Characterization of the infective properties of a new genetic group of Trypanosoma cruzi associated with bats

(1)

ContentslistsavailableatSciVerseScienceDirect

Acta

Tropica

j o u r n al hom ep age : 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

Characterization

of

the

infective

properties

of

a

new

genetic

group

of

Trypanosoma

cruzi

associated

with

bats

Fernando

Yukio

Maeda,

Renan

Melatto

Alves,

Cristian

Cortez,

Fabio

Mitsuo

Lima,

Nobuko

Yoshida

∗

DepartamentodeMicrobiologia,ImunologiaeParasitologia,UniversidadeFederaldeSãoPaulo,SãoPaulo,Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received19July2011

Receivedinrevisedform23August2011 Accepted1September2011

Available online 7 September 2011

Keywords: Trypanosomacruzi Newgenotype Metacyclicforms Cellinvasion,Bats

a

b

s

t

r

a

c

t

AnewgenotypeofTrypanosomacruzi,associatedwithbatsfromanthropicareas,wasrecentlydescribed. HerewecharacterizedaT.cruzistrainfromthisnewgeneticgroup,whichcouldbeapotentialsource ofinfectiontohumans.Metacyclictrypomastigotes(MT)ofthisstrain,hereindesignatedBAT,were comparedtoMTofwellcharacterizedCLandGstrains,asregardsthesurfaceprofileandinfectivity towardhumanepithelialHeLacells.BATstrainMTexpressedgp82,thesurfacemoleculerecognizedby monoclonalantibody3F6andknowntopromoteCLstraininvasionbyinducinglysosomalexocytosis, aswellasmucin-likemolecules,butlackedgp90,whichfunctionsasanegativeregulatorofinvasionin Gstrain.AsetofexperimentsindicatedthatBATstraininternalizationisgp82-mediated,andrequires theactivationofhostcellphosphatidylinositol3-kinase,proteinkinaseCandthemammaliantargetof rapamycin.MTofBATstrainwereabletomigratethroughagastricmucinlayer,apropertyassociated withp82andrelevantfororalinfection.Gp82wasfoundtobeahighlyconservedmolecule.Analysis oftheBATstraingp82domain,containingthecellbinding-andgastricmucin-bindingsites,showed91 and93%sequenceidentitywithGandCLstrains,respectively.HelacellinvasionbyBATstrainMTwas inhibitedbypurifiedmucin-likemolecules,whichwereshowntoaffectlysosomeexocytosisrequired forMTinternalization.AlthoughMTofBATstraininfectedhostcellsinvitro,theywerelesseffectivethan GorCLstrainsininfectingmiceeitherorallyorintraperitoneally.

1. Introduction

Trypanosomacruzi,theprotozoanparasitethatcausesChagas’ disease,isconstitutedofgenotypicallyheterogeneouspopulations thatmaydifferconsiderablyintheirphenotypiccharacteristics.In 2009,byreviewingtheavailableknowledge,anexpertcommittee reachedanewconsensusforT.cruziintraspecificnomenclature: theknownT. cruziisolates andstrainsshouldbereferredtoby sixdiscretetypingunits,TcI-TcVI(Zingalesetal.,2009).Recently, phylogeneticanalysesusingSSUrDNA,cytochromebandhistone H2Bgenes,andgenotypingmethodtargetingITS1rDNA,revealed anewgenotypeofT.cruzi,which isassociatedwithbatsandis notclusteredwithinanyofthepreviouslydefinedlineages(Marcili etal.,2009).Thisnewgroupisformedexclusivelybyhighly homo-geneousbatisolatesfromanthropicareasthatwereendemicfor Chagas’disease,indicatingthatbatsmaybeimportantreservoirs andpotentialsourceofT.cruziinfectiontohumans(Marcilietal., 2009).Itisthereforerelevanttoinvestigatetheinfectiveproperties oftheparasiteisolatedfrombat.

∗Correspondingauthor.

E-mailaddress:nyoshida@unifesp.br(N.Yoshida).

Metacyclic trypomastigotes (MT) of T. cruzi from bats were foundtoinvadeculturedcells,followedbyintracellular develop-mentofparasites (Marciliet al.,2009).Howdotheyenter host cells,whichMTmoleculesareinvolvedintheprocess,what sig-nalingpathwaysaretriggeredduringMT-targetcellinteraction,to whatextentdoesthisnewgeneticgroupdifferfromotherT.cruzi lineages?Thesearequestionsthatremaintobeelucidated. Differ-entT.cruzistrainsmayvarygreatlyintheirinfectivityinvitroand invivo,andthesedifferencesareassociatedwiththedifferential engagementofsurfacemoleculesandtriggeringofdistinct signal-ingpathwaysinbothcells(Neiraetal.,2002;Ferreiraetal.,2006; Cortezetal.,2006a;Covarrubiasetal.,2007).Forinstance,strains G(TcI)andCL(TcVI),belongingtohighlydivergentgeneticgroups andassociatedwithmarsupialandhumaninfection,respectively (Brionesetal.,1999),arecharacterizedbytheirdifferential infec-tivitytowardculturedmammaliancellsaswellasinmice(Yoshida, 2006).MTofpoorlyinfectiveGstrainapparentlyusethemucin-like glycoproteinstoenterhumanepithelialcellswhereasMTofhighly invasiveCLstrainrelyonthesurfacemoleculegp82(Ruizetal., 1998),whichinducesaCa2+_-dependent_disruption_of_the_host_cell actincytoskeleton(Cortezetal.,2006b)thatfacilitateslysosomal exocytosisandparasiteinternalization(Martinsetal.,2011).There arealsoevidencesthatsuccessfulestablishmentofCLstraininmice bytheoralroute,whichisthemainmodeofT.cruzitransmissionin

Open access under the Elsevier OA license.

