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Immunology. May 2003; 109(1): 147–155.
PMCID: PMC1782932

Peptide-based analysis of the amino acid sequence important to the immunoregulatory function of Trypanosoma cruzi Tc52 virulence factor

Abstract

The intracellular protozoan parasite Trypanosoma cruzi is the aetiological agent of Chagas' disease. We have previously identified a T. cruzi-released protein called Tc52, which is crucial for parasite survival and virulence. In the present study, we attempted to define the Tc52 epitope(s) responsible for its immunoregulatory function. A naturally occurring major peptide fragment of molecular mass 28 kDa (Tc28k) was identified, which was localized in the C-terminal portion of Tc52 and was inhibitory for T-cell activation. Synthetic peptides corresponding to amino acid sequences in Tc52 were evaluated for their ability to modulate T-cell proliferation and cytokine production. Results obtained using five peptides spanning the N-terminal or C-terminal domain of the Tc52 protein indicated that the activity mapped to Tc52 residues 432–445. Moreover, it was found that the peptide, when coupled to a carrier protein (ovalbumin), exhibited increased inhibitory activity on T-lymphocyte activation. Incubation with 8 nm ovalbumin-coupled peptide 432–445 resulted in approximately the same levels (>75%) of inhibition of T-cell proliferation as 5 µg/ml Tc28k. Furthermore, we showed that the coupled peptide significantly down-regulated the secretion of interferon-γ (IFN-γ) and interleukin-2 (IL-2). Likewise, in immunized mice, the coupled peptide 432–445 was a very poor B- and T-cell antigen compared with the other Tc52-derived peptides. These results suggest that the immunomodulatory portion of the T. cruzi Tc52 virulent factor may reside, at least in part, in a conserved sequence within its C-terminal domain, which could minimize its antigenicity.

Introduction

The ability of parasites to survive and multiply in their host often depends upon their capacity to inhibit or suppress the host immune response. Trypanosoma cruzi, the aetiological agent of Chagas' disease, is an obligate intracellular parasite, which causes chronic infections in humans and a large number of other mammalian species.1 This protozoan parasite is transmitted to humans and other vertebrate hosts in the faeces of haematophagous bugs of the Reduviid family. The complex life cycle of T. cruzi includes different stages in the insect vector and the vertebrate host. There are two parasite stages in the vector: epimastigotes and metacyclic trypomastigotes, whereas the vertebrate stages are bloodstream trypomastigotes and intracellular amastigotes. The parasite leaves the phagolysosome shortly after invasion of the host cell and multiplies in the cytosole as an amastigote. Studies have shown that the phagolysosome has an acidic pH and that the parasite secretes an acid-active, transmembrane pore-forming protein implicated in the process of vacuole disruption and parasite escape into the cytoplasm.2,3

Chagas' disease is associated with many immunological and immunopathological reactions. During the chronic phase, parasites are not readily detectable in the circulation, but are sequestered in various tissues.4 The acute phase is associated with an immunosuppression state, believed to facilitate the dissemination of the parasite in the host.5 The impairment of immune response has been attributed to a wide range of mechanisms, including decreased interleukin (IL)-2 production; increased suppressor activity by splenic T cells and macrophages; down-regulation of CD3, CD4, CD8 and T-cell receptors; inhibition of IL-2 receptor (IL-2R) expression; T-cell apoptosis; and increased nitric oxide (NO) production by macrophages.68 Furthermore, the search for parasite-derived molecules that might subvert immune regulation allowed the identification of a number of parasite molecules with potent activities:

  1. The glycosylphosphatidylinositol-anchored mucin-like glycoproteins isolated from T. cruzi trypomastigotes which induced synthesis of IL-12 and tumour necrosis factor-α (TNF-α) by macrophages.9
  2. The T. cruzi antigen molecule SAPA (shed acute-phase antigen), which exhibited a neuraminidase-transsialidase activity that down-regulated T-lymphocyte proliferation as a consequence of T-suppressor/cytotoxic cell activation and secretion of prostaglandin E2 (PGE2), an immunoregulator effector substance.10
  3. A T. cruzi membrane glycoprotein which inhibited the expression of IL-2R chains and secretion of cytokines by subpopulations of activated human T lymphocytes,11 among others.

