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Infection and Immunity, March 2003, p. 1561-1565, Vol. 71, No. 3
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.3.1561-1565.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Cell Adhesion and Ca²⁺ Signaling Activity in Stably Transfected Trypanosoma cruzi Epimastigotes Expressing the Metacyclic Stage-Specific Surface Molecule gp82

Patricio M. Manque,^1, Ivan Neira,¹ Vanessa D. Atayde,¹ Esteban Cordero,¹ Alice T. Ferreira,² José Franco da Silveira,¹ Marcel Ramirez,^1, and Nobuko Yoshida^1*

Departamento de Microbiologia, Imunologia e Parasitologia,¹ Departamento de Biofísica, Escola Paulista de Medicina, Universidade Federal de Sao Paulo, Sao Paulo, Brazil²

Received 12 August 2002/ Returned for modification 3 October 2002/ Accepted 13 December 2002

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Metacyclic trypomastigotes of Trypanosoma cruzi express a developmentally regulated 82-kDa surface glycoprotein (gp82) that has been implicated in host cell invasion. gp82-mediated interaction of metacyclic forms with target cells induces in both cells activation of the signal transduction pathways, leading to intracellular Ca²⁺ mobilization, which is required for parasite internalization. Noninfective epimastigotes do not express detectable levels of gp82 and are unable to induce a Ca²⁺ response. We stably transfected epimastigotes with a T. cruzi expression vector carrying the metacyclic stage gp82 cDNA. These transfectants produced a functional gp82, which bound to and triggered a Ca²⁺ response in HeLa cells, in the same manner as the metacyclic trypomastigote gp82. Such properties were not found in epimastigotes transfected with the plasmid vector alone. Epimastigotes expressing gp82 on the surface adhered to HeLa cells but were not internalized. Treatment of gp82-expressing epimastigotes with forskolin, an activator of adenylyl cyclase that increases the metacyclic trypomastigote entry into target cells, did not promote parasite internalization. P175, an intracellular tyrosine phosphorylated protein, which appears to play a role in gp82-dependent signaling cascade in metacyclic forms, was undetectable in epimastigotes, either transfected or not with pTEX-gp82. Overall, our results indicate that gp82 is required but not sufficient for target cell invasion.

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Trypanosoma cruzi is the causative agent of Chagas' disease, a major public health problem in Latin America. A key process for the successful establishment of T. cruzi infection in mammalian hosts is the penetration of metacyclic trypomastigote forms into target cells. Metacyclic forms synthesize a developmentally regulated 82-kDa surface glycoprotein (gp82) that has been implicated in the parasite entry into host cells (17, 19), an event that requires intracellular Ca²⁺ mobilization (5, 12, 23). gp82 is an adhesion molecule that binds to host cells in a receptor-mediated manner (11, 17, 20) and triggers Ca²⁺ mobilization both in the parasite and in the target cell (19, 30). It remains to be determined whether gp82 alone is sufficient to promote parasite internalization, as is the case of penetrin, a heparin-binding protein of T. cruzi tissue culture trypomastigotes that, when engineered into noninvasive Escherichia coli, allows bacterial entry into fibroblastic cells (15), and of some bacterial proteins, such as Listeria monocytogenes internalin, which confers invasiveness to both Enterococcus faecalis and internalin-coated latex beads (9).

gp82 is encoded by a multigene family whose members are organized in subsets distributed throughout the genome (1). This precludes the functional genetic analysis by gene knockout. Among the alternative strategies used to circumvent that problem is the stable transformation with episomes and overexpression of the protein of interest in a developmental stage that does not express it. Norris (14) showed that transfection of epimastigotes with a trypomastigote-stage specific complement regulatory protein gene conferred resistance to the complement action to epimastigotes that are otherwise susceptible to complement-mediated lysis. Overexpression of cruzipain, a major cysteine proteinase of T. cruzi, enhanced differentiation of epimastigotes into metacyclic trypomastigotes (25).

Ramirez et al. (18) have shown the correct processing and the expression of gp82 on the surface of epimastigotes stably transfected with plasmid pTEX carrying the complete open reading frame (ORF) of gp82 gene. In the present study, we further investigated the phenotypic characteristics acquired by transfected T. cruzi epimastigotes expressing gp82, with particular emphasis on the properties associated with host cell invasion. We have found that the homologous expression of gp82 augments the cell adhesion capacity of epimastigotes and confers to these developmental forms the ability to induce target cell Ca²⁺ mobilization.

