Molecular analysis of surface glycoprotein multigene family TrGP expressed on the plasma membrane of Trypanosoma rangeli epimastigotes forms

Acta Tropica 111 (2009) 255–262

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Molecular analysis of surface glycoprotein multigene family TrGP expressed on the plasma membrane of Trypanosoma rangeli epimastigotes forms夽 ˜ a , N. Lander b , E. Rodríguez b , G. Crisante c , N. Anez ˜ c , J.L. Ramírez b , M.A. Chiurillo a,∗ C.P. Pena a b c

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

a r t i c l e

i n f o

Article history: Received 5 February 2009 Received in revised form 12 April 2009 Accepted 5 May 2009 Available online 9 May 2009 Keywords: Trypanosoma rangeli gp85/trans-sialidase Multigenic family TrGP

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 transsialidase superfamily (TrGP). This superfamily, collectively known in T. cruzi as gp85/TS, includes members that are involved in host cell invasion and infectivity. To confirm the presence of this superfamily in the genome of T. rangeli and obtain a better knowledge of its characteristics, we designed a PCR and RT-PCR cloning strategy to allow sequence analysis of both genomic and transcribed copies. We identified two full-length copies of TrGP, some pseudogenes, and N- and C-terminal sequences of several genes. We also analyzed the expression and cellular localization of these proteins in epimastigote forms of a Venezuelan T. rangeli isolate using polyclonal antibodies made against a recombinant peptide from the N-terminal region of a TrGP member. We confirmed that TrGP is a multigenic family that shares many features with T. cruzi gp85/TS, including the telomeric location of some of its members, and by immunofluorescence analysis that its location is at the surface of the parasite. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Trypanosoma rangeli despite being non-pathogenic to humans shares a wide range of vertebrate hosts and triatomine vectors with Trypanosoma cruzi, the etiological agent of Chagas’s disease, and produces serological cross-reactivity with this parasite (Grisard et al., 1999). Furthermore, the morphological similarity of these two parasites, the lack of an appropriate specific diagnostic procedure, and the absence of clinical manifestations, contribute to the underestimation of infections caused by T. rangeli (Guhl and Vallejo, 2003). During the T. rangeli life cycle, the triatomine vectors become infected after feeding with the blood of infected animals. The parasite subsequently replicates within the insect’s gut, and at some point, the epimastigote forms cross the midgut epithelium to reach the haemocoel. Once in the haemolymph, epimastigotes either invade and multiply within hemocytes, or divide as free parasites in the haemolymph. Finally parasites invade and multiply within the salivary glands transforming into infective metacyclic

夽 Note: Nucleotide sequence data reported in this paper are available in the GenBankTM database under the accession numbers FJ404790–FJ404809. ∗ Corresponding author. Tel.: +58 251 2591985; fax: +58 251 2591886. E-mail address: [email protected] (M.A. Chiurillo). 0001-706X/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.actatropica.2009.05.003

tripomastigote forms (Grisard et al., 1999). Recent data based on kDNA and spliced leader (SL) gene markers, indicate a complex parasite–vector relationship, and suggest a co-evolution of the vector with T. rangeli isolates, such that each triatomine species would select the sub-population that can be transmitted to the vertebrate host (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 glycoproteins family named trans-sialidase (TS) superfamily, that according to sequence identity, molecular weight, and function are classified into a variable number of groups by different authors (Colli, 1993; Cross and Takle, 1993; Frasch, 2000), although they can be gathered into two main groups (Frasch, 2000): Group I includes genes encoding 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 enzymes that release sialic acid residues from the host cell surface glycoconjugates (Pontes de Carvalho et al., 1993; Buschiazzo et al., 1997). Group II molecules are devoid of enzymatic activity and include the 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 of identity, characterized by the presence of two conserved neuraminidase motifs: ASP box (SxDxGxTW), and the VTV motif (VTVxNVfLYNR), but lacking critical residues in the FRIP motif (Phe-

