Trypanosoma cruzi: analysis of two different strains after piplartine treatment

brazjinfectdis2018;22(3):208–218

w w w . e l s e v ie r . c o m / l o c a t e / b j i d

The

Brazilian

Journal

of

INFECTIOUS

DISEASES

Original

article

Trypanosoma

cruzi:

analysis

of

two

different

strains

after

piplartine

treatment

Gabriela

Alves

Licursi

Vieira

_,

_Marco

_Túlio

_Alves

_da

_Silva

_,

_Luis

_Octávio

_Regasini

_,

Fernando

Cotinguiba

_,

_Helen

_Julie

_Laure

_,

_José

_César

_Rosa

_,

_Maysa

_Furlan

_,

Regina

Maria

Barretto

Cicarelli

a_{UniversidadeEstadualPaulista–UNESP,InstitutodeQuímica,Araraquara,SP,Brazil}

b_{UniversidadeEstadualPaulista–UNESP,InstitutodeBiociências,LetraseCiênciasExatas,SãoJosédoRioPreto,SP,Brazil} c_{UniversidadeEstadualPaulista–UNESP,FaculdadedeCiênciasFarmacêuticas,Araraquara,SP,Brazil}

d_{UniversidadedeSãoPaulo,FaculdadedeMedicinadeRibeirãoPreto,CentrodeQuímicadeProteínas,RibeirãoPreto,SP,Brazil} e_{UniversidadedeSãoPaulo,InstitutodeFísicadeSãoCarlos,SãoCarlos,SP,Brazil}

f_{InstitutodePesquisasdeProdutosNaturais(IPPN),CentrodeCiênciasdaSaúde,UniversidadeFederaldoRiodeJaneiro(UFRJ),Riode} Janeiro,RJ,Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received23October2017 Accepted18February2018 Availableonline5June2018

Keywords: Trypanosomatids Proteome Piplartine Therapeutictargets

a

b

s

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Thehemoflagellateprotozoan,Trypanosomacruzi,mainlytransmittedbytriatomineinsects throughbloodtransfusionorfrommother-to-child,causesChagas’disease.Thisisa seri-ousparasiticdiseasethatoccursinLatinAmerica,withconsiderablesocialandeconomic impact.Nifurtimoxandbenznidazole,drugsindicatedfortreatinginfectedpersons,are effectiveintheacutephase,butpoorlyeffectiveduringthechronicphase.Therefore,it isextremelyurgenttofindinnovativechemotherapeuticagentsand/oreffectivevaccines. Sincepiplartinehasseveralbiologicalactivities,includingtrypanocidalactivity,thepresent studyaimedtoevaluateitontwoT.cruzistrainsproteome.Considerablechangesinthe expressionofsomeimportantenzymesinvolvedinparasiteprotectionagainstoxidative stress,suchastryparedoxinperoxidase(TXNPx)andmethioninesulfoxidereductase(MSR) wasobservedinbothstrains.Thesefindingssuggestthatblockingtheexpressionofthetwo enzymescouldbepotentialtargetsfortherapeuticstudies.

©2018SociedadeBrasileiradeInfectologia.PublishedbyElsevierEditoraLtda.Thisisan openaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/ by-nc-nd/4.0/).

Introduction

Chagas’ disease, caused by the parasite Trypanosoma cruzi,

isanendemicillnessinLatinAmerica,mainlyprevalentin

∗ _{Correspondingauthors.}

E-mailaddresses:silvamta@gmail.com(M.T.Silva),cicarell@fcfar.unesp.br(R.M.Cicarelli).

1 _{GALVandMTAScontributedequallytothismanuscript.}

Brazil,Argentina,Chile,Colombia, andVenezuela.The cur-renttreatmentforChagas’diseaseisbasedonbenznidazole andnifurtimox,whichareeffectiveagainstacuteinfections, butpoorlyeffectiveduringthechronicphase.InBrazil, nifur-timoxhaditsproductionandusediscontinued.Inthiscontext,

https://doi.org/10.1016/j.bjid.2018.02.009

1413-8670/©2018SociedadeBrasileiradeInfectologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

brazj infect dis.2018;22(3):208–218

209

Fig.1–Chemicalstructureofpiplartine.

majoreffortsarenecessarytoidentifynewtargetsanddevelop noveltrypanocidaldrugs.1–5

Phytochemical studies of Piper species have led to the isolation of typical classes of secondary metabolites such as amides, terpenes, benzoic acid derivatives, and hydro-quinones,lignans, neolignans, and flavonoids.6–9 _Piplartine

(Fig.1)isanamideisolatedfromPiperspecies.Anexcellent review described its biological activities,10 _{such as}

cyto-toxic, genotoxic, antitumor, antiangiogenic, antimetastatic, antiplateletaggregation,antinociceptive, anxiolytic, antide-pressant, anti-atherosclerotic, antidiabetic, antibacterial, antifungal, leishmanicidal, trypanocidal, schistosomicidal, and larvicidal activities.11–19 _{Administration of piplartine}

inhibitedsolidtumordevelopmentinmicetransplantedwith Sarcoma 180 cells and histopathological analysis showed thatliverandkidneys oftreatedanimalswereonlyslightly and reversibly affected.20 _{Anticancer activity of piplartine}

was reinforced when it reduced human glioblastoma (SF-295) and colon carcinoma (HCT-8) cell viability.21 _Acute

toxicity studies performed in rats and mice treated with different piplartine doses by oral route demonstrated no obvious clinical alterations.22 _{Viability assay carried out}

against T. cruzi epimastigote forms demonstrated growth inhibition higher than that observed with benznidazole,23

and activity against Leishmania donovani was observed, withconsiderablereductioninparasiticburdenand spleen weightinvivo.24

The low systemic cytotoxicity of piplartine associated withactivity againsttrypanosomecells broughtnew ques-tionsaboutthepossibilityofitsuseasatrypanocidaldrug. Therefore,theobjectiveofthisstudywastoevaluateprotein expressionchangesinresponsetopiplartinetreatmentonthe epimastigoteformofT.cruzi.Thecapacityoftheparasiteto grow,associatedtohighlevelofcellsobtained,allowfor per-formingdifferentialproteomicandmassspectrometryassays. Theobtainedresultsareconsistentwiththosedescribed in theliteratureandmaycontributewithfuturesimilaranalysis usingotherformsofT.cruzi.