(2)

someregions(Coura,2006),iscriticallydependentongp82(Neira etal.,2003;Cortezetal.,2003).Inthisstudyweaimedat charac-terizingaT.cruzistrainfrombat,hereindesignatedBAT,focusing ontheexpressionofsurfacemoleculesimplicatedinparasite–host cellinteractionandonthemechanismsofcellinvasion.

2. Materialsandmethods

2.1. Parasites,mammaliancellcultureandinvasionassays

AstrainofanewlineageofT.cruzi(Marcilietal.,2009),isolated fromMyotislevisinSãoPaulo,waskindlyprovidedbyDr.Marta M.G.TeixeirafromUniversidadedeSãoPaulo,Brazil.Inaddition tothisstrain,designatedBAT,weusedT.cruzistrainsG,isolated fromanopossumin theBrazilianAmazon(Yoshida,1983), and CL,isolatedfromthedomiciliaryinsectvectorTriatomainfestans inthesouthern stateofRio Grandedo Sul,Brazil,in adwelling wherepeoplewereinfected(BrenerandChiari,1963).Theparasites weremaintainedcyclicallyinmiceandinliverinfusiontryptose mediumcontaining5%fetalbovineserum.Fordifferentiationof epimastigotesintoMT, Grace’s medium(Invitrogen)and TC100 medium(Vitrocell,Brazil)werealsoused.MTfromculturesatthe stationarygrowthphasewerepurifiedbypassagethrough DEAE-cellulosecolumn,asdescribed(TeixeiraandYoshida,1986).HeLa cells,thehumancarcinoma-derivedepithelialcells,weregrown at37◦_C_in_Dulbecco’s_Minimum_Essential_Medium_(DMEM)

sup-plementedwith10%fetal calfserum, streptomycin(100␮g/ml) andpenicillin(100U/ml)inahumidified5%CO2atmosphere.Cell invasionassayswerecarriedoutasdetailedelsewhere(Yoshida etal.,1989), byseedingtheparasitesontoeachwellof24-well platescontaining13mmdiameterroundglasscoverslipscoated with1.5×105_HeLa_cells._The_multiplicity_of_infection_(MOI)_was 10:1forCLstrainand20:1forGandBATstrains.After1h incuba-tionwithMT,theduplicatecoverslipswerewashedinPBS,fixedin Bouinsolution,stainedwithGiemsa,andsequentiallydehydrated inacetone,agradedseriesofacetone:xylolandxylol.Thenumber ofintracellularparasiteswascountedin250stainedcells.

2.2. IsolationofacDNAclonecontainingtheC-terminaldomain ofBATstraingp82

ComplementaryDNA(cDNA)fromBATstrainMT(1×108₎_was obtainedusingtheAccessQuickTM _RT-PCR_System_(Promega)_on totalRNAextractedbyTRIzol(Invitrogen).FollowingcDNA synthe-sis,thestrategyfortheamplificationofC-terminaldomainofBAT straingp82wasbasedonitspresumedsimilaritywithCL/Gstrain gp82.Theforwardprimer5′

-GGATCCATGTTCGTCAGCAGCCTGCTG-3′ _corresponded _to _a _sequence _that _precedes _the _epitope _for

mAb3F6and containedATG plusan artificialBamHI site;the reverseprimer5′_-_{GAATTCGTTCAGTGGGCGGTTGTACAAGAAGA-3}′

corresponded to a sequence that follows the highly con-servedVTVKNVFLYNRmotifcharacteristicofallmembersofthe gp85/trans-sialidadesuperfamilyandcontainedastopcodonplus anartificialEcoRIsite.Atotalof40cyclesofdenaturing, anneal-ingandelongationat 94◦_C _for₂₀_s,₅₅◦_C_for ₃₀_s_and₇₂◦_C _for

1min,respectively,wereperformed.Afterpurification,using Pure-Linkkit(Iinvitrogen)thePCRproductwasclonedintheplasmid vectorpGEM-TEasy(Promega).Followingligationtothevector, theproductwastransformedinEscherichiacolistrainDH5␣,and thecolonies growninLBbroth.Clonescontainingtheexpected 771bpfragmentafterrestrictionanalysiswithEcoRIandBamHI weresequencedusingABI3130XLGeneticAnalyzerandBigDye Terminatorv3.1(AppliedBiosystems).

2.3. Southernblotanalysisandpulsedfieldgelelectrophoresis

ForSouthernblotanalysis,T.cruziDNAwasdigestedwith dif-ferentrestrictionenzymes,separatedbyelectrophoresison0.8% agarosegelandblottedontonylonmembranes.Hybridizationwith theprobe,whichconsistedofaDNAfragmentcorrespondingtoORF ofgp82gene(wholeinsertofgp82cDNAclone)labeledwith[32_P], andwashingswereperformedasdetailed(Arayaetal.,1994).For pulsedfieldgelelectrophoresis,agaroseblockscontaininggenomic DNAwereprepared,incubatedat50◦_C_for₁₆_h_in_lysis_solution

containing10mM Tris–HCl,pH8.0, 500mM EDTA,1%sarkosyl, 1mg/mlproteinaseK,equilibratedin TE,washed andstored in 0.5MEDTAat4◦_C._Small_portions_(equivalent _to₁₀7 _parasites) wereelectrophoresed(1.2%agarosegelin0.5×TBE)at80Vfor 132hinGeneNavigatoreSystem(Pharmacia),frompulsetimes varyingfrom90to800s.DNAfromHansenulawingeiwasusedas reference.Aftertransfertonylonmembranes,chromosomalDNA bandswerehybridizedwiththe[32_P]-labeled_insert_of_gp82_cDNA cloneandrevealedbyexposuretoX-rayfilm(Hyperfilm-MP, Amer-sham).

2.4. ProductionandpurificationofJ18andGST

TherecombinantproteinJ18,containingthefull-lengthT.cruzi gp82sequence(GenBankTM_data_base,_accession_number_L14824) inframewithglutathioneS-transferase (GST),wasproduced in E.coliDH5-␣bytransformingthebacteriawithapGEX-3construct comprisingthegp82gene.Detailsoftheconstructionandthe purifi-cationofJ18,aswellasofGST,aredescribedelsewhere(Cortezet al.,2006b).

