Acta
Tropica
j ou rna l h o me p ag e : w w w . e l s e v i e r . c o m / l o c a t e / a c t a t r o p i c a
Tridimensional
ultrastructure
and
glycolipid
pattern
studies
of
Trypanosoma
dionisii
Miriam
Pires
de
Castro
Oliveira
a,∗,
Thiago
Cesar
Prata
Ramos
a,
Adriana
Maria
V.N.
Pinheiro
a,
Silvio
Bertini
b,
Helio
Kiyoshi
Takahashi
b,
Anita
Hilda
Straus
b,
Edna
Freymuller
Haapalainen
caDepartamentodeBiologiaEstruturaleFuncional,UniversidadeFederaldeSãoPaulo,RuaBotucatu,740,SãoPaulo,SP,04023-900,Brazil bDepartamentodeBioquímica,UniversidadeFederaldeSãoPaulo,RuaBotucatu,862,1◦andar,SãoPaulo,SP,04023-900,Brazil
cCentrodeMicroscopiaEletrônica,UniversidadeFederaldeSãoPaulo,RuaBotucatu,862,térreo,SãoPaulo,SP,04023-900,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory: Received1April2013
Receivedinrevisedform17July2013 Accepted1August2013
Available online 8 August 2013
Keywords: Trypanosomadionisii Tridimensionalultrastructure 3Dreconstruction
Epimastigote Glycolipid
a
b
s
t
r
a
c
t
Trypanosoma(Schizotrypanum)dionisii isanon-pathogenicbat trypanosomeclosely relatedto Try-panosomacruzi,theetiologicalagentofChaga’sdisease.Bothkinetoplastidspresentsimilarmorphological stagesandareabletoinfectmammaliancellsinculture.Inthepresentstudyweexamined3D ultrastruc-tureaspectsofthetwospeciesbyserialsectioningepimastigoteandtrypomastigoteforms,andidentified commoncarbohydrateepitopesexpressedinT.dionisii,T.cruziandLeishmaniamajor.Amajordifferencein 3DmorphologywasthatT.dionisiiepimastigoteformspresentlargermultivesicularstructures,restricted totheparasiteposteriorregion.ThesestructurescouldberelatedtoT.cruzireservosomesandarealso richincruzipain,themajorcysteine-proteinaseofT.cruzi.Weanalyzedthereactivityoftwomonoclonal antibodies:MEST-1directedtogalactofuranoseresiduesofglycolipidspurifiedfromParacoccidioides brasiliensis,andBST-1directedtoglycolipidspurifiedfromT.cruziepimastigotes.Bothantibodieswere reactivewithT.dionisiiepimastigotesbyindirectimmunofluorescense,butwenoteddifferencesinthe locationandintensityoftheepitopes,whencomparedtoT.cruzi.Insummary,despitesimilarfeaturesin cellularstructureandlifecycleofT.dionisiiandT.cruzi,weobservedauniquemorphologicalcharacteristic inT.dionisiithatdeservestobeexplored.
© 2013 Elsevier B.V. All rights reserved.
1. Introduction
Trypanosoma(Schizotrypanum)dionisiiisanon-pathogenicbat trypanosomecloselyrelatedtoTrypanosomacruzi,theetiological agentofChaga’sdisease.In1978,Bakeretal.suggestedthe phy-logeneticproximitybetweenT.dionisiiandT.cruzi,andin1987,
Petryetal.(1987a,b,c)demonstratedthatthesespeciesshare epi-topes,definedbymonoclonalantibodies(MAbs)anti-epimastigote forms.Thephylogeneticproximitybetweenthesespecieswas con-firmedyearslater,basedongenesofthesmallsubunitribosomal RNA,and theglycosomalglyceraldehyde phosphate dehydroge-nase(Hamiltonetal.,2007).
AsT.cruzi,duringitslifecycle T.dionisiialternatesdifferent developmental forms between hosts: epimastigotes and meta-cyclictrypomastigotesintheinvertebratehostandbloodstream trypomastigotesandamastigotesinthemammalianhost(Baker etal.,1971;WilkinsandBaker,1975;Molyneux,1991).Invitro,
∗Correspondingauthor.Tel.:+551155764528.