In this paper, two-dimensional gel electrophoresis (2-DE) and mass spectrometry were used to identify different expressed proteins as a result of piplar-tine effect on two T. cruzi strains, Y25 _{and Bolivia,}26

respectively.

Materials

and

methods

Piplartineisolation

IsolationofpiplartinefromPipertuberculatumleaveswas car-riedoutaccordingtoamethodologypreviouslypublished.23

Briefly,specimensofPipertuberculatumwerecultivatedfrom seedsundergreenhouseconditionsattheInstituteof Chem-istry, UniversidadeEstadual Paulista,Araraquara,SãoPaulo State, Brazil.Plantmaterialwas collected inMay 2005and identified by Dr.Guillermo E. D. Paredes from Universidad PedroRuizGallo,Kambayeque,Peru.Voucher(Cordeiro-1936) wasdepositedattheherbariumoftheInstituteofBiosciences, UniversidadedeSãoPaulo,SãoPaulo,SãoPauloState,Brazil. Shade-driedandpowderedleavesofP.tuberculatum(200g) were extracted with ethyl acetate (Synth) and methanol (Synth)(9:1),forthreeweeksatroomtemperature.After fil-tering,thesolventwasevaporatedunderreducedpressureto yieldcrudeextract(11g).Crudeextract(10g)wassubjected tochromatographycolumnoversilicagel 60(70–230mesh, Merck) and eluted with hexane (Synth) and ethyl acetate (Synth)(0–100%ethylacetate),andethylacetate(Synth)and methanol (Synth) (0–100% methanol)to afford 12 fractions (E1–E12). Fractions E6 and E7 (2.17g) was further purified bychromatographycolumnoversilicagel60(70–230mesh, Merck)andelutedwithhexane(Synth)andacetone(Synth) (9:1)toprovide21fractions(F1–F21),includingpiplartine (frac-tionF11,532mg).Structureofpiplartinewasidentifiedby1_H

and13_{CNMRspectraanalysis.}

Trypanosomacruzicellviabilityassay

Epimastigoteforms(YandBoliviastrains)weremaintainedin LiverInfusionTryptose(LIT)medium(68.4mMNaCl;5.4mM KCl;56.3mMNa2HPO4;111mMDextrose;0.3%LiverInfusion

Broth;0.5%Tryptose)27 _{andthecellswereharvestedduring}

theexponentialgrowthphase.Theparasites(1×107_cells/mL)

weretreatedfor48hinLITat28◦Cwith13.7␮M(Ystrain)and 10.5␮M(Boliviastrain)piplartine,whichcorrespondstohalfof IC50/72hpreviouslydeterminedforthiscompound.Triplicates

oftreatedandcontrol(withoutpiplartine)parasiteswere pre-pared.CalculatedIC50in72hofexperimentforpiplartineinY

andBoliviastrainswere27.45␮Mand20.95␮M,respectively. ForBalb/cmacrophagesIC50was305.79␮M.Safetyindexwas

11.14and14.59,incomparisonwithpiplartineIC50forYand

Boliviastrains,respectively(datanotpublished).Thechosen concentrationofthepiplartinefor2DE-DIGEtestswashalfthe IC50previouslydetermined,sincetheintentionwastoinduce

proteinexpressionchanges,maintainedconsiderablenumber ofviableparasites.

Samplepreparation,labelingandtwo-dimensional electrophoresis

Treatedandcontrolepimastigotesparasitesfrombothstrains were centrifuged at 3000×g for 5min at 4◦C, the pellet was washed three times with tryps wash buffer (100mM NaCl,3mMMgCl2,20mMTris–Hcl,pH7.5)andincubatedin

210

Table1–SampleschematiclabelingwithCyDyeDIGEkit.

Labelingsamples

GEL1 Pool(50␮gofproteins+1␮LofCy2) Control1(50␮gofproteins+1␮LofCy3) Treated1(50␮gofproteins+1␮LofCy5) GEL2 Pool(50␮gofproteins+1␮LofCy2) Control2(50␮gofproteins+1␮LofCy5) Treated2(50␮gofproteins+1␮LofCy3) GEL3 Pool(50␮gofproteins+1␮LofCy2) Control3(50␮gofproteins+1␮LofCy3) Treated3(50␮gofproteins+1␮LofCy5) GEL4 Preparative(geltocutthespots)

(500␮gofproteinsofallsamples appliedintheexperiment-control andtreatedtriplicates)

lysisbuffer(7MUrea,2MThiourea,4%CHAPS,40mMTris, 0.1mg/mLPMSF;1MDTT,1mg/mLPepstatin,10mg/mL Apro-tinin and 10mg/mLLeupeptin; all enzyme inhibitors from Sigma-Aldrich) for two hours. Protein extraction was per-formedusingthreeindependentbiologicalreplicatesofeach sample(treatedand control).To obtain thesoluble protein fraction,thecelllysatewascentrifugedat14,000×gfor10min at4◦Candtheproteincontentwasdeterminedusingthe Brad-fordmethod.

