infectivemetacyclic formsreleasevesiclescarryingvirulencefactorssuchasGP82 gly-coproteins and mucins, while in contact with HeLa cells, thus suggestingthatparasite-derivedEVscouldbeusedascarriersto delivervirulenceandmodulatoryfactorsintohostcells[4].Inthe light ofrecent findings showingthat mammaliancells are able tousemembranevesiclestotransfermRNAsandmicroRNAsto neighboring cells[5],togetherwiththeidentificationofa great numberofproteinscontainingnucleicacid-bindingdomainsinthe proteomicanalysisofT.cruzi-derivedvesicles[4],wedecidedto analyzewhetherT.cruzi-secretedEVsalsocontainRNAmolecules andperformtheircharacterization.

ToobtaintheT.cruziEVs,epimastigotesandmetacyclic trypo-mastigotes(cloneDm28c)werecultivatedasdescribedpreviously withminormodifications[4].Epimastigotesformswerecollected at exponential growthphase (∼2.5×107cells/mL), while

meta-cyclictrypomastigoteswerecollectedfromtheculturesupernatant ofTAU3AAGmediumafter72hofdifferentiation(5×106cells/mL).

To obtainthe sufficient amountof EVs-derived small RNAsfor

http://dx.doi.org/10.1016/j.molbiopara.2014.02.004

72 E.Bayer-Santosetal./Molecular&BiochemicalParasitology193(2014)71–74

libraryconstruction,wecombinedthetotal RNAextractedfrom twodistinct biologicalpreparations.Briefly, twodistinct prepa-rations of 3×109 epimastigotes or metacyclic trypomastigotes

(1×108parasites/mL)wereincubatedfor12hinDMEMwithout

fetalbovineserum(FBS)orinTAU3AAGmedium,respectively. Par-asiteviabilitywasassessedbypropidiumiodideincorporationand showedthatmorethan98%ofcellswereviable.Afterthe incuba-tion,cellswereremovedbycentrifugationat3000×gfor10min

(1stpellet),andthesupernatantwasfilteredin0.45-␮msyringe

filtersandultracentrifugedat100,000×gfor2h(2ndpellet).The

pelletscontainingcells(1stpellet)andEVs(2ndpellet)weremixed with1mLofTriReagent(Sigma)andRNAextractionwasperformed asdescribed bythemanufacturerwithminormodifications. To improvetherecoveryofsmallRNAs,30␮gofglycogen

(Invitro-gen)wasaddedfollowedbyisopropanolprecipitationfor16hat

−20◦C.

TotalRNAs extracted from two distinct biological replicates were mixed 1:1 (1␮g each) before small RNA isolation and

librarygeneration. Such procedure wasperformed for all sam-ples(eVes,mVesandmCell)soeachoneofthemwascomposed oftwodistinctbiologicalreplicates,thusresultspresentedinthe manuscriptare anaverageof independentbiologicalreplicates. SmallRNAsweresequencedbyLCSciences(Houston,TX).Briefly, thesmallRNAfractionof16–40ntwasisolatedfromtotal RNA ofepimastigote-andmetacyclic-derivedvesicles(eVesandmVes, respectively),andmetacyclictrypomastigoteparentalcells(mCell) ina15%Tris–borate–EDTA–ureapolyacrylamidegel.Fig.1Ashows adenaturingformaldehydeagarosegeldisplayingthedifferences observedbetweentotalRNAofeVesandtotalRNAofparentaleCell fromwhichvesicleswereisolated.ItisinterestingtonotethateVes seemtobecomposedofawidevarietyofRNAmolecules,including rRNA,mRNAs,andsmallRNAs.TheprofileofmVesisquitesimilar totheeVesinitsRNAcomposition(datanotshown).However,as wewereunabletoperformthecharacterizationofalltypesofRNA moleculescontainedintheseT.cruzi-derivedEVs,wedecidedto focusonthesmallnoncodingRNAs,whichhavethepotentialtobe regulatorymolecules.