In the mid-1990s, a T. cruzi-released protein was identified of molecular mass (Mr) 52 kDa (Tc52), composed of two homologous domains and sharing significant homology to glutathione S-transferases.12 In further studies, Tc52 was shown to contribute to immune dysfunction by modulating T-cell and macrophage activation.13,14 Moreover, it was shown that under conditions of experimental infection, Tc52 appears immunologically silent during the early acute phase, failing to elicit antibodies and lymphocyte proliferation.14 Many of the biological properties of Tc52 have been attributed to its capacity to ‘scavenge’ free and macrophage-released cysteine, as well as glutathione, in vitro and in vivo, and through its capacity to modulate cytokine gene expression in macrophages, as well as dendritic cells.14,15 Therefore, the present study was performed using a major Tc52 cleavage fragment and synthetic peptides corresponding to the amino acid sequence of Tc52 molecule to begin to identify the region(s) of Tc52 important to its immunoregulatory function(s).

Materials and methods

Animals

Male BALB/c (H-2d) and CBA (H-2k) mice (5–6 weeks old) were obtained from IFFA-CREDO (Charles River Co., L'Arbresle, France) bred and maintained in a clean conventional colony at the Centre IRD (Montpellier, France), in accordance with the local ethical guidelines.

Immunization and production of antibodies against synthetic peptides

Synthetic peptides derived from the Tc52 primary sequence [amino acids: 41–60 (P1); 65–80 (P2); 115–128 (P3); 192–205 (P4) and 432–445 (P5)], used either coupled (P1, 2, 3 and 5) or uncoupled (P4 and P5) to ovalbumin (OVA), were obtained from Eurogentec (Hestral, Belgium). The mass ratio of peptide to carrier protein was determined from amino acid composition and found to be as follows: 5, 10·3, 1·64 and 14·2 moles of peptide/mole of OVA for P1, P2, P3 and P5, respectively.

Seven-week-old male BALB/c mice were inoculated three times intraperitoneally at 2-week intervals with three 50-µg doses of synthetic peptides (P1, P2, P3 or P5) coupled to OVA in association with either 50 µg of N-acetylmuramyldipeptide (MDP) or 30 µl of Bordetella pertussis and Alum (BpAl) as adjuvant (VAXICOQ; Institut Merieux, Lyon, France: 4 IU of B. pertussis and 1·25 mg of aluminium hydroxide/500 µl). Two weeks after the last immunization, mice were bled from the orbital sinus and their sera were stored at −20° until used. Control sera were obtained from OVA- and adjuvant-immunized mice.

Immunoblotting analysis

Tc52 native protein was purified from parasites, as described previously.15 Briefly, epimastigote (T. cruzi TcY7 clone) soluble extracts obtained by sonication and centrifugation were prepared and adjusted to 5 mg of proteins/ml in buffer [20 mm HEPES, pH 7·25, 1 mm EDTA, 0·15 m KCl supplemented with a 0·5-mm final concentration of phenylmethylsulphonyl fluoride (PMSF)], and passed through S-hexyl glutathione affinity matrix (Sigma, St Louis, MO). The column was then washed with 30 ml of the same buffer, and bound material was eluted using 20 ml of buffer containing 2·5 mm S-hexyl glutathione. All the eluates were dialysed against phosphate-buffered saline (PBS) (10 mm sodium phosphate, pH 7·2, 0·15 m NaCl) and passed through a column containing polymixin B coupled to resin, as previously described.15 The endotoxin level, determined by the Limulus amebocyte assay, was < 0·06 ng/ml.

Native Tc52 was incubated in 10 mm Tris–HCl buffer, of varying pH, from 4 to 7, for 30 min at 37° at a concentration of 1 mg/ml. Aliquots of 10 µg of protein (10 µl) were mixed with 10 µl of sample buffer [62·5 mm Tris–HCl, pH 6·8, containing 3% sodium dodecyl sulphate (SDS), 10% sucrose and 0·005% Bromophenol blue], boiled for 5 min, fractionated by SDS-polyacryamide gel electrophoresis (SDS–PAGE), electroblotted onto nitrocellulose, and then incubated with mouse immune serum to Tc52, OVA-coupled peptides or OVA. Antibody binding was detected with specific peroxidase-labelled anti-mouse immunoglobulin G (IgG) (Diagnostics Pasteur, Marnes-la Coquette, France).

