Cell adhesion and Ca2+ signaling activity in stably transfected Trypanosoma cruzi epimastigotes expressing the metacyclic stage-specific surface molecule gp82

Abstract

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 Ca(2+) mobilization, which is required for parasite internalization. Noninfective epimastigotes do not express detectable levels of gp82 and are unable to induce a Ca(2+) 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 Ca(2+) 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.

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.

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.

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.

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.