AsmallRNAlibrarywasgeneratedusingtheIlluminaTruseqTM

SmallRNAPreparationkit,whichisspecificallydesignedto iso-latesmallRNAshaving5′ _{phosphateand 3}′ _{hydroxyl ends.The}

purified cDNA library was used for cluster generation on Illu-mina’s Cluster Station and sequenced on Illumina GAIIx. Raw sequencingreadswereobtainedusingIllumina’sPipelinev1.5 soft-ware,following sequencingimageanalysisbypipelineFirecrest moduleand base-callingby pipelineBustard module. Sequenc-ingdataanalysiswasperformedbyaproprietarypipelinescript (LCSciences).Aftertherawsequencereadswereextractedfrom image data,a series of digital filters was employed toremove unmappable/low quality reads and adaptors. Those remaining filtered reads were grouped and used to map with the refer-ence database that was composed of T. cruzi transcripts from TriTrypDB 4.0 (http://tritrypdb.org/tritrypdb/). Filtered unique reads were aligned against the reference database using Bowtie2(http://bowtie-bio.sourceforge.net/bowtie2/index.shtml). Onemismatchwasallowedinaseedalignment,theseedlength wassetto20andthestrategyoflocalalignmentwasusedfor map-ping.Theuniquereadswereclusteredintosevenclasses(rRNA, tRNA,snRNA,snoRNA,coding-sequences(CDS),pseudogene,and unspecified)basedontheirproductsannotatedintheNCBIGene database.Normalizationofsequencecountsineachsamplewas performedbytheDESeqmethod.

Alluniquereadsweredistributedintosevencategoriesusing genomeannotations(Table1)andresultsrevealedsome differ-encesbetweenEVsderivedfrombothdevelopmentalforms(eVes vs.mVes)aswellasbetweenEVsandcells(mVesvs.mCell).The lengthdistributionofalluniquereadsseparatedaccordingtotheir

Table1

ClassesofsmallRNAs.

RNA eVes mVes mCell

#ofreads % #ofreads % #ofreads %

rRNA 2,373,222 55.80 2,717,445 76.46 984,404 57.95

tRNA 1,496,361 35.18 234,005 6.58 337,837 19.89

CDS 56,245 1.32 187,876 5.29 80,085 4.71

snoRNA 3068 0.07 18,129 0.51 67,930 4.00

Pseudogene 14,125 0.33 31,433 0.88 13,970 0.82

snRNA 413 0.01 2954 0.08 972 0.06

Unspecified 48 0.00 419 0.01 77 0.00

Nohit 466,011 10.96 473,791 13.33 255,694 15.05

Totalreads 4,252,955 100.00 3,554,196 100.00 1,698,747 100.00

categoriesand a summaryof alldataobtainedand analyzedin

thepresentworkcanbefoundinTablesS1A-IandS2A-G,

respec-tively.rRNA-derivedsmallRNAswerethemostabundantclassin

thethreesamples(56–76%),followedbytRNA-derivedsmallRNAs

thatwerethesecondmostabundantclassineVes(35%)andmCell

(20%),andpresentinlowerproportioninmVesfraction(6%).Reads

thatcouldnotbealignedtothegenome(nohit)werethesecond

mostabundantclassinmVes(13%) andthethirdineVes(11%)

andmCell(15%).SmallRNAsderivedfromcoding-sequenceswere

slightlymoreabundantinmetacyclic-derivedsamples(5%)as

com-paredtoepimastigotes(1%),andsnoRNA-derivedsmallRNAswere

mainlyfoundinthemCellsample(4%).Severalstudieshaveshown

thatRNAsareselectively packedintomembranevesicles asthe

RNAprofilesinthesevesiclesdonotfullyreflecttheRNAprofiles

observedintheparentalcells[5].AlistofsmallRNAsfoundin

theEVsofepimastigotesandmetacyclictrypomastigotes contain-ingtheirrelativedifferentialexpressionisshowninTableS3A-G. Thus,inaccordancewiththeseresultswefoundseveralsequences thataredifferentiallyexpressedinEVsderivedfrombothparasite forms(eVesvs.mVes)aswellasdifferentiallyexpressedinEVsand parentalcells(mVesvs.mCell)(TableS3),whichsupportsthe dif-ferentialpackagingofthesesmallRNAsintoparasite-derivedEVs.It isworthmentioningthatsincetherearenoreplicates,significance couldnotbeassessed(TableS3).