Purification of Tc28k fragment

The Tc28k protein was further purified by preparative electrophoresis by use of 7·5% SDS–PAGE in a Bio-Rad Prep Cell (Bio-Rad, Richmond, CA). The purity of the preparation was controlled by analytical SDS–PAGE and Western blotting using anti-Tc52-specific immune serum. The protein samples were pooled, concentrated by using Centriprep-10 concentrators (Amicon, Beverley, MA), and stored at −80°. Protein used in the in vitro proliferation assays was passed over an Extracti-Gel D column (Pierce Chemical Co., Rockford, IL) to remove SDS. Sequencing of Tc28k peptide was performed using standard methods, as reported previously.16

Cell culture and proliferation assays

After cervical dislocation, the spleens were removed and homogenized in a Petri dish. After two to three washes in RPMI-1640, the cells were adjusted to 107/ml in RPMI-1640, supplemented with 2 mm glutamine (Sigma Chemical Co.), penicillin and streptomycin (100 U/ml and 100 µg/ml, respectively; Sigma Chemical Co), 0·05 mm 2-mercaptoethanol (2-ME), 20 mm HEPES (Gibco BRL, Cergy Pontoise, France) and 10% fetal calf serum (FCS) (Gibco BRL). Spleen cells were cultured in 96-well flat-bottomed plates in a 200-µl volume at a concentration of 2 × 105 cells/well. The cells were stimulated with either 0·1 µg/ml rat IgG2a monoclonal antibody (mAb) to mouse CD3 (Cliniscience, Montrouge, France) or 5 µg/ml of concanavalin A (Con A) in the presence or absence of tested molecules (Tc28k, OVA-coupled or uncoupled synthetic peptides at different concentrations). After 48 hr of incubation at 37° in 5% CO2, 1 µCi of [3H]thymidine (Amersham, Arlington Heights, IL) was added to the wells. Pulsed splenocytes were harvested on a glass filter, using an automated multiple sample harvester, and then dried. Incorporation of radioactive thymidine was determined by liquid scintillation counting, following a standard protocol. Assays were carried out in triplicate and the stimulatory index (SI) was calculated by dividing the arithmetic mean of counts per minute (c.p.m.) obtained from stimulated cultures by the arithmetic mean of c.p.m. obtained from control cultures without stimulation.

In some experiments, we attempted to determine the immunogenicity of P1-, P2-, P3- and P5 peptides coupled to OVA. Groups of three mice were immunized subcutaneously in each hind leg footpad with 100 µg of peptide mixed with 50 µg of MDP and, 10 days later, the draining lymph nodes were removed. Single-cell suspensions were prepared and stimulated with either 10 µg of peptide or 10 µg of Tc52 protein. Anti-CD3 antibodies were used as a positive control.

Cytokine enzyme-linked immunosorbent assays

The production of cytokine was determined by two-site sandwich enzyme-linked immunosorbent assays (ELISAs) in supernatants of spleen cells stimulated with either 0·1 µg/ml anti-CD3 or 5 µg/ml of Con A after 24, 48 and 72 hr of incubation, at 37° under 5% CO2. Ninety-six-well flat-bottomed microtitre plates (Immune Plate Maxisorp; Nunc, Roskilde, Denmark) were coated with unlabelled rat antibodies to the following cytokines: interferon-γ (IFN-γ) (R4-6A2 cell line), IL-2 (JES6-1A12 cell line), IL-4 (BVD4-1D11 cell line) or IL-10 (JES5-2A5 cell line) in carbonate buffer, pH 8·5, overnight at 4°. The plates were washed with PBS containing 0·1% Tween-20 (PBS-T; Calbiochem; San Diego, CA) and blocked with PBS (200 µl/well) containing 1% gelatin for 2 hr at room temperature. The plates were incubated with each supernatant for 2 hr at room temperature. After washing with PBS-T, the plates were incubated for 1 hr at room temperature with biotinylated-labelled rat antibodies to the following cytokines: IFN-γ (XMG1·2 cell line), IL-2 (JES6-5H4 cell line), IL-4 (BVD6-24G2 cell line) or IL-10 (SXC-1 cell line). Antibody pairs specific for interleukins were purchased from PharMingen (San Diego, CA). After washing with PBS-T, the plates were incubated for 1 hr at room temperature with streptavidin-peroxidase (Sigma) and developed with o-phenylenediamine (OPD; Sigma) in citrate buffer. The optical densities were recorded at 450 nm. The concentration of specific interleukins was determined by comparison to a standard curve generated with different recombinant interleukins: recombinant (r)IFN-γ, rIL-2, rIL-4 and rIL-10 (R & D Systems, Abingdon, UK).