E-mailaddress:castro.oliveira@unifesp.br(M.P.d.C.Oliveira).
metacyclictrypomastigoteformsareabletoinvadeandreplicate withinalargenumber ofmammaliancells.Theseforms remain inthecytoplasmandtransformintoamastigotes,thereplicative forms.Afteranintensemultiplication phase,amastigotes trans-formintotrypomastigotesandaftercellularrupturearereleased intothemedium(Bakeretal.,1971;Thorneetal.,1979;Bakerand Selden,1981;Glauertetal.,1982;Molyneux,1991).
ManyT.cruzistudieshaveindicatedthatparasiteandhostcell glycoconjugatesareinvolvedinthecellularinvasionprocesses(De Arrudaetal.,1989;Mingetal.,1993;Ramirezetal.,1993;Giordano etal.,1994;Silvaetal.,2006;Cortezetal.,2006a,b;Ferreiraetal., 2006).Glycoconjugatescontaininggalactofuranose(Galf)residues have been described in fungi, bacteria, and parasites such as: polysaccharidesofMycobacteriumsp.,Streptococcussp.,Aspergillus
sp;lipopolysaccharidesofEscherichiacoli;glycoproteinsofCrithidia
sp.,Leptomonas samueliand T.cruzi; and glycolipidsof T. cruzi,
T.dionisii,Leishmaniasp.,Histoplasmacapsulatumand Paracoccid-ioidesbrasiliensis(AlvesandColli,1975;Mendonc¸a-Previatoetal., 1983;LederkremerandColli,1995;Toledoetal.,1995;Branquinha etal.,1999;PedersenandTurco,2003;Takahashietal.,2009;Tefsen etal.,2012)
M.P.d.C.Oliveiraetal./ActaTropica128 (2013) 548–556 549
Fig.1. T.dionisiiepimastigoteG1/Sphase(3Dreconstructionofmicrographsshowedatsupplementarydata,Fig.S1).(A)ageneralviewoforganellesdistribution.Green: mitochondrion;lightgreen:kinetoplast;darkyellow:flagellarpocket;orange:basalbodies;red:flagellum;royalblue:Golgi;lightyellow:electron-densevesicles;blue: nucleus;lightblue:nucleolus;lightgray:electron-densechromatin;brown:reservosomes;darkgray:membrane;pink:nonelectron-densevesicles(acidocalsisomes); white:contractvacuole.(B)Detailofthediskshapedkinetoplast(lightgreen)inthemiddleoftheuniquemitochondrion(green),thenucleusinblueandnucleolusinlight blue.(C)Detailofthecytostome-cytopharynx(lightpink)emergingfromtheflagellarpocket(darkyellow)andcontouringthekinetoplast(lightgreen)andthenucleus (blue).Parasiteswereprocessedfortransmissionelectronmicroscopyandthe3DmodelwasgeneratedwithBlender®software.Scalebar:2m.
Althoughthe biological role of Galfresidues is still unclear, thepostulatedabsenceofGalfandgalactofuranosidasesin mam-malianspecies suggeststheintriguinghypothesis that terminal Galfresidues play a central role in survival of fungiand para-sites,bypreventingtheactionofthehost’sglycosidasesontheir glycoconjugates. If this hypothesis is correct, Galf residues are potentiallyusefulasspecifictargetmoleculesfortherapyof par-asiticand fungal diseases.But,there is littleinformation about differences of these glycoconjugates containing Galf residues, betweenpathogenicandnon-pathogenictrypanosomatides.
