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Acta Tropica 111 (2009) 255–262

Contents lists available at ScienceDirect

Acta Tropica

journa l homepage: www.e lsev ier .com/ locate /ac ta t ropica

olecular analysis of surface glycoprotein multigene family TrGP expressed onhe plasma membrane of Trypanosoma rangeli epimastigotes forms�

.P. Pena a, N. Lander b, E. Rodríguez b, G. Crisante c, N. Anez c, J.L. Ramírez b, M.A. Chiurillo a,∗

Laboratorio de Genética Molecular “Dr. Yunis-Turbay”, Decanato de Ciencias de la Salud, Universidad Centroccidental Lisandro Alvarado, Barquisimeto, VenezuelaCentro de Biotecnología, Fundación Instituto de Estudios Avanzados, Caracas, VenezuelaCentro de Investigaciones Parasitológicas “J.F. Torrealba”, Universidad de los Andes, Mérida, Venezuela

r t i c l e i n f o

rticle history:eceived 5 February 2009eceived in revised form 12 April 2009ccepted 5 May 2009vailable online 9 May 2009

eywords:

a b s t r a c t

Trypanosoma rangeli, a non-pathogenic hemoflagelate that in Central and South America infects humans,shares with Trypanosoma cruzi reservoirs and triatomine vectors, as well as geographical distribution.Recently, we have described in T. rangeli a truncated gene copy belonging to the group II of the trans-sialidase superfamily (TrGP). This superfamily, collectively known in T. cruzi as gp85/TS, includes membersthat are involved in host cell invasion and infectivity. To confirm the presence of this superfamily in thegenome of T. rangeli and obtain a better knowledge of its characteristics, we designed a PCR and RT-PCRcloning strategy to allow sequence analysis of both genomic and transcribed copies. We identified two

rypanosoma rangelip85/trans-sialidaseultigenic family

rGP

full-length copies of TrGP, some pseudogenes, and N- and C-terminal sequences of several genes. We alsoanalyzed the expression and cellular localization of these proteins in epimastigote forms of a VenezuelanT. rangeli isolate using polyclonal antibodies made against a recombinant peptide from the N-terminalregion of a TrGP member. We confirmed that TrGP is a multigenic family that shares many features withT. cruzi gp85/TS, including the telomeric location of some of its members, and by immunofluorescence

is at t

analysis that its location

. Introduction

Trypanosoma rangeli despite being non-pathogenic to humanshares a wide range of vertebrate hosts and triatomine vectorsith Trypanosoma cruzi, the etiological agent of Chagas’s disease,

nd produces serological cross-reactivity with this parasite (Grisardt al., 1999). Furthermore, the morphological similarity of thesewo parasites, the lack of an appropriate specific diagnostic proce-ure, and the absence of clinical manifestations, contribute to thenderestimation of infections caused by T. rangeli (Guhl and Vallejo,003).

During the T. rangeli life cycle, the triatomine vectors becomenfected after feeding with the blood of infected animals. The par-site subsequently replicates within the insect’s gut, and at someoint, the epimastigote forms cross the midgut epithelium to reach

he haemocoel. Once in the haemolymph, epimastigotes eithernvade and multiply within hemocytes, or divide as free para-ites in the haemolymph. Finally parasites invade and multiplyithin the salivary glands transforming into infective metacyclic

� Note: Nucleotide sequence data reported in this paper are available in theenBankTM database under the accession numbers FJ404790–FJ404809.∗ Corresponding author. Tel.: +58 251 2591985; fax: +58 251 2591886.

E-mail address: mchiurillo@ucla.edu.ve (M.A. Chiurillo).

001-706X/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.actatropica.2009.05.003

he surface of the parasite.© 2009 Elsevier B.V. All rights reserved.

tripomastigote forms (Grisard et al., 1999). Recent data based onkDNA and spliced leader (SL) gene markers, indicate a complexparasite–vector relationship, and suggest a co-evolution of the vec-tor with T. rangeli isolates, such that each triatomine species wouldselect the sub-population that can be transmitted to the vertebratehost (Vallejo et al., 2003; Maia Da Silva et al., 2007).

