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Peña Et Al., 2009 PDF
Peña Et Al., 2009 PDF
Peña Et Al., 2009 PDF
Acta Tropica
journal homepage: www.elsevier.com/locate/actatropica
Laboratorio de Gentica Molecular Dr. Yunis-Turbay, Decanato de Ciencias de la Salud, Universidad Centroccidental Lisandro Alvarado, Barquisimeto, Venezuela
Centro de Biotecnologa, Fundacin Instituto de Estudios Avanzados, Caracas, Venezuela
Centro de Investigaciones Parasitolgicas J.F. Torrealba, Universidad de los Andes, Mrida, 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 hemoagelate 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 conrm 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 identied 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 conrmed 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 immunouorescence
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 Chagass 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 specic 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 insects 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
Note: Nucleotide sequence data reported in this paper are available in the
GenBankTM database under the accession numbers FJ404790FJ404809.
Corresponding author. Tel.: +58 251 2591985; fax: +58 251 2591886.
E-mail address: mchiurillo@ucla.edu.ve (M.A. Chiurillo).
0001-706X/$ see front matter 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.actatropica.2009.05.003
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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 (:) (5075%)
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 .
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 nal 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 TrisHCl, 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 conrmed
by SDS-PAGE. Finally, the protein was puried by passive dialysis
from acrylamide strips with 50 mM NaHCO3 , 0.1% SDS under constant shaking for 24 h at room temperature. Puried fractions were
reanalyzed by SDS-PAGE (MW 50-KDa). The same procedure was
performed for GST purication.
according to Anez-Rojas
et al. (2006).
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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 (2530%)
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
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259
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 amplied 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).
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 amplied 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. rangelis 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) Immunouorescence 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 agellar 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 gure legend, the reader is referred to the web version of the article.)
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 (7583 kDa) could be
explained by post-translational modications, 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 immunouorescense confocal
microscopy using anti-TrGPNLast antibodies, and in permeabilized
parasites we found that they reacted exclusively with cell surface
components (Fig. 4B). The immunouorescent label was evenly
distributed over the entire cellular surface of the parasite with a
granular appearance, including the agellar pocket and the agel-
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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 conrmed 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 signicant 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 nding was the high percentage of identity among cDNA clones from T. rangeli recovered from triatomine
haemolymph. Although several forms and stages of the parasite
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