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Molecular & Biochemical Parasitology xxx (2007) xxx-xxx

Proteome analysis of Leishmania (Viannia) braziliensis by two-dimensionalgel electrophoresis and mass spectrometry

u Patricia Cuervou,b,t, Jose Batista de Jesusd, Magno Junqueirae, Leila Mendonga-Limad,Luis Javier Gonzdlezr,Lilzaro Betancourtl', Gabriel Grimaldi Jr. a,

Gilberto Barbosa Domonte, Octavio Fernandesb, Elisa Cupolilloa'*n Departamento de Imunologia, Instituto Oswaldo Cruz, FIOCRUZ, Av. Brasil4365, Manguinhos,21045-900 Rio de Janeiro, RJ, Brazil

b Departamento Medicina Tropical, Instituto Oswaldo Cruz, FIOCRUZ, Av. Brasil 4365, Manguinhos, 21045'900 Rio de Janeiro, RJ, Brazilc Centro Internacional de Entrenamiento e Investigaciones Mddicas, CIDEIM, Cali, Colombiu

d Departarnento de Bioquimica e Biologia Molecular, Instituto Oswaldo Cruz, FIOCRIIZ, Av. Brasil4365, Manguinhos, 21045-900 Rio de Janeiro, RJ, Brazile Departamento de Bioquimica, Instituto de Quimica, UFRJ/Rede Protedmica de Rio de Janeiro, Rio de Janeiro, Brazil

t Departamento de Prote6mica, Centro de Ingenieria GenCtica y Biotecnologia, Ciudad de la Habana, Cuba

Received 2l December 2006; received in revised form !2 March 2007; accepted2l March 2007

Abstract

Leishmania (Viannia) braziliensir, a protozoan parasite widespread in the New World, is responsible for the infection of different mammal orders,

including humans. This species is considered to be a major etiological agent of American cutaneous leishmaniasis. A proteomic study was carried

out to identify proteins expressed by L. (V.) braziliensis. One hundred and one spots representing 75 protein entries were identified by MALDI-

TOF-TOF. Isoelectric point values estimated by gel electrophoresis matched closely with predicted values, although some discrepancies existed

suggesting that post-translational protein modifications may be commonin L. braziliensis. Moreover, 20 hypothetical proteins were experimentally

identified. Identified proteins were classified into 15 groups according to biological process. Among the proteins identified, approximately 407o

have not been previously reported in a proteomic map of Leishmania. ln addition, a number of potential virulence factors and drug targets were

identified in this protein map, including some proteins associated with the metastatic phenotype. This study describcs the first compilation of a

proteomic reference map for L. braziliensis (pI 4-7, M, 10-130kDa) and provides a very useful tool for comparative studies of strains isolated

from patients presenting different clinical manifestations of leishmaniasis as well as a potential tool to identify markers for clinical diagnosis,

therapeutics, and prognosis.A 2OO7 Published by Elsevier B.V.

Keywords: Leishmania (V) brazitiensis; Leishmaniasis; Proteome; Mass spectrometryi Leishmania braziliensis 2D ret'erence map

ffiELSEVIER

M O L E C U L A R& B I O C H E M I C A LPARAS ITO LOGY

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1.. Introduction

Leishmania (Viannia) braziliensis is a parasite autochthonousto the New World and responsible for most of the cutaneousleishmaniasis cases in at least l5 countries of the American Con-tinent [ 1 l. The disease encompasses a broad spectrum of clinicalmanifestations ranging from simple cutaneous lesions, which

Abbreviations: GO, gene ontology; HSP70, heat shock protein 70;

EF-lp, elongation factor 1-beta; GPI-, glycosyl phosphatidylinositol-; IgE,

immunoglobulin E; PCNA, proliferative cell nuclear antigen; CP, cysteine

proteinase; TryP, tryparedoxin peroxidase; TSA, thiol-specific-antioxidant* Corresponding author. Tel.: +55 2 I 3865 8 177; fax: +55 21 2290 047 9.

E-mail address : ecupoli C@ioc.fi ocruz.br (8. Cupolillo)'

0166-6851/$ - see front matter @ 2007 Published by Elsevier B.V.

doi: I 0. | 0 I 6/j.molbiopara.2007.03.0 | 3

may heal spontaneously [I,2], to mucosal lesions, a hyperergicinvasive and destructive form ofleishmaniasis [3 l. L. braziliensisexhibits wide genetic polymorphisms at the isoenzymatic level

12,4-11, in surface epitope expression [ti-l I I and protease activ-ity [121. However, the role of this parasite in the development ofdistinct clinical forms is still obscure. It has been suggested thatthe disease phenotype may be related to both the host immuneresponse and parasite gene expression. Currently, there are nounambiguous genotypic or phenotypic markers of the parasiteassociated with specific clinical form of the disease.

Since the regulation of gene expression in kinetoplastidsdiffers from higher eukaryotic species and control at the post-transcriptional level is the major mechanism of gene regulationin these protozoa [131, proteomic approaches should be useful

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P. Cueruo et al. / Molecular & Biochemical Parasitology xxx (2007) xn-xxx

zr in the identification of potential clinical-associated phenotypic22 markers of Leishmanio. Previous studies on Leishmania sp.zo proteome profiling include the generation of partial proteomic2a maps of Old- and New-World Leishmania species I I 4- 1 6] andzs analysis of developmentally regulated proteins in these para-26 sites 111-211. Of particular importance is the demonstrationzz that proteomic tools are valuable for the study of potential drugm targets, drug resistance mechanisms and the identification of2e putative virulence factors in Leishmania parasites L16,22-23J.so The above studies also revealed significant inter-species dif-sr ferences in protein expression, indicating the need for detailed32 expression analysis ofindividual Leishmania species.ss Karyotype analysis of L. braziliensis has shown that this par-34 asite has 35 chromosome pairs, with chromosomes 20 and 34ss fused, whereas Old-World species have 36. The G + C content ofsa L. braziliensls is lower than L. major (57 and63%o,respectively),gz ond it has been reported that 45.3Vo of the obtained predicted3s protein sequences displayed similarity to annotated proteins ofss L. major [24]. Among the proteomic studies of Leishmania40 parasites, only a few studies have been conducted analyz-o' ing Leishmania (Viannia) species. Specifically, these studiesrz focused on identifying developmentally regulated proteins in L.as (V.) panameisis [20] and virulence factors in L. (V) guyanensisu 1221. The genome project of L. (U.) braziliensis is underwayrs (www.sanger.ac.uk/Projects/L-braziliensis/), but as far as weao know, there are no available data related to proteomic anaiysisaz of this parasite.aa Considering the epidemiological and medical importance ofcg L. braziliensiso the first reference proteomc map (p1 4-7, Mrm 10-130 kDa) of promastigote forms of this parasite was obtainedsr by combining two-dimensional gel electrophoresis (2DE), mass52 spectrometry and datamining. The reference map of L. brazilien-ss sis would be useful for differential expression studies ofpotentialuo drug targets and identiflcation ofputative virulence markers and5s vaccine candidates in this parasite species as well as other Leish-s6 mania (Viannia) species considering the high genetic similarity57 among these parasites [6,3 I