Strain G (T. cruzi I), isolated from an opossum in the Amazon region (27), was used throughout this study. Parasites were maintained alternately in mice and liver infusion tryptose (LIT) medium containing 5% fetal calf serum (FCS). Epimastigotes were grown in LIT medium at 28°C. Metacyclic trypomastigotes were harvested from LIT cultures at the stationary growth phase and were purified by chromatography on a DEAE-cellulose column, as described previously (24). Construction of pTEX-gp82 vector was carried out by subcloning a BamHI/HindIII fragment containing the entire ORF of the gp82 gene (1) into the BamHI and HindIII sites of plasmid pTEX (8). The pTEX-gp82 constructs were checked by sequencing and restriction mapping analysis. For transfection of T. cruzi epimastigotes, parasites in the mid-log phase were washed in phosphate-buffered saline (PBS) and resuspended in electroporation buffer (137 mM NaCl, 5 mM KCl, 5.5 mM Na₂HPO₄, 0.77 mM glucose, 21 mM HEPES; pH 7.2) at a final concentration of 10⁸ cells/ml. Aliquots of 0.45 ml were dispensed into disposable 0.4-mm cuvettes (Bio-Rad Laboratories) containing 30 µg of plasmid DNA (pTEX or pTEX-gp82). The cells were electroporated by using a Bio-Rad gene pulser at 350 V and 500 mF, with two consecutive pulses. After 1 min on ice, the samples were diluted fivefold with LIT medium containing 10% FCS plus 0.5% human blood and allowed to recover for 24 h. Geneticin (G418 sulfate; Life Technologies) was added, at a concentration of 200 µg/ml, and parasites were incubated at 28°C. After selection, transfected epimastigotes were grown in the presence of 400 µg of Geneticin/ml. For Southern blot analysis, T. cruzi DNA was isolated as previously described (1), digested with restriction enzyme, separated by electrophoresis on 0,8% agarose gel, and blotted onto nylon membranes. RNA for Northern blot analysis was isolated by treating parasites with 1 ml of Trizol reagent (Gibco-BRL). After complete dissolution and the addition of 0.2 ml of chloroform, the suspension was centrifuged for 15 min at 12,000 x g to recover the aqueous phase. An equal volume of isopropanol was added to the aqueous phase to precipitate the RNA overnight at -20°C. The RNA was denatured with 50% formamide and 2.2 M formaldehyde and subjected to electrophoresis in a 1.0% agarose gel containing formaldehyde. After being stained with ethidium bromide, RNA was transferred to nylon membranes. The membranes were prehybridized in a solution containing 50% formamide, 5x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 5x Denhardt solution, 0.5% sodium dodecyl sulfate (SDS), 5 mM EDTA, and 0.1 mg of tRNA/ml at 42°C for 2 h and then hybridized overnight 42°C with ³²P-labeled probe. The probes consisted of a DNA fragment corresponding to ORF of gp82 gene or to the 500-bp fragment of the neo gene that confers resistance to the drug G418, obtained by digestion of the plasmid pTEX with KpnI and PstI. After hybridization, the membranes were subjected to three washes at 60°C in 2x SSC containing 0.1% SDS, followed by two washes in 0.1x SSC containing 0.1% SDS, and then exposed to X-ray film. Western blot analysis was performed essentially as described previously (28), and the final reaction was revealed by chemiluminescence by using the ECL detection reagent and Hyperfilm-MP (Amersham). Expression of gp82 on parasite surface was analyzed by flow cytometry as detailed elsewhere (19).