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Arg-Ile-Pro) that are important in determining catalytic activity (Colli, 1993; Cross and Takle, 1993; Frasch, 2000; Buschiazzo et al., 2000). In T. cruzi, gp85/TS members are expressed in infective trypomastigotes forms and intracellular amastigotes stages, and by their capacity to adhere to the host cell surface and the extracellular matrix, some of these members are implicated in cell invasion (Magdesian et al., 2001; Yoshida, 2006; Alves and Colli, 2007). Recently, we have described that T. rangeli contains and ˜ expresses genes of gp85/TS superfamily (Anez-Rojas et al., 2005). The first ORF of gp85/TS described in T. rangeli (TrGP-1) was a telomeric truncated copy that conserves the motifs that characterize the family, but lacks the GPI anchor site, and the C-terminal hydropho˜ bic tail (Chiurillo et al., 2002; Anez-Rojas et al., 2005). In this work we have further studied T. rangeli’s gp85/TS members and we have confirmed that they represent a multigenic family. Additionally, we evaluated the expression and cellular localization of these proteins in T. rangeli epimastigotes using immunodetection methods. 2. Materials and methods 2.1. Parasites T. rangeli isolates, provided by Drs. Palmira Guevara from Universidad Central de Venezuela (Caracas, Venezuela), and Nés˜ tor Anez from Universidad de los Andes (Mérida, Venezuela) were cultured in biphasic blood–agar/NNN media at 25 ◦ C. We used T. rangeli Venezuelan strains: M/CAN/VE/82/DOG82 and MHOM/Ve/99/CH-99 for DNA isolation; MHOM/Ve/99/CH-99 for RNA isolation from experimental infections and parasitic cultures; and IRHO/Ve/98/Triat-1 for protein expression experiments. Fourth instar nymphs of Rhodnius prolixus used in this study were obtained from the Centro de Investigaciones Parasitológicas “J.F. Torrealba”, Universidad de los Andes (Mérida, Venezuela). Triatomines were artificially fed with cultures containing exponentially grown T. rangeli. After the infective meal, triatomines were kept at 27 ◦ C, at 70% humidity. Triatomine’s haemolymph was collected by sectioning a leg from the bugs. The presence of flagellates in the collected haemolymph was observed under light microscopy in fresh and Giemsa stained preparations. 2.2. Nucleic acid isolation DNA from T. rangeli culture epimastigote forms was isolated using Wizard Genomic DNA Purification Kit (Promega). Total RNA was purified from epimastigote parasites obtained from culture or infected triatomines using TRIzol reagent (Invitrogen) following manufacturer’s instructions. 2.3. Cloning of TrGP genes by PCR and reverse transcriptase-PCR Genomic DNA or total RNA, from M/CAN/VE/82/DOG82 and MHOM/Ve/99/CH-99 T. rangeli strains, respectively, were used as template in reactions with Platinum Taq DNA Polymerase High Fidelity (Invitrogen), or one-step AccessQuickTM RT-PCR System (Promega), respectively. A set of forward (atgF: 5 -CACGTGCCCAACATGTCCCGGCAT-3 ; atgF2: 5 -ATGGCCTTTGGCAGTACGGC-3 ; slF: 5 -CTAACGCTATTATTGATACAGTTTCTG-3 ), and reverse primers (nR1: 5 -GATGATACCCTCGGCAAGTG-3 ; nR2: 5 -TTTGTTGCCCTTTGCAATTG-3 ; nR3: 5 -GGCCTGCATCACAAATAC-3 ; nR4: 5 TCATGGAGACAAGCCCTTTTC-3 ) were used to clone sequences encoding N-terminal TrGP regions by PCR and RT-PCR reactions. While vtvF: 5 -GTCTTTTTGTACAACCGCCC-3 and oligo-dT: 5 -CCCCCCCCCCCTTTTTTTTTTTTTTTTTTTTT-3 ) primers were employed to clone C-terminal sequences by RT-PCR. We designed atgF, atgF2, nR1, nR2, nR3, nR4 and vtvF primers based on the nucleotide sequence of the TrGP-1 gene (GenBank accession no.