.l.

m 2. Material and methods

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2. l. Parasite culture

Promastigotes ot L. (V.) braziliensis IOC-L 2463 (MHOM/BR/2001/JOLIVAL) were grown at 25 "C in Schneider'smedium supplemented with 20Vo (v/v) heat-inactivated fetalbovine serum and passed every 4 days until a density ofI x 109 cells/ml was reached (promastigotes at late log phase).Parasite density was estimated by counting in a hemocytome-ter. At the start of the study, the strain was characterized asL. (V.) braziliensis through multilocus enzyme electrophoresisfollowing protocols described elsewhere {61.

2.2. Sample solubilization and protein concentrationdetermination

Parasites were harvested by centrifugation at 2000 x g forl0min at 4"C, washed twice in PBS pH 1.2 and lysed by 10

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cycles of freezing and thawing in PBS buffer (13.6 mM NaCl,0.27 mM KCl, 0.4mM NazHPO4, 0.15 mM KHzPO+) contain-ing a cocktail of protease inhibitors (Protease Arrest GenoTechnology, Inc.), The lysate was centrifuged at 14,000 x g for15 min at 4 "C to remove insoluble material and the proteins inthe resulting supernatant were precipitated with I07o (vlv)TCAand washed with cold acetone in the same conditions. Finally,the pellet was resuspended in IEF buffer (9 M urea, 4Vo CHAPS,40mM dithiothreitol (DTT) and}.5Vo ampholytes 3-10). Resus-pension was allowed to proceed for t h at room temperature.Protein concentration was determined using the 2D Quant Kit(GE Healthcare), Proteins were aliquoted into single-use sam-ples of 500 p.g and stored at -80'C until analysis.

2.3. 2D electrophoresis

For the first dimension, aliquots of 500 pg were diluted to afinal volume of 300 pL in rehydration solution (8M urea,2VoCHAPS, 40mM DTT,0.5Vo ampholytes 3-10). Samples wereapplied to the IPG-strip (17 cm pH 4-1 linear; Bio-Rad) by in-gel rehydration. All isoelectric focusing was performed on aProtean@ IEF cell (Bio-Rad) with a temperature of 20'C anda maximum current of 50 pA/strip. Focusing parameters wereas follows: active rehydration (50V) for 12h; step l-gradientfrom 1 to 250V over 30min; step 2-gradient from 250 to1000V over 30min; step 3-constant 1000V for th; step4-gradient from 1000 to 4000 V over 45 min; step 5-4000 Vconstant for t h; step 6-8000V until complete 55,000Vh.Focused strips were stored at -80'C for a maximum of I weekbefore use, or applied directly to the second dimension gel.Before the second dimension, proteins were reduced (10 mg/mlDTT) and alkylated (25mglmL iodoacetamide) in equilibra-tion buffer (6 M urea, 2Vo SDS, 300 mM Tris-Cl pH 8.8, 20Voglycerol). Equilibrated IPG strips were separated across l2%oSDS-PAGE gels using a vertical system (Bio-Rad) and standardTris/glycine/SDS buffer. Gels were run at 40mAigel at 15'Cuntil the tracking dye left the gel.

2.4. Protein visualization and imase analysis

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Gels were stained with colloidal Coomassie Brilliant Blue r's

G-250 following procedures described elsewhere [25]. Gel 110

images were documented using a GS-800TM calibrated imag- 11r

ing densitometer (Bio-Rad) and image analysis was performed ",

using PDQuestrM software (Bio-Rad). Comparison of 2DE r1s

maps derived from five independent protcin preparations, each 114

one obtained from five independent parasite cultures, was per- 115

formed. To determine experimentai pI and M, coordinates for 116

each single spot, 2DE gels were calibrated using a select set 1i7

of reliable identification landmarks distributed throughout the 110

entire gel. The theoretical pI and M. of identified proteins were 1re

calculated using Masslynx software v 3.5 (Waters). rzo

2.5. Protein digestion, peptide extraction and mass p1

spectrometric analysis 122

Protein spots were manually excised and treated for digestion. p3

Gel pieces were washed three times with 50Vo (v/v) acetonitrile p4

P. Cueruo et al. / Molecular & Biochemical Parasitology xxx (2007) xxx-xx-x

12s in 25mM ammonium bicarbonate for 15min each, shrunk by126 dehydration in acetonitrile, and dried in a vacuum centrifuge.127 Gel pieces were rehydrated in 15 pL of 50 mM ammonium bicar-pa bonate containing 200 ng of sequencing grade modified trypsin12s (Promega). After 15min, 20pL of 50mM ammonium bicar-r3o bonate were added to keep the gel pieces wet during tryptic13r digestion (37 " C, overnight). To extract peptides, 20 p"L of 0.5Vo1s2 (v/v) trifluoroacetic acid (TFA) in 50Vo (vlv) acetonitrile werei3s added and samples were sonicated for 30 min. The separated liq-134 uid was concentrated under a vacuum to an approximate volumer3s of l0 pL. Tryptic peptides were purified using ZipTip C18, fol-1s6 lowing the manufacturer's instructions (Millipore), eluted with137 3.0 pL of 0.1Vo (v/v) TFA in 50Vo (v/v) acetonitrile and co-r38 crystallized with matrix (7 mglmL a-cyano-4-hydroxycinnamic13e acid) on a stainless steel plate using 0.5 pL of matrix and 0.5 pLr4o of sample. Mass spectra were acquired on a 4700 Proteomics141 Analyzer mass spectrometer (MALDI-TOF/TOFrM, Applied142 Biosystems) operating in delayed reflector mode with an accel-14s erated voltage of 20 kV. MS/MS spectra corresponding to the144 five most intense signals were obtained automatically using ther4s CID acquisition mode.