Among the most prominent genes shared by T. rangeli and T.cruzi are those encoding for a large GPI-anchored glycoproteinsfamily named trans-sialidase (TS) superfamily, that according tosequence identity, molecular weight, and function are classifiedinto a variable number of groups by different authors (Colli, 1993;Cross and Takle, 1993; Frasch, 2000), although they can be gath-ered into two main groups (Frasch, 2000): Group I includes genesencoding proteins with trans-sialidase and sialidase activity in T.cruzi and T. rangeli, respectively. The sialidases expressed in T.rangeli epimastigotes forms (TrSial) are strict hydrolytic enzymesthat release sialic acid residues from the host cell surface glycocon-jugates (Pontes de Carvalho et al., 1993; Buschiazzo et al., 1997).Group II molecules are devoid of enzymatic activity and includethe gp85 family or gp85/TS (80–90 KDa), FL-160 (160 kDa) and Tc13

subgroups of proteins (Frasch, 2000).

The gp85/TS family includes proteins with variable degrees ofidentity, characterized by the presence of two conserved neu-raminidase motifs: ASP box (SxDxGxTW), and the VTV motif(VTVxNVfLYNR), but lacking critical residues in the FRIP motif (Phe-

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56 C.P. Pena et al. / Acta T

rg-Ile-Pro) that are important in determining catalytic activityColli, 1993; Cross and Takle, 1993; Frasch, 2000; Buschiazzo et al.,000). In T. cruzi, gp85/TS members are expressed in infective try-omastigotes forms and intracellular amastigotes stages, and byheir capacity to adhere to the host cell surface and the extracellu-ar matrix, some of these members are implicated in cell invasionMagdesian et al., 2001; Yoshida, 2006; Alves and Colli, 2007).

Recently, we have described that T. rangeli contains andxpresses genes of gp85/TS superfamily (Anez-Rojas et al., 2005).he first ORF of gp85/TS described in T. rangeli (TrGP-1) was a telom-ric truncated copy that conserves the motifs that characterize theamily, but lacks the GPI anchor site, and the C-terminal hydropho-ic tail (Chiurillo et al., 2002; Anez-Rojas et al., 2005). In this worke have further studied T. rangeli’s gp85/TS members and we have

onfirmed that they represent a multigenic family. Additionally, wevaluated the expression and cellular localization of these proteinsn T. rangeli epimastigotes using immunodetection methods.

. Materials and methods

.1. Parasites

T. rangeli isolates, provided by Drs. Palmira Guevara fromniversidad Central de Venezuela (Caracas, Venezuela), and Nés-

or Anez from Universidad de los Andes (Mérida, Venezuela)ere cultured in biphasic blood–agar/NNN media at 25 ◦C. Wesed T. rangeli Venezuelan strains: M/CAN/VE/82/DOG82 andHOM/Ve/99/CH-99 for DNA isolation; MHOM/Ve/99/CH-99 for

NA isolation from experimental infections and parasitic cultures;nd IRHO/Ve/98/Triat-1 for protein expression experiments.

Fourth instar nymphs of Rhodnius prolixus used in this studyere obtained from the Centro de Investigaciones Parasitológi-

as “J.F. Torrealba”, Universidad de los Andes (Mérida, Venezuela).riatomines were artificially fed with cultures containing exponen-ially grown T. rangeli. After the infective meal, triatomines wereept at 27 ◦C, at 70% humidity. Triatomine’s haemolymph was col-ected by sectioning a leg from the bugs. The presence of flagellatesn the collected haemolymph was observed under light microscopyn fresh and Giemsa stained preparations.

.2. Nucleic acid isolation

DNA from T. rangeli culture epimastigote forms was isolatedsing Wizard Genomic DNA Purification Kit (Promega). Total RNAas purified from epimastigote parasites obtained from culture

r infected triatomines using TRIzol reagent (Invitrogen) followinganufacturer’s instructions.