AF426022). slF and oligo-dT primers were designed based on T. rangeli SL sequence (GenBank accession no. M62864) and poly-A tail, respectively. The information of the 3 region of TrGP acquired from the cDNA recombinants obtained by using oligo-dT primer allowed us to design a cR primer (5 -TCCACTGTGCCCCACTCA-3 ), which was used with atgF as forward primer and genomic DNA as template to amplify full-length TrGP gene sequences. The PCR products were then cloned into pGEM-T Easy vector (Promega), and transformed into Escherichia coli strain TOP10F’. According to the portion of TrGP included in the recombinants they were classified in three groups: (1) containing the N-terminal (trgpN); (2) containing the C-terminal (trgpC); and (3) a full-length gene copy. From each group we sequenced and analyzed several clones. 2.4. DNA sequence analysis Nucleotide sequences of TrGP recombinants were obtained using BigDye® Terminator v3.1 Cycle Sequencing Kit in an ABI PRISM 310 Genetic Analyzer (Applied Biosystems). Nucleotide and protein sequence alignments were performed using DNAMAN v. 5.2.2 software (Lynon BioSoft). BLAST algorithms were used to search for homologous nucleic acid or protein TrGP sequences in GenBank and T. cruzi GeneDB databases, at http://www.ncbi.nlm.nih.gov and http://www.genedb.org. Genomic or cDNA sequences were annotated and submitted to the GenBank (accession nos.: FJ404790–FJ404809). Motif scanning for predicted protein sequences 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 common feature in TrGP copies, we designed a simple PCR assay based on the conserved T. rangeli subtelomeric sequences (SubTr) described by Chiurillo et al. (2002), who using Balb-31 digestion and hybridization experiments showed its exclusive subtelomeric location. We used vtvF as forward primer, which anneals at 3 end of TrGP copies (VTV motif), and as reverse primer TrF3: 5 -CCCCATACAAAACACCCTT-3 that anneals at SubTr. Sequences were 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 polymerase (Invitrogen) and the following cycling conditions: 94 ◦ C for 3 min, followed by 35 cycles of 94 ◦ C for 1 min, 57 ◦ C for 30 s, 72 ◦ C for 2 min, and a final elongation at 72 ◦ C for 10 min. Amplified products were separated in a 0.8% agarose gel, and visualized with UV light after stain with ethidium bromide. DNA of TrTel-4 recombinant (GenBank accession no. AF426022) containing a confirmed TrGP telomeric copy was used as positive control (Chiurillo et al., 2002). Some PCR fragments were purified from agarose gel using Wizard SV Gel, and PCR Clean-Up System (Promega) following the manufacture’s recommendations. Then, the fragments were cloned into pGEM-T Easy vector (Promega), and transformed into E. coli strain TOP10F’. The nucleotide sequence of these recombinants was obtained by automatic sequencing. 2.6. Expression and purification of recombinant TrGP For indirect immunofluorescence microscopy assays we generate antibodies against TrGP. To avoid any cross-reaction with TrSial, the anti-TrGP antibody was prepared from the expression of a 706 bp fragment of the TrGP-1 gene encoding the N-terminal domain (235 aa) of the putative protein. In a PCR reaction using TrTel-4 as template (Chiurillo et al., 2002), we used a forward 5 -TAGGATCCATGGCCTTTGGCAGTACGGC-3 and reverse 5 -TCATGGACTCGAGCCCTTTTCCTCTCC-3 primers, contain-

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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. cruzi gp85/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%) symbols. Alignments were done with DNAMAN v. 5.2.2 (Lynnon Biosoft) software, and then manually corrected to include T. cruzi sequences. (B) Alignments of deduced signal peptide sequences from members of TrGP gene family and the N-terminal of a T. cruzi gp85/TSA representative. The signal peptide predicted using SignalP 3.0 program (Bendtsen et al., 2004) in TrGP proteins are enclosed in a light gray box. The potential initiator methionines are indicated by 夽.

ing BamHI and XhoI restriction sites, respectively. The PCR product was digested with BamHI and XhoI restriction enzymes and for the expression of a recombinant peptide fused to Schistosoma japonicum glutathione S-transferase (GST), the digested fragment was cloned into pGEX-5X-2 vector (Amersham Biosciences). Following electroporation with the TrGP construct, the recombinant protein, named TrGPNLast , was expressed in E. coli BL21 (DE3) pLysS (Invitrogen). After growing the recombinant bacteria in LB medium, protein expression was induced by adding isopropyl␤-d-thiogalactopyranoside to a final concentration of 1 mM, and incubating for 6 h at 37 ◦ C. Cells were collected by centrifugation and the pellet was resuspended in lysis buffer (25 mM Tris–HCl, pH 7.8; 2 mM MgSO4 ; 50 mM NaCl, 0.1% Triton X-100, 10 mM lysozyme) plus Set VII protease inhibitors (Calbiochem) and 1 U/ml DNase I (Calbiochem), and then incubated for 30 min at 4 ◦ C. The lysate was centrifuged at 10,000 × g for 10 min at 4 ◦ C. The recombinant protein was recovered from the pellet and its expression confirmed by SDS-PAGE. Finally, the protein was purified by passive dialysis from acrylamide strips with 50 mM NaHCO3 , 0.1% SDS under constant shaking for 24 h at room temperature. Purified fractions were reanalyzed by SDS-PAGE (MW ∼50-KDa). The same procedure was performed for GST purification.

2.7. Production of anti-TrGP antibodies Anti-TrGPNLast polyclonal antibodies were obtained by immunizing New Zealand rabbits with four doses (15 days each) of the purified protein as antigen. Each rabbit received a first dose consisting of 200 ␮g of the antigen with Freund’s complete adjuvant (1:1). The following doses were 100 ␮g/rabbit with Freund’s incomplete adjuvant.

2.8. GPI-anchored proteins analysis T. rangeli GPI-anchored membrane proteins were isolated using the partition Triton X-114 method previously described by Ko and Thompson (1995). Rabbit immunization and production of polyclonal serum against GPI-anchored proteins were carried out ˜ according to Anez-Rojas et al. (2006).