r46 2.6. Databatse search

147 An automatic database search was performed using ther4o GPS ExplorerTM Protein Analysis Software (Applied Biosys-14e tems), which performs a combined search using the MS and150 MS/MS data against the National Center for Biotechnology1sr non-redundant database (NCBInr) by means of the Mascot@1s2 database search engine. The Mascot search parameters were as1ss follows: only tryptic peptides with one missed cleavage werers4 considered, no taxonomic restrictions were used; fixed and vari-1ss able modifications were carbamidomethylation of Cys residues156 and Met-oxidation, respectively; mass accuracy of 100 ppm and1s7 0.5 Da were acceptable for the matching peptides (MS mode)1ss and their corresponding fragments (MS/MS mode), respectively.lse A global Mascot score greater than 50 was considered signif-160 icant (p < 0.05). In addition, the combination of the number of16r matching peptides (>2) in MS mode and pcptides identified by162 MS/MS (>1) was taken into account to classify the identification163 as a positive hit. Finally, all peptides automatically identified byi64 homology with other Leishmania species were confirmed by:16s (i) search for the homologue protein in GeneDB L. braziliensis166 databases and (ii) manual sequencing ofadditional peptides not167 identified by the GPS sottware. All MSA4S spectra of peptides168 that were not automatically identified by Mascot were manually16s interpreted in order to: (1) determine the presence of post-17o translational modif,cations, (2) identify amino acid changes with171 respect to sequences reported in the data bases, (3) reconfirm1i2 the identifications (4) detect the presence of protein mixtures17s in the analyzed spot and (5) perform the protein identification174 itself in cases where automatic identification was unsuccessful.r75 De novo sequenced peptides were blast searched against the176 peptide database available on GeneDB L. braziliensis databases17i (http://www.genedb.org/genedb/lbraziliensis/blastjsp) of thena Wellcome Trust Sanger Centre (http://wwwsanger.ac.uk/).17s GeneDB server search parameters (Matrix, pam30; filter, none;

expect, 100) were optimized for aligning short sequences (6-20 r8oamino acid residues). Gene ontology (GO) annotations (biolog- 181ical process) for L. braziliensls proteins were assigned based 1a2on sequence similarity searches against GO annotated L. major 183proteins in the l. major GeneDB database. Poor quality MSMS r84spectra were not considered for either manual de novo sequenc- r85ing or identification in the protein sequence database, Only 186reliable partial sequences of de novo sequenced peptides were ft7used for alignment-based identification. The sequence ofthose 188partially sequenced peptides was extended to the nearest tryptic 1oecleavage site and the coincidence between the experimental and reotheoretical mass in addition to the quality of the alignment were 1e1considered as identification criteria. B2

3. Results and discussion 1s3

3.1. 2D electrophoresis and image analysis 1e4

Soluble proteins of late log phase promastigotes of l. re5braziliensis were separated by 2DE in pH range 3-10 (data 1e6not shown), demonstrating that most spots were concentrated 1s7in the pH 4-7 region, Therefore, this pH range was selected reafor constructing the 2DE reference map of L. (V.) braziliensis. lsePDQuestTM software was used for the image analysis of five 2oorepresentative 2DE gels obtained fiom five independent exper- 201iments. The 2DE protein spot profiles obtained from these five 2o2separate protein preparations were highly reproducible in terms 2oaof both the total number of protein spots and their relative posi- 204tions and intensities (Fig. 1). Approximately 641 spots were 205detected in the Coomassie G-stained gels. Proteins detected in 206the pI 4-7 2DE gels had a molecular mass range between l0 and 207l3OkDa with most of the spots predominantly located above 2082lkDa. 2oe

3.2. Protein identification 21o

Similarity among Leishmania species together with advances 211in mass spectrometry technologies and protein identification 212programs [26.2'/] allowed for the idcntification of L. braziliensis 213proteins by homology. One hundred and sixty spots of varying 214intensity and molecular weight were excised and digested with 21strypsin. Resultant peptides were submitted to MS and MS/MS 216analyses and combined data were used for database searches. 21iAutomatic search and/or manual interpretation of mass spec- 21atra and blast searching allowed the identification of 101 spots 21s(Tables I and 2). Identified proteins had a molecular mass 220between l0 and 120kDa (Fig. l). Seventy-five different pro- 221teins were found among the 101 identiflcations (Fig. I and 222Tables I and2). The genes corresponding to the identifled pro- 22steins were located in 25 of the 35 L. braziliensis chromosomes. 224Chromosomes 35, 30 and 25 concentrated approximately 387o of 22sthe genes encoding the identified proteins (18, 10, and 10 pro- 226teins, respectively). Remaining genes (62Vo) were distributed 22iover 22 other chromosomes. The presence of elongation fac- 22ators, HSP70 and other chaperones among the most abundant 22sproteins, was in good agreement with the proteome analysis of 230other Leishmania species f l6,l8l.

P. Cueno et al. / Molecular & Biochemical Parasitology xxx (2007) xxx-xxx

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E TA E, ET= E - l = . = F

r . ! . e € : a =E ! "

u & E3 i : EF €EEE EFsE E .E €sb i' : E E : i ' ! . = R

; € i . . :Etre-&* Ss ?EEH.#EFi ' :F S.s Ef ; rE= H a 2 y , 6 G 6F E g E a o : = € i E Ee - E a 6 4 J a. t r . 4 E t v : : U i j E t r o8 . : . E ; 4 . = F € : > E E€ ! d ? : . , 8 . =E i l H . E E g g t s N d E . EFE;ET ; . qJ JEFsF d 9 . F Ug :J t r , 8 : EE : ; .E ;?€ iE i l a €3 e , \> , t r:E ; iFAX€ i l F EsE?E P 9 r i : . !g ! ts 9 ,^E E € E = t i F i i s E pS s n l l S o o i A e { g I

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ai!Egii,iiE,E;s:iE= iF .=€* -=* ' f i *EEEFrE! ;ErEEEi t !E8 .q€ E igg"i;E si€ E =7ztiSIrFi ; i I I€€t+BaE€?gEEEFEEFEEEFEE d-. E E + E ee- E E i E t E t EEts rs '€ i E€€E{ig€g: 5 4 . 9 e ? A F I e a ; . E t r o : o .