In T. cruzi studies, Golgher et al. (1993), showed that Galf
residuespresentinT.cruziglycoconjugatesarehighlyantigenic, sincepatientswithChagas’diseasepresent anti-Galfantibodies andBertinietal.(2003a,b)demonstratedthatT.cruzi trypomas-tigotesGalf-containingglycoproteinsareimportantforVerocells invasion.Moreover,Nogueiraetal.(2007),demonstratedthatGalf -containingGIPCs are important for T. cruzi epimastigote forms differentiation,proliferationandinteractionwithhostmidgutcells, andSuzukietal.(2002,2008)suggestedthat,inother trypanoso-matids,asLeishmaniamajor,glycoinositolphospholipidscontaining
Galf residues could be involved in promastigote-macrophage attachment and invasion, using Fab fragments of MAb MEST-1 (whichrecognizesterminalGalfresidue presentonGIPL-1ofL. majorpromastigotes),andp-nitrophenyl--d-galactofuranoside.
Ourrecentstudydescribedsimilaritiesonhostcellinteraction andinvasionprocessesofT.dionisiiandT.cruzi,andalsoimportant differences,suchasthereducedtrans-sialidaseactivityinT. dion-isii,whichmaybeoneofthefactorsresponsibleforintracellular retentionofsomeamastigotesintotheparasitophorousvacuoles, preventingtheirscapeintothecytoplasm,andsubsequent differ-entiationtotrypomastigotes(infectiveforms)(Oliveiraetal.,2009). Webelievethatthesimilaritiesanddifferencesbetweentheseso closelyspeciesdeservetobebetterexplored,inordertoidentify newtargetstoChagas’diseasevaccines.
Fig.2. T.dionisiiepimastigotemitosisphase(3Dreconstructionofmicrographsshowedatsupplementarydata,Fig.S2).(A)Ageneralviewoforganelledistribution.Green: mitochondrion;lightgreen:kinetoplast;yellow:flagellarpocket;red:twoflagella;pink:nonelectron-densevesicles(acidocalsisomes);blue:nucleus;lightgray:chromatin; brown:multivesicularstructures(reservosomes);darkgray:membrane.(B)Azoomofthekinetoplastdivision(lightgreen).(C)Aviewofthekinetoplast(lightgreen)inthe middleoftheuniquemitochondrion(green).Parasiteswereprocessedfortransmissionelectronmicroscopyandthe3DmodelwasgeneratedwithBlender®software.Scale
bar:2m.
3Dreconstructionsusingserialsectionsofthematerialunder study,isapowerful methodtovisualizemorphologicalaspects. Recentstudiesshowedtrypanosomatidentirecells,atthe3Dlevels, givinginformationaboutorganellesdistributionindifferentcell stages(Porto-Carreiroetal.,2000;Alberioetal.,2004;Eliasetal., 2007;Ferreiraetal.,2008).
Inthisstudyweexamined3DultrastructureofT.dionisii,and comparedwiththeresultsrecentlydescribedforT.cruzi(Eliasetal., 2007;Ferreiraetal.,2008;Ramosetal.,2011),andalsoidentified glycogonjugateepitopes,containingGalfresiduesrecognizedby MAbMEST-1,sharedbyT.dionisii,T.cruzi,P.brasiliensis,andL.major
(Suzukietal.,1997,Takahashietal.,2009),andrecognizedbyMAb BST-1(directedtoT.cruziglycolipids,Bertinietal.,2003a,b)shared byT.dionisiiandT.cruzi.
2. Materialsandmethods
2.1. Parasites
M.P.d.C.Oliveiraetal./ActaTropica128 (2013) 548–556 551
Fig. 3. T. dionisii trypomastigote forms processed for transmission electron microscopy.Organellesdistributioninanultrathincentralcutoftheparasite. Kine-toplast(blackarrow),reservosome(yellowarrow),nucleus(greenarrow),lipid inclusions(blackarrowheads),acidocalsisomes(greenarrowheads)and mitochon-drion(redarrow).Scalebar:2m.
miceandinLITcontaining5%FBS.MetacyclicformsofCLstrain wereobtainedafteronepassageinGrace’s medium(Gibco),by seeding5mlLITcultureinto35mlmedium.Metacyclicformsfrom culturesatthestationarygrowthphasewerepurifiedbypassage throughDEAE-cellulosecolumn(Camargo,1964;Yoshida,1983). Allanimalexperimentalprocedureswereapprovedbythe Institu-tionalEthicalCommitteeofUNIFESP/EPM.