Statistic analysis

The data were analysed using the Student's t-test.

Results

Characterization of a 28-kDa Tc52 major cleavage fragment

We have previously shown that Tc52, a T. cruzi-released protein, is composed of two homologous domains [the Tc52 N-terminal region (Tc52N, amino acids: 1–224) and the Tc52 C-proximal region (Tc52C, amino acids: 225–445)] sharing about 27% identity and an additional 27% similarity when substitution of homologous amino acids was included.12 During our studies we observed that Tc52, when incubated at 37° in an acidic pH buffer which corresponds to that encountered by the parasite in the phagolysosome, was split, generating a number of peptides varying in size, a major degradation product being a 28-kDa peptide, named Tc28k (Fig. 1a). When the excised band was applied to an amino acid sequencer, the N-terminal amino acid sequence was determined to be QRTTVKETIXXXE, which coincides completely in its first nine amino acids with the amino acid sequence 193–201 of the Tc52 molecule. A theoretical Mr of 30·240 Da would be expected for the peptide comprising amino acids 193–445 of the Tc52 protein, which is slightly higher than the Mr of Tc28k.

Figure 1
Molecular and immunological characterization of a major Tc52 cleavage fragment (Tc28k). (a) Tc52 native protein samples, dissolved in 10 mm Tris–HCl buffer of increasing pH (lane 1, pH 4; lane 2, pH 5; lane 3, pH 6; and lane 4, pH 7), were reacted ...

Complementary experiments were conducted using mouse antibodies directed against three synthetic peptides (P1, amino acids 41–60; P2, amino acids 65–80; and P3, amino acids 115–128) derived from Tc52N and coupled to OVA. The peptides were selected on the basis of divergent internal sequences within Tc52N and Tc52C. The calculated similarity between P1, P2 and P3 and their Tc52C homologous sequences were 65, 52 and 35%, respectively (Fig. 1b).

As shown in Fig. 1(c), mice immunized with P1 or P2 peptides reacted against Tc52 native protein and its Tc28k cleavage fragment. In contrast, anti-P3-OVA immune serum recognized only the Tc52 native protein. Taking into account the high level of similarity among P1, P2 and their homologues in the Tc52C domain and the low score shared by P3, the profiles of Tc52 recognition by anti-peptide antibodies suggests that Tc28k comprises the C-terminal domain of Tc52 molecule. These observations, together with the sequencing data, suggested that Tc28k, localized within the Tc52C part of the molecule, constitutes its major cleavage product.

Effect of Tc28k on lymphocyte activation

In initial experiments, we studied the effects of Tc28k on the activation of T lymphocytes. Splenocytes from BALB/c mice were cultured with different concentrations of Tc28k in the presence of Con A, and the level of proliferation was determined. As the concentration of Tc28k in culture increased, there was a corresponding decrease in the lymphoproliferative response (Fig. 2). Con A-stimulated [3H]thymidine incorporation was inhibited to approximately 50% at 1 µg/ml Tc28k when compared with the control, virtually almost-complete inhibition was observed at 5 µg/ml. The Tc28k-induced inhibition of lymphocyte activation was not the result of cell toxicity, as cell viability at the end of the culture period (assessed by Trypan Blue dye exclusion) ranged between 75 and 90%. Moreover, the same range of viability was observed when increasing concentrations of Tc28K up to 15 µg/ml were used. Furthermore, the inhibition was not caused by endotoxin because the Tc28k preparation contained endotoxin at < 0·06 ng/ml, and endotoxin itself did not inhibit blastogenesis. Therefore, it is reasonable to assume that the down-regulation of T-cell proliferation by Tc28k is not the result of a toxic effect of the protein on spleen cells.