.3. Cloning of TrGP genes by PCR and reverse transcriptase-PCR

Genomic DNA or total RNA, from M/CAN/VE/82/DOG82 andHOM/Ve/99/CH-99 T. rangeli strains, respectively, were used as

emplate in reactions with Platinum Taq DNA Polymerase Highidelity (Invitrogen), or one-step AccessQuickTM RT-PCR SystemPromega), respectively. A set of forward (atgF: 5′-CACGTGCCCAA-ATGTCCCGGCAT-3′; atgF2: 5′-ATGGCCTTTGGCAGTACGGC-3′; slF:′-CTAACGCTATTATTGATACAGTTTCTG-3′), and reverse primersnR1: 5′-GATGATACCCTCGGCAAGTG-3′; nR2: 5′-TTTGTTGCC-TTTGCAATTG-3′; nR3: 5′-GGCCTGCATCACAAATAC-3′; nR4: 5′-CATGGAGACAAGCCCTTTTC-3′) were used to clone sequencesncoding N-terminal TrGP regions by PCR and RT-PCR reac-

ions. While vtvF: 5′-GTCTTTTTGTACAACCGCCC-3′ and oligo-dT:′-CCCCCCCCCCCTTTTTTTTTTTTTTTTTTTTT-3′) primers weremployed to clone C-terminal sequences by RT-PCR. We designedtgF, atgF2, nR1, nR2, nR3, nR4 and vtvF primers based on theucleotide sequence of the TrGP-1 gene (GenBank accession no.

111 (2009) 255–262

AF426022). slF and oligo-dT primers were designed based on T.rangeli SL sequence (GenBank accession no. M62864) and poly-Atail, respectively. The information of the 3′ region of TrGP acquiredfrom the cDNA recombinants obtained by using oligo-dT primerallowed us to design a cR primer (5′-TCCACTGTGCCCCACTCA-3′),which was used with atgF as forward primer and genomic DNAas template to amplify full-length TrGP gene sequences. The PCRproducts were then cloned into pGEM-T Easy vector (Promega),and transformed into Escherichia coli strain TOP10F’. Accordingto the portion of TrGP included in the recombinants they wereclassified in three groups: (1) containing the N-terminal (trgpN);(2) containing the C-terminal (trgpC); and (3) a full-length genecopy. From each group we sequenced and analyzed severalclones.

2.4. DNA sequence analysis

Nucleotide sequences of TrGP recombinants were obtained usingBigDye® Terminator v3.1 Cycle Sequencing Kit in an ABI PRISM 310Genetic Analyzer (Applied Biosystems). Nucleotide and proteinsequence alignments were performed using DNAMAN v. 5.2.2software (Lynon BioSoft). BLAST algorithms were used to searchfor homologous nucleic acid or protein TrGP sequences in GenBankand T. cruzi GeneDB databases, at http://www.ncbi.nlm.nih.govand http://www.genedb.org. Genomic or cDNA sequences wereannotated and submitted to the GenBank (accession nos.:FJ404790–FJ404809). Motif scanning for predicted proteinsequences was performed using the ExPASy proteomic server(http://www.expasy.org).

2.5. Assessing the presence of TrGP copies in the telomere

To determine whether the telomeric location was a com-mon feature in TrGP copies, we designed a simple PCR assaybased on the conserved T. rangeli subtelomeric sequences (SubTr)described by Chiurillo et al. (2002), who using Balb-31 digestionand hybridization experiments showed its exclusive subtelom-eric location. We used vtvF as forward primer, which anneals at3′ end of TrGP copies (VTV motif), and as reverse primer TrF3:5′-CCCCATACAAAACACCCTT-3′ that anneals at SubTr. Sequenceswere amplified in a 50 �l final volume, using 0.4 mM each primer,0.2 mM dNTP, 1.5 mM MgCl2, 1.25 U of Taq Platinum DNA poly-merase (Invitrogen) and the following cycling conditions: 94 ◦C for3 min, followed by 35 cycles of 94 ◦C for 1 min, 57 ◦C for 30 s, 72 ◦Cfor 2 min, and a final elongation at 72 ◦C for 10 min. Amplified prod-ucts were separated in a 0.8% agarose gel, and visualized with UVlight after stain with ethidium bromide. DNA of TrTel-4 recombi-nant (GenBank accession no. AF426022) containing a confirmedTrGP telomeric copy was used as positive control (Chiurillo et al.,2002). Some PCR fragments were purified from agarose gel usingWizard SV Gel, and PCR Clean-Up System (Promega) following themanufacture’s recommendations. Then, the fragments were clonedinto pGEM-T Easy vector (Promega), and transformed into E. colistrain TOP10F’. The nucleotide sequence of these recombinants wasobtained by automatic sequencing.