For T. cruzi adhesion assay, HeLa cells were grown at 37°C in 24-well plates on a 13-mm-diameter round glass coverslip at a density of 2 x 10⁵ cells/well in Dulbecco modified Eagle medium supplemented with 10% FCS, streptomycin (100 µg/ml), and penicillin (100 U/ml) in a humidified 5% CO₂ atmosphere. Parasites were seeded onto HeLa cells (5 x 10⁶ parasites/well). After 1 h of incubation at 37°C, the coverslips were washed in PBS, fixed with methanol, and stained with Giemsa. Host cell invasion assay has been detailed elsewhere (28). To determine the target cell binding of gp82, HeLa cells (5 x 10⁴), grown in 96-well microtiter plates, were fixed with 4% paraformaldehyde in PBS, washed with PBS, and blocked with PBS containing 10% FCS (PBS-FCS) for 1 h at room temperature. Sonicated extracts of parasites at various protein concentrations were then added. After 1 h of incubation at 37°C and several washes in PBS, HeLa cells were sequentially incubated for 1 h at 37°C with monoclonal antibody (MAb) 3F6 in PBS-FCS and anti-mouse immunoglobulin G (IgG) conjugated to peroxidase. After several washes in PBS, the bound enzyme was revealed with o-phenylenediamine. The cytosolic free Ca²⁺ concentration in HeLa cells was measured as described previously (5). Detection of tyrosine phosphorylated T. cruzi proteins has been detailed elsewhere (30).

The parasites transfected with the construct pTEX-gp82, or with the vector alone, were first analyzed by Southern blot. Upon digestion with the restriction enzyme XhoI, the parasite DNA was hybridized with the neo gene probe. In epimastigotes carrying pTEX-gp82, the probe hybridized to a 7.2-kb band that corresponds to the linearized plasmid plus the ORF of gp82 gene, whereas a band of 5.6 kb was detected in epimastigotes transfected with pTEX alone, and no hybridization signal was visualized in the nontransfected control (Fig. 1A). When the DNA of epimastigotes transfected with pTEX-gp82 was partially digested with XhoI, several bands higher than 7.2 kb could be detected, confirming the presence of multiple episomal copies of plasmid pTEX-gp82 (data not shown). The Northern blot analysis with the gp82 gene probe revealed a 2.1-kb transcript in epimastigotes carrying pTEX-gp82 and a 2.4-kb transcript in metacyclic forms (Fig. 1B). This difference in the size of gp82 transcripts was expected, provided that in the pTEX-gp82 construct the 5' and 3' untranslated regions of gp82 mRNA were replaced by the 5' untranslated region and intergenic spacer of glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) gene (8). Confirming previous results showing the presence of low amounts of gp82 transcripts in epimastigotes (1), a weak 2.4-kb hybridization signal was seen in wild-type epimastigotes and in parasites transfected with pTEX alone (Fig. 1B).

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FIG. 1. Characterization of T. cruzi epimastigotes transfected with pTEX constructs. The samples analyzed were wild-type epimastigotes (Epi), epimastigotes transfected with the plasmid vector alone (pTEX), epimastigotes transfected with the pTEX-gp82 construct (pTEX-gp82), or metacyclic trypomastigotes (Meta). (A) Southern blot of T. cruzi genomic DNA digested with XhoI hybridized with the 32P-labeled fragment of neomycin phosphotransferase gene (neo). (B) Steady-state levels of gp82 transcripts in transfected parasites. A total of 10 µg of total RNA was blotted onto nylon membrane and hybridized with the gp82 gene probe. (C) Western blot containing soluble parasite extracts was probed with the MAb 3F6 directed to metacyclic stage gp82 molecule. (D) Flow cytometric analysis of gp82 expression. Live parasites were incubated with the MAb 3F6, followed by reaction with fluorescence-labeled goat anti-mouse IgG, and were then analyzed by fluorescence-activated cell sorting.

In Western blots, the gp82 band, which is revealed by MAb 3F6 as a doublet or a triplet in T. cruzi G strain, was visualized in epimastigotes carrying pTEX-gp82 but not in epimastigotes transfected with pTEX, in a manner indistinguishable from that in metacyclic forms, although of lower intensity (Fig. 1C). We confirmed by flow cytometry that the levels of gp82 on the surface of epimastigotes transfected with pTEX-gp82 were lower than in metacyclic trypomastigotes (Fig. 1D). Higher levels of gp82 expression could not be achieved by increasing the concentration of Geneticin in the growth medium. We did not find any evidence of rearrangement of plasmid molecules, such as reported in pTEX transformed parasites (8, 26), that might explain the reduced gp82 expression. As in epimastigotes transfected with pTEX-gp82 the gp82 transcript is devoid of the 5' and 3' untranslated regions, a possibility exists that its stability is reduced. Gene expression in trypanosomatids is largely controlled at the posttranscriptional level, with trans-splicing, polyadenylation, and binding of protein factors to the 3' untranslated portions of the transcripts promoting either mRNA stability or increase in the translational efficiency (2-4, 6, 7). Another possibility is the presence of a positive regulator effector in metacyclic trypomastigotes but not in epimastigotes.

