Infection by Trypanosoma cruzi metacyclic forms deficient in gp82 but expressing a related surface molecule, gp30

Abstract

Trypanosoma cruzi metacyclic trypomastigotes invade and replicate in the gastric mucosal epithelium after oral infection. In this study we analyzed the process of infection by T. cruzi isolates deficient in the expression of gp82, the metacyclic stage-specific surface glycoprotein implicated in target cell entry in vitro and in promoting mucosal infection in mice after oral challenge. Mice infected by the oral route with metacyclic forms of gp82-deficient isolate 569 or 588 developed patent parasitemia but at greatly reduced levels compared to those infected with the gp82-expressing isolate CL. Metacyclic forms of both isolates expressed gp30, a surface glycoprotein detectable by monoclonal antibody (MAb) 3F6 directed to gp82. Otherwise, the gp82-deficient isolates displayed a surface profile similar to that of the CL isolate and also entered epithelial HeLa cells in a manner inhibitable by MAb 3F6 and dependent on the parasite signal transduction that involved the activation of protein tyrosine kinase and Ca(2+) mobilization from thapsigargin-sensitive stores. Like gp82, gp30 triggered the host cell Ca(2+) response required for parasite internalization. Purified gp30 and the recombinant gp82 inhibited HeLa cell invasion of metacyclic forms of isolates 569 and 588 by approximately 90 and approximately 70%, respectively. A cell invasion assay performed in the presence of gastric mucin, mimicking the in vivo infection, showed an inhibition of 70 to 75% in the internalization of gp82-deficient isolates but not of the CL isolate. The recombinant gp82 exhibited an adhesive capacity toward gastric mucin much higher than that of gp30. Taken together, our findings indicate that target cell entry of metacyclic trypomastigotes can be mediated either by gp82 or gp30 but that efficient mucosal infection depends on the expression of gp82.

FIG. 1.

Expression of surface glycoprotein gp30 in T. cruzi isolates 569 and 588. (A) Metacyclic trypomastigotes were analyzed by immunoblotting using MAb 3F6. Note the component of ∼30 kDa recognized by MAb 3F6 in both isolates and the absence of gp82, which is predominant in isolate CL. Positions of molecular size markers are shown on the left in kilodaltons. (B) Detergent extracts of metacyclic forms of isolate 569 were subjected to treatment with endoglycosidase H, as described in Materials and Methods. The immunoblot with MAb 3F6 shows the conversion of the 30-kDa molecule into a smaller component, indicating its glycoprotein nature. (C) Live parasites were incubated for 1 h on ice with MAb 3F6 or with unrelated control, MAb 1C3, and processed for analysis by flow cytometry. Note that gp30 is similarly expressed on the surface of isolates 569 and 588.

FIG. 2.

Oral infection of mice with gp82-expressing or gp82-deficient T. cruzi isolates. Each mouse received 4 × 10⁵ metacyclic trypomastigotes of gp82-expressing isolate CL or gp82-deficient isolate 569. Each datum point corresponds to the mean parasitemia of six animals in each group of mice.

FIG. 3.

Host cell invasion of gp82-expressing or gp82-deficient T. cruzi isolates. (A) Metacyclic forms of isolates 569, 588, and CL were incubated with HeLa cells for 1 h at 37°C, and the intracellular parasites in at least 500 Giemsa-stained cells were counted. Values are means and standard deviations (SD) of six experiments. (B) Parasites were left untreated or treated for 30 min at room temperature with 500 μg of MAb 3F6 per ml or 250 μg of the corresponding Fab fragments per ml before being added to HeLa cells. After a 1-h incubation, the HeLa cells were washed in PBS and stained with Giemsa. The reference value of 100% was ascribed to the untreated control. Values are means and SD of three experiments.

FIG. 4.

Inhibitory effect of gp30 and recombinant gp82 on host cell invasion of gp82-deficient T. cruzi isolates. Metacyclic forms of isolates 569 and 588 were added to HeLa cells that had been left unincubated or had been preincubated for 15 min with 60 μg of native gp30 per ml (A) or with 80 μg of J18 per ml (B), the recombinant protein containing the entire gp82 sequence. After 1 h at 37°C, the intracellular parasites in a total of at least 500 Giemsa-stained cells were counted. A reference value of 100% was ascribed to the control without the T. cruzi protein. Values are the means and SD of three experiments performed in duplicate. (C) HeLa cells were preincubted with purified gp30 or J18 at the indicated concentrations before the addition of metacyclic forms of T. cruzi isolate 569. Representative results of a set of experiments are shown. Values are the means and SD of triplicate determinations. (D) SDS-PAGE gel containing the extract of T. cruzi isolate 569 (Ext), purified gp30, and molecular size markers (M), stained with Coomassie blue.

FIG. 5.

Effect of inhibition of parasite PTK and Ca²⁺ mobilization on host cell invasion by gp82-deficient T. cruzi isolates. (A) Metacyclic forms of isolates 569 and 588, pretreated (+) or not pretreated (−) with the PTK inhibitor genistein, were incubated with HeLa cells at 37°C for 30 min. After the cells were washed in PBS, the intracellular parasites in at least 500 Giemsa-stained cells were counted. The values are the means and SD of three experiments performed in duplicate. (B) Parasites were incubated for 20 min at 37°C in the absence (−) or presence (+) of HeLa cell extract, washed with PBS, and processed for immunoblotting using antiphosphotyrosine antibodies. Note the increase in p175 phosphorylation levels in parasites exposed to HeLa cell extract. (C) Metacyclic forms were either untreated or treated with 1 μM thapsigargin before being incubated with HeLa cells for 30 min. Values are the means and SD of three experiments performed in duplicate.

FIG. 6.

Differential interaction of T. cruzi metacyclic-stage surface molecule gp30 and 82 with gastric mucin. (A) Gastric mucin at 20 mg/ml was added to HeLa cells 15 min before they were incubated with metacyclic trypomastigotes. After a 1-h incubation, the cells were washed, fixed, and stained for intracellular parasite counting. The values are the means and SD of three experiments performed in duplicate. (B) J18, the recombinant gp82, or purified gp30 at 10 μg/ml was added to microtiter plates coated with porcine gastric mucin. After being washed, the plates were sequentially incubated with anti-J18 antibody and anti-mouse immunoglobulin conjugated to peroxidase. The bound enzyme was revealed using o-phenylenediamine. The values are the means and SD of triplicate samples. O.D.₄₉₂, optical density at 492 nm.