2.2. Monoclonalantibodiesandimmunofluorescencelabeling
MAbBST-1(IgG3)andMAbMEST-1(IgG3)werepreviously pro-duced,byimmunizationofBALB/cmicewithpurifiedglycolipid fractionofT.cruzi(CLstrain)epimastigotes(MAbBST-1) (unpub-lisheddata),andpurifiedglycolipidfractioncontainingterminal residuesofGalfofP.brasiliensis(MAbMEST-1),whichcross-reacts withglycolipidscontainingGalf1–3terminalresiduesexpressed inT.cruziandL.major(Suzukietal.,1997).
Anti-cruzipain MAb, which recognizes a major cysteine-proteinaseof T. cruzi present in the reservosomes, was kindly providedbyDr.SergioSchenkman(DepartmentofMicrobiology, Parasitology,andImmunolgy,UNIFESP,SãoPaulo,Brazil).
2.3. Immunofluorescencelabeling
Formaldehyde (4%) fixed parasites and live parasites, were adsorbed onto coverslips treated with 0.1% poly-lysine. The adsorbed parasites were blocked with 5% bovine serum albu-min(BSA) inphosphate-bufferedsaline(PBS)and incubatedfor 1h, at roomtemperature, withMAb culturesupernatants (pri-mary antibody). Bound IgGs were developed with anti-mouse IgGconjugatedtoAlexa-Fluor488(SigmaChemicalCo.,St.Louis, MO, EUA) and DNA-rich structures were stained with 10M DAPI (4,6-diamidino-2-phenylindoledihydrochloride, Molecular Probes,EugeneOR,USA).Thecoverslipswereexaminedona Bio-Rad1024-UVconfocalmicroscopy.Controlsperformedusingan irrelevantMAb(IgG3).
Fixed parasites adsorbed onto coverslips were delipidated ornot witha mixture of isopropanol–hexane–water (55:20:25,
vol/vol/vol,upperphasediscarded)atroomtemperature,blocked withBSA/PBS,andusedforimmunofluorescenceassay.
2.4. Transmissionelectronmicroscopy
Parasiteswerefixedfor1hatroomtemperature,withamixture of2%formaldehydeand2.5%glutaraldehydeincacodylatebuffer (0.1Msodium cacodylate buffer, pH 7.2). Then, parasites were decantedovernightonACLAR®(ElectronMicroscopySciences)film precoatedwith0.01% poly-l-lysine. Thefilmwaswashed three timeswithcacodylatebuffer,andmorethree timeswithwater. Afterthat,theparasiteswerefixedwith1%osmiumtetroxide,in thesamebuffer, for30minatroomtemperature.Thefilm was thenwashedwithcacodylatebuffer,followedbythreewasheswith waterandtreatedfor30minwith0.4%uranylacetateinwater(for membranecontrastenhancement).Then,thefilmwasgradually dehydratedinaseriesofethanolsolutions,andfinallyembedded inEponTMresin(ElectronMicroscopySciences).Aftersectioning, toproduceconsecutivessections,thematerialwasstainedwith uranylacetateand leadcitrate,mountedonFormvargrids, and observedinaJeol1200EXIItransmissionelectronmicroscopy.
2.5. 3Dreconstructions
3D reconstructions were made with electron micrographs, obtainedwithacameraattachedtothemicroscopy,andscanned athighresolution.TheimageswerealignedusingtheReconstruct® (version 1.1.0.0) software (Fiala, 2005) by selecting congruent points in alternate images. After alignment, the morphological structuresweremanuallytracedusingthedrawclosedpointby pointfunction,andthe3Dimagesgeneratedbytheprogram. Sub-sequently,modelsofallstructuresweremergedintheBlender® 3D(version2.49b)modelingsoftware(http://www.blender.org/).