2.6. Expression and purification of recombinant TrGP

For indirect immunofluorescence microscopy assays we gen-erate antibodies against TrGP. To avoid any cross-reaction withTrSial, the anti-TrGP antibody was prepared from the expres-

sion of a 706 bp fragment of the TrGP-1 gene encoding theN-terminal domain (235 aa) of the putative protein. In a PCRreaction using TrTel-4 as template (Chiurillo et al., 2002), weused a forward 5′-TAGGATCCATGGCCTTTGGCAGTACGGC-3′ andreverse 5′-TCATGGACTCGAGCCCTTTTCCTCTCC-3′ primers, contain-

C.P. Pena et al. / Acta Tropica 111 (2009) 255–262 257

Fig. 1. (A) Comparison of nucleotide sequences of the 5′ UTR region in trgpN recombinants, obtained by RT-PCR using slF primer. Conserved nucleotides are shaded in black(100%), and light gray (>66% identity). The ATG initiation codons are shown in italics. As an example, the nucleotide sequence of the 5′-terminal coding region of a T. cruzigp85/TS family member (GenBank accession no. XM 807627) is also aligned. In this last case, conserved nucleotide with trgpN are indicated with (*) (100%), and (:) (50–75%)s , and ts T. cru( al init

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coverslip and left to air-dry. Preparations were mounted with

ymbols. Alignments were done with DNAMAN v. 5.2.2 (Lynnon Biosoft) softwareignal peptide sequences from members of TrGP gene family and the N-terminal of aBendtsen et al., 2004) in TrGP proteins are enclosed in a light gray box. The potenti

ng BamHI and XhoI restriction sites, respectively. The PCR productas digested with BamHI and XhoI restriction enzymes and for

he expression of a recombinant peptide fused to Schistosomaaponicum glutathione S-transferase (GST), the digested fragment

as cloned into pGEX-5X-2 vector (Amersham Biosciences). Fol-owing electroporation with the TrGP construct, the recombinantrotein, named TrGPNLast, was expressed in E. coli BL21 (DE3)LysS (Invitrogen). After growing the recombinant bacteria in LBedium, protein expression was induced by adding isopropyl--d-thiogalactopyranoside to a final concentration of 1 mM, and

ncubating for 6 h at 37 ◦C. Cells were collected by centrifugationnd the pellet was resuspended in lysis buffer (25 mM Tris–HCl, pH.8; 2 mM MgSO4; 50 mM NaCl, 0.1% Triton X-100, 10 mM lysozyme)lus Set VII protease inhibitors (Calbiochem) and 1 U/ml DNase ICalbiochem), and then incubated for 30 min at 4 ◦C. The lysate wasentrifuged at 10,000 × g for 10 min at 4 ◦C. The recombinant pro-ein was recovered from the pellet and its expression confirmedy SDS-PAGE. Finally, the protein was purified by passive dialysisrom acrylamide strips with 50 mM NaHCO3, 0.1% SDS under con-tant shaking for 24 h at room temperature. Purified fractions wereeanalyzed by SDS-PAGE (MW ∼50-KDa). The same procedure waserformed for GST purification.

.7. Production of anti-TrGP antibodies

Anti-TrGPNLast polyclonal antibodies were obtained by immu-izing New Zealand rabbits with four doses (15 days each) of theurified protein as antigen. Each rabbit received a first dose consist-

ng of 200 �g of the antigen with Freund’s complete adjuvant (1:1).he following doses were 100 �g/rabbit with Freund’s incompletedjuvant.