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P. Cuervo et al. / Mole:cular & Biochemical Parasitology xxx (2007) xnt-xxx

6 0 r o Nh r 9 9 €

h I 6 NN d N N

t t l

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h ad

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N

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d

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f r qq q9 r h h

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P, Cuervo et al. / Molecular & Biochemical Parasitology xxx (2007) xn-xxx

4 p l

Fig. l. Proteome of the soluble fraction of Leishmania braziliensis IOC-L 2463 strain. Gel obtained aller separation of soluble proteins (500 pg) by 2-DE (firstdimension: IEF pH range 4-7, second dimension: 127o SDS-PAGE) and staining with colloidal Coomassie Brilliant Blue G-250. Protein spots identified by PMFand/or de novo sequencing are numbered and their identities are given in Tables I and 2. This figure is a reliahle representation of five independent gels. Spots 5, I 9,108, I 19 and 121 were used for gel calibration.

231

232

233

234

235

236

238

239

240

241

242

243

244

245

246

247

244

249

250

251

252

253

254

255

3.2.1. Automatic identification by GPS sojtwareGPS ExplorerrM Protein Analysis Software with an inte-

grated Mascot search engine allows sequence database searchesbased on masses of peptides generated by protein digestion onfragment ion spectra recorded from peptides or on a combinationof mass and tandem mass spectra. Cross-species protein search-ing with the combined data using this software allowed for theidentification of 63 protein spots (Table I ). All identifled pep-tides were confirmed by manual interpretation of tandem massspectra. This strategy allowed us to verify that the GPS softwaremade correct assignations by homology with other Leishmaniaspecies in l00%o (63 | 63) of the ,|' braziliensis proteins assessed.In the case of spot J29 ('I'able 1). assigned as B-tubulin of t . majorby automatic identification of two peptides (FPGQLNSDLRand VGEQFTGMFR), the manual sequencing assigned both theabove mentioned peptides and a third one (MNVYFDESAGGR)to three very similar predicted proteins of L. brazilienri.r, twohypothetical proteins (LbrM33;0880, LbrM33.0890) and B-tubulin (LbrM2l.1630). In fact, the three peptides matched thehypothetical sequences better than P-tubulin. raising doubtsabout the automatic assigtunentirr,However, B-tubulin genes ofLeishmania are multi-copy genes, which are affanged in directtandem repeat units or in singletons in different chromosomesL28,291, resulting in intraspecific heterogeneity [301. Thesehypothetical proteins identifled and assigned to chromosome 33

may represent the B-tubulin tandem arrays recently described byJackson et al. [29]in L. braziliensis, indicating that the automaticassignation used in this study was correct,

For several spots, only one or two ofthe five peptides submit-ted to MS/I\4S were identified with GPS software. Other peptideswere not identified, even with good quality tandem mass spec-tra. These spectra were manually sequenced and blast searched,confirming previous identifications by the GPS ExplorerrMSoftware in all cases (Table l). In some spots, peptide assig-nation was not possible due to amino acid changes occurringbetween sequences of L. braziliensis-derived peptides and theirhomologues in other Leishmania species (Table l, spots J65 andJ103). This result is not surprising given the reported differencesbetween predicted protein sequences of L. braziliensis and anno-tated proteins of L. major l24l as well as in the genotypic andphenotypic markers between these two species [8,3 I ].

3.2.2. Manual identification by de novo sequencing andblast searching

When automatic identification was unsuccessful, assigna-tion was achieved by manual de novo sequencing and blastsearching 126,321. Total or partial peptide sequences were ana-lyzed with GeneDB BLAST (http://www.genedb.org/genedb/lbrazilierrsis/blastjsp) search engines setting parameters forshort. nearlv exact matches. Given that information on Z.

257

260

261

262

263

264

266

267

268

269

270

271

273

274

275

276

277

274

279

P. Cueno et al. / Molecular & Biochemical Parasitology xxx (2007) i:xx-xtcx

Table 2Peptides manuafly sequenced and blast searched against L. braziliensis predicted proteins GeneDBu

Code Protein name Accession No. GeneDB Mr theoretical, Da (Mr pl theoretical (pI Peptide sequence

L. b. Proteins Database experimental, kDa) experimental)Enor*ppm

J26 PDI, protein disulfideisomerase. Leishmaniabraziliensis

J27 PDI, protein disulfideisomerase. Leishmantabraziliensis

J32 NADH:flavinoxidoreductaseA'{ADHoxidase, putative.Leishmania rnajor

J34 Isocitratedehydrogenase,putative. Leishmaniabraziliensis

J40 Hypothetical protein,

conserved. Leishmaniabraziliensis

J4l Hypotheticalprotein,conserved. Leishmaniabraziliensis

J43 TPR domain protein,conserved. Leishmaniabraziliensis

J44 TPR domain protein,

conserved. Leishmaniabraziliensis

J4'7 Adenylate kinase,putative. Leishmaniabraziliensis

J50 Hypotheticalprotein,conserved. Lei s htnaru.abraziliensis

J I J

Similarity to endo-llikeprotein. LeishmaniabraziliensisHypothetical protein.Le is hmania b razilie n s is

Hslvu complexproteolytic subunit-like.L e i s hrnani a b r a zilie n s i sHypothetical protein; ,, ,conserved. LeishmaniabraziliensisHypothetical protein,

conserved. Leishmaniabraziliensis

LbrM35.6670

LbrM35.6670

LmjFl2. l 130brazill998bl0.p lkc

LbrM33.2570

LbrM30.05l0

LbrM07.0640

LbrM30.2470

LbrM30.2470

LbrM2l. l l00

LbrM20.39l0

LbrM35.6300

LbrM33,0850

LbiM35.3770

LbrM30.2850

LbrMl4.0190

52812.8 (50.3)

s28 r2.8 (49.6)

41981.5 (44.8)

46s39.5 (43.7)

36828.8 (43.0)

38228.4 (41.8)

4s738.9 (42.2)

45738.9 (41.5)

30972.8 (39.4)

2rr37.3 (38.4)

26426.8 (30.7)

26934.4 (29.3)

23874.4 (27.9)

36401.6 (30.5)

22305.t (27.8)

5.07 (4.81)

5.07 (4.8s)

5.75 (5.01)