2.6. PAGE-SDSandWesternblotting
T.dionisii andT. cruzi(CL andG strains)epimastigoteforms (8×109 cells) were solubilized in 0.5ml of sample buffer and submitted toPAGE-SDS (Laemmli,1970). Afterelectrophoresis, antigensweretransferredtoanitrocellulosemembrane(NC)that was blocked with5% BSA, and incubated with MAb (BST-1 or MEST-1)for 2h. Then, NCwasincubatedfor 2h, atroom tem-perature,withhorseradishperoxidase-conjugatedtoanti-mouse immunoglobulins(Sigma)and for 5minwithSuperSignal West PicoChemiluminescentSubstrate(Pierce,IL,USA).Reactive anti-genswerevisualizedbythephotodocumentationsystemG-BOX HR-16(Syngene)(Tagliarietal.,2012).
3. Resultsanddiscussion
G1,S,G2,M(mitosis)andC(cytokinesis)T.cruziepimastigote cellularcycle,phaseswererecentlymorphologicallycharacterized (Eliasetal.,2007).UltrathinserialcutsofT.dionisiiepimastigote G1/Sphase (Supplementary dataFig. S1) wereanalyzed by 3D reconstruction.T.dionisiiepimastigoteG1/Sphaseasoccurswith
T.cruzi,hasauniqueflagellum,onenucleusandonekinetoplast (Fig. 1). We have observed, that unlike happens with T. cruzi
Fig.4.T.dionisiiexpressesglycoepitopesharedwithT.cruziwhichisrecognizedbyMAbBST-1.T.dionisiiepimastigoteswerefixedandlabeledwithMAbBST-1.(Aand C)Phasecontrastimage.(B)MergedimageofMAbBST-1ingreenandDAPI(nucleusandkinetoplast)inblue.Whitearrowsindicateposteriorstructures(reservosomes) labeledwithMAbBST-1andwhitearrowheadsshowapunctuallabelingofparasitessurface.(D)AcloseupofT.dionisiiBST-1labeled.YellowarrowshowsDAPI(nucleus andkinetoplast)inblueofanonBST-1labeledparasite.Whitearrowspointtothereservosomeslabelingandwhitearrowheadpointstosurfacepunctuallabeling.Images acquiredunderconfocalmicroscopy.Scalebar:10m.(Forinterpretationofthereferencestocolorinthisfigurelegend,thereaderisreferredtothewebversionofthe article.)
-lucentinclusions,whicharenotlimitedbyaunitmembraneand presentsanelectron-densematrix.
The distribution of the acidocalcisomes occurs throughout the cytoplasm of T. dionisii epimastigotes at G1/S and mitosis phases(pinkvesicles,inFigs.1Aand2A).Thesestructuresappear inthe electron-micrographsasempty organelles,containingan electron-denseconstricted region(datanot shown). The acido-calcisomes are acid organelles, rich in calcium, polyphosphate and othersions, and mayplay animportant rolein osmoregu-lationand maintainingpHhomeostasis (Docampo et al.,2005).
T.dionisiimitosisultrathinserialcuts(Supplementary data,Fig. S2)wereanalizedby3Dreconstruction,andwedidnotobserve either lipid inclusions or electron dense vesicles, it was only observed some acidocalcisomesscattered throughout the cyto-plasm(Fig.2A).
Thereservosomesarestructuresresponsibleforprotein,lipids andproteasessorting,andprobablyhavelysosomaltypical func-tions(Cunha-e-Silvaetal.,2006;Sant’Annaetal.,2008;DeSouza etal.,2009a).AmastigoteandtrypomastigoteformsofT.cruzido notpresentatypicalreservosome,butreservosome-likestructures (Sant’Annaetal.,2008;DeSouzaetal.,2009a).Intrypomastigotes ofT.dionisiiweidentifiedauniquelargereservosome,nexttothe kinetoplast(Fig.3).Wealsodetectedpresenceofcruzipain,the majorT.cruzicytseine-proteinase(Cazzulloetal.,1997,2001),inT.
dionisiiepimastigotesreservosomesbyimmunofluorescence(data notshown).