.8. GPI-anchored proteins analysis

T. rangeli GPI-anchored membrane proteins were isolated usinghe partition Triton X-114 method previously described by Kond Thompson (1995). Rabbit immunization and production ofolyclonal serum against GPI-anchored proteins were carried outccording to Anez-Rojas et al. (2006).

hen manually corrected to include T. cruzi sequences. (B) Alignments of deducedzi gp85/TSA representative. The signal peptide predicted using SignalP 3.0 programiator methionines are indicated by �.

2.9. Western blot analysis

Proteins were resolved by SDS-PAGE and electro-transferred toHybondTM ECL nitrocellulose membranes (Amersham Biosciences).Blots were blocked with a solution of 5% non-fat milk in TBST(50 mM Tris, pH 8.0, 150 mM NaCl, 0.1% Tween-20) and incubatedwith primary antibody (1:5000) for 1 h at room temperature. Then,blots were washed three times for 15 min with TBST and incu-bated with goat anti-rabbit HRP (Calbiochem) (dilution 1:10000)for 1 h. Finally, blots were washed three times again with TBST andimmunodetections were developed using 3,3′ diaminobenzidine assubstrate.

2.10. Indirect immunofluorescence

The immunofluorescence assays were carried out following themethodology described by Figarella et al. (2007) with some modi-fications. Briefly, for each preparation 1 × 106 parasites were fixedwith 4% paraformaldehyde solution and 0.1% glutaraldehyde in PBSovernight at 4 ◦C. The next day parasites were washed with PBSand resuspended in 0.5 ml of blocking solution (100 mM Na2HPO4,100 mM glycine, pH 7.2) for 15 min at room temperature. For per-meabilization, parasites were incubated in 0.5 ml of 0.2% TritonX-100 in PBS during 5 min. Immediately, parasites were washedwith 1% BSA in PBS, and they were incubated overnight at 4 ◦C with200 �l of rabbit anti-TrGPNLast (1:1000). Parasites were washedtwice again with 1% BSA and incubated for 1 h at 4 ◦C in darknesswith a secondary antibody (Alexa Fluor 488 goat anti-rabbit, Invit-rogen) diluted 1:1000. Then, 4′,6-diamidino-2-fenilindol (DAPI,Santa Cruz Biotechnologies) was added to a final concentrationof 0.25 �g/ml and the preparation was incubated for 20 min atroom temperature. Finally, cells were washed once with 1% BSAin PBS and twice with dH2O. Cells were resuspended in 15 �lof dH2O, and 5 �l of the suspension was placed onto a clean

2 �l of Fluoromount-GTM (SouthernBiotech) and examined witha confocal microscope D-Eclipse C1 (Nikon). Preparations with-out primary antibody were used as a negative control. Imageswere processed with software EZ-C1 FreeViewer Silver Version 3.00(Nikon).

258 C.P. Pena et al. / Acta Tropica 111 (2009) 255–262

Fig. 2. (A) Multiple amino acid sequence alignment of three TrGP genes. Alignments were done by Clustal W. Conserved residues are shaded in black (100% conservation)and gray (67% conservation). Continuous line boxes enclose the conserved motifs that characterize proteins of the gp85/TS superfamily. Discontinuous line boxes includethe amino acids sequence of TrGPNLast recombinant protein used to produce anti-TrGP serum. The dotted line marks the C-terminal hydrophilic domain of TrGP-4 deducedprotein. (B) Homology tree representing Clustal W multiple alignment of deduced amino acid sequences from members of group I and II of T. cruzi and T. rangeli TS genesuperfamily. The length of the pathway connecting each pair of nodes roughly indicates the level of dissimilarity between sequences. A rule of homology level is placed on topo -3 (FJ4( ide seG

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f the graph. Sequences are: T. rangeli: TrGP-4 (FJ404803), TrGP-1 (AF426022), TrGPAY186573), TcTS/gp85 (XP820450), TcTS 1 (X57235) and TcTS 2 (L26499). NucleotenBank accession numbers.