5.49 (5.30)

5.59 (5.83)

s.s4 (s.69)

s. l9 (s.Os)

s.l9 (4.93)

s.89 (s .21)

5.31(5 .03)

5.67 (s.65)

5.33 (5. lo)

5 .88 (5 . l6 )

6.83 (4.62)

4.7s (4.63)

EMEEFVR

FYAPWC-GHC-KrAryvc.GHC-QKb

EMEEFVR

FYAPWC-GHC-KYAPWC-GHC'QKb

GYSTFC'HEGGIYGKd

ATVPSNVDEGVRE

WGYALAER

W-GYALAERGPLGTPVGEGLR

pQVDIDAFER

QVDIDAFERKGQIYALAM-AR

LEVDAFHR

YDSLVSTER

DYSNAIIDCER

DYSNAIIDCER

YNPPPEDqIALTEB

YVHEUTASSLPPR

TEVTYEVDADGNR

YDPDAYTPEER

GVNTFSPEGR

VHM"YAPAEIK

LNDFPDQLSR

NTANIADLYGR

QQFLQAHTVNQSNFR

LSPPHLVYDEGK

-30-30

-2r

-30-30

- J 6

2026

-65

-40

20

167

J70

17 l

-36-56

-40

- 5 1

-44

0

-34

q

0

-8

177

12

Table 2 (Continued)

P. Cuervo et al. / Molecular & Biochemical Parasitology xxx (2007) xxx-xxx

Code Protein name Accession No. GeneDBL. b. Proteins Database

M, theoretical, Da (M, pI theoretical (pIexperimental, kDa) experimental)

Peptide sequence Error t ppm

J78 Hypotheticalprotein,conserved. Leishmaniabraziliensis

J79 hoteasomeactivatorprotein pa26, putativeLe i shma n ia b razi lien s i s

J8O PMM,phosphomannomutase,putative. Leishmaniabraziliensis

J83 Proteasome beta 2subunit, putative.Lei s h ma n i a b razi I i e n s i s

J87 hoteasome alpha 2subunit, putative.Le i s hmania b razilien s t s

J94 ATP synthase, epsilonchain, putative.Le i s hmania b razi I ie ns rs

J97 IgE-dependenthistamine-releasingfactor, putative.Leishmania major

Jl04 40S ribosomal proteinSl2, putative. Leishmaniamajor

J105 Maoc familydehydratase-like protein.Leishmania braziliensis

J106 Ribonucleoproteinpl8,mitochondrial precursor,putative. Leishmaniamajor

Jl07 Prefoldin 5Jike protein.Leis hmania braziLiensis

J109 Ubiquitin-conjugatingenzyme E2, putative.Leis hmania b razi I ie n s i s

Ill2 Cofilin-likeprotein.Le is hmania b razi lie ns i s

Jl13 Prefoldin subunit 2,putative. Leishmaniabraziliensis

JIl4 Hypotheticalprotein,conserved. Leishmania.braziliensis

J115 Calpain-likecysteinepeptidase, putative.Leis hmania braziliensis

LbrM14.0l90

LbrM34.0660

LbrM35.l910

LbrM34.3330

LbrM2l. l550

LbrM30.3390

LmjF24.1500brazil672c09.p I k"

LmjF13.0570brazil 1484b05.qlkc

LbrM07.0400

LmjF15.0280brazilST5cl l.qlkc

LbrM22.0490

LbrM34.l1l0

LbrM29.0500

LbrM09'0440

LbrM23.l77Oa,

LbrMl4.0750

2230s.r (27.r)

18190.1 (27.3)

28230.r (28.s)

27970.r (27.2)

2s360.6 (2s.6\

20433.r (23.4)

19s78.2 (23.0)

15826.1 (17.5)

28044.s (r7.6)

21628.5 (r9.7)

17866.9 (16.5)

16783 .s ( 15 .1 )

15875.8 (14 .1)

26405.2 (15.8) (5.00)

r692s.s (r4.9)

1316r.2 (r4.7)

4.7s (4.64)

s.05 (5.88)

5.91 (s.92)

6.35 (6.05)

5.s5 (5.46)

s.20 (4.6s)

4.39 (4.42)

4.80 (4.70)

6.2s (4.86)

7.48 (5.90)

5.51 (s.24)

6.46 (5.95)

5.2s (5.01)

7.00

s.r7 (4.93)

4.73 (4.48)

LSPPHLVYDEGK -59

EFQGELHDIYR _56

TSEGGNDYEIYCDSR _50

NIHFFGDK -5I

DKTEVMIGYDKPNER _5

EGFDGQMTPENTQVGR -43

EPIPISQLVR _50

ALAQQQSALSSAYDEK 6

VVDVVHNNRf -I9

AFQANAAAFVKC -35

ASYMAHIR _21

QGNIDLINVEER _5'I

pQGNIDLINVEER -58

LAQW'AGLABI -49

MSTEIAPLTIRi'J _43

EMPGPGTVYLSQNLR

NC-PPDLETYNATLQRK

pQQNQVIDALQQK

QQNQVIDALQQK

DQWSPAUTISK

DQW-SPALTISK

VHTAINDLR

IAELENESHEHDLVAR

VSEGSLTEQEIVQQYNRI

vl-!x!s'IqIB

YENAQPTYSGNTWK

DNGDGLLFR'

-47

-48

5

t4

0

- 5 L

- 3 1

-38

-48

-50

- + J

-49

P. Cuervo et al. / Molecular & Biochemical Parasitology xxx (2007) xxx-xxx l 3

Table2 (Conrinued)

Code Protein name Accession No. GeneDBL. b. Proteins Database

Mrtheoretical,Da(M, pltheoretical(pI Peptidesequenceexperimental, kDa) experimental)

Error* ppm

ISFEANPIPK

4.73 (4.48) YENAQPTYSGNTVVK -47

-44

J116 Calpain-likecysteinepeptidase, putative.Le i s hmania b razi Lie ns is

Jl19 605 acidic ribosomalprotein P2. Leishmaniabntziliensis

Il2l EndoribonucleaseL-PSP(pb5), putative.Le is hmania b razi I i e n s i s

Jl22 Hypotheticalprotein,conserved. Leishmaniabraziliensis

Jl37 Hypotheticalprotein,conserved. Leishmaniabraziliensis

Il47 Hypotheticalprotein,conserved. Leishmaniubraziliensis

Jl5l Dihydroorotatedehydrogenase, putative,

Leishmania tnaior

LbrMl4.0750

LbrMl5.0780

LbrM23.0210

LbrM25.2l90

LbrM20.0930

LbrM32.0790

LmjFl6.0530brazil673h04.p lkc

13161 .2 (14 . r )

|177.2 (n.2)