2.9. Western blot analysis Proteins were resolved by SDS-PAGE and electro-transferred to HybondTM 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 incubated with primary antibody (1:5000) for 1 h at room temperature. Then, blots were washed three times for 15 min with TBST and incubated with goat anti-rabbit HRP (Calbiochem) (dilution 1:10000) for 1 h. Finally, blots were washed three times again with TBST and immunodetections were developed using 3,3 diaminobenzidine as substrate.

2.10. Indirect immunofluorescence The immunofluorescence assays were carried out following the methodology described by Figarella et al. (2007) with some modifications. Briefly, for each preparation 1 × 106 parasites were fixed with 4% paraformaldehyde solution and 0.1% glutaraldehyde in PBS overnight at 4 ◦ C. The next day parasites were washed with PBS and resuspended in 0.5 ml of blocking solution (100 mM Na2 HPO4 , 100 mM glycine, pH 7.2) for 15 min at room temperature. For permeabilization, parasites were incubated in 0.5 ml of 0.2% Triton X-100 in PBS during 5 min. Immediately, parasites were washed with 1% BSA in PBS, and they were incubated overnight at 4 ◦ C with 200 ␮l of rabbit anti-TrGPNLast (1:1000). Parasites were washed twice again with 1% BSA and incubated for 1 h at 4 ◦ C in darkness with a secondary antibody (Alexa Fluor 488 goat anti-rabbit, Invitrogen) diluted 1:1000. Then, 4 ,6-diamidino-2-fenilindol (DAPI, Santa Cruz Biotechnologies) was added to a final concentration of 0.25 ␮g/ml and the preparation was incubated for 20 min at room temperature. Finally, cells were washed once with 1% BSA in PBS and twice with dH2 O. Cells were resuspended in 15 ␮l of dH2 O, and 5 ␮l of the suspension was placed onto a clean coverslip and left to air-dry. Preparations were mounted with 2 ␮l of Fluoromount-GTM (SouthernBiotech) and examined with a confocal microscope D-Eclipse C1 (Nikon). Preparations without primary antibody were used as a negative control. Images were processed with software EZ-C1 FreeViewer Silver Version 3.00 (Nikon).

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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 include the 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 deduced protein. (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 gene superfamily. 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 top of the graph. Sequences are: T. rangeli: TrGP-4 (FJ404803), TrGP-1 (AF426022), TrGP-3 (FJ4044802), TrSial 1 (L14943) and TrSial 2 (U83180); T. cruzi: Tcasp (U77951), TcASP-2 (AY186573), TcTS/gp85 (XP820450), TcTS 1 (X57235) and TcTS 2 (L26499). Nucleotide sequences were analyzed using the DNAMAN version 5.2.2 software. In parenthesis GenBank accession numbers.

3. Results 3.1. Cloning and sequence analysis of TrGP Considering that N-terminal region of TrGP-1 has a lower identity with T. cruzi gp85/TS (≤50%), and T. rangeli sialidase (25–30%) than the full-length translated gene, we decided to characterize several fragments of this region. All trgpN recombinants corresponded to non-interrupted ORFs (between 450 and 748 bp), and 12 out the 14 recombinants showed sequence variations. A GenBank BLASTN search with trgpN sequences revealed identities between 85 and

88% with TrGP-1, and of 50–55% with T. cruzi gp85/TS members. On the other hand, when an analysis by BLASTX was conducted, they showed 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 group of trgpN cDNA clones obtained from parasites recovered from the triatomine haemolymph resulted in an identity of ≥90% among them. However, when sequences from cDNA recombinants derived from culture epimastigotes were included in the analysis, the overall percentage of identity dropped to 67%. The same trend was observed at the nucleotide level.

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Fig. 3. PCR detection of TrGP telomeric copies. (A) Schematic representation of T. rangeli telomere organization. The sense of primers used to amplify TrGP telomeric copies is shown. Discontinuous blocks mean variable sequence length. (B) Agarose gel electrophoresis of amplified products with vtvF and TrF3 primers. Lanes: 1, 1 Kb DNA ladder (Promega); 2, TrTel-4 recombinant; 3, T. rangeli M/CAN/VE/82/DOG82; 4, T. rangeli MHOM/Ve/99/CH-99; 5, T. cruzi YBM (M/HOM/VE/92/YBM).