Eachfraction(50␮g ofproteinofcontroland of treated triplicates)wasmarkedwithDIGEKit(GEHealthcare) accord-ingTable1.Briefly:DIGEusesdirectlabelingofproteinswith fluorescentdyes(knownasCyDyes:Cy2,Cy3,andCy5)prior toIEF(isoelectricfocusing).Dyeconcentrationsarekeptlow, suchthatapproximatelyonedyemoleculeisaddedper pro-tein.TheimportantaspectoftheDIGEtechnologyisitsability tolabeltwoormoresampleswithdifferentdyesand sepa-ratethemonthesamegel,eliminatinggel-to-gelvariability. Thismakesspot matching andquantitation muchsimpler andmoreaccurate.Cy2isusedforanormalizationpool cre-atedfrom amixtureofallsamplesintheexperiment.This Cy2-labeledpool(50␮gofproteinofall samplesappliedin theexperiment-controlandtreatedtriplicates)isrunonall gels, allowing spot matching and normalization of signals fromdifferentgels.28–32_{Thelabelproteinswerethen}

solubi-lizedintheDeStreakfollowingthemanufacturerinstructions (GEHealthcare).Eachstripwasrehydratedwiththesample solutionfor20hatroomtemperatureandtheproteinswere separatedoverimmobilizedlinearpH3–10gradients(IPGphor system,GEHeathcare)usingthefollowingprotocol:200V/12h, 1000V/1000Vh,8000V/8000Vh,8000V/20,000Vh.ThepHwide rangewasappliedtoobtainthelargestnumberofprotein dif-ferentialexpression.Thestripswereincubatedfor15minin 5mLofequilibratebuffer(6Muréia,50mMTris–Hcl1.5M,pH 8.8,30%glicerol,2%SDS,1%bromophenolblue)containing 50mgDTT,followedbyasecondincubationfor15minin5mL ofthesamebuffercontaining125mgiodoacetamide.

Second dimension separation was performed by 12.5% SDS-PAGE polyacrylamide gels. Gels were run in a SE 600 Ruby (GE Healthcare) at10mA/gel for45min and then at 30mA/gel until the end ofthe gel wasreached and 100W and 600V. Molecular massmarkers were prepared accord-ingtotheinstructionsofmanufacturer(GEHealthcare)and appliedtofilterpaperbeforepositioningthestrips.Thegels werescannedinTyphoonVariableMode(GEHealthcare)and detectedspotswerequantifiedandanalyzedwiththeImage Master Platinum software (GE Healthcare).The differential expressionanalysiswasperformedcomparingthetreatedand controlgroupsbytheStudent’sttest(>1.3foldandp≤0.05).

Thecut-offlimitemployedwasbasedonstatistically signifi-cantdifferences(p≤0.05).

Preparativegels(containing500␮gofproteinofcontroland treatedtriplicates)werestainedwithCoomassiecolloidalas follows:fixationin45%v/vethanoland10%v/vaceticacidfor 15min,washedindeionizedwaterandtheadditionof0.1% w/vCoomassiebrilliantblueG(Sigma-Aldrich)overnight.The gelsweredestainedwith40%v/vethanoland10%v/vacetic acid until visualization of spots.Ten spots were manually excised from preparative gel, being five spots, which pro-teinweremoreexpressedthancontrol,andfivespotswhich treatedpresentedmoreexpression.

Gelinsitutrypsindigestionandmassspectrometry analysis

Thegelspotscorresponding totagged-proteinwereexcised anddestainedwith50%acetonitrile/0.1Mammonium bicar-bonate pH8.0.Gelswere dehydratedwithneatacetonitrile andvacuumdriedfor30min.Gelswereswollenwith0.5␮g of modify trypsin (Promega) in 20␮L of 0.1M ammonium bicarbonate pH 8.0, and the gels were covered by adding 100␮L of 0.1M ammonium bicarbonate pH 8.0 for 22h at 37◦C. Trypsin hydrolysis was stopped with 5␮L of formic acid. The tryptic peptides were extracted and desalted in microtipfilledwithPOROS50R2(PerSeptiveBiosystems) pre-viouslyequilibratedin0.2%formicacidandelutedwith60% methanol/5% formic acid for mass spectrometry analysis. SamplesweredilutedinmatrixCHCA5mg/mL (alfa-cyano-4-hydroxicinnamic acid, Sigma) with 50% acetonitrile/0.1% TFA(trifluoroaceticacid)and2–3␮LweredepositedonMALDI plates using drop driedmethod. Massspectrometry analy-siswasperformedinMALDI-TOF/TOF(AXIMAperformance, Kratos-Shimadzu)andtheMS/MSspectraoftrypticpeptides were submitted directly to MASCOT program against the NCBInrandlocaltrypanosomatidsdatabases.

Results

SeveralT.cruziproteomicstudieshavebeenperformedto gen-erate2DEproteinmapsofdifferentlifecyclestages,tostudy development-inducedchanges,identifyproteinsinvolvedin drugresistanceandeffectsoftrypanocidalagents.33–36

Fig.2AandB,fromYandBoliviastrains,respectively,show the 10 protein spots differently overexpressed inthe cells, beingfivespotsincontrol(Tables2and3,column“control”, spots1–5),andfivespotsintreatedcondition(Tables2and3, column“treated”,spots6–10).

brazj infect dis.2018;22(3):208–218

211

A

B

Fig.2–Preparative2-DEgelimageofT.cruzi(A.Ystrain;B.Boliviastrain)treatedwithpiplartinefromP.tuberculatumand control.IEFwasperformedwith500␮gofsolubleproteinfromtheparasitesusing13cmstripswithapHrangeof3–10. SDS-PAGEwasperformedwith12.5%polyacrylamidegels,whichwerestainedwithColloidalCoomassieBlue.Arrows indicatemodulatedproteinsofcontrolsandaftertreatmentwiththecompound;numbersrefertospotidentificationin

Tables1and2,respectively.