2.5. Purificationofmucin-likemoleculesfromT.cruzi

WefollowedtheproceduredescribedbyAcosta-Serranoetal. (2001).Cultures(totalof5×1010_parasites_for_each_preparation) were centrifuged, and the pellet was freeze-dried and placed in a sonicating water bath for 10min with 10ml of chloro-form/methanol/water(1:2:0.8,byvolume).Aftercentrifugationat 2000×gfor5min,andtwomoreextractionofthepellet,the insol-ublematerialservedasasourceofdelipidatedparasiteswhereas thepooledfractions(30ml)wereplacedinaround-bottomflask and dried by rotatory evaporation. The residue was extracted with20mlofbutan-1-ol/water(2:1,byvolume)Thebutan-1-ol phasecontainedthelipidfraction(F1)andtheaqueousphase(F2) containedepimastigotemucins.F2waswashedtwicewith water-saturatedbutan-1-olandconcentrated.Thedelipidatedparasites wereextracted(threetimes)bysonicationwith10mlof9%butanol inwater,andthepooledsolublematerialcontainingmucins(F3) wasconcentrated.Themucinswereresuspendedin2mlofbuffer A(0.1Mammoniumacetatein5%ppropan-1-ol(v/v))and fraction-atedonanoctyl-Sepharosecolumn(10×0.5cm),pre-equilibrated inbufferA.AfterwashingthecolumnwithbufferA,andelution withalineargradientover100mlataflowrateof12ml/h, start-ingwith15mlofbufferAandendingwith60%(v/v)propan-1-olin water,fractions(2ml)wereanalyzedbysilverstainingofSDS-PAGE gels,aswellasbyimmunoblottingusingtheavailablemonoclonal antibodies.

2.6. Parasitemigrationthroughthegastricmucinlayer

(3)

transwellfilterswereplacedontoparasite-containingwells,and 100␮lPBS wereadded tothefilterchamber. Atdifferent time pointsofincubationat37◦_C,₁₀_␮_l_were_collected_from_the_filter

chamberfordeterminationofparasitenumberandthevolumein thischamberwascorrectedbyadding10␮lPBS.

2.7. Exocytosisassay

ConfluentmonolayersofHeLacells,grownin24-wellplatesin DMEMwerewashedtwiceinPBSandincubatedin300␮lDMEM withoutphenolred.After1h,thesupernatantswerecollectedand thecellswerelysedinDMEMorPBS++_containing_1%_NP-40,_and 30␮lof1MsodiumacetatepH4.0wasaddedtodecreasepH. Sam-pleswerecentrifugedfor5minat13,000×gandthesupernatants werecollected,20␮laliquotsweredilutedwith60␮lcitratebuffer and160␮lof100mM4-nitrophenylN-acetyl-␤-d-glucosaminide (Sigma) wereadded. After1h incubationat 37◦_C, _the_reaction

wasstoppedbyadding720␮lof200mMsodiumboratepH9.8 andabsorbance wasmeasuredat405nmin aLabsystems Mul-tiskanMS plate reader. Exocytosis wasexpressedas % of total

␤-hexosaminidaseactivity(supernatant+cellextract).

2.8. Indirectimmunofluorescenceassays

Tovisualizeparasitesco-localizedwithlyososomemarker,MT wereincubatedwithadherentHeLacellsfor1hat37◦_C._After

fix-ationwith4%p-formaldehydein PBSfor30min,thecellswere incubatedwith50mMNH4ClinPBSfor30min,washed3times andpermeabilizedby30mintreatmentwith1%saponininPGN (PBS containing 0.15% gelatin and 0.1% sodium azide). Follow-ing1hincubationatroomtemperaturewithmouseanti-human Lamp-2(H4B4monoclonalantibody),diluted1:5inPGN,and3 washesinPBS,thecellswereincubatedfor1hinPGNwithAlexa Fluor568-conjugatedanti-mouseIgG(Invitrogen),diluted1:200, and10␮g/ml DAPI(4′_,6′_{1-diamino-2-phenylindole}

dihydrochlo-ride)forvisualizationofnucleus.ImageswereacquiredinOlympus BX51,equippedwithanOlympusDP71CCDcamera,usingImage ProPlus6.2software(MediaCyberneticInc.).

2.9. OralinfectionofmicewithBATstrainMT

ToexaminetheinfectivityofBATstrainMTinvivo,fourtofive week-oldfemaleBalb/cmice,bredintheanimalfacilityat Uni-versidade Federal deSão Paulo,were used. Allprocedures and experimentsconformed withtheregulation of theinstitutional EthicalCommitteeforanimalexperimentation,andthestudywas approvedbytheCommittee(#CEP0117/11).Micewereinfected withmetacyclicformsbytheoralroute(106_parasites_per_mouse), using a plastic tubeadapted to a 1ml syringe.Starting on day 10post-inoculation,parasitemiawasmonitoredtwiceaweekby examining5␮lbloodsamplescollectedfromthetail,atthephase contrastmicroscope.

2.10. Statistics

TodeterminesignificanceofdataStudent’sttest,theprogram GraphPadInStatwasused.