Figure 2
Proliferative responses of spleen cells from normal BALB/c mice after stimulation with concanavalin A (Con A) or anti-CD3. The cells were cultured for 48 hr (2·5 × 105/well) in the presence of Con A (5 µg/ml) or anti-CD3 (0·1 ...

To rule out the possibility that the inhibition of T-cell proliferation was caused by a cytotoxic effect of the buffer in which the Tc28k was contained, spleen cells from BALB/c mice were stimulated with Con A in the presence of a T. cruzi protein, the Tc24 flagellar calcium-binding protein,17 isolated following the same procedure as Tc28k. Under these experimental conditions, no inhibitory effect on T-cell proliferation occurred (data not shown), suggesting therefore that the buffer in which the proteins were contained have no cytotoxic effects on T cells.

Lymphocyte proliferation in response to anti-CD3 was then used to evaluate if the inhibitory response was a result of the effect of mitogen stimulation on the responding lymphocytes. As shown in Fig. 2, 62% inhibition was observed when the cells were stimulated with anti-CD3 in the presence of 1 µg/ml Tc28k. These results indicate that T-cell inhibition was not dependent on the Con A-mediated response and suggest that Tc28k is an important mediator of the suppressive phenomenon. Furthermore, different preparations of Tc28k were examined in the assay (data not shown). As they were found to have a similar pattern of lymphocyte suppression, for clarity, the data corresponding to a standard preparation of Tc28k are shown in this study.

Identification of a Tc28k peptide sequence carrying the T-cell inhibitory activity

Several reports have successfully demonstrated that synthetic peptides could be used to either induce or down-regulate T-cell responses.18 On account of the inhibitory effect of Tc28k on anti-CD3-induced T-cell proliferation, we thought that synthetic peptides modelled from the sequence of Tc28k would be expected to be capable of modulating the T-cell response. We used computer-assisted epitope analysis [BIMAS (http://bimas.dcrt.nih.gov)] to determine the areas of Tc28k exposed on the surface and thus accessible to the cells. Two predicted sequences were found, and the synthetic oligopeptide derivatives (P4, amino acids 193–226; P5, amino acids 432–445) were tested for inhibitory activity on anti-CD3-induced T-cell activation. As shown in Fig. 3, spleen cells from BALB/c mice were cultured with or without increasing concentrations of P4 or P5 and stimulated by anti-CD3 antibodies. Non-stimulated cells had a low rate of replication and incorporated low amounts of [3H]thymidine into DNA (42 ± 14 c.p.m.). Addition of P4 at concentrations of 3 and 15 µm had no significant effect on T-cell proliferation (SI = 185 ± 26·4 and 190·2 ± 10·5, respectively, when compared with the control SI = 195·5 ± 5·2). In contrast, at 3 µm P5, a significant inhibition (P < 0·05) of anti-CD3-induced T-cell proliferation was seen. No significant inhibition of T-cell proliferation was observed below and above 3-µm concentration of P5. Although these findings were unusual, they were obtained in independent experiments. This apparent unusual feature of P5 peptide needs further investigation.

Figure 3
Proliferative responses of spleen cells from normal BALB/c mice after stimulation with anti-CD3. The cells were cultured for 48 hr (2·5 × 105/well) in the presence of anti-CD3 (0·1 µg/ml), with or without P4 or P5 peptides ...

A number of other synthetic peptides derived from the Tc52 primary sequence (P1, P2 and P3), when used in the assay at different concentrations had no effect on T-cell activation by anti-CD3 antibodies (data not shown). The inhibitory activity of P5 was not caused by a toxic effect of the peptide towards T cells, or to a binding of P5 to anti-CD3 antibodies. The average viability of cells, which were cultured for 48 hr, with or without P5, was > 85%, as assessed by Trypan Blue dye-exclusion testing, and no binding of anti-CD3 antibodies to P5 or P5-OVA was observed (see below). Furthermore, the inhibitory activity of P5 was also observed in the case of spleen cells from CBA mice (data not shown).