The differential approach using T. cruzi cells demon-stratedtryparedoxinperoxidase,involvedinoxidativestress response,hadimportantdecreasedexpressionintreated(that is,moreexpressedincontrol),asshowedinTables2and3.It waspossibletoobservetwospotsdifferentialexpressedbyY strain,withreductionof2.194and2.349folds(Table2,spots2 and3);andtwospotsbyBoliviastrain,withreductionof1.310 and1.982folds(Table3,spots1and4).

Methylthioadenosinephosphorylase(MTAP,Table2,spot 6 and Table 3, spot 2) catalyzes the reversible phospho-rolytic cleavage of methylthioadenosine, a by-product of the polyamine biosynthesis, producing methylthioribose-1-phosphate(MTRP)andadenine.37_{Interesting,intheBolivia}

strain, MTAP was identified in spots 2and 9, one overex-pressed(spot9)andtheother(spot2)representingreduction oftheexpression.InFig.2B,itispossibletoobservethatthese spotspresentedsmallshiftingelandshouldrepresenta post-translationmodification,suggestingthatthismechanismcan regulatetheexpressionofthismember.

Somestudiesdemonstratedtheinteractionbetween try-paredoxin and cystathionine ␥-lyase (CGL),38 _{although the}

enzyme found in our experiment was cystathionine ␤-synthase(CBS)(Tables2and3,spots5).Previousstudiesin mammalcellsdemonstratedthatCBSandCGLactinthesame pathwayand areprimarilyresponsibleforhydrogensulfide biogenesis.39

EnzymeD-isomer specific2-hydroxyaciddehydrogenase, whoseexpressionlevelswasalteredonlyintheBoliviastrain (Table 3, spot 3), wasone ofthe proteins identified inthe present work This enzyme is responsible to catalyze the NAD-dependentoxidationofR-lactatetopyruvateandispart ofthe detoxification pathwayof methylglyoxal (dependent ofglyoxalase I, glyoxalase II and low molecular mass thi-ols)intrypanosomatids,anddatafromTrypanosomaconorhini

andCrithidiafasciculatashowedactivationinthepresenceof cysteine.40_{Altogether,thesedatastronglysuggestaredox}

reg-ulationofthisenzymeintrypanosomatids.

Other protein overexpressed only in the Bolivia strain treatedwithpiplartinewasmethionine sulfoxidereductase (MSR,Table3,spot10).Severalaminoacidresidueswere sus-ceptibletooxidationsuchascysteine,histidine,tryptophan, tyrosineandmethionine.

Proteomics studies usually identified proteins highly expressedsuchaschaperones,structuralandribosomal pro-teins and others. In the present work, it was possible to observe heat shock protein 70 and tubulin differentially expressed(Tables2and3).

Discussion

Variousarticlesexploredtheactivityofdifferentcompounds againstYandBoliviaTrypanosomacruzistrains,inspecial com-paringbenzinidazoleactivity.Alvesandcollaboratorsfound higherIC50ofbenzinidazoleforY(11.77␮g/mL)comparedto Bolivia(0.99␮g/mL)strain.Thenaturalextractstestedshowed moderate activity againstBolivia strains, whileinY strain therewassatisfactoryactivity.41_{Inotherstudies,Boliviastrain}

provedtobemoreresistanttobeniznidazolethanYstrain. Kohatsu and collaborators observed an IC50 almost three times higherin Bolivia than inYstrain and demonstrated thatthiscompoundincreasedtheexpressionofthe mitochon-drialtryparedoxinperoxidaseonlyinYstrain.42_{Additionally,}

Boliviastrainwasmoreresistant totreatmentoftwo nitro-furansderivatives(NFandNFOH-121)whencomparedtoY strain. Also,no changes at mRNAprocessing levels in the Boliviastrainwasobserved.43_{MiceinfectedwithYandBolivia}

212

Table2–T.cruzi(Ystrain)proteinidentificationincontrolandtreatedparasiteswithpiplartine.

Spot X-folddifferentialexpression NCBI MASCOT

Control Treated Protein Accessnumber Peptides Mass(Da) Score pI Aminoacid

sequencecover(%) 1 +2.111 CytochromeC oxidasesubunitIV gi|71660419 FTGEPPSWMR ACTDLNQLEEWSR 39,119 123 5.72 6.90 2 +2.194 Tryparedoxin peroxidase gi|71408703 ANEYFEK EAAPEWAGK VVQAFQYVDK HITVNDLPVGR DYGVLIEEQGISLR 25,734 338 5.96 22.60 3 +2.349 Tryparedoxin peroxidase gi|17224953 QITVNDLPVGR SYGVLKEEDGVAYR HGEVCPANWKPGDK 22,660 183 5.96 19.60 4 +2.483 Beta-tubulin gi|1220547 LREEYPDR LAVNLVPFPR FPGQLNSDLR KLAVNLVPFPR FPGQLNSDLRK INVYFDEATGGR AVLIDLEPGTMDSVR AVLIDLEPGTMDSVR GHYTEGAELIDSVLDVR 50,126 513 4.74 16.70 5 +2.853 Cystathionine beta-synthase gi|71404306 GYVLLDQYR SADKESFALASEVHR AHYEGTAQEIYDQCGK 47,830 308 6.43 9.60

b r a z j i n f e c t d i s . 2 0 1 8; 2 2(3) :208–218

213

Spot X-folddifferentialexpression NCBI MASCOT

Control Treated Protein Accessnumber Peptides Mass(Da) Score pI Aminoacid

sequencecover(%) 6 +1.890 Methylthioadenosine phosphorylase gi|71655964 VGGVDCAFLPR TFPGLAAGTEGWR HGVGHVLNPSEVNYR LHEQGTSVTMEGPQFSTK LHEQGTSVTMEGPQFSTK YYAIETPFGRPSGDICVK 33,523 456 6.34 24.80 7 +1.951 ProstaglandinF2␣ synthase gi|25006239 YDFEEADQQIR ETYGVPEELTDDEVR AQQNWPLNEPRPETYR 42489 235 5.83 11.60 8 +1.962 ProstaglandinF2␣ synthase gi|25006239 FIANPDLVER YDFEEADQQIR KIEPLSLAYLHYLR ETYGVPEELTDDEVR AQQNWPLNEPRPETYR 42,489 391 5.83 17.90