3. Results

3.1. Surfaceprofileandinfectivityofmetacyclictrypomastigotes (MT)ofBATstrain

TocomparetheMTsurfaceprofileofBATstrainwiththatof GandCLstrains,weusedmonoclonalantibodies(mAbs)directed eithertogp90,gp82ormucin-likemolecules,whichareexpressed

onthe parasitesurface and areimplicated in interactions with host cells. Gp90, which acts as a negative modulator of cell invasion (Málaga and Yoshida, 2001), wasundetectable in BAT strainwhereastheinvasion-promotingandmAb3F6-reactivegp82 (Ramirezet al.,1993)wasexpressedat levelscomparable toG andCLstrains(Fig.1A).Mucin-likeglycoproteinswererevealed inBATstrainbymAb2B10butnotbymAb10D8(Fig.1A), indi-catingthattheylackgalactofuranoseresiduesthatarepartofthe epitopeformAb10D8(Yoshida,2006),anantibodythatinhibits Gstraininfectivity(Yoshidaetal.,1989).Overall,thesurface pro-fileofBATstraindisplayedhighersimilaritytoCLstrainthantoG strain.TodeterminetheinfectivityofBATstrain,ascomparedto GandCLstrains,MTwereincubatedwithHeLacellsfor1hand thenumber ofintracellularparasiteswascountedafterfixation, stainingwithGiemsaand serial dehydration.The rateof inter-nalizationofBATstrain(MOI=20)wassignificantlyhigherthan thatofGstrain(MOI-20)andcomparabletothatofCLstrainat MOI=10(Fig.1B).Next,weexaminedwhethergp82was impli-cated.Inonesetofexperiments,MTwerepre-incubatedfor15min withmAb3F6andthenaddedtoHelacells.After1hincubation,the cellswereprocessedforparasitecounting.MAb3F6significantly inhibitedparasiteinternalization(Fig.1C).Tofurtherassessthe involvementofgp82,Helacellswerepre-incubatedfor15minin absenceorinthepresenceofJ18,therecombinantprotein contain-ingthefulllengthgp82sequencefusedtoGST,orGSTascontrol, at40␮g/ml,andMTofBATstrainwereadded.After1hincubation, inthepresenceofJ18orGST,thecellswereprocessedasabove. ParasiteinvasionwasinhibitedbyJ18,butnotbyGST(Fig.1D). Theseresultsindicatethat,similartoCLstrain(Ramirezetal.,1993), BATstrainreliesongp82moleculetoenterhostcells.Inanother setofexperiments,wetestedtheeffectofdrugsthataffectcell signalingandwerepreviouslyshowntoinhibitCLstrainMT inva-sion(Martinsetal.,2011).HeLacellsweretreatedfor30minwith 100nMofwortmannin,aninhibitoroflipidkinasephosphoinositol 3kinase(PI3K),phorbolmyristateacetate(PMA),adrugthatcan downregulateproteinkinaseC(PKC),orrapamycin,whichinhibits mammaliantargetofrapamycin(mTOR).Afterremovalofthedrug, theparasiteswereadded.Following1hincubation,alongwiththe untreatedcontrols,thecellswereprocessedforcountingof inter-nalizedparasites.AllthreedrugsdiminishedinvasionofBATstrain (Fig.1E).Asthegp82-mediatedinvasionofCLstrainMTisinhibited bythereferreddrugs,andisassociatedwithlysosomalexocytosis thatcontributesforparasitophorousvacuoleformation(Martins etal.,2011),wecheckedwhetherBATstrainMTco-localizedwith lysosomemarkerduringinvasion.HeLacellswereincubatedwith MTfor1handwerethenprocessedforimmunofluorescenceusing anti-Lamp-2antibody.Parasitesco-localizedwithLamp-2couldbe visualized(Fig.1F).

3.2. Highidentityofgp82sequencesdeducedfromcDNAclones ofBAT,GandCLstrains

(4)

Fig.1.Surfaceprofileandinfectivityofmetacyclictrypomastigotes(MT)ofT.cruziBATstrain.(A)MTofBATstrainandreferencestrainsGandCLwereprocessedfor Westernblotanalysis,usingtheindicatedmonoclonalantibodiesdirectedtosurfacemoleculesgp90,gp82andmucin-likegp35/50.(B)Cellinvasionassayswereperformed byincubatingHeLacellswithBATstrain(MO=20),Gstrain(MOI=20)orCLstrain(MOI=10)for1h.AfterfixationandGiemsastaining,thenumberofintracellularparasites wascountedinatotalof250cells.Thevaluesarethemeans±SDoffourindependentexperimentsperformedinduplicate.(C)BATstrainMT,untreatedorpretreatedwith mAb3F6,wereincubatedfor1hwithHeLacells,whichwerethenprocessedasin(B)forparasitecounting.(D)HeLacells,untreatedorpretreatedwiththerecombinant proteinJ18orGST,at40␮g/ml,wereincubatedwithBATstrainMTandprocessedforparasitecounting.(E)HeLacellsweretreatedwiththeindicateddrug,at50nM.After washingoutthedrugs,thecellswereincubatedfor1hwiththeparasites,fixedandstainedwithGiemsa.Valuesin(B–E)arethemeans±SDofthreeindependentassays performedinduplicate.Inallcases,MTinvasionwassignificantlyinhibited(*_p_<_0.05)_by_the_indicated_treatment._(F)_HeLa_cells_were_incubated_with_BAT_strain_MT_for₁_h andthenprocessedforimmunofluorescenceusinganti-Lamp-2antibodyandDAPI.ParasiteassociatedwithLamp-2areindicatedbywhitearrow.Scalebar=10␮m.

p3,afewaminoacidchangeswasdetectedinBATascomparedto GandCLstrains(Fig.2B).Theobservedsubstitutionspresumably donotresultinsubstantialchangesinthepropertiesofBATstrain gp82,asjudgedbytheability ofmAb3F6andtherecombinant proteinJ18ininhibitingBATstrainentryintohostcells(Fig.1C andD).Itshouldbenotedthatthepairofcontiguousglutamicacid residuesandofasparticacidsinp4andp8sequences,previously showntoberequiredforcellbindingofgp82(Manqueetal.,2000), wasconserved inthe3strains(Fig.2B).Thegenomic organiza-tionofBATstraingp82genefamilywasalsoexamined.Southern blotofgenomicDNAdigestedwithrestrictionenzymeBamHI,Eco RI,HindIIIorXhoI,washybridizedwiththeinsertofcDNAclone J18.TheprofileofBATstrain differedconsiderablyfromthat of GandCLstrains(Fig.3A).Chromosomalmappingofgp82genes,

performedbyhybridizingthesameprobewithchromosomalsize fragmentsseparatedbypulsedfieldgelelectrophoresis,revealed markeddifferencesofBATstrainascomparedtoGandCLstrains (Fig.3B).