. Results

.1. Cloning and sequence analysis of TrGP

Considering that N-terminal region of TrGP-1 has a lower iden-ity with T. cruzi gp85/TS (≤50%), and T. rangeli sialidase (25–30%)

han the full-length translated gene, we decided to characterize sev-ral fragments of this region. All trgpN recombinants correspondedo non-interrupted ORFs (between 450 and 748 bp), and 12 out the4 recombinants showed sequence variations. A GenBank BLASTNearch with trgpN sequences revealed identities between 85 and

044802), TrSial 1 (L14943) and TrSial 2 (U83180); T. cruzi: Tcasp (U77951), TcASP-2quences were analyzed using the DNAMAN version 5.2.2 software. In parenthesis

88% with TrGP-1, and of 50–55% with T. cruzi gp85/TS members. Onthe other hand, when an analysis by BLASTX was conducted, theyshowed 70–80% of identity with TrGP-1, and of 40–45% (55–64%considering similarities) with T. cruzi gp85/TS.

The alignment of amino acid sequences deduced from the groupof trgpN cDNA clones obtained from parasites recovered from the

triatomine haemolymph resulted in an identity of ≥90% amongthem. However, when sequences from cDNA recombinants derivedfrom culture epimastigotes were included in the analysis, the over-all percentage of identity dropped to 67%. The same trend wasobserved at the nucleotide level.

C.P. Pena et al. / Acta Tropica 111 (2009) 255–262 259

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ig. 3. PCR detection of TrGP telomeric copies. (A) Schematic representation of T. ras shown. Discontinuous blocks mean variable sequence length. (B) Agarose gel elecPromega); 2, TrTel-4 recombinant; 3, T. rangeli M/CAN/VE/82/DOG82; 4, T. rangeli M

The analysis of trgpN sequences obtained by RT-PCR using SLerived primer allowed the detection of the 5′ UTR of TrGP genesaverage length: 153 bp). A BLASTN analysis with TrGPs 5′ UTRsevealed a high percentage of identity (80%) with the 5′ regionemarked by the putative first and second methionine codonsresent in most of T. cruzi gp85/TS genes (Fig. 1A). Although in T. cruzip85/TS, it is not possible to assure which one of these two methion-nes is the real initiation codon, the predicted first methionine inrGP corresponds to the second one in T. cruzi gp85/TS (Fig. 1B). Dif-erent from T. cruzi gp85/TS proteins, where no N-terminal signaleptide was predicted, the in silico analysis of the N-terminal regionf T. rangeli TrGP protein family showed this feature (Kozak, 1989).

Three full-length TrGP clones, namely TrGP-2 (2069 bp), TrGP-(2088 bp) and TrGP-4 (2292 bp) were sequenced. The alignment

f these three sequences with TRGP-1 showed 72% identity atucleotide level, which reached 80% when only TrGP-1, 3 and 4ere aligned. The in silico translation of TrGP-2 and one of the

rgpC-cDNA recombinants revealed that they were interrupted byany stop codons in all its possible frames, and therefore they

hould be regarded as a TrGP pseudogenes. A BLASTX search withrGP-3 and TrGP-4 nucleotide sequences resulted in a sequencedentity/similarity of 43–48%/57–63% to T. cruzi gp85/TS members,

eing the highest percentage of identity with the T. cruzi amastig-te surface protein-2 (ASP-2) subfamily (GenBank accession nos.Y186573 and U77951).

As shown in Fig. 2A, the deduced amino acid sequences of TrGP-, 3 and 4 shared many features with all members of gp85/TS

elomere organization. The sense of primers used to amplify TrGP telomeric copiesoresis of amplified products with vtvF and TrF3 primers. Lanes: 1, 1 Kb DNA ladder/Ve/99/CH-99; 5, T. cruzi YBM (M/HOM/VE/92/YBM).

family: the putative N-terminal signal peptide, two highly con-served copies of the sialidase motif SxDxGxTW, a complete copyof the subterminal element VTVxNVfLYNR, the hydrophobic tail,the potential GPI anchor signal sequence, and the absence of manycritical residues for catalytic activity. Within the C-terminal regionof TrGP-4 deduced protein there is an amino acid tandem repeat(TR) composed by seven partially conserved copies of eleven highlyhydrophilic residues (Fig. 2A). Using the translated amino acidsequences of TrGP-3 and TrGP-4, and several members of groupI and II of TS superfamily from T. rangeli and T. cruzi, we did a ClustalW alignment to construct the homology tree shown in Fig. 2B. Thistree shows that although TrGP sequences share the branch with T.cruzi gp85/TS members, they make their own cluster. Sequences ofthe group I of the TS superfamily, both TrSial as TcTS, are groupedat a second branch.