1667 4.4 (16.7)

23662.s (3r.r)

3965t.r (44.4)

56163.1 (32 .1)

35095.4 (36.9)

4.29 (4.29)

6.88 (6.80)

4.48 (4.39)

s.8s (5.87)

8.4s (s.85)

s.89 (6.2r)

DNGDGLLFR*ISFEANPIPK

M-SAM-PAAGSGAAAGGAAPAAGAAAPAAAAAK

ASPSQADVEAIC-R

M'VYISGQLPK

-49-44

I

LM'VGQLGDPLTIEEGK _34

eaeesc@QM-K -37

LTSHLTNTQLLNEEADR _46

APVTQSGLDLSNFEDK _40

MTEC-WYIPEEVADRi'.J 87

YPC-TDEGDBIDIETVQY 29DFIASR

YLAANSEAYPHPFGVKN _453

YVFPTALANVNAFYRO 80

-7

-+J

M':Methionine modified by oxidation, C*:cysteine modified by carbamidomethyl, W":tryptophan modified by oxidation, pQ: peptide with modif,cation of glutamine

to pyroglutamic acid in N-terminal end.a Amino acid sequence manually deduced is underlined.b Fragmented peptide.c Protein is not available in L. b. predicted proteins GeneDB. Peptides were blast searched against I. major predicted proteins GeneDB. L. braziliensis Accession

No. reported in this table correspond to the best alignment against L. b. unassembled shotgun reads GeneDB data base.d Peptide manually sequenced in L. braziliensis presents a C modified residue in the 4th position whereas I,. major shows a A residue in the same position.e Peptide manually sequenced in tr. braziliensis presents a G residue in the 8th position whereas L. maior shows a D residue in the same position.f Peptide manually sequenced in L, braziliensis presents a H residue in the 6th position whereas Z. major sbows a Y residue in the same position.8 Peptide manually sequenced in L. braziliensis presents a A residue in the 4th position whereas Z. najor shows a T residue in the same position,h Peptide manually sequenced in L. braziliensis presents a A residue in the 8th position whereas Z. major shows a V residue in the same position.i N-terminal peptide with acetylation..j Peptide with methionine processed.k Peptide manually sequenced in L. braziliensis presents a R residue in the l5th position whereas I. maior shows a K residue in the same position.I Peptide manually sequenced in L. braziliensis IOC-L 2463 presents Q and E residues in the 9th and lOth positions, respectively, whereas tr. braziliensis M2904

shows E and Q residues in the same positions.m Peptide with modification of asparagine (4th position) to aspartic acid, by deamidation.n Peptide manually sequenced in L, braziliensis.presents nearly exact matching with brazi1673h04.plk sequence but several amino acid changes with respect to L.

rnajor sequence: Y A, N, A and P residues in the lst, 3th, 5th, 8th, and lOth positions, respectively in L. b., whereas Z. maior shows C, T, V, V and S residues in the

same positions.o Peptide manually sequenced in L. braziliensis presents F and V residues in the 3th and l0th positions, respectively, whereas L. maior shows L and I residues in

the same positions,

2ao braziliensis predicted proteins is available for blast searching,2a1 this strategy allowed us to unambiSuously identify 38 spotsza2 (Table 2). A representative manually interpreted tandem mass2a3 spectrum is shown in Supplementary Fig. l. Several peptides

2a4 could not be identified by blast searching against the pre-

2as dicted protein database, but showed nearly exact matches with2a6 sequences in the t. braziliensis unassembled shotgun reads

database. Assignment of these protein spots was made after 287

comparison between the L. braziliensir translated read and the 288

L. major annotated protein database. All these peptides exhib- 2as

ited amino acid changes between the two Leishmania species, 2so

but the alignment of L. brazilien.ti,s translated sequences to Z. 2sl

major annotated proteins was very significant in all cases (see 2s2

Supplementary Fig. 2). Although Version I of the L. brazilien- 2s3

P. Cuervo et al. / Moleculqr & Biochemical Parasitology xxx (2007) xxx-xxx

294

295

296

257

298

299

300

301

302

303

304

305

306

307

308

309

3 1 0

3 1 1

312

3 1 4

3 1 5

317

3 1 8

3 1 9

320

324

325

326

327

324

329

330

331

332

334

335

336

338

339

340

341

342

343

344

345

346

347

348

349

350

3 5 1

352

353

354

356

358

359

360

361

362

363

364

365

366

368

{A) 100

(!

o&

7 q

xt!q 5 0I

Eo! } 2 5x

UJ

I 9CL

; 8(Eg 'o. 86oO . a

lu

0

(B) 10

50 75 100

Theoretical il?, (kDa)

3 4

Iheoret ical p, {pH}

Fig. 2. Comparison of experimentally determined and theoretical (A) M, and(B) pI values of identified protein spots from L. braziliensls 2DE gels. Overall,a good conelation (M, Spearman r = 0.86, P < 0.0001 and pl Spearman r =0.72,

P < 0.0001) is observed for the majority of the data, which fall in the M. rangeof l0 and 80 kDa and the pI range between 4.2 and 6.5.

.si.r genome is currently available, the dataset is still preliminaryand several genes are not annotated or are misannotated (e.9.,protein spot J65, corresponding to putative proteasome alpha3 subunit, whose sequence in the L. braziliensis database isapparently truncated or mistranslated, as shown by lack ofalignment of the manually sequenced peptide mlz 1610.67,SELKPHAQCADQAR). In addition, directMS gene annotationfor sequences in the shotgun reads database of the L. brazilien-sis genome was achieved in approximately l07o of the proteinsidentified in this study (Tables I and 2).

3.3. Peptide modifications

Protein mass and isoelectric point values estimated by gelelectrophoresis showed a good correlation with predicted values,although some discrepancies were detected (Fig. 2) suggest-ing that post-translational protein modifications are commonin L. braziliensis. Post-translational modifications may regu-late protein function in cells by altering either localization,molecular interactions or activity of proteins. About l17o ofthe identifled spots showed post-translational modifications suchas acetylation, N-terminal pyroglutamylation and"/or N-terminal

processing of methionine. Modifications such as deamidationand oxidation of tryptophan (about9%o), which can occur eitheras post-translational modifications or during sample preparation,were also observed (Tables 1 and 2). These protein modificationsseem to be common in trypanosomatids [331.