InaccordancewithpreviousresultsshowingthatrRNA-derived smallRNAfragmentsarepresentinalmostallsmallRNAsequence libraries,thesemoleculesconstitutedthemostabundantclassin thethreelibrariesanalyzedinourwork(eVes,mVes,andmCell). AlthoughtheseresultsmightsuggestthatthesesmallRNAsare randomlydegradedproducts, more recent studies inmammals andyeasthaverevealedthatrRNA-derivedsmallRNAsare func-tionalmoleculesprocessedbyspecialenzymes[6]andtheymight playimportantrolesinheterochromatinassemblyand transcrip-tionregulation[7].Moreover,inapreviousworkwehaveshown thepresenceofarelativelyhighpercentage(6-11%)ofribosomal proteinsandnucleicacid-bindingproteinsintheT.cruzisecretome

[4].Thus,therRNA-derivedsmallRNAscouldoriginatefromthe turnoverofribosomesand/ortheycouldbealsoassociatedwith polysomescarryingmRNAswhichmaybemobilizedintosecreted vesicles[5].

SimilarlytowhatwasobtainedinadifferentstudyanalyzingT. cruzismallRNAs[8],inourworkaround11–15%ofthesequenced readscouldnotbealignedtothegenome(nohit)usingthedefault alignmentprocedures.Onepossiblereasonisduetogenomic dif-ferencesbetweenthereferencecloneCLBrenerusedasdatabase andthecloneDm28cusedtoconstructthecDNAlibrary. tRNA-derivedsmallRNAfragments(tsRNA)havebeenidentifiedinother

E.Bayer-Santosetal./Molecular&BiochemicalParasitology193(2014)71–74 73

Fig.1.ProfileofthesmallRNAsofT.cruziextracellularvesicles.(A)Denaturingformaldehydeagarosegel(1%)showingthedistinctRNAbandpatternbetween

epimastigote-derivedvesicles(eVes)andparentalepimastigotecells(eCell).(B)LengthdistributionoftRNA-derivedsmallRNAs.

viewofbisectionalcleavageofmaturetRNA(∼70–80nt)nearthe

anticodon.MosttRNAisoacceptorswerefoundtobeprecursorsof tsRNAidentifiedinparasite-derivedEVs,butspecificisoacceptors weredetectedin higherlevels ascompared toothers, suggest-ingthattsRNAaredifferentiallypackedintomembranevesicles (Table2andTableS3).ConsistentwithresultspublishedbyReifur et al. [10], we foundthat themost abundanttsRNAs in meta-cyclicforms(mCell)weretRNAGlu_(42%),tRNAThr_{(21%),andtRNA}Val

(15%).Moreover,contrastingwithtsRNAidentifiedinmCell,the most abundanttsRNAs found in mVes were quite diverse and werecomposedof tRNAGlu _(33%),tRNAThr _(17%),tRNAGly _(10%),

tRNAVal_(9%),andtRNAPhe_{(9%).Inaddition,Garciaetal.[9]showed}

thattRNAGlu_(60%)andtRNAAsp_{(35%)werethemostabundantly}

expressedtsRNAs in epimastigote cells (eCell), while themost abundanttsRNAs identified ineVes by thepresent study com-prisedtRNAGlu_(49%),tRNAVal_(31%),tRNAThr_{(12%),andtRNA}Asp

(3%).Takentogether,thesedatareinforcethedifferential packag-ingofsmallRNAsintoparasite-derivedEVsandshowthedifference invesiclecomposition betweenthetwoparasitedevelopmental forms.

Ineukaryotes,tRNAcleavagehasbeenproposedasamechanism todown-regulateorinhibitproteinsynthesisduringdifferentiation

Table2

SmallRNAsmappedontRNAisoacceptors.