AcentralcutoftheT.dionisiitrypomastigoteformshowthat these evolutionaryforms appear tobe very poorin intracellu-larstructureswhencomparedtotheepimastigoteform.Weonly observed thecharacteristic rounded kinetoplast,a unique large reservosome,themitochondrion,theelongatednucleus,somelipid inclusions,andafewvesicleswithacidocalcisomecharacteristics (Fig.3).ThesinglemitochondrionofT.dionisiiwasidentifiedby thepresenceofitsdoublemembrane.Itisextensivelybranched and resembles a tubular network, as described in T. cruzi (De Souzaet al.,2009b;Ramos etal., 2011)however witha differ-ence,inT.dionisiiepimastigoteformsitseemstobedistributed throughoutthecytoplasmandnotrestrictedtotheparasitesurface (Figs.1A–Cand2AandC).Thekinetoplastwaspositionedinside theuniquemitochondrion,andlocatedinaseparated regionof thisorganelle(Figs.1A–Cand2Aand2C)aspreviousdescribedin
T.cruzi(O’DalyandBretana,1976;DeSouzaetal.,2009b;Ramos etal.,2011).
Intrypanosomes,Morrisetal.(2001)andLiuetal.(2005)have proposedthemechanismsofkinetoplastdivision.Morerecently,
M.P.d.C.Oliveiraetal./ActaTropica128 (2013) 548–556 553
Fig.5. T.cruzi(CLstrain)BST-1andMEST-1labeling.AandC:phasecontrastimages.B:mergedimageofMAbBST-1ingreenandDAPI(nucleusandkinetoplast)inblue. Whitearrowsindicateastrongreservosomeslabelingandwhitearrowheadastrongpunctuallabelingoftheflagellum.D:mergedimageofMAbMEST-1ingreenandDAPI (nucleusandkinetoplast)inblue.Whitearrowsindicateapunctuallabelingatparasitebodiesandwhitearrowheadapunctuallabelingoftheflagellum.Imagesacquired underconfocalmicroscopy.Scalebars:10m.(Forinterpretationofthereferencestocolorinthisfigurelegend,thereaderisreferredtothewebversionofthearticle.)
earlystepsofT.dionisii,showedakinetoplastconstriction(Fig.2B), suggesting that in this parasite the division occurs from the peripherytothecenter,similarasoccursinT.brucei(Ogbadoyi etal.,2003),anddistinctfromT.cruzi.
TheGolgicomplex,nucleusand electron-densechromatinof
T. dionisiiforms, were identified bytheir typicalmorphologies, as occurs with T. cruzi 3D reconstruction (Ramos et al., 2011) (Figs.1Aand2A).
RegardingthedensechromatindistributioninthenucleusofT. dionisiiepimastigotesatG1/S,wehavenotedthatitis intercon-nectedandlocatedmainlyinthenuclearperiphery,closetothe nuclearmembrane(Fig.1A),asdescribedforT.cruzi(Ramosetal., 2011).Thedensechromatinleavesaninternalspaceforacentrally locatednucleolus,whichissphericalandcentral(Fig.1A),similarly asinT.cruzi(DeSouza,2002).InT.dionisiimitosis3D reconstruc-tion,weobservedthecharacteristicdispersedchromatin,theintact nucleusmembrane,andtheabsenceofthenucleolus(Fig.2A),as previouslydescribedbySolari(1995)inT.cruzi.