9 +1.982 Heatshock70kDa

protein, mitochondrial precursor gi|71407515 DAGTIAGLNVIR EISEVVLVGGMTR EISEVVLVGGMTR RFEDSNIQHDIK KVSNAVVTCPAYFNDAQR 71,503 242 5.75 8.40

10 +2.024 70kDaheatshock

protein

gi|119394467 FEELCGDLFR AVHDVVLVGGSTR TTPSYVAFTDTER

214

Table3–T.cruzi(Boliviastrain)proteinidentificationincontrolandtreatedparasiteswithpiplartine.

Spot X-folddifferentialexpression NCBI MASCOT

Control Treated Protein Accessnumber Peptides Mass(Da) Score pI Aminoacidsequence

cover(%) 1 +1.310 Tryparedoxin peroxidase gi|17224953 QITVNDLPVGR SYGVLKEEDGVAYR HGEVCPANWKPGDK 22,660 219 5.96 19.60 2 +1.796 Methylthioadenosine phosphorylase gi|71655964 VGGVDCAFLPR TFPGLAAGTEGWR HGVGHVLNPSEVNYR YYAIETPFGRPSGDICVAK 33,523 398 6.34 18.90 3 +1.928 D-isomerspecific 2-hydroxyacid dehydrogenase-protein gi|71420052 EVDEYGVR TANLVPDSVR FIGLVNEYAK TANLVPDSVRR RLDTYLVDPVR GITQNEDDLACALR ESDFVVNILPGTEETKR 38,795 479 6.04 20.90 4 +1.982 Tryparedoxin peroxidase gi|17224953 QITVNDLPVGR SYGVLKEEDGVAYR HGEVCPANWKPGDK 22,660 221 5.96 19.60 5 +2.144 Cystathionine beta-synthase gi|71404306 GYVLLDQYR SADKESFALASEVHR AHYEGTAQEIYDQCGGK TETALPWDHPDSLIGMAR 47,830 421 6.43 13.80 6 +1.529 ChaperoninHSP60, mitochondrial protein gi|3023478 NVIIEQSYGAPK GLIDGETSDYNR GYISPYFVTDAK AVGVILQSVAEQSR YVNMFEAGIIDPAR AAVQEGIVPGGGVALLR ALDSLLGDSSLTADQR VLENNDVTVGYDAQR KVTSTENIVQVATISANGDEELGR 59,639 801 5.38 24.20

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Spot X-folddifferentialexpression NCBI MASCOT

Control Treated Protein Accessnumber Peptides Mass(Da) Score pI Aminoacidsequence

cover(%)

7 +1.544 Heatshock70kDa

protein, mitochondrial precursor gi|71407515 EISEVVLVGGMTR RFEDSNIQHDIK KVSNAVVTCPAYFNDAQR 71,503 184 5.75 6.60

8 +1.683 Heatshockprotein

70 gi|10626 TTPSYVAFTDTER ARFEELCGDLFR AVVTVPAYFNDSQR DCHLLGTFDLSGIPPAPR SQIFSTYADNQPGVHIQVFEGER 74,564 437 5.64 11.80 9 +1.864 Methylthioadenosine phosphorylase gi|71655964 VGGVDCAFLPR TFPGLAAGTEGWR HGVGHVLNPSEVNYR LHEQGTSVTMEGPQFSTK YYAIETPFGRPSGDICVAK 33,523 516 6.34 24.80 10 +2.570 Peptidemethionine sulfoxidereductase gi|71405176 IHDPTTVNR QGVDVGTQYR AFGDAQVVTSLEK SAIFYHDDQQLK LHVAEDYHQMYLEK LHVAEDYHQMYLEK 20,204 386 6.12 32.80

216

differences inmortalityrates. All groupsinfected withthe Ystrain(treatedandnon-treated)presented100% survival. However,only60%survivalwasobservedinthegroupinfected with the Bolivia strain, while non-treated mice had 100% survival.Thesefindingsindicatedthattreatmentwith ben-zinidazolecouldbedetrimentaltoanimalsinfectedwiththe Boliviastrain.44_{Altogether,theseresultsreinforcethatwide}

geneticheterogeneityofT. cruzistrains affectthe response againsttrypanocidalcompounds.Ourfindingsshowedsimilar proteomevariationsinbothstrains,alteringtheexpressionof proteinsinvolvedinoxidativestressresponse.Inotherhands, twoproteinspresentedproteinexpressionvariationsonlyin theBoliviastrain,D-isomerspecific2-hydroxyacid dehydro-genaseandmethioninesulfoxidereductase.