tRNAtype eVes mVes mCell

#ofreadsa _{% #ofreads}a _{% #ofreads}a _%

Ala 4997 0.0 2520 2.0 2190 1.0

Arg 8009 0.0 2686 2.0 5405 1.0

Asn 14 0.0 26 0.0 17 0.0

Asp 67,610 3.0 9113 7.0 20,542 5.0

Cys 274 0.0 339 0.0 351 0.0

Glu 1,028,208 49.0 43,706 33.0 182,381 42.0

Gln 2954 0.0 1988 2.0 2515 1.0

Gly 39,870 2.0 12,713 10.0 38,617 9.0

His 11,475 1.0 3287 2.0 11,926 3.0

Ile 80 0.0 107 0.0 62 0.0

Leu 2490 0.0 4180 3.0 2302 1.0

Lys 145 0.0 303 0.0 329 0.0

Met 1270 0.0 785 1.0 626 0.0

Phe 15,584 1.0 12,129 9.0 3354 1.0

Pro 9972 0.0 1490 1.0 2656 1.0

Ser 96 0.0 259 0.0 171 0.0

Thr 255,589 12.0 22,855 17.0 93,032 21.0

Trp 1448 0.0 331 0.0 4760 1.0

Tyr 257 0.0 320 0.0 314 0.0

Val 650,606 31.0 12,364 9.0 65,375 15.0

2,100,948 100.0 131,501 100.0 436,925 100.0

a_{NormalizedvaluesderivedfromTableS3wereused.}

andunderoxidativestress,aswellasundernutrientstarvation.In

T.cruzi,themachineryrequiredforRNAinterference(RNAi)seems tobeeitherentirelylostorextensivelysimplifiedastheparasiteis unabletousedsRNAtotriggerdegradationoftargetmRNAs[12].In thiscontext,tsRNAshavegainedspecialattentionasitwasshown thatthesemoleculescanassociatewithArgonaute/Piwiprotein familymembers,leadingtotheassemblyofspecific ribonucleo-proteincomplexesinvolvedinsilencinggeneexpression[6,13,14]. Itisinteresting tonotethatRNA-bindingproteinsconstitutean importantfractionofT.cruzisecretome[4]andtheseproteinsare knowntoplayanessentialroleinthecontrolofgeneexpression intrypanosomes,modulatingRNAprocessing,stability,turnover, andtranslation[15].Moreover,additionalexperimentalevidence demonstratedthatthetRNA-maturingenzymetRNaseZwasable todown-regulateexpressionofhumangenesbydegradingmRNAs underthedirectionof small-guidetsRNA[16,17].ThetRNaseZ belongstothemetallo-lactamasefamilyanditwasreportedthat trypanosomatidgenomesencodeforatleast4metallo-lactamases

[9].Furthermore,tsRNAswerereportedtoberecruitedto cyto-plasmicgranulesthatpartiallycolocalizedwithreservosomes[9], whichsuggeststhattheycouldbepackedintovesiclesforsecretion. SmallRNAs derived fromCDS(1–5%)and pseudogenes (1%) comprised a minor portion of the libraries and could simply represent degradation products derived from mRNAs due to theconstitutivepolycistronictranscriptionandpost-transcription gene expression regulationof theseorganisms. However, these smallRNAsmightalsorepresentfunctionalmoleculesthatform sense-antisensedsRNAsbycomplementarybasesthusgenerating naturalantisensesiRNAs(NAT-siRNAs),whichcandisplay regula-toryfunctionsasalreadyshowninTrypanosomabrucei[18].NATs canbedividedintotwotypes:cis-NATsinwhichthetwotranscripts derive fromthe samegenomic locusbut opposite strands, and

74 E.Bayer-Santosetal./Molecular&BiochemicalParasitology193(2014)71–74

aboutthe possiblefunctionsthat these smallRNAs might play oncetransferredtomammalianhostcellsand/ortoother para-sites.Garcia-Silvaetal.[21]showedthatepimastigotessubmitted tonutrientstarvationsecreteextracellularvesiclescarryingsmall tRNAsandTcPIWI-trypproteinsascargo,whichcouldbe trans-ferredtootherparasitesandtomammaliancells,thusincreasing metacyclogenesisandsusceptibilityofmammaliancellsto infec-tion[21].Thesefindingsmakeusspeculatewhetherthesevesicles couldbeusedasvehiclestoexchangegeneticmaterialbetween parasites,andbetweenparasites andhostcells assuggestedby Hechtetal.[22].Furtherworkwillbeneededtoclarifythe molec-ularmechanismsthatunderlaythesevesicles-mediatedexchange eventsandtheirfurtherimplicationsinhost–pathogeninteractions andquorumsensing.

Acknowledgments

ThisworkwassupportedbytheNationalInstitutesofHealth (NIH)grantsR01AI070655-A5andR01AI070655-A5S1(toI.C.A.), and5G12MD007592(toBBRC/UTEP),andbygrantsfromFAPESP (2011/51475-3)andCNPq(Brazil)toJ.F.S.andE.B.S.Wewouldlike tothankDr.Marjorie MendesMarinieSouzaforherhelpwith dataanalysis.We arethankful totheBiomoleculeAnalysisand GenomicAnalysisCoreFacilitiesatBBRC/UTEP(NIH/NIMHDgrant 5G12MD007592).

AppendixA. Supplementarydata

Supplementarymaterialrelated tothis article canbefound, in the online version, at http://dx.doi.org/10.1016/j.molbiopara. 2014.02.004.

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