The analysis of trgpN sequences obtained by RT-PCR using SL derived primer allowed the detection of the 5 UTR of TrGP genes (average length: 153 bp). A BLASTN analysis with TrGPs 5 UTRs revealed a high percentage of identity (80%) with the 5 region demarked by the putative first and second methionine codons present in most of T. cruzi gp85/TS genes (Fig. 1A). Although in T. cruzi gp85/TS, it is not possible to assure which one of these two methionines is the real initiation codon, the predicted first methionine in TrGP corresponds to the second one in T. cruzi gp85/TS (Fig. 1B). Different from T. cruzi gp85/TS proteins, where no N-terminal signal peptide was predicted, the in silico analysis of the N-terminal region of T. rangeli TrGP protein family showed this feature (Kozak, 1989). Three full-length TrGP clones, namely TrGP-2 (2069 bp), TrGP3 (2088 bp) and TrGP-4 (2292 bp) were sequenced. The alignment of these three sequences with TRGP-1 showed 72% identity at nucleotide level, which reached 80% when only TrGP-1, 3 and 4 were aligned. The in silico translation of TrGP-2 and one of the trgpC-cDNA recombinants revealed that they were interrupted by many stop codons in all its possible frames, and therefore they should be regarded as a TrGP pseudogenes. A BLASTX search with TrGP-3 and TrGP-4 nucleotide sequences resulted in a sequence identity/similarity of 43–48%/57–63% to T. cruzi gp85/TS members, being the highest percentage of identity with the T. cruzi amastigote surface protein-2 (ASP-2) subfamily (GenBank accession nos. AY186573 and U77951). As shown in Fig. 2A, the deduced amino acid sequences of TrGP1, 3 and 4 shared many features with all members of gp85/TS

family: the putative N-terminal signal peptide, two highly conserved copies of the sialidase motif SxDxGxTW, a complete copy of the subterminal element VTVxNVfLYNR, the hydrophobic tail, the potential GPI anchor signal sequence, and the absence of many critical residues for catalytic activity. Within the C-terminal region of TrGP-4 deduced protein there is an amino acid tandem repeat (TR) composed by seven partially conserved copies of eleven highly hydrophilic residues (Fig. 2A). Using the translated amino acid sequences of TrGP-3 and TrGP-4, and several members of group I and II of TS superfamily from T. rangeli and T. cruzi, we did a Clustal W alignment to construct the homology tree shown in Fig. 2B. This tree shows that although TrGP sequences share the branch with T. cruzi gp85/TS members, they make their own cluster. Sequences of the group I of the TS superfamily, both TrSial as TcTS, are grouped at a second branch. 3.2. Presence of TrGP in telomere PCR reactions combining primers based on the conserved structures of T. rangeli subtelomeric sequences (Chiurillo et al., 2002) and VTV motif of TrGP amplified many fragments (Fig. 3A). These amplicons formed a smear from ∼1 to >10 Kb in two T. rangeli isolates, with some discrete fragments ranging between 1 and 2 Kb (Fig. 3B, lanes 3 and 4). This result indicates that TrGP copies are abundant at T. rangeli’s telomeres, and the different size bands can represent the characteristic length polymorphism of the telomeric regions. The PCR fragment obtained with the

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Fig. 4. (A) Detection of TrGPNLast recombinat protein using anti-GPI-anchored proteins of T. rangeli. Lanes: 1, GST; 2, TrGPNLast . (B) Immunofluorescence microscopy analysis of T. rangeli permeabilized epimastigotes. (a) DAPI stain of nucleus (n) and kinetoplast (k) is shown in blue. (b) Immunodetection of TrGP (green) performed with polyclonal anti-TrGPNLast antibodies. Fluorescense is observed at the surface membrane and the flagellar pocket of T. rangeli epimastigotes. (c) Overlap of a and b images. Scale bar: 5 ␮m. T. rangeli strain: IRHO/Ve/98/Triat-1. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

positive control (lane 2, 960 bp) and the failure of amplification when T. cruzi DNA was used as template (lane 5) indicate the high specificity of the reaction. When cloned and sequenced, some of the most abundant PCR products from TrGP genes displayed the SubTr telomeric sequence associated with them, confirming their telomeric/subtelomeric location. Different to TrGP-1, one of these fragments showed a telomeric TrGP that includes a TGA stop codon which coincides with the 3 end of full-length gp85/TS genes (not shown). 3.3. TrGP expression and cellular localization Since the DNA sequence used to generate the recombinant peptide TrGPNLast has only <20% of identity with TrSial gene, antiTrGPNLast antibodies should be specific for TrGPs. Similar to the ˜ report of Anez-Rojas et al. (2005) with a different antibody generated from the N-terminal region of TrGP-1, when the anti-TrGPNLast antibodies were used in Western blot assays against T. rangeli epimastigotes extracts they detected a ∼73-kDa protein band (not shown). The size differences between the native TrGP and the deduced protein from the DNA sequences (75–83 kDa) could be explained by post-translational modifications, and the removal of the signal peptide and the hydrophobic tail. When the fused protein TrGPNLast was incubated with an antibody generated against the GPI-anchored proteins fraction of T. rangeli, the fused peptide, but not a recombinant GST fragment, was recognized by these antibodies (Fig. 4A). The cellular localization of TrGP proteins in T. rangeli epimastigotes cells was assessed by immunofluorescense confocal microscopy using anti-TrGPNLast antibodies, and in permeabilized parasites we found that they reacted exclusively with cell surface components (Fig. 4B). The immunofluorescent label was evenly distributed over the entire cellular surface of the parasite with a granular appearance, including the flagellar pocket and the flagel-