Although P5 seems to have an inhibitory effect at low concentrations, we hypothesized that coupling of P5 to a carrier protein, such as OVA, might confer upon peptide some tertiary structure required for efficient biological activity. In order to evaluate this concept, spleen cells from BALB/c mice were stimulated with Con A in the presence of 4 or 8 nm P5-OVA. As shown in Fig. 4, P5-OVA significantly suppressed Con A-induced T-cell proliferation, the inhibitory effect being more efficient at an 8 nm concentration of P5-OVA. A similar inhibitory effect was observed when using anti-CD3 mAb (Fig. 4).

Figure 4
Proliferative responses of spleen cells from normal BALB/c mice after stimulation with concanavalin A (Con A) or anti-CD3. The cells were cultured for 48 hr (2·5 × 105/well) in the presence of Con A (5 µg/ml) or anti-CD3 (0·1 ...

Previous studies have shown that the degree of resistance to T. cruzi varied among mice strains.19 Therefore, we examined the effect of P5-OVA on spleen cells from CBA mice. As shown in Fig. 5, P5-OVA at a concentration of 8 nm significantly (P < 0·05) suppressed spleen T-cell proliferation induced by Con A or anti-CD3 mAb. Controls consisting of P1-, P2- and P3-OVA did not inhibit Con A-induced T-cell proliferation (SI = 182 ± 23, 198 ± 25 and 195 ± 35, respectively, compared with the control spleen cells stimulated with Con A in the absence of coupled peptides: SI = 201 ± 22). Therefore, the biological activity of P5-OVA is not strain-dependent. These results suggest that the inhibitory activity of Tc52 could be linked to the P5 sequence. Coupling to OVA, however, seems to confer to the peptide some structural requirement necessary for the expression of its inhibitory activity.

Figure 5
Proliferative responses of spleen cells from normal CBA mice after stimulation with concanavalin A (Con A) and anti-CD3. The cells were cultured for 48 hr (2·5 × 105/well) in the presence of Con A (5 µg/ml) or anti-CD3 (0·1 ...

P5-OVA down-regulates the cytokine-producing capability of spleen cells

Complementary investigations were carried out in order to determine the effect of P5-OVA on the cytokine production by spleen cells upon stimulation with anti-CD3 or Con A. The levels of IFN-γ, IL-2, IL-10 and IL-4 were determined in the supernatants of spleen cells (2 × 105 cells/well) from normal BALB/c or CBA mice cultured for 24, 48 and 72 hr, respectively. We determined, by ELISA, the levels of these cytokines with reference to a standard curve calculated using recombinant cytokines. As shown in Table 1 and Table 2, although a certain degree of inhibition of IL-10 secretion could be seen, P5-OVA at 8 nm induced a highly significant reduction of IFN-γ and IL-2 production by spleen cells from BALB/c mice and CBA mice when compared with non-treated cells (P < 0001).

Table 1
P5-coupled ovalbumin (P5-OVA) down-regulates the cytokine-producing capability of spleen cells from normal BALB/c mice
Table 2
P5-coupled ovalbumin (P5-OVA) down-regulates the cytokine-producing capability of spleen cells from normal CBA mice

Immunogenicity of OVA-coupled peptides

We have already shown that during experimental infection, the Tc52 native protein appears immunologically silent, failing to elicit antibody response and lymphocyte proliferation in the acute phase of the disease. However, following immunization, Tc52 was able to stimulate both arms of the immune system.14 Therefore, experiments were conducted in order to examine the antibody and cellular responses to Tc52-derived synthetic peptides following immunization. When serum specimens from P1-, P2- and P3-OVA-immunized mice were reacted against Tc52 in a Western blot, a strong signal was observed (see Fig. 1). In contrast, no reactivity was seen when using anti-P5-OVA immune serum (data not shown).