Rajandco-workers22_{identified12potentialpiplartine}

tar-getsusing differentialproteomicsapproachon twohuman celllines,bladdercarcinomaandosteosarcoma.Interestingly, theseauthors identifiedvarious proteinsinvolvedinstress oxidative response such as glutathione S-transferases and peroxiredoxin.Inaddition, itwasfoundthatpiplartinecan interact directly with purified recombinant glutathione S-transferasep1 (GSTp1), inhibiting the enzyme activity and decreasingthelevels ofreduced glutathione.Theseactions ledtoincreaseofoxidizedglutathionelevelsintumorcells. Thesedatasuggestthatpiplartinecaninteractdirectlywith componentsofthe oxidativestress responsein trypanoso-matidscells.Tryparedoxinperoxidasepresentscrucialrolein theregulationoftheparasiteROSlevelsanditisimportant tohighlighttheimportanceofthisenzymeasChagas’disease drugtarget.45

Mitochondrial and cytosolic tryparedoxin peroxidase detoxifyperoxynitrite,hydrogenperoxideandsmall hydroper-oxideorganiccompounds.Ascorbateheme-dependent perox-idaseoccursintheendoplasmicreticulumandpresentsaction againsthydrogenperoxide. Otherenzymes thatactagainst oxidizingagentsarethesuperoxidedismutases(SOD) occur-ringinthecytosol,mitochondriaandglicosomaandworkin thedetoxificationofsuperoxide.46

Themainresultsobtainedcould beexplainedby poten-tialinteraction amongtryparedoxin peroxidaseandseveral otherproteins,whicharedifferentiallyexpressedand iden-tified.Pi ˜neyroandco-workers38_{usingproteomicapproaches}

described potential interactions between tryparedoxin 1 (TcTXN1) and tryparedoxin peroxidase in vitro and in vivo.

Methylthioadenosinephosphorylase(MTAP)isresponsibleto catalyzethereversiblephosphorolyticcleavageof methylth-ioadenosine,apolyaminebiosynthesissubproduct,producing methylthioribose-1-phosphate(MTRP)andadenine.In mam-malcells,MTAPisredoxregulatedandcanbeinactivatedby specificoxidationoftwoconservedcysteinestosulfenicacid, anditshouldbepointedoutthattheseaminoacidswere con-servedinT.cruziprotein.35

Therefore, two hypotheses for the difference in MTAP expressionfollowingpiplartinetreatmentcanbepostulated: first,thedirectinteractionbetweentryparedoxinand trypare-doxinperoxidasewasaffected,modifyingMTAPlevels.These resultssuggestasecondaryroleofpiplartineinpolyamines metabolism.Secondly,redoxunbalancingcausedfor trypare-doxinperoxidasechange theexpressionofMTAP. Sincefor the Bolivia strain two different spots (2 and 9) were

iden-tified, one overexpressed (spot 9) and other (spot 2) with down-expression,andthesespotspresentedlittleshiftingel probably representing a post-translationmodification.This mechanismcouldregulatetheexpressionofthisenzyme.

T. cruzi appears to be the first protist proven to

have two independent pathways for cysteine production; sulfur-assimilation from sulfate and transsulfuration from methionineviacystathionine.Extracellularl-cysteine,butnot l-cystine,hasbeenshowntobeessentialforgrowthoftheT.

bruceibloodstreamform.Trypanosomesrequirecysteinenot onlyforproteinbiosynthesis,butalsoforformationof trypan-othioneandglutathione.Theymayrequiremultiplecysteine acquiringpathwaystoensurethisaminoacidisavailableto fulfilltheneedsoftheirredoxmetabolism.38_{Therefore,itis}

possibletocorrelatetryparedoxinperoxidase,cystathionine beta-synthaseandredoxbalance.Recently,thesame interac-tome approachwasusedtodemonstratethepartnersofT. cruzitryparedoxinII.40

Oxidationofmethioninetomethioninesulfoxideaffects thebiologicalfunctionofoxidizedproteins.47_{Consequently,}

theoccurrenceofasystemforreducingmethioninesulfoxide to methionine is important for cell integrity and its reac-tion ispresentinvirtuallyallorganisms.48,49_{Therefore, the}

presenceofa systemforreducing methionine sulfoxideto methionineguaranteescellintegrityandispresentin virtu-allyallorganisms.38,50_{Methioninesulfoxidereductase(MSR)}

protectstheorganismfromoxidativestressandisinvolved withresistanceagainstbioticandabioticstressinplantsand animals.38,51_{Ariasandco-authors}52_{studiedMSRproduction}

inT.cruziand T.brucei,indicatingacrucialfunctionofthis enzyme in the redox metabolismin trypanosomatids and couldhelptheparasitestofaceoxidativestress.Thisenzyme isnotdirectlyinvolvedintheremovalofoxidizingagents,but actmainlyinrepairingoxidizedproteins,maintaining cellu-larfunctionality.Theyfoundthatthereversaloftheoxidized stateofMSRrecombinantisdependentonthe presenceof reduced tryparedoxin(TXN),probablyreducing substrateof theMSR.52

Conclusion

TheoxidativestressresponseisanimportantdefenseofT. cruziagainstthehostimmunesystemandothermechanisms of parasite clearance. Therefore, activity moleculesable to interfere inthis parasiticmetabolism representapotential therapeutictarget inChagas’disease.TheDIGEtechnology eliminatinggel-to-gel variabilityimprovedthe obtentionof proteinspots.Altogether,ourresultssuggestedthatpiplartine activityintrypanosomespromotedimbalanceofsome impor-tantenzymesrelatedtoredoxmetabolismintheseparasites. Itshouldbefurtherinvestigatedwhethertheseenzymescould serveaspotentialbiomarkerstoevaluatediseasetreatment.

Conflicts

of

interest

brazj infect dis.2018;22(3):208–218

217

Acknowledgments

Financial support: FAPESP (2007/02076-3; 2007/56140-4;

2011/24017-4)CAPESandPADC(Proc.2018/02-II).