lum tip. The same result was observed using non-permeabilized epimastigotes (data not shown). Preimmune serum did not react with these parasites. 4. Discussion Since the T. rangeli genome sequence has not yet been completed, the analysis by a low throughput sequencing strategies can generate valuable information about multigene families encoding surface antigens. Herein we proved that TrGP constitutes a multigenic family, and showed for the first time the existence of complete copies of the gp85/TS gene family in T. rangeli’s genome. Moreover, the information obtained from 5 and 3 UTR regions of TrGP genes may help to the optimization of T. rangeli gene expression vectors. Using a new PCR strategy based on subtelomeric sequences, we also confirmed the presence of more TrGP copies at T. rangeli’s telomeres. In protozoan parasites, telomeres are usually enriched in contingency genes such as surface antigenic determinants, and in the case of T. cruzi, gp85/TS or related sequences, together with retrotransposon elements, are a structural part of its telomeres (Chiurillo et al., 1999; Kim et al., 2005). Like many trypanosomatids’s surface protein gene families, the TrGP family contains pseudogenes, which through recombination could contribute to the TrGP repertoire variability, a hypothesis that has been proposed to explain the existence of numerous pseudogenes of gp85/TS in the genome of T. cruzi (El-Sayed et al., 2005; Azuaje et al., 2007). On the other hand, herein we demonstrated that TrGP pseudogenes could be transcribed to mRNA. This fact can be explained by the polycistronic nature of trypanosomatids transcription (Worthey et al., 2003), however, some evidences indicating a role for pseudogenes in the control of gene expression in kinetoplastid parasites have been reported for other genes (Taylor and Rudenko, 2006; Durand-Dubief et al., 2007).

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Trypanosomatid parasites have a high prevalence of genes coding for proteins containing TR domains such as TS, mucins, and mucin-associated surface proteins (MASP), and although TR do not have identical sequences, their hydrophilic properties have been retained (Goto et al., 2008). Many of the T. cruzi proteins that have been confirmed as serological antigens have TR domains (Da Silveira et al., 2001), and some of them have been implicated in evasion of the host immune response, thus contributing to parasite survival (Buscaglia et al., 1999; Goto et al., 2008). Considering that the in silico analysis of TR domain described in TrGP-4 revealed immunogenic properties and no significant amino acid sequence identity with those reported in T. cruzi available databases, we could speculate that they could be exploited to develop more accurate diagnostic methods to distinguish mixed infection by T. cruzi and T. rangeli. Another interesting finding was the high percentage of identity among cDNA clones from T. rangeli recovered from triatomine haemolymph. Although several forms and stages of the parasite ˜ coexist in this compartment (Anez, 1983; Paláu et al., 2001), our results suggest a uniform expression of TrGP proteins. However, we cannot rule out a bias caused by the primers used to amplify these sequences. The high degree of conservation of T. cruzi gp85/TS genes and T. rangeli TrGPs suggests that these proteins play an important role in the parasite’s life cycle. The key question is why T. rangeli needs to express gp85/TS proteins when this parasite does not seem to be very effective in invading or multiplying within mammalian cells. In T. rangeli vector infections pathological effects are mainly observed during its multiplication in the haemolymph and hemocytes, and the invasion of the salivary glands of the triatomine bug (Guhl and Vallejo, 2003). We can speculate that TrGP proteins may be necessary during the migration and multiplication of the parasite through and in different triatomine tissues and compartments. In the case of TrSial, it has been proposed that its enzymatic activity may constitute a mechanism to regulate the attachment of T. rangeli to the vector’s salivary glands (Basseri et al., 2002). Nevertheless, since its natural host is unknown, we cannot discard that TrGP expression may also be implicated in the survival of the parasite in the vertebrate host. Acknowledgements This work was supported by FONACIT grant N◦ S1-2002000542 and CDCHT-UCLA 007-ME-2007. To Mrs. M. E. Camargo for technical assistance. M.G. Rojas and M. Sayegh for performing DNA automatic sequencing. Mrs. Sharon Sumpter for revising the English of the MS. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.actatropica.2009.05.003. References Alves, M.J.M., Colli, W., 2007. Trypanosoma cruzi: adhesion to the host cell and intracellular survival. IUBMB Life 59, 274–279. ˜ Anez, N., 1983. Studies on Trypanosoma rangeli Tejera 1920. VI. Developmental pattern in the haemolymph of Rhodnius prolixus. Mem. Inst. Oswaldo Cruz 78, 413–419. ˜ ˜ Anez-Rojas, N., García-Lugo, P., Crisante, G., Rojas, A., Anez, N., 2006. Isolation, purification and characterization of GPI-anchored membrana proteins from Trypanosoma rangeli and Trypanosoma cruzi. Acta Trop. 97, 140–145. ˜ ˜ Anez-Rojas, N., Peralta, A., Crisante, G., Rojas, A., Anez, N., Ramírez, J.L., Chiurillo, M.A., 2005. Trypanosoma rangeli expressed a gene of the group II trans-sialidase superfamily. Mol. Biochem. Parasitol. 142, 133–136. Azuaje, F.J., Ramirez, J.L., Franco Da Silveira, J., 2007. In silico, biologically-inspired modelling of genomic variation generation in surface proteins of Trypanosoma cruzi. Kinetoplastid Biol. Dis. 6, 6.