An assay to measure the in vitro stimulatory property of coupled peptides on a primed T-cell population was also performed. The data obtained showed that, in contrast to P1-, P2- and P3-OVA, P5-OVA did not elicit significant T-cell proliferation (Table 3). However, the absence of proliferation was not a result of the inability of cells to respond to an external stimulus. Indeed, a significant level of cell proliferation occurred in lymph node cells from BALB/c mice immunized with P5-OVA and stimulated in vitro with anti-CD3 antibodies (SI = 212 ± 30). Moreover, comparable levels of stimulation were observed in the case of lymph node cells from BALB/c mice immunized with P1-, P2- or P3-OVA and stimulated with anti-CD3 antibodies (SI = 190 ± 12; 215 ± 11; 189 ± 22, respectively). Similar observations were recorded when using lymph node cells from CBA mice (data not shown).

Table 3
Proliferative response of lymph node cells from peptide-immunized BALB/c mice against the peptides

The draining lymph node cells obtained from mice injected with P1-, P2- or P3-OVA peptides exhibited significant proliferative responses to in vitro challenge with Tc52 (SI = 9·5 ± 2·1; 8·3 ± 1·5; 7·9 ± 2·3, respectively). In contrast, no significant secondary in vitro response to Tc52 challenge was observed when using lymph node cells from mice immunized with P5-OVA peptide. Moreover, lymph node cells from P1-, P2- or P3-OVA-immunized mice could be restimulated in vitro with peptides, P1, P2 or P3, or the carrier alone (i.e. OVA) (data not shown). This observation, together with the fact that an antibody response against Tc52 upon immunization with the P1-, P2- or P3-OVA peptides, could be obtained, is in agreement with the notion that T-cell help was provided.

Discussion

The Tc52 protein of T. cruzi is present among parasite-released antigens and expressed principally by parasite-dividing forms (e.g. epimastigotes and amastigotes).12 Tc52 protein functions in vitro as a thioltransferase16 and has been reported to be among factors crucial for parasite survival and virulence.20 Moreover, immunological investigations have shown that Tc52 exerts immunoregulatory activity towards the cells of the immune system (i.e. macrophages and dendritic cells).14,15 Unexpectedly, under conditions of experimental infections, the Tc52 protein appears to be immunologically relatively silent during the early acute phase, failing to elicit significant levels of antibodies and lymphocyte proliferation, whereas immunization with Tc52 stimulated both arms of the immune system.13,14

We have hypothesized that analysis of the structure–function relationship in the Tc52 molecule could reveal discrete domains that might be involved in different functions. The results obtained in this study showed that Tc52, under a physiological pH similar to that found in the phagolysosome of the host cell, undergoes a spontaneous cleavage process giving rise to a major peptide of Mr 28 kDa (Tc28k). Tc28k, corresponding to the fragment in position 193–445, was able to mimic the inhibitory activity of Tc52 on T-cell proliferation in vitro. To determine the portion of the Tc28k peptide necessary for expression of immunomodulatory activity, a series of peptides derived from the Tc28k sequence and the other portion of the Tc52 molecule have been synthesized, based on a computer-assisted epitope analysis, allowing the identification of putative sequences expected to be exposed on the surface and thus accessible to the cells. The results obtained here, using five synthetic peptides spanning the Tc52 amino acid sequence, indicate that the inhibition of T-cell proliferation localizes to a peptide sequence within the Tc28k fragment at its C terminus (P5, amino acids 432–445). However, it is noteworthy that the inhibitory effect of P5 was very low. Considering that the conformation of this peptide might be different from that assumed by the sequence within the native protein, P5 was coupled to OVA. The resulting compound, P5-OVA, exerted a significant level of suppressive activity in a dose-dependent manner. Evidence for the specificity of P5, as a biologically active molecule, was the lack of activity of the other synthetic peptides coupled to OVA.

The selective relevance of the P5 sequence within the Tc52 molecule for its immunomodulatory capacity was further stressed by the finding that spleen cells treated with P5-OVA showed a considerable decrease in their capacity to secrete IL-2 and IFN-γ upon stimulation with anti-CD3 mAbs. These observations might have some relevance in vivo. In fact, it has been known for many years that spleen cells from acutely infected mice are unable to produce IL-2 in vitro in response to mitogens21 and this could account for the transient state of immunosuppression that occurs during T. cruzi acute infection. Moreover, several investigators have reported the involvement of T. cruzi-secreted molecules in the development of immunosuppression phenomenon.17