We examined the ability of epimastigotes expressing gp82 to bind to HeLa cells. A threefold increase in the number of adhered parasites was found in gp82-expressing epimastigotes compared to vector-transfected or wild-type epimastigotes (Fig. 2A). To ascertain that the increased cell adhesion of epimastigotes carrying pTEX-gp82 was mediated by gp82, we performed a binding assay in which HeLa cells, immobilized on the bottom of the microtiter plates, were incubated with increasing concentrations of the sonicated extract of the parasites and the bound gp82 was detected by MAb 3F6. The gp82 expressed in epimastigotes transfected with pTEX-gp82 bound to HeLa cells in the same manner as its metacyclic stage counterpart (Fig. 2B). We have previously shown that, when engineered to express the outer membrane protein LamB fused to peptides based on gp82 sequences implicated in host cell attachment (20), an otherwise nonadherent E. coli bound to HeLa cells (16). These data, plus the observation that MAb 3F6 inhibits the binding of metacyclic forms to HeLa cells (data not shown), reinforce the role of gp82 in cell adhesion.

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FIG. 2. Gp82-mediated binding of T. cruzi to host cells. (A) Live parasites were incubated with HeLa cells for 3 h at 37°C. After washes in PBS, fixation with methanol, and staining with Giemsa, the number of adherent epimastigotes was counted in a total of 500 Giemsa-stained cells. (B) Increasing concentrations of sonicated parasite extracts were added to wells in enzyme-linked immunosorbent assay plates containing paraformaldehyde-fixed HeLa cells. After washes, the cells were sequentially incubated with MAb 3F6 and anti-mouse IgG conjugated to peroxidase. o-Phenyldiamidine was used to reveal the bound enzyme. Representative results of one of three experiments are shown. Values are the means ± the standard deviation of triplicates. Epimastigotes (; Epi), epimastigotes transfected with the vector alone (; pTEX) or with the pTEX-gp82 construct (•; pTEX-gp82), or metacyclic trypomastigotes (; Meta) were evaluated.

As the metacyclic trypomastigote gp82 has been shown to induce Ca²⁺ mobilization in HeLa cells (5, 19), we investigated whether the gp82 generated in epimastigotes could induce a Ca²⁺ response in target cells by measuring the increase in HeLa cell cytosolic free Ca²⁺ concentration, [Ca²⁺]_i, upon addition of sonicated T. cruzi extracts. As shown in Fig. 3, extracts of gp82-expressing epimastigotes, but not of pTEX-transfected or wild-type epimastigotes, triggered in HeLa cells a Ca²⁺ signal similar to that induced by metacyclic trypomastigotes.

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FIG. 3. Ca2+ signaling activity of T. cruzi toward host cells. A total of 25 µl of sonicated extract of parasites, equivalent to 109 cells, were added at the indicated times (arrow) to fura 2-loaded HeLa cells in a cuvette containing 2.5 ml of Tyrode solution. The results representative of three experiments are presented.

Considering that the host cell Ca²⁺ mobilization is an important requirement for T. cruzi internalization (5, 12, 23), we sought to determine whether the expression of gp82 with Ca²⁺ signaling activity was sufficient to confer to an otherwise-noninvasive epimastigote the ability to enter target cells. To clarify the point, the epimastigotes expressing gp82 were incubated with HeLa cells at 37°C for 1 h at a parasite/cell ratio of 20:1, and the number of intracellular parasites was counted in at least 500 Giemsa-stained cells. Epimastigotes carrying pTEX-gp82 failed to enter HeLa cells. In five independent experiments, the means ± the standard deviation of intracellular parasites per 500 cells was 104.2 ± 16.3 in HeLa cells seeded with metacyclic trypomastigotes, whereas in HeLa cells incubated with epimastigotes transfected with pTEX or pTEX-gp82 we only occasionally saw a few (one to four) parasites that appeared to be intracellularly located even when the incubation time was extended to 3 h, a finding indicating that components other than gp82 are necessary for infection.