3.3. MigrationofBATstrainMTthroughgastricmucinand invasionofhostcells

(5)

Fig.2. Sequencesofgp82carboxy-terminaldomainofdifferentT.cruzistrains.(A)ShownaretheaminoacidsequencesdeducedfromcDNAclonesF11(BATstrain),J18(G strain)andR31(CLstrain).Overall,BATstrainsequencewas91%and93%identicaltoGandCLsequences.(B)Thesequencesrepresentedbypeptidesp4andp8,identified asthehostcellbindingsiteofgp82,aswellasthesequencesp3andp7,identifiedastheepitopeformAb3F6andthegastricmucin-bindingsite,areshown,withasterisks indicatingtheaminoacidresiduesofBATstraingp82thatdifferfromGandCLstrains.

thefilterchamberwerecollectedatdifferenttimepointsandthe numberofparasitescounted.Alongthetime,increasingnumberof MTwasrecoveredfromthetranswellchamber(Fig.4A).Next,cell invasionassaysinthepresenceofgastricmucinwereperformed. Ithasbeenshownthatgp82-expressingCLstrainmetacyclicforms efficientlyinvadeHeLacellsregardlesswhethergastricmucinis presentornot,incontrasttogp82-deficientT.cruzistrainswhose internalizationisimpairedbygastricmucin(Cortezetal.(2003). AlsoshownwasthatCLstrainmetacyclicforms,aswellasgp82, aredevoidofsubmaxillarymucin-bindingproperty,andparasite invasionisreducedinthepresenceofthismucin(Staquicinietal., 2010).InassaysinwhichBATstrainMTwereincubatedwithHeLa cellsinthepresenceof2mg/mlofgastricorsubmaxillarymucin,a markeddecreaseinparasiteinvasionwasobservedinthepresence ofsubmaxillarybutnotofgastricmucin(Fig.4B).

Fig.3.Genomicorganizationofgp82genesinT.cruzistrains.(A)Southernblot ofgenomicDNAdigestedwiththeindicatedrestrictionenzymeswashybridized withthewholeinsertofgp82cDNAclone(J18)labeledwith[32_P]._(B)_Chromosomal bandsofparasiteswereseparatedbypulsedfieldgelelectrophoresis,transferredto nylonmembraneandhybridizedwiththe[32_P]-labeled_probe_as_above._Numbers correspondtomolecularsizes.NotethedifferencesbetweenBATstrainandthe othertwostrains.

3.4. InhibitionofMTinvasionofhostcellsbyT.cruzimucin moleculesthatexhibitlysosomalexocytosis-inhibitingproperties

Mucin-likemoleculesexpressedonthesurfaceofmetacyclic forms have been implicated in target cell invasion of Gstrain (Yoshidaetal.,1989).Asmucinsareexpressedathighlevelsin BATstrain(Fig.1A),weinvestigatedwhethertheyplayedarole in invasion. HeLa cells were incubated withMT in absence or inthepresenceofmucinspurifiedfromBAT,aswellasfromG andCLstrains.InternalizationofBATstrainMTwasinhibitedby mucinsfromallthreestrains,thehomologousmucinsexhibiting thehighesteffect(Fig.5A).Next,thelysosomalexocytosis-inducing activityofmucinmoleculeswasexamined.HeLacellswere incu-batedwithmucin,at20␮g/ml,and1hlaterthelysosomalenzyme

␤-hexosaminidasewasmeasuredinthesupernatantaswellasin thecellextract.ExocytosiswassignificantlyreducedbyBATstrain mucin(Fig.5B).AlthoughGandCLstrainmucinsalsodiminished

(6)

Fig.5. EffectofT.cruzimucinsonhostcellexocytosisandinvasionbyBATstrainMT. (A)TheindicatedT.cruzimucinswereaddedtoHeLacells15minbeforeparasites. After1hincubation,thecellswerefixedandGiemsa-stainedforparasitecounting. Valuesaremeans±SDoffourindependentassaysperformedinduplicate.Invasion wassignificantlyinhibited(*_p_<_0.01,**_p_<_0.05)_by_mucins_of_different_strains._(B) Helacellswereincubatedfor1hwiththeindicatedT.cruzimucinsandthereleased lysosomalenzyme␤-hexosaminidasewasmeasured.Valuesaremeans±SDofthree independentassaysperformedinduplicate.Thedifferencebetweenexocytosisof HelacellstreatedwithBATstrainmucinandthecontrolwassignificant(*_p_<_0.05).

lysosomalexocytosis,thedifferencefromthecontrolwasnot sta-tisticallysignificant.

3.5. InvivoinfectionbyBATstrainMT

TodeterminetheinfectivityofBATstrainMTbytheoralroute,a groupofmice(n=5)wasinfectedorally(106_parasites_per_mouse) andthecourseofinfectionwasmonitored.Bloodsampleswere examinedforthepresenceofparasitesupto30daypostinfection. Parasitemiawasnotdetectable,whatis incontrasttoinfection byCLstrainMTthatconsistentlyresultedinpatentparasitemias (Cortezetal.,2003,2006a;Covarrubiasetal.,2007).Intraperitoneal injectionwasalsoquiteinefficient.Onaverage,from10infected micewecouldrecoverparasitesinhemoculturefromtwotothree mice,whereaspositivehemocultureisinvariablyobtainedfromall miceinfectedwithGorCLstrain.