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1. SouzaW.Fromthecellbiologytothedevelopmentofnew chemotherapeuticapproachesagainsttrypanosomatids: dreamsandreality.KinetoplastidBiolDis.2002;1:1–21.

2. SoeiroMN,deCastroSL.Trypanosomacruzitargetsfornew chemotherapeuticapproaches.ExpertOpinTherTargets. 2009;13:105–21.

3. UrbinaJA.NewinsightsinChagas’diseasetreatment.Drugs Future.2010;35:409–19.

4. UrbinaJA.SpecificchemotherapyofChagasdisease: relevance,currentlimitationsandnewapproaches.Acta Trop.2010;115:55–68.

5. GraelCFF,AlbuquerqueS,LopesJLC.Chemicalconstituents ofLychnophorapohliiandtrypanocidalactivityofcrudeplant extractsandofisolatedcompounds.Fitoterapia.

2005;76:73–82.

6. LagoJHG,RamosCS,CasanovaDCC,etal.Benzoicacid derivativesfromPiperspeciesandtheirfungitoxicactivity againstCladosporiumcladosporioidesandC.sphaerospermum.J NatProd.2004;67:1783–8.

7. NavickieneHMD,AlécioAC,KatoMJ,etal.Antifungalamides fromPiperhispidumandPipertuberculatum.Phytochemistry. 2000;55:621–6.

8. NavickieneHMD,MorandimAA,AlécioAC,etal.Composition andantifungalactivityofessentialoilfromPiperaduncum, PiperarboretumandPipertuberculatum.QuímNova. 2006;29:467–70.

9. RegasiniLO,CotinguibaF,SiqueiraJR,BolzaniVS,SilvaDHS, FurlanM.RadicalscavengingcapacityofPiperarboreumand Pipertuberculatum(Piperaceae).LatAmJPharm.2008;27:900–3.

10.BezerraDP,PessoaC,deMoraesMO,Saker-NetoN,Silveira ER,Costa-LotufoLV.Overviewofthetherapeuticpotentialof piplartine(piperlongumine).EurJPharmSci.2013;48:453–63.

11.Barbosa-FilhoJM,PiuvezamMR,MouraMD,etal. Anti-inflammatoryactivityofalkaloids:atwentycentury review.RevBrasFarmacogn.2006;16:109–39.

12.NakamuraCV,SantosAO,VendramettoMC,etal.Atividade antileishmaniadoextratohidroalcoólicoedefrac¸õesobtidas defolhasdePiperregnellii(Miq.)C.DC.Var.Pallescens(C.DC.). YunckRevBrasFarmacogn.2006;16:61–6.

13.AmorimMFD,DinizMFFM,AraújoMST,etal.The controvertibleroleofkava(PipermethysticumG.Foster),an anxiolyticherb,ontoxichepatitis.RevBrasFarmacogn. 2007;17:448–54.

14.Saúde-GuimarãesDA,FariaAR.Substânciasdanaturezacom atividadeanti-Trypanosomacruzi.RevBrasFarmacogn. 2007;17:455–65.

15.deMoraesJ,NascimentoC,LopesPO,etal.Schistosoma mansoni:invitroschistosomicidalactivityofpiplartine.Exp Parasitol.2011;127:357–64.

16.Quintans-JúniorLJ,AlmeidaJRGS,LimaJT,etal.Plantswith anticonvulsantproperties–areview.RevBrasFarmacogn. 2008;18:798–819.

17.deMoraesJ,NascimentoC,YamaguchiLF,KatoMJ,NakanoE. Schistosomamansoni:invitroschistosomicidalactivityand tegumentalalterationsinducedbypiplartineon schistosomula.ExpParasitol.2012;32:222–7.

18.Araújo-VilgesKM,OliveiraSV,CoutoSCP,etal.Effectof piplartineandcinnamidesonLeishmaniaamazonensis,

PlasmodiumfalciparumandonperitonealcellsofSwissmice. PharmBiol.2017;55:1601–7.

19.CampeloYDM,MafudAC,VérasLMC,etal.Synergisticeffects ofinvitrocombinationsofpiplartine,epiisopiloturineand praziquantelagainstSchistosomamansoni.Biomed Pharmacother.2017;88:488–99.

20.BezerraDP,CastroFO,AlvesAP,etal.Invivogrowth-inhibition ofSarcoma180bypiplartineandpiperine,twoalkaloid amidesfromPiper.BrazJMedBiolRes.2006;39:801–7.

21.BezerraDP,FerreiraPMP,MachadoCML,etal.Antitumour efficacyofPipertuberculatumandpiplartinebasedonthe hollowfiberassay.PlantaMed.2015;81:15–9.

22.RajL,IdeT,GurkarAU,FoleyM,SchenoneM,LiX.Selective killingofcancercellsbyasmallmoleculetargetingthestress responsetoROS.Nature.2011;475:231–4.

23.CotinguibaF,RegasiniLO,DebonsiHM,etal.Piperamidesand theirderivativesaspotentialanti-trypanosomalagents.Med ChemRes.2009;18:703–11.

24.BodiwalaHS,SinghG,SinghR,DeyCS,SharmaSS,Bhutani KK.AntileishmanialamidesandlignansfromPipercubeba andPiperretrofractum.JNatMed.2007;61:418–21.

25.SilvaLHP,NussenzweigV.SobreumacepadeTrypanosoma cruzialtamentevirulentaparaocamundongobranco.Folia clinicaetbiológica.1953;20:191–208.

26.FunayamaGK,PradoJC.Estudodecaracteresdeumaamostra bolivianadoTrypanosomacruzi.RevSocBrasMedTrop. 1974;8:75–81.

27.FernandesJF,CastellaniO.Growthcharacteristicsand chemicalcompositionofTrypanosomacruzi.ExpParasitol. 1966;18:195–202.