Cell invasion by T. cruzi requires the activation of signal transduction pathways in host cells as well as in the parasites. The gp82-mediated interaction of metacyclic forms with HeLa cells triggers the signaling cascade, leading to Ca²⁺ mobilization in both cells (19, 30). Despite the acquisition of the capacity to induce a gp82-mediated Ca²⁺ signal in HeLa cells, it is possible that the epimastigotes transfected with pTEX-gp82 are unable to transduce the signal to the parasite interior for lack of some essential components present in metacyclic trypomastigotes. An externally added activator of signaling processes could eventually complement that deficiency. Metacyclic forms of T. cruzi G strain have been found to have their invasive capacity augmented 100% when pretreated with forskolin (13), an activator of adenylyl cyclase (21, 22), suggesting the involvement of cyclic AMP-dependent processes. We tested the effect of forskolin on the infectivity of epimastigotes carrying pTEX-gp82. Parasites were pretreated with 10 µM forskolin for 15 min at 37°C before cell invasion assays. Epimastigotes transfected with pTEX or pTEX-82 remained noninvasive after treatment with forskolin, whereas the infectivity of the metacyclic trypomastigotes was increased by the drug (data not shown).

We have found that metacyclic forms of a T. cruzi clone, expressing gp82 at lower levels than the recombinant epimastigotes as measured by fluorescence-activated cell sorting but otherwise exhibiting the same molecular profile of the parental strain, do enter HeLa cells to a lower degree (data not shown), a finding consistent with the poor gp82 expression. The factor missing in epimastigotes may be the tyrosine phosphorylated protein p175, an intracellular component of the signaling cascade not detectable in epimastigotes (Fig. 4A). Binding of MAb 1G7, which reacts with the metacyclic stage-specific surface glycoprotein gp90 and hampers the parasite Ca²⁺ response (19), as well as its internalization (29), greatly reduced the p175 phosphorylation levels (Fig. 4B). This finding is compatible with the role played by gp90 as the negative regulator of G strain metacyclic trypomastigote entry into target cells (10). No decrease in p175 phosphorylation was observed with MAb 3F6. In T. cruzi CL strain, that do not express MAb 1G7-reactive gp90, the phosphorylation levels of p175 are increased and concomitantly the intracellular Ca²⁺ mobilization is induced, upon recognition of gp82 by the host cell receptor or by MAb 3F6 (30). All of these data, taken together, reinforce the notion that the surface molecule gp82 is required but not sufficient for parasite internalization.

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FIG. 4. Analysis of expression of tyrosine phosphorylated p175 in T. cruzi. (A) Metacyclic trypomastigotes (Meta), epimastigotes (Epi), and recombinants carrying the pTEX-gp82 construct (pTEX-gp82) were processed for anti-phosphotyrosine immunoblotting. (B) Parasites were incubated at 37°C for 20 min in the absence (-) or in the presence of MAb 1G7, washed in PBS, and detergent lysed, and the total lysates were subjected to SDS-polyacrylamide gel electrophoresis and analyzed by immunoblotting with anti-phosphotryrosine antibodies. Note the decrease in p175 phosphorylation levels upon reaction with MAb 1G7.

 
This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). P.M. Manque was recipient of a posdoctoral fellowship from FAPESP.

 
* Corresponding author. Mailing address: Escola Paulista de Medicina, Universidade Federal de São Paulo, R. Botucatu, 862-6o Andar, 04023-062, Sao Paulo, SP, Brazil. Phone: 55-11-5576-4532. Fax: 55-11-5571-1095. E-mail: nyoshida{at}ecb.epm.br.

Editor: J. M. Mansfield

Present address: Department of Microbiology and Immunology, Medical College of Virginia, Virginia Commonwealth University, Richmond, Va.

Present address: Departamento de Genética, Universidade Federal de Pernambuco, Recife, PE, Brazil.

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Infection and Immunity, March 2003, p. 1561-1565, Vol. 71, No. 3
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.3.1561-1565.2003
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