4. Discussion

OurresultshaveindicatedthatBATstrainMTinvadehostcells inamannersimilartoCLstrainMT,i.e.,theyengagethesurface moleculegp82andtriggertheactivationofmTOR,PI3KandPKC topromotetheirinternalizationthroughlysosomeexocytosis.The involvementofgp82inBATstrainMTentryintohostcellswas impliedfromthefindingsthatparasiteinternalizationwas inhib-itedbymonoclonalantibodydirectedtogp82,aswellasbythe recombinantproteinbasedongp82.Asregardsthelysosome exo-cytosis,itsroleininvasionwasdeducedfromexperimentsshowing thatdownregulationofmTOR,PI3KandPKC,previouslyshownto affectmobilizationoflysosomesfromtheperinuclearregiontothe cellperiphery(Martinsetal.,2011),diminishedMTinternalization. OfinterestwasthefindingthatBATstraingp82shareshigh sequence identitywith gp82of genetically divergent Gand CL strains,isolatedfromdifferentsourcesinwidelydistant geograph-icalregions. Gstrainderived froma marsupial captured inthe BrazilianAmazonwhileCLstrain,associatedwithhuman infec-tion,wasisolatedinthefarsouthofthecountry.Theconservation of p82moleculemay beassociated withits crucial role in the establishmentofinfectionbytheoralroute,whichispossiblya modeoftransmissionprevalentamonginsectivorousmammalian hostssinceancienttimes.Insectstagemetacyclicformsarewell equippedtoefficientlyinfectbytheoralroute,selectivelyinvading thegastricmucosalepithelium(Hoft,1996;Hoftetal.,1996),and

gp82playsacentralroleinselectivelybindingtogastricmucin,a propertycriticalfortheparasitemigrationthroughthemucuslayer towardtheunderlyingtargetcells(Staquicinietal.,2010).Alsoof noteisthatgp82isresistanttodegradationbypepsin(Cortezetal., 2006a).

Inadditiontogp82,BATstrainMTexpressmucin-likemolecules athighlevels.Thesemoleculesmayalsoplayaroleinparasite inter-nalization.Hostcellinvasionwasreducedinthepresenceofmucins purified from BAT isolate. The finding that BAT strain mucins reducedthelevelsoflyosomalexocytosis,i.e.,theyhadan oppo-siteeffectofgp82,furtherreinforcestheroleplayedbyexocytosis in MTinvasion. We presumethat gp82-mediatedparasite–host cellinteractionprevailsoverthatmediatedbymucinsduringBAT straininvasion.Ifthesituationweretheotherwayaround,the impairmentoflysosomal exocytosis,which contributesto para-sitophorousvacuoleformation(Tardieuxetal.,1994;Rodríguez etal.,1999;Fernandesetal.,2011),wouldresultinlowinfection rate.GstrainMT,whichrelypredominantlyonmucinmolecules, arepoorlyinvasive(Yoshida,2006).Bycontrast,highlyinvasiveCL strainMTdependmostlyongp82andminimally,ifatall,onmucin molecules(Ramirezetal.,1993).BATandCLstrainmucinslack recognitionbymAb10D8,whichreactswithanepitopecontaining galactofuranoseresiduesinGstrainmucins(Yoshida,2006)and reducesparasiteinfectivity(Yoshidaetal.,1989).Itispossiblethat thisstructuraldifferencedeterminesthedifferentialinteractionof mucinsfromBAT,CLandGstrainswithhostcells.

Wehaveanalyzedhereonlyonestrainfromeachofthethree geneticgroups,thereforewecannotassertthatthesestrainsare representativesoftherespectivelineages.AsregardsTcI,the meta-cyclicformsof7strainsthatwehaveanalyzedsofar,including thoseisolatedfrommarsupialorfromwildtriatominein differ-entgeographicalregions,displayedsimilarsurfaceprofileandtheir ability toinfecthumanepithelialcells was associatedwiththe expressionofgp90onthesurface(Ruizetal.,1998;Yoshida,2006). Thus,Gstrainthat originatedfromamarsupial intheBrazilian AmazonmaybeabonafiderepresentativeofTcIassociatedwith thewildtransmissioncycle.However,asTcIthat predominates innorthernSouthAmericaisalsoassociatedwithhumandisease, chagasiccardiomyopathybeingcommonplaceincountriessuchas Venezuela(Milesetal.,2009),itwouldbeofinterestto investi-gatetheinfectivepropertiesofTcIstrainsisolatedfromChagasic patients.Wehavefoundinapreviousstudythatmetacyclicforms ofT.cruzistrains(presumablyTcII),isolatedfromacutecasesof Chagas’diseaseinBrazilandexpressinggp90athighlevels, exhib-ited reducedcapacity toenter hostcells in vitrobut efficiently infected mice by the oral route, provided that they expressed pepsin-susceptiblegp90isoform,whichwasdigesteduponcontact withgastricjuice(Cortezetal.,2006a;Covarrubiasetal.,2007). Theinter-lineagehybrids TcVandTcVIareapparentlythemain causesofsevereacuteandchronicChagasdiseaseinthegreater GranChacoregionandneighbouringcountrieswhereT.infestansis theprincipaldomesticvector(Milesetal.,2009).Metacyclicforms ofCLstrain(TcVI),isolatedfromT.infestans,wascharacterizedby expressinggp90atlow levelsandbyhighinfectivityinvitroas wellasinvivo(Yoshida,2006).IfthisisacommonfeatureofTcV andTcVI,itremainstobeinvestigated.BATstraindistinguished fromallT.cruzistrainsexaminedtodatebylackofreactionwith monoclonalantibodiesdirectedtogp90.Morestrainshavetobe analyzedtoassesswhetherthisisageneralcharacteristicwithin thisnewgeneticgroup.