TheT.cruzicytostomeisaninvaginationoftheplasma mem-brane,whichcanpenetratedeeplyintothecytoplasm(Milderand Deane,1969;De Souza,1984,2008), and forma tubular struc-turecalledcytostome-cytopharynx. Themacromoleculescanbe
capturedinthecytostome,followingintothecytopharynxandthen internalizedintoendocyticvesicles,whichmergedinthe reservo-somes(DeSouzaetal.,2009a).T.dionisiicytostome-cytopharynx, asitwasdescribedinT.cruzi3Dreconstruction(Ramosetal.,2011), progressesfromtheparasitesurfacetowardtheposteriorend, con-touringthekinetoplast,andthenucleus(Fig.1C).Aswellascited inT.cruzi,byPorto-Carreiroetal.(2000),theT.dionisiicytostome extendstowardthereservosomes.WealsoobservedthatinT. dion-isii,thecomplexcytostome-cytopharynxisassociatedinitsorigin withtheflagellarpocket(Fig.1C),aswasdescribedinT.cruzi,by
Okudaetal.(1999),andrecentlyshowedon3Dreconstructionof
T.cruziepimastigotes(Ramosetal.,2011).Oursobservations cor-roboratetheendocyticandexocyticsiteoftheflagellarpocket(De Souzaetal.,2009a).
Fig.6.T.dionisiiexpressesglycolipidpresentingterminalresiduesofGalfrecognizedbyMAbMEST-1.Fixedparasitesweredelipidatedornotwithamixtureof iso-propanol/hexane/water(55:20:25;vol/vol/vol).AfterT.dionisiidelipidationMAbMEST-1reactivitywasabolished(PanelD).(A)Controlparasites,phasecontrastimage.(B) controlparasites,mergedimageofMAbMEST-1ingreenandDAPI(nucleiandkinetoplasts)inblue.Whitearrowsindicatedparasiteswithastrongpunctuallabelingof surfaceorvesiclesotherthenreservosomes,andwhitearrowheadsindicatedparasitesweaklylabeled.(C)Delipidatedparasites–phasecontrast.(D)Delipidatedparasites mergedimageofMAbMEST-1(itshouldappearingreen)andDAPI(nucleiandkinetoplasts)inblue.ImageshowtheabolishedMAbMEST-1reactivity.ImagesAandB acquiredunderconfocalmicroscopy.ImagesCandDacquiredunderfluorescencemicroscopy.Scalebar:10m.(Forinterpretationofthereferencestocolorinthisfigure legend,thereaderisreferredtothewebversionofthearticle.)
togetherwithsharingmechanismsofcellularinvasionprocessand differencesintrans-sialidaseexpression,betweenT.dionisiiandT. cruzi,thatwe describedinpreviouswork(Oliveiraetal.,2009), canenrichthenew‘batseeding’hypothesisofT.cruzievolution (Hamiltonetal.,2012).
Inadditiontothethree-dimensionalmorphologicalstudiesofT. dionisiiweverified,usingMAbsBST-1andMEST-1,thatthis para-sitesharescarbohydrateepitopes,suchasglycoepitopescontaining Galfresidues,withT.cruziepimastigotes.
InT.dionisiiepimastigotes,theMAbBST-1(whichwasraised against T. cruzi CL epimastigoteglycolipids), showed reactivity withmultivesicularstructures(reservosomes,indicatedbywhite arrowsinFig.4BandD).Also,aweakBST-1labelingwasobserved intheparasitesurfaceandflagellum(arrowheads).However,not allparasitesarelabeledwithBST-1.Theseresultssuggestthe exist-enceofthreesubpopulationsofT.dionisiiepimastigotes:parasites thatexpressweakBST-1 labelingat surface/vesiclesotherthan reservosomes,parasitesthatexpressglycoepitopesrecognizedby BST-1atsurfaceandinreservosomevesicles,andparasiteswhich arenotlabeledbyBST-1.T.cruziepimastigoteheterogeneitywas previouslyreportedbySilvaetal.,2006,analyzingdifferent epi-mastigoteepitopes,andalsocitedbyusinT.dionisii,usingdistinct MAbsanti-T. cruzi (Oliveira et al., 2009).BST-1 reacts withall
populationofT.cruziepimastigotes(strainsCL),astrong fluores-cenceisobservedinreservosomes(arrow),parasitesurfaceand flagellum(Fig.5B).