Peptides with N-terminal pyroglutamate and oxidized tryp-tophan were found, in the majority of cases, in pairswith unmodified glutamine and tryptophan, respectively(Terbles I and 2). These observations are in agreement witha previous report [34] and are commonly found in peptidesanalyzed by MS [34,35]. Cleavage of N-terminal methion-ine residues and N-terminal acetylation are, by far, the mostcommon modifications, occurring on the vast majority ofeukaryotic proteins t361. By searching of the Z. brazilien-sis genome at the GeneDB database, at least 2 methionineaminopeptidases (LbrM19-V2.0860 and LbrM2l-V2.0950), 1peptide deformylase (LbrM32-Y2.1460) and at least 11 entriesfor acetyltransferases were found, indicating that this Leish-mania species express enzymes involved in these classes ofmodifications. The biological significance of N-terminal acety-lation varies according to the particular protein [36]. N-terminalacetylated residues are important for the activity of diverseproteins, such as actin l37l and proteasome subunits [381.N-terminal pyroglutamate residues derived from N-terminal glu-taminyl residues, as detected in this study, could function inreducing susceptibility to degradation by aminopeptidases [39-|and, hence, play an important role in biological function [401,The glutaminyl cyclase, enzyme involved in physiological N-terminal pyroglutamylation, is also encoded by the genome ofL. braziliensis (LbrM05-V2.0930). Among the herein identifiedL. braziliensis proteins with acetylation, N-terminal pyroglu-tamylation and/or N-terminal processing of methionine therewere metabolic enzymes, proteins involved in DNA replication,proteins involved in protein synthesis and in the proteasome-ubiquitin pathway, as well as a number of hypothetical proteins(Tables I and 2). Several deaminated proteins (such as initi-ation factors, calpain-like cysteine peptidase and hypotheticalproteins), which probably occurred under physiological condi-tions, were found in this study. Deamidation of proteins has beenassociated with important biological processes such as proteinturnover, development, and molecular agingl4l,42l.It would beinteresting to invcstigate if this modification could be associatedwith the life cycle of Leishmania parasites.

3.4. Biologicalfunction of the identified peptides

According to GO annotation of biological processes, theidentified proteins ofZ. braziliensis could be classified in 15 cat-egories (Table 3). One-fourth of them (including multipleproteinspecies) were clustered into a single category, protein fold-ing, representing the most commonly found group in this map.Included in this group were HSP70 and disulfide isomerase,which are implicated to be potential virulence factors of Leish-mania {43451. Another large set of proteins identified in thisstudy belongs to the protein synthesis category. Thirteen spots.corresponding to 10 proteins, were identified in this group. Ofparticular interest is elongation factor I -beta (EF- I B). Besides its

4

J

Table 3Biological process categories of the identified proteins

P. Cueno et al, / Molecular & Biochemical Parasitology tcxx (2007) xxx-xxx

Number ofidentified spots

242772II2II

132IIIII

I3I

III

IIII

l 5

Categorieso

Table3 (Continued)

Categories' Number ofidentified spots

ATPase beta subunit, putative

Transitional endoplasmic reticulum ATPase,putauve

DephosphorylationProtein phosphatase-2C; PP-2C

Fatty acid metabolismMaoc t'amily dehydratase-like protein

Electron transportNADH:fl avin oxidoreductase/NADH oxidase,

putativeDNA replication

Proliferative cell nuclear antigen (PCNA),

putativeApoptosis

Activated protein kinase C receptor LACK

Unknown biological processTPR domain protein, conserved

. Similarity to endo-l-like protein

IgE-dependent histamine-releasin g factor,putanve

Hypothetical protein, conservedHypothetical proteinRibonucleoprotein p I 8, mitochondrial precursor,

putauveEndoribonuclease L-PSP (pb5), putative

II

,,

1I

1I

1I

LI

262II

19II

I

Bold and underlined values indicate total number of spots in each category.a Categories were taken from gene ontology annotations of biological pro-

cess, based on sequence similarity searches against the GO annotated L' maior

proteins.

role in protein synthesis, the EF-l complex is implicated in othercellular processes such as signal transduction and apoptosis [461as well as survival of amastigotes in macrophages [47]. A recentreport demonstrated that the EF-18 complex has trypanothioneS-transferase and trypanothione-peroxidase activities [48]. Inaddition, it was identified as a polypeptide associated with themetastatic phenotype of a L. guyanensis clone and, hence, oneof the potential factors involved in parasite dissemination [22].

L. braziliensir proteins with proteolysis activity were thethird most abundant category found in this analysis. Eight ofthe nine different proteins identified in this group are compo-nents related to the mechanism of proteins degradation throughthe ubiquitin-proteasome pathway. This pathway is a highly spe-cific and regulated system fbr targeted proteolysis, responsiblenot only for the selective degradation of damaged and misfoldedproteins but also for the regulated turnover of critical cell regula-tors and partial proteolysis of propeptides [49] . A previous studyhas reported that replication of Leishmanla requires intact func-tion of the proteasome system [50]. Carbohydrate metabolismrepresented the fourth most abundant category, accounting forseven idcntified proteins. Within this class, we detected theexpression of enzymes involved in glycolysis and the tricar-boxylic acid cycle pathways, all involved in the catabolism ofglucose. We also observed the expression of phosphomannomu-

tase, an enzyme involved in the synthesis of mannose-containingglycoconjugates such as lipophosphoglycan, proteophosphogly-

Protein folding/chaperonsPDI, protein disulfide isomeraseHeat shock protein 70 and related