3.2. Presence of TrGP in telomere

PCR reactions combining primers based on the conserved struc-tures of T. rangeli subtelomeric sequences (Chiurillo et al., 2002)and VTV motif of TrGP amplified many fragments (Fig. 3A). Theseamplicons formed a smear from ∼1 to >10 Kb in two T. rangeli

isolates, with some discrete fragments ranging between 1 and2 Kb (Fig. 3B, lanes 3 and 4). This result indicates that TrGPcopies are abundant at T. rangeli’s telomeres, and the differentsize bands can represent the characteristic length polymorphismof the telomeric regions. The PCR fragment obtained with the

260 C.P. Pena et al. / Acta Tropica 111 (2009) 255–262

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ig. 4. (A) Detection of TrGPNLast recombinat protein using anti-GPI-anchored protef T. rangeli permeabilized epimastigotes. (a) DAPI stain of nucleus (n) and kinetoplnti-TrGPNLast antibodies. Fluorescense is observed at the surface membrane and the. rangeli strain: IRHO/Ve/98/Triat-1. (For interpretation of the references to color in

ositive control (lane 2, 960 bp) and the failure of amplificationhen T. cruzi DNA was used as template (lane 5) indicate the high

pecificity of the reaction. When cloned and sequenced, some ofhe most abundant PCR products from TrGP genes displayed theubTr telomeric sequence associated with them, confirming theirelomeric/subtelomeric location. Different to TrGP-1, one of theseragments showed a telomeric TrGP that includes a TGA stop codon

hich coincides with the 3′ end of full-length gp85/TS genes (nothown).

.3. TrGP expression and cellular localization

Since the DNA sequence used to generate the recombinanteptide TrGPNLast has only <20% of identity with TrSial gene, anti-rGPNLast antibodies should be specific for TrGPs. Similar to theeport of Anez-Rojas et al. (2005) with a different antibody gener-ted from the N-terminal region of TrGP-1, when the anti-TrGPNLast

ntibodies were used in Western blot assays against T. rangeli epi-astigotes extracts they detected a ∼73-kDa protein band (not

hown). The size differences between the native TrGP and theeduced protein from the DNA sequences (75–83 kDa) could bexplained by post-translational modifications, and the removal ofhe signal peptide and the hydrophobic tail. When the fused pro-ein TrGPNLast was incubated with an antibody generated againsthe GPI-anchored proteins fraction of T. rangeli, the fused peptide,ut not a recombinant GST fragment, was recognized by these anti-odies (Fig. 4A).

The cellular localization of TrGP proteins in T. rangeli epi-astigotes cells was assessed by immunofluorescense confocal

icroscopy using anti-TrGPNLast antibodies, and in permeabilized

arasites we found that they reacted exclusively with cell surfaceomponents (Fig. 4B). The immunofluorescent label was evenlyistributed over the entire cellular surface of the parasite with aranular appearance, including the flagellar pocket and the flagel-

T. rangeli. Lanes: 1, GST; 2, TrGPNLast. (B) Immunofluorescence microscopy analysisis shown in blue. (b) Immunodetection of TrGP (green) performed with polyclonal

llar pocket of T. rangeli epimastigotes. (c) Overlap of a and b images. Scale bar: 5 �m.gure legend, the reader is referred to the web version of the article.)

lum tip. The same result was observed using non-permeabilizedepimastigotes (data not shown). Preimmune serum did not reactwith these parasites.