(7)

lineagemaycausesevereclinicalmanifestations.IncentralBrazil, TcIwasidentifiedin12acutecasesofChagas’disease(Luquetti etal.,1986),andA ˜nezetal.(2004)havefoundthepredominance ofTcIhumanisolatesinVenezuelaalliedtothehigherprevalence ofseveresymptomsofthedisease.Anotherreportthatcontradicts theassumptionofinnocuousnessofTcIreferstoaBolivianpatient withChagasdiseasewithaccompanyingAIDS,andseverecentral nervoussysteminvolvement,whosecerebrospinalfluidshowed TcIparasitepopulation(Burgosetal.,2008).Aninterestingquestion iswhetherT.cruziinfectioninbatsisharmlesstothehostand whetherthis couldbeassociatedwiththeirancient association. Thefirstfossilevidenceoftriatomine-trypanosomatidassociation hasbeenreportedbyPoinar(2005),whosuggestedthatbatswere theoriginalvertebratehostsofT.cruzi-liketrypanosomes.Amatter ofdebateiswhetherT.cruziinfectionspreadfrombatstoother mammals,includinghumans.

5. Conclusions

MetacyclicformsofBATstrain,belongingtoanewgenotypeof T.cruziassociatedwithbatsfromanthropicareas,havethe abil-itytoinvadeculturedhumanepithelialcellsthroughamechanism dependentonthesurfacemoleculegp82.Inthesamemanneras thehighlyinfectiveT.cruziCLstrain,whichalsoreliesongp82for internalization,BATstraintriggersinthetargetcellsthesignaling cascadesinvolvingphosphatidylinositol 3-kinase,proteinkinase Candthemammaliantargetofrapamycin.Thisleadsto lysoso-malexocytosis,aneventrequiredforparasiteinternalization.BAT strainwasnotveryeffectiveininfectingmice,butthepotentialof transmissiontohumansofthenewT.cruzigenotypeharboredby batshasstilltobeevaluated.

Acknowledgements

ThisworkwassupportedbyFundac¸ãodeAmparoàPesquisa do Estadode SãoPaulo (FAPESP#2006/61450-0) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq #301409/2007-2and#470726/2007-5),Brazil.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,atdoi:10.1016/j.actatropica.2011.09.001.

References

Acosta-Serrano,A.,Almeida,I.C.,Freitas-Junior,L.H.,Yoshida,N.,Schenkman,S., 2001.Themucin-likeglycoproteinsuper-familyofTrypanosomacruzi:structure andbiologicalroles.Mol.Biochem.Parasitol.114,143–150.

A ˜nez,N.,Crisante,G.,daSilva,F.M.,Rojas,A.,Carrasco,H.,Umezawa,E.S.,Stolf, A.M.,Ramírez,J.L.,Teixeira,M.M.,2004.PredominanceoflineageIamong Try-panosomacruziisolatesfromVenezuelanpatientswithdifferentclinicalprofiles ofacuteChagas’disease.Trop.Med.Int.Health9,1319–1326.

Araya,J.E.,Cano,M.I.,Yoshida,N.,FrancodaSilveira,J.,1994.Cloningand characteri-zationofageneforthestage-specific82-kilodaltonsurfaceantigenofmetacyclic trypomastigotesofTrypanosomacruzi.Mol.Biochem.Parasitol.65,161–169. Brener, Z., Chiari, E., 1963. Variac¸ões morfológicas observadas em diferentes

amostrasdeTrypanosomacruzi.Rev.Inst.Med.Trop.SãoPaulo5,220–224. Briones,M.R.S.,Souto,R.P.,Stolf,B.S.,Zingalez,B.,1999.Theevolutionoftwo

Try-panosomacruzisubgroupsinferredfromrRNAgenescanbecorrelatedwiththe interchangeofAmericanmammalianfaunaintheCenozoicandhasimplications topathogenicityandhostspecificity.Mol.Biochem.Parastiol.104,219–232. Burgos,J.M.,Begher,S.,Silva,H.M.,Bisio,M.,Duffy,T.,Levin,M.J.,Macedo,A.M.,

Schijman,A.G.,2008.MolecularidentificationofTrypanosomacruziItropism forcentralnervoussysteminChagasreactivationduetoAIDS.Am.J.Trop.Med. Hyg.78,294–297.

Cortez,M.,Neira,I.,Ferreira,D.,Luquetti,A.O.,Rassi,A.,Atayde,V.D.,Yoshida,N., 2003.InfectionbyTrypanosomacruzimetacyclicformsdeficientingp82but expressingarelatedsurfacemoleculegp30.Infect.Immun.71,6184–6191. Cortez,M.,Silva,M.R.,Neira,I.,Ferreira,D.,Sasso,G.R.S.,Luquetti,A.O.,Rassi,A.,

Yoshida,N.,2006a.Trypanosomacruzisurfacemoleculegp90downregulates

invasionofgastricmucosalepitheliuminorallyinfectedmice.MicrobesInfect. 8,36–44.

Cortez, M., Atayde, V., Yoshida, N., 2006b. Host cell invasion mediated by Trypanosoma cruzisurfacemoleculegp82is associatedwith F-actin disas-semblyandisinhibitedbyenteroinvasiveEscherichiacoli.MicrobesInfect.8, 1502–1512.

Coura,J.R.,2006.Transmissionofchagasicinfectionbyoralrouteinthenatural historyofChagas’disease.Rev.Soc.Bras.Med.Trop.39(Suppl.3),113–117. Covarrubias,C.,Cortez,M.,Ferreira,D.,Yoshida,N.,2007.Interactionwithhost

fac-torsexacerbateTrypanosomacruzicellinvasioncapacityuponoralinfection.Int. J.Parasitol.37,1609–1616.

Fernandes,M.C.,Cortez,M.,Flannery,A.R.,Tam,C.,Mortara,R.A.,Andrews,N.W., 2011. Trypanosoma cruzi subverts the sphingomyelinase-mediated plasma membranerepairpathwayforcellinvasion.J.Exp.Med.208,909–921. Ferreira,D.,Cortez,M.,Atayde,V.D.,Yoshida,N.,2006.Actincytoskeleton-dependent

and– independenthostcellinvasionbyTrypanosomacruziismediatedby dis-tinctparasitesurfacemolecules.Infect.Immun.74,5522–5528.