Likewise, in immunized mice, the P5-OVA peptide was a very poor B- and T-cell antigen compared to the native Tc52 protein and its N-terminal part-derived peptides. In fact, in the sera of mice immunized with P1, P2 or P3 OVA-coupled peptides, the relative antibody titres against Tc52 protein increased by greater than 100-fold after the booster injection (data not shown), indicating a T-cell-driven B-cell response. In contrast, mice immunized with P5 coupled to OVA did not develop an antibody response to Tc52. Moreover, P5-OVA failed to restimulate lymph node cells from P5-OVA-immunized mice. The lack of reactivity in this system could be a result of inappropriate concentrations of P5-OVA used in vitro, or to suppressed T-cell responses in P5-OVA immunized mice. Another possible explanation could be the direct effect of P5-OVA on antigen-presenting cells, blocking cellular events and leading to the down-regulation of T-cell proliferation. Furthermore, lymph node cells from P5-OVA-immunized mice responded to stimulation in vitro with anti-CD3 mAb. Therefore, lymph node cells from P5-OVA-immunized mice lack T cells against the peptide, and this is not the consequence of a generalized suppression of their responses. These results suggested that the immunomodulatory portion of T. cruzi Tc52 may reside, at least in part, in a sequence within its C-terminal domain, which also appears to minimize its antigenicity. Further studies are needed to explore the possible interference of P5-OVA with signal-transduction pathways.

A number of reports have documented that infectious organisms [e.g. human immunodeficiency virus (HIV); hepatitis B; Plasmodium strains; T. cruzi]2225 express variant T-cell epitopes that improve their survival by inhibiting T-cell responses. In general, cellular immune responses are specific for a very limited set of peptides within the context of the major histocompatibility complex (MHC) molecules.26 T lymphocytes require a processed foreign antigen presented on a cell surface in association with a MHC molecule for effective recognition.27 The MHC genes are polymorphic and their products are receptors for peptides which, while bound, are displayed to T lymphocytes.28 Whether peptide P5 interaction with MHC molecules and subsequent MHC blockage or T-cell receptor (TCR) antagonism29 occurs, awaits further investigations.

In previous studies it was shown that Tc52, like other proteins belonging to the thioredoxin and glutaredoxin families, exerts immunoregulatory functions.30 Indeed, we found that Tc52 had the ability to promote the maturation of dendritic cells and elicit NO release by macrophages. Moreover, the data obtained suggested that the glutathione (GSH) binding site of Tc52 is involved in Tc52-induced dendritic cell (DC) activation and signalling via Toll-like receptor 2, but is not involved in binding to DC.15 Therefore, on first examination, the fact that Tc52 carries stimulatory/inhibitory activities towards the cells of the immune system may appear paradoxical. Yet, it is interesting that a common feature of GSTs is that they are composed of an N-terminal region also named glutathione binding site (G-site) and a non-specific hydrophobic C-terminal region (H-site), which accommodates the electrophilic substrate. The P5-OVA is localized in the C-proximal portion of Tc52 protein. Therefore, it is probable that discrete Tc52 domains might be involved in different functions.

The relationship between structure and function has been studied in detail for human IL-1β, a cytokine with multiple biological effects which plays an important role in the development of the host immune and inflammatory reactions.31 The synthetic-peptide approach allowed to demonstrate that different moieties of the IL-1β protein are involved in distinct biological activities.32,33 By means of short synthetic peptides corresponding to the Tc52 sequences, we were able to approach the identification of minimal Tc52 structure, which might carry its immunomodulatory activity in vivo. Such molecules may permit parasites to escape immune surveillance and to grow unimpeded by normal immune responses. Moreover, by impairing multiple immune-effector functions as a result of blocking the signal-transduction pathways utilized by cytokines such IL-2 and IFN-γ, the host may also become more susceptible to opportunistic infections. Consequently, it is reasonable to assume that these molecules, which may confer a selective advantage to the pathogen, represent potential targets for developing therapeutic strategies to blunt the host immune-system dysfunction.

Acknowledgments

This work received support from IRD and INSERM. M. Borges is funded by ‘Fundação para a Ciência e Tecnologia’, Portugal Grant PRAXIS XXI/BD/20093/99.

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