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Basseri, H.R., Tew, I.F., Ratcliffe, N.A., 2002. Identification and distribution of carbohydrate moieties on the salivary glands of Rhodnius prolixus and their possible involvement in attachment/invasion by Trypanosoma rangeli. Exp. Parasitol. 100, 226–234. Bendtsen, J.D., Nielsen, H., von Heijne, G., Brunak, S., 2004. Improved prediction of signal peptides: SignalP 3.0. J. Mol. Biol. 340, 783–795. Buscaglia, C.A., Alfonso, J., Campetella, O., Frasch, A.C., 1999. Tandem amino acid repeats from Trypanosoma cruzi shed antigens increase the half-life of proteins in blood. Blood 93, 2025–2032. Buschiazzo, A., Tavares, G.A., Campetella, O., Spinelli, S., Cremona, M.L., Paris, G., Amaya, M.F., Frasch, A.C.C., Alzari, P.M., 2000. Structural basis of sialyltransferase activity in trypanosomal sialidases. EMBO J. 19, 16–24. Buschiazzo, A., Campetella, O., Frasch, A.C.C., 1997. Trypanosoma rangeli sialidase: cloning, expression and similarity to T. cruzi trans-sialidase. Glycobiology 7, 1167–1173. Chiurillo, M.A., Cano, I., Da Silveira, J.F., Ramirez, J.L., 1999. Organization of telomeric and sub-telomeric regions of chromosomes from the protozoan parasite Trypanosoma cruzi. Mol. Biochem. Parasitol. 100, 173–183. Chiurillo, M.A., Peralta, A., Ramírez, J.L., 2002. Comparative study of Trypanosoma rangeli and T. cruzi telomeres. Mol. Biochem. Parasitol. 120, 305–308. Colli, W., 1993. Trans-sialidase: a unique enzyme activity discovered in the protozoan Trypanosoma cruzi. FASEB J. 7, 1257–1264. Cross, G.A.M., Takle, G.B., 1993. The surface trans-sialidase family of Trypanosoma cruzi. Ann. Rev. Microbiol. 47, 385–411. Da Silveira, J.F., Umezawa, E.S., Luquetti, A.O., 2001. Chagas disease: recombinant Trypanosoma cruzi antigens for serological diagnosis. Trends Parasitol. 17, 286– 291. Durand-Dubief, M., Absalon, S., Menzer, L., Ngwabyt, S., Ersfeld, K., Bastin, P., 2007. The argonaute protein TbAGO1 contributes to large and minichromosome segregation and is required for control of RIME retroposons and RHS pseudogene-associated transcripts. Mol. Biochem. Parasitol. 156, 144–153. El-Sayed, N.M., Myler, P.J., Bartholomeu, D.C., Nilsson, D., Aggarwal, G., Tran, A.N., Ghedin, E., Worthey, E.A., Delcher, A.L., Blandin, G., Westenberger, S.J., Caler, E., Cerqueira, G.C., Branche, C., Haas, B., Anupama, A., Arner, E., Aslund, L., Attipoe, P., Bontempi, E., Bringaud, F., Burton, P., Cadag, E., Campbell, D.A., Carrington, M., Crabtree, J., Darban, H., da Silveira, J.F., de Jong, P., Edwards, K., Englund, P.T., Fazelina, G., Feldblyum, T., Ferella, M., Frasch, A.C., Gull, K., Horn, D., Hou, L., Huang, Y., Kindlund, E., Klingbeil, M., Kluge, S., Koo, H., Lacerda, D., Levin, M.J., Lorenzi, H., Louie, T., Machado, C.R., McCulloch, R., McKenna, A., Mizuno, Y., Mottram, J.C., Nelson, S., Ochaya, S., Osoegawa, K., Pai, G., Parsons, M., Pentony, M., Pettersson, U., Pop, M., Ramirez, J.L., Rinta, J., Robertson, L., Salzberg, S.L., Sanchez, D.O., Seyler, A., Sharma, R., Shetty, J., Simpson, A.J., Sisk, E., Tammi, M.T., Tarleton, R., Teixeira, S., Van Aken, S., Vogt, C., Ward, P.N., Wickstead, B., Wortman, J., White, O., Fraser, C.M., Stuart, K.D., Andersson, B., 2005. The genome sequence of Trypanosoma cruzi, etiologic agent of Chagas disease. Science 309, 409– 415. Figarella, K., Uzcategui, N., Zhou, Y., LeFurgey, A., Ouellette, M., Bhattacharjee, H., Mukhopadhyay, R., 2007. Biochemical characterization of Leishmania major aquaglyceroporin LmAQP1: possible role in volume regulation and osmotaxis. Mol. Microbiol. 