4. Discussion

Since the T. rangeli genome sequence has not yet been com-pleted, the analysis by a low throughput sequencing strategies cangenerate valuable information about multigene families encodingsurface antigens. Herein we proved that TrGP constitutes a multi-genic family, and showed for the first time the existence of completecopies of the gp85/TS gene family in T. rangeli’s genome. More-over, the information obtained from 5′ and 3′ UTR regions of TrGPgenes may help to the optimization of T. rangeli gene expressionvectors.

Using a new PCR strategy based on subtelomeric sequences,we also confirmed the presence of more TrGP copies at T. rangeli’stelomeres. In protozoan parasites, telomeres are usually enrichedin contingency genes such as surface antigenic determinants, andin the case of T. cruzi, gp85/TS or related sequences, together withretrotransposon elements, are a structural part of its telomeres(Chiurillo et al., 1999; Kim et al., 2005). Like many trypanoso-matids’s surface protein gene families, the TrGP family containspseudogenes, which through recombination could contribute to theTrGP repertoire variability, a hypothesis that has been proposed toexplain the existence of numerous pseudogenes of gp85/TS in thegenome of T. cruzi (El-Sayed et al., 2005; Azuaje et al., 2007). Onthe other hand, herein we demonstrated that TrGP pseudogenescould be transcribed to mRNA. This fact can be explained by the

polycistronic nature of trypanosomatids transcription (Worthey etal., 2003), however, some evidences indicating a role for pseudo-genes in the control of gene expression in kinetoplastid parasiteshave been reported for other genes (Taylor and Rudenko, 2006;Durand-Dubief et al., 2007).

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C.P. Pena et al. / Acta T

Trypanosomatid parasites have a high prevalence of genes cod-ng for proteins containing TR domains such as TS, mucins, and

ucin-associated surface proteins (MASP), and although TR do notave identical sequences, their hydrophilic properties have beenetained (Goto et al., 2008). Many of the T. cruzi proteins thatave been confirmed as serological antigens have TR domains (Dailveira et al., 2001), and some of them have been implicated invasion of the host immune response, thus contributing to parasiteurvival (Buscaglia et al., 1999; Goto et al., 2008). Considering thathe in silico analysis of TR domain described in TrGP-4 revealedmmunogenic properties and no significant amino acid sequencedentity with those reported in T. cruzi available databases, we couldpeculate that they could be exploited to develop more accurateiagnostic methods to distinguish mixed infection by T. cruzi and T.angeli.

Another interesting finding was the high percentage of iden-ity among cDNA clones from T. rangeli recovered from triatomineaemolymph. Although several forms and stages of the parasiteoexist in this compartment (Anez, 1983; Paláu et al., 2001), ouresults suggest a uniform expression of TrGP proteins. However, weannot rule out a bias caused by the primers used to amplify theseequences.

The high degree of conservation of T. cruzi gp85/TS genes and T.angeli TrGPs suggests that these proteins play an important role inhe parasite’s life cycle. The key question is why T. rangeli needs toxpress gp85/TS proteins when this parasite does not seem to beery effective in invading or multiplying within mammalian cells. In. rangeli vector infections pathological effects are mainly observeduring its multiplication in the haemolymph and hemocytes, andhe invasion of the salivary glands of the triatomine bug (Guhl andallejo, 2003). We can speculate that TrGP proteins may be neces-ary during the migration and multiplication of the parasite throughnd in different triatomine tissues and compartments. In the casef TrSial, it has been proposed that its enzymatic activity may con-titute a mechanism to regulate the attachment of T. rangeli to theector’s salivary glands (Basseri et al., 2002). Nevertheless, sincets natural host is unknown, we cannot discard that TrGP expres-ion may also be implicated in the survival of the parasite in theertebrate host.

cknowledgements

This work was supported by FONACIT grant N◦ S1-2002000542nd CDCHT-UCLA 007-ME-2007. To Mrs. M. E. Camargo for technicalssistance. M.G. Rojas and M. Sayegh for performing DNA automaticequencing. Mrs. Sharon Sumpter for revising the English of the MS.

ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at doi:10.1016/j.actatropica.2009.05.003.

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