28.AlbanA,DavidSO,BjorkestenL,etal.Anovelexperimental designforcomparativetwo-dimensionalgelanalysis: two-dimensionaldifferencegelelectrophoresisincorporating apooledinternalstandard.Proteomics.2003;3:36–44.

29.MarougaR,DavidS,HawkinsE.ThedevelopmentoftheDIGE system:2Dfluorescencedifferencegelanalysistechnology. AnalBioanalChem.2005;382:669–78.

30.TongeR,ShawJ,MiddletonB,etal.Validationand

developmentoffluorescencetwo-dimensionaldifferentialgel electrophoresisproteomicstechnology.Proteomics.

2001;1:377–96.

31.WestermeierR,ScheibeB.Differencegelelectrophoresis basedonlys/cystagging.MethodsMolBiol.2008;424:73–85.

32.MindenJ.Comparativeproteomicsanddifferencegel electrophoresis.Biotechniques.2007;43:739–41.

33.Parodi-TaliceA,DuránR,ArrambideN,etal.Proteome analysisofthecausativeagentofChagas’disease: Trypanosomacruzi.IntJParasitol.2004;34:881–6.

34.AndradeHM,MurtaSMF,ChapeaurougeA,PeralesJ,NirdéP, RomanhaAJ.ProteomicanalysisofTrypanosomacruzi resistancetobenznidazole.JProteomeRes.2008;7:2357–67.

35.AtwoodJA,WeatherlyDB,MinningTA,BundyB,CavolaC, OpperdoesFR.TheTrypanosomacruziproteome.Science. 2005;309:473–6.

36.Menna-BarretoRFS,BeghiniDG,FerreiraATS,PintoAV,De CastroSL,PeralesJ.Aproteomicanalysisofthemechanism ofactionofnaphthoimidazolesinTrypanosomacruzi epimastigotesinvitro.JProteomics.2010;73:2306–15.

37.BertinoJR,WaudWR,ParkerWB,LubinM.Targetingtumors thatlackmethylthioadenosinephosphorylase(MTAP) activity.CancerBiolTher.2011;11:627–32.

38.Pi ˜neyroMD,Parodi-TaliceA,PortelaM,AriasDG,GuerreroSA, RobelloC.Molecularcharacterizationandinteractome analysisofTrypanosomacruziTryparedoxin1.JProteomics. 2011;74:1683–92.

39.SinghS,BanerjeeR.PLP-dependentH2Sbiogenesis.Biochim BiophysActa.2011;1814:1518–27.

40.AriasDG,Pi ˜neyroMD,IglesiasAA,GuerreroSA,RobelloC. Molecularcharacterizationandinteractomeanalysisof

218

TrypanosomacruzitryparedoxinII.JProteomics. 2015;120:95–104.

41.AlvesRT,RegasiniLO,FunariCS,etal.Trypanocidalactivityof BrazilianplantsagainstepimastigoteformsfromYand BoliviastrainsofTrypanosomacruzi.RevbrasFarmacogn. 2010;22:528–33.

42.KohatsuAAN,SilvaFAJ,FranciscoAI,etal.Differential expressiononmitochondrialtryparedoxinperoxidase (mTcTXNPx)inTrypanosomacruziafterferrocenyldiamine hydrochloridestreatments.BrazJInfectDis.2017;21:125–32.

43.BarbosaCF,OkudaES,ChungMC,FerreiraEI,CicarelliRMB. Rapidtestfortheevaluationoftheactivityoftheprodrug hydroxymethylnitrofurazoneintheprocessingof TrypanosomacruzimessengerRNAs.BrazJMedBiolRes. 2007;40:33–9.

44.SantosCD,LoriaRM,OliveiraLG,etal.Effectsof

dehydroepiandrosterone-sulfate(DHEA-S)andbenznidazole treatmentsduringacuteinfectionoftwodifferent

Trypanosomacruzistrains.Immunobiology.2010;215:980–6.

45.FinziJK,ChiavegattoCW,CoratKF,etal.Trypanosomacruzi responsetotheoxidativestressgeneratedbyhydrogen peroxide.MolBiochemParasitol.2004;133:

37–43.

46.PiacenzaL,PeluffoG,AlvarezMN,MartínezA,RadiR. Trypanosomacruziantioxidantenzymesasvirulencefactorsin Chagasdisease.AntioxidRedoxSignal.2013;19:723–34.

47.FriguetB.Oxidizedproteindegradationandrepairinageing andoxidativestress.FEBSLett.2006;580:2910–6.

48.MoskovitzJ.Methioninesulfoxidereductases:ubiquitous enzymesinvolvedinantioxidantdefense,proteinregulation, andpreventionofaging-associateddiseases.Biochim BiophysActa.2005;1703:213–9.

49.Boschi-MullerS,GandA,BranlantG.Themethionine sulfoxidereductases:catalysisandsubstratespecificities. ArchBiochemBiophys.2008;474:266–73.

50.MoskovitzJ,FlescherE,BerlettBS,AzareJ,PostonJM, StadtmanER.Overexpressionofpeptide-methionine sulfoxidereductaseinSaccharomycescerevisiaeandhumanT cellsprovidesthemwithhighresistancetooxidativestress. ProcNatlAcadSciUSA.1998;95:14071–5.

51.BechtoldU,MurphyDJ,MullineauxPM.Arabidopsispeptide methioninesulfoxidereductasepreventscellularoxidative damageinlongnights.PlantCell.2004;16:908–19.

52.AriasDG,CabezaMS,ErbenED,etal.Functional

characterizationofmethioninesulfoxidereductaseAfrom Trypanosomaspp.FreeRadicBiolMed.2011;50:37–46.