TheMAbMEST-1,previouslyraisedagainstP.brasiliensis gly-colipidscontainingterminalresiduesofGalf(Suzukietal.,1997), whichalsoreactsstronglywithT.cruziglycolipidspresentin reser-vosomes(Suzukietal.,1997,2001)andL.majorsurfaceglycolipids (Suzukietal.,1997),alsorecognizesT.dionisiiepimastigotes.The MEST-1 labelingwaspunctual,appearing tobeconcentratedat theperipheryoftheparasitebody(Fig.6B),wealsonotedsome parasitesweaklylabeled,showingagainparasitesubpopulations (arrowheads).T.dionisiiMAbMEST-1labelingwastotallydifferent fromthatshowedforepimastigotesofT.cruziYstrain,whereitwas observedastronglabelingofparasitereservosomes(Suzukietal., 2001).Ontheotherhandsimilarpatternoflabelingwasobserved betweenT.cruziCLstrainandT. dionisii(Figs.5Dand 6B).InT. dionisiiepimastigotesMEST-1reactivegalactofuranosecontaining antigensseemtobeconcentratedatparasitesurfaceprobablyin membranemicrodomainsorinvesiclesotherthanreservosomes (Fig.6B).
M.P.d.C.Oliveiraetal./ActaTropica128 (2013) 548–556 555
Fig.7. ProfileofT.cruziandT.dionisiiepimastigotecomponentsrecognizedbyMAb BST-1.Parasites(8×109cells)weresolubilizedinsamplebuffer,submittedto
PAGE-SDS8%electrophoresisandanalyzedbyWesternblotting.MAbBST-1recognized inT.cruziCLstrain(CL)alowmolecularweightcomponent(lessthan26kDa)and inGstrain(G)twomaincomponentsbetween80and115kDa.InT.dionisii(Tdio) MAbBST-1recognizedalowmolecularweightcomponent(lessthan26kDa)and componentsbetween72and115kDa.
a mixture of isopropanol–hexane–water (55:20:25; vol/vol/vol) andlabeledwithMEST-1.UndertheseconditionstheMEST-1 label-ingwasabolished(Fig.6D),confirmingthelipidcharacteristicof therecognizedantigen.Moreover,Westernblotresultsshowthat MAbBST-1 recognizedinT. dionisiiepimastigotes, aswellin T. cruziGstrainepimastigotes,highmolecularweightglycoproteins (72–115kDa).BST-1alsorecognizeslowmolecularweightantigens (lowerthan26kDa)inT.dionisiiepimastigotes(Fig.7).The pres-enceoftheselowmolecularweightantigenscouldberelatedto theNETNESglycoproteinsofT.cruzi(MacRaeetal.,2005),which containsanunusuallysmallmaturepeptidecomponent(13amino acids)andmolecularweightbetween13and15kDa,orcorrespond toreactiveglycolipidantigens.Thesedatashouldbebetterstudied. ThefactofT.dionisiiglycolipidepitopesrichinGalfresidues appearstobelessabundantthaninT.cruzi,inadditionto previ-ousobservationsthatT.dionisiiislessinvasiveandpresentslow infectivecapacity(invitro),exceptundercontrolled environmen-talconditions(Oliveiraetal.,2009;Maedaetal.,2012),corroborate thehypothesisthatglycoconjugatesrichinGalfresiduesmaybe relatedtoparasitesinfectivityandwesuggestthatmaybegood targetsforthedevelopmentsofnewdrugsandvaccinesagainst Chagas’disease.
Acknowledgments
ThisworkwaspartofMiriamPiresdeCastroOliveira’sPhD the-sis.ThefinancialsupportfromtheBrazilianagenciesCoordenac¸ão
deAperfeic¸oamentodePessoaldeNívelSuperior(CAPES),Conselho NacionaldeDesenvolvimentoCientíficoeTecnológico(CNPq),and Fundac¸ãodeAmparoàPesquisadoEstadodeSãoPaulo(FAPESP, 06/07005-4).
AppendixA. Supplementarydata
Supplementarydataassociatedwiththisarticlecanbefound, in the online version, at http://dx.doi.org/10.1016/j.actatropica. 2013.08.001.
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