Hoft,D.F., 1996.Differentialmucosalinfectivityofdifferentlife stagesof Try-panosomacruzi.Am.J.Trop.Med.Hyg.55,360–364.

Hoft,D.F.,Farrar,P.L.,Kratz-Owens,K.,Shaffer,D.,1996.Gastricinvasionby Try-panosomacruziandinductionofprotectivemucosalimmuneresponses.Infect. Immun.64,3800–3810.

Luquetti,A.O.,Miles,M.A.,Rassi,A.,deRezende,J.M.,deSouza,A.A.,Póvoa,M.M., Rodrigues,I.,1986.Trypanosomacruzi:zymodemesassociatedwithacuteand chronicChagas’diseaseincentralBrazil.Trans.R.Soc.Trop.Med.Hyg.80, 462–470.

Málaga,S.,Yoshida,N.,2001.TargetedreductioninexpressionofTrypanosoma cruzisurfaceglycoproteingp90increasesparasiteinfectivity.Infect.Immun.69, 353–359.

Manque,P.M.,Eichinger,D.,Juliano,M.A.,Juliano,L.,Araya,J.,Yoshida,N.,2000. CharacterizationofthecelladhesionsiteofTrypanosomacruzimetacyclicstage surfaceglycoproteingp82.Infect.Immun.68,478–484.

Marcili,A.,Lima,L.,CavazzanaJr.,M.,Junqueira,A.C.V.,Velduo,H.H.,Silva,F.M., Campaner,M.,Paiva,R.,Nunes,V.L.B.,Teixeira,M.M.G.,2009.Anewgenotype ofTrypanosomacruziassociatedwithbatsevidencedbyphylogeneticanalyses usingSSUrDNA,cytochromebandhistoneH2Bgenesandgenotypingbaseson ITS1rDNA.Parasitology136,641–655.

Martins,R.M.,Alves,R.M.,Macedo,S.,Yoshida,N.,2011.Starvationandrapamycin differentially regulatehost celllysosome exocytosisand invasionby Try-panosomacruzimetacyclicforms.Cell.Microbiol.13,943–954.

Miles,M.A.,Llewellyn,M.S.,Lewis,M.D.,Yeo,M.,Baleela,R.,Fitzpatrick,S.,Gaunt, M.W.,Mauricio,I.L.,2009.Themolecularepidemiologyandphylogeographyof TrypanosomacruziandparallelresearchonLeishmania:lookingbackandtothe future.Parasitology136,1509–1528.

Neira,I.,Ferreira,A.T.,Yoshida,N.,2002.Activationofdistinctsignaltransduction pathwaysinTrypanosomacruziisolateswithdifferentialcapacitytoinvadehost cells.Int.J.Parasitol.32,405–414.

Neira,I.,Silva,F.A.,Cortez,M.,Yoshida,N.,2003.InvolvementofTrypanosomacruzi metacyclictrypomastigotesurfacemoleculegp82inadhesiontogastricmucin andinvasionofepithelialcells.Infect.Immun.71,557–561.

PoinarJr.,G.,2005.Triatomadominicanasp.n(Hemiptera:Reduviidae:Triatominae), andTrypanosomaantiquussp.n.(Stercoraria:Trypanosomatidae),thefirst fos-silevidenceofatriatomine-trypanosomatidvectorassociation.VectorBorne ZoonoticDis.5,72–81.

Ramirez,M.I.,Ruiz,R.C.,Araya,J.E.,FrancodaSilveira,J.,Yoshida,N.,1993. Involve-mentofthestage-specific82-kilodaltonadhesionmoleculeofTrypanosomacruzi metacyclictrypomastigotesinhostcellinvasion.Infect.Immun.61,3636–3641. Rodríguez,A.,Martinez,I.,Chung,A.,Berlot,C.H.,Andrews,N.W.,1999.cAMP reg-ulatesCa2+_-dependent_exocytosis_of_lysosomes_and_{lysosome-mediated}_cell invasionbytrypanosomes.J.Biol.Chem.274,16754–16759.

Ruiz,R.C.,FavoretoJr.,S.,Dorta,M.L.,Oshiro,M.E.M.,Ferreira,A.T.,Manque,P.M., Yoshida,N.,1998.InfectivityofTrypanosomacruzistrainsisassociatedwith differentialexpressionofsurfaceglycoproteinswithdifferentialCa2+_signaling activity.Biochem.J.330,505–511.

Staquicini,D.I.,Martins,R.M.M.,Macedo,S.,Sasso,G.R.S.,Atayde,V.D.,Juliano,M.A., Yoshida,N.,2010.Roleofgp82intheselectivebindingtogastricmucinduring infectionwithTrypanosomacruzi.PLoSNegl.Trop.Dis.4(3),613.

Tardieux,I.,Nathanson,N.H.,Andrews,N.W.,1994.Roleinhostcellinvasionof Trypanosomacruzi-inducedcytosolicfreeCa2+transients.J.Exp.Med.179, 1017–1022.

Teixeira,M.M.G.,Yoshida,N.,1986.Stage-specificsurfaceantigensofmetacyclic trypomastigotesofTrypanosomacruziidentifiedbymonoclonalantibodies.Mol. Biochem.Parasitol.18,271–282.

Yoshida,N.,1983.SurfaceantigensofmetacyclictrypomastigotesofTrypanosoma cruzi.Infect.Immun.40,836–839.

Yoshida,N.,Mortara,R.A.,Araguth,M.F.,Gonzalez,J.C.,Russo,M.,1989.Metacyclic neutralizingeffectofmonoclonalantibody10D8directedtothe35and 50-kilodaltonsurfaceglycoconjugatesofTrypanosomacruzi.Infect.Immun.57, 1663–1667.

Yoshida,N.,2006.MolecularbasisofmammaliancellinvasionofTrypanosomacruzi. An.Acad.Bras.Ciênc.78,87–111.