Chaperonin Hsp 60Chaperonin containing t-complex protein

Prefoldin 5Jike protein

Prefoldin subunit 2, putative

Glucose-regulated protein 78Prefoldin-like protein

Heat shock protein 83-l

Protein synthesis40S ribosomal protein Sl2, putative

605 acidic ribosomal protein P2

Eukaryotic translation initiation factor 3, putative

Putative elongation factor l-beta

Eukaryotic translation initiation factor 6, putative

Possible nascent polypeptide associated complex

subunit, copy I

GTP-binding protein, putative

Eukaryotic Initiation Factor 5a

Eukaryotic Fanslation initiation factor lA,

PutativeAcidic ribosomal protein Pl

ProteolysisHs lvu complex proteolytic subunit-like

Proteasome activator protein pa26, putative

Proteasome beta 2 subunit, PutativeProteasome alpha 2 subunit, putative

Proteasome alpha 3 subunit, putative

Proteasome alpha 7 subunit, putative

Proteasome regulatory ATPase subunit 5, putative

Ubiquitin-conjugating enzyme E2, putative

CalpainJike cysteine peptidase, putative

Carbohydrate metabolismPMM, phosphomannomutase, putatlve

EnolaseIsocitrate dehydrogenase, putative

PGAM 2,3-bisphosphoglycerate-independentphosphoglycerate mutase

Pyruvate kinasePyruvate dehydrogenase El beta subunit, putative

Succinyl-CoA ligase [GDP-forming] beta-chain,

PutativeAmino acid metabolism

S-Adenosylmethionine synthetaseS-Adenosylhomocysteine hydrolase

Glutamine synthetase, Putative

Nucleoside, nucleoride and nucleic acid metabolism

Adenylate kinase, putative

ATP synthase, epsilon chain, putative

Dihydroorotate dehydrogenase. putative

Antioxidant/detoxi fi cationMitochondrial perox iredoxinTryparedoxin peroxidase 2

Cytoskeleton associatedAct in aCofllinJike proteinBeta tubulin

ATP hydrolysis

I

1 t

2)

zIIII

9III

II2

3

369

370

371

373

374

375

376

377

378

379

380

381

383

384

385

386

387

388

389

390

391

392

393

394

l 6 P. Cueruo et al. / Molecular & Biochemical Parasitology xxx (2007) nx-xxx

396

397

398

399

400

401

402

403

404

405

406

407

408

409

4 1 0

4 1 1

412

4 1 3

4 t 4

4 1 5

4 1 6

417

4 1 8

4 1 9

420

421

422

423

424

425

426

424

429

430

431

432

433

434

435

436

437

438

439

440

441

cans, GPl-anchored proteins, glycoinositol-phospholipids andN-glycan glycoproteins, all of which have been implicated asvirulence factors in Leishmania sp. [5 I l.

Several proteins belonging to different categories (suchas calpain-like cysteine peptidase, tryparedoxin peroxidase 2,S-adenosylhomocysteine hydrolase, and dihydroorotate dehy-drogenase) that were identified in this study are recognizedas potential virulence factors or drug targets in protozoanparasites 122,52-58J. However, several other proteins iden-tified in this study were assigned for the first time to aproteome map of Leishmania parusites. Among them, the puta-tive IgE-dependent histamine-releasing factor and the putativeproliferative cell nuclear antigen (PCNA) deserve special atten-tion. IgE-dependent histamine-releasing factor causes histaminerelease from human basophils or mast cells and IL-8 secretionfrom eosinophils. Histamine regulates Th cell differentiation[59] and possibly drives Th2 responses during the chronic phaseof L. major infection [60.|. Furthermore, eosinophily has beenreported in patients with cutaneous leishmaniasis [61], andinduction of IL-S by monocytes infected with l. majorhasbeendescribed [62]. This factor has also been identified in Plasmod-ium falciparun, another intracellular parasite [63]. PCNA, alsoknown as cyclin, is a stable cell cycle regulated nuclear proteinwhose rate of synthesis correlates directly with the prolifera-tive state of normal cultured cells and tissues [64,65 1. It hasbeen reported that cyclin-dependent kinases regulate the cellcycle of Leishmania during its life cycle [661 and representimportant targets for antileishmanial drug development [67].Finally, this proteome analysis of L. braziliensis validates pre-vious gene predictions [13] by confirming for the first time theexpression of 20 hypothetical proteins whose existence has notbeen previously demonstrated. Their identification in 2DE gelsoffers the possibility to study the regulation of such proteinsunder specific environmental conditions or in various mutantbackgrounds.

4. Conclusions

The development of proteomic studies in L. braziliensis hasbeen hampered by the lack of extensive genome sequence datafor this parasite. Nevertheless, our results made possible the con-struction of the first reference map of L. braziliensis proteinsusing promastigote forms of the IOC-L 2463 strain. This iso-late belongs to the most widely distributed zymodeme in Brazilf7,68f representing the same zymodeme (IOC/Z 27) of the L.b ra zi li e n s i s r eference strain (MHOM /BR/ 197 5 1M2904) used inthe genome sequencing project at the Sanger Centre. This strainwas isolated from a patient living in an endemic region (Corte dePedra, Bahia, Brazil) where strains assigned to this zymodemehave been found associated w.ith different clinical forms of thedisease, providing an excellent opportunity to conduct furtherstudies on comparative proteomic analysis of different parasiteisolates. Although the amastigote stage is responsible for mam-malian host infection, the promastigote proteomic map is helpfulbecause -90Vo of the proteome remains qualitatively unchangedthroughout the parasite life cycle [19,691. Further investigationon the potential role of EF-IB as a factor involved in parasite

dissemination might be of particular interest, considering that 4sostrain IOC-L 2463 was isolated from a patient with mucosal dis- 4s1ease. A comparative proteomic study of the parasite with other 4s2Leishmania strains isolated from patients presenting distinct 4s3clinical manifestations could help understand the relationship 4s4existing between EF-lp and the biological behavior of this 455parasite. 456

Acknowledgements 4s7

We thank Dr, Nancy G. Saravia (CIDEIM) for encourage- rsament to do this study, Marise Fonseca dos Santos (IQ-UFRJ) 4ssand Rodrigo Mexas (Laborat6rio de Produglo e Tratamento de 460Imagen-FIOCRUZ) for their precious contributions to this work. 461This work has been supported by the following Brazilian agen- 462cies: CNPq, FIOCRUZ/PDTIS and FAPERJ (E.C,). P.C. is a 463Ph.D. student of the Cellular and Molecular Biology Program at 464the Instituto Oswaldo Cruz and is a CNPq (PEC/PG Program, 46sBrazil) fellowship recipient. 466

Appendix A. Supplementary data 46i

Supplementary data associated with this article can be found, 468in the online version, at doi: I 0. I 0 1 6/j.nrolbiopara.2007.03.0l 3. 46s

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