65, 1006–1017. Frasch, A.C.C., 2000. Functional diversity in the trans-sialidase and mucin families in Trypanosoma cruzi. Parasitol. Today 16, 282–286. Goto, Y., Darrick, C., Reed, S.G., 2008. Immunological dominance of Trypanosoma cruzi tandem repeat proteins. Infect. Immun. 76, 3967–3974. Grisard, E.C., Steindel, M., Guarnieri, A.A., Eger-Mangrich, I., Campbell, D.A., Romanha, A.J., 1999. Characterization of Trypanosoma rangeli strains isolated in Central and South America: overview. Mem. Inst. Oswaldo Cruz 94, 203– 209. Guhl, F., Vallejo, G.A., 2003. Trypanosoma (Herpetosoma) rangeli Tejera 1920: an updated review. Mem. Inst. Oswaldo Cruz 98, 435–442. Kim, D., Chiurillo, M.A., El-Sayed, N., Jones, K., Santos, M.R., Porcile, P.E., Andersson, B., Myler, P., da Silveira, J.F., Ramírez, J.L., 2005. Telomere and subtelomere of Trypanosoma cruzi chromosomes are enriched in (pseudo)genes of retrotransposon hot spot and trans-sialidase-like gene families: the origins of T. cruzi telomeres. Gene 346, 153–161. Ko, Y., Thompson, G., 1995. Purification of glycosylphosphatidylinositol anchored proteins by modified Triton X-114 partitioning and preparative gel electrophoresis. Anal. Biochem. 224, 166–172. Kozak, M., 1989. The scanning model for translation: an update. J. Cell. Biol. 108, 229–241. Magdesian, M.H., Giordano, R., Ulrich, H., Juliano, M.A., Juliano, L., Schumacher, R.I., Colli, W., Alves, M.J.M., 2001. Infection by Trypanosoma cruzi: identification of a parasite ligand and its host-cell receptor. J. Biol. Chem. 276, 19382– 19389. Maia Da Silva, F., Junqueira, A.C., Campaner, M., Rodrigues, A.C., Crisante, G., Ramirez, ˜ L.E., Caballero, Z.C., Monteiro, F.A., Coura, J.R., Anez, N., Teixeira, M.M., 2007. Comparative phylogeography of Trypanosoma rangeli and Rhodnius (Hemiptera: Reduviidae) supports a long coexistence of parasite lineages and their sympatric vectors. Mol. Ecol. 16, 3361–3373. ˜ Paláu, M.T., Montilla, M., Zúniga, C.A., 2001. Estudio del comportamiento de dos cepas de Trypanosoma rangeli. MedUNAB 4, 166–172. Pontes de Carvalho, L.C., Tomlinson, S., Nussenzweig, V., 1993. Trypanosoma rangeli sialidase lacks trans-sialidase activity. Mol. Biochem. Parasitol. 62, 19– 26. Taylor, J.E., Rudenko, G., 2006. Switching trypanosome coats: what’s in the wardrobe? Trends Genet. 22, 614–620.

262

C.P. Pe˜ na et al. / Acta Tropica 111 (2009) 255–262

Vallejo, G.A., Guhl, F., Carranza, J.C., Moreno, J., Triana, O., Grisard, E.C., 2003. Parity between kinetoplast DNA and mini-exon gene sequences supports either clonal evolution or speciation in Trypanosoma rangeli strains isolated from Rhodnius colombiensis, R. pallescens and R. prolixus in Colombia. Infect. Genet. Evol. 3, 39–45. Worthey, E.A., Martinez-Calvillo, S., Schnaufer, A., Aggarwal, G., Cawthra, J., Fazelinia, G., Fong, C., Fu, G., Hassebrock, M., Hixson, G., Ivens, A.C., Kiser, P., Mar-

solini, F., Rickel, E., Salavati, R., Sisk, E., Sunkin, S.M., Stuart, K.D., Myler, P.J., 2003. Leishmania major chromosome 3 contains two long convergent polycistronic gene clusters separated by a tRNA gene. Nucleic Acids Res. 31, 4201– 4210. Yoshida, N., 2006. Molecular basis of mammalian cell invasion by Trypanosoma cruzi. An. Acad. Bras. Cienc. 78, 87–111.