Expression and cellular localization of molecules of the gp82 family in Trypanosoma cruzi metacyclic trypomastigotes

Abstract

A member of the Trypanosoma cruzi gp82 family, expressed on metacyclic trypomastigote surface and identified by monoclonal antibody (MAb) 3F6, plays a key role in host cell invasion. Apart from the gp82 defined by MAb 3F6, no information is available on members of this protein family. From cDNA clones encoding gp82 proteins sharing 59.1% sequence identity, we produced the recombinant proteins J18 and C03, the former containing and the latter lacking the epitope for MAb 3F6. Polyclonal antibodies to J18 and C03 proteins were generated and used, along with MAb 3F6, to analyze the expression and cellular localization of gp82 family members in metacyclic forms of CL and G strains, which belong to highly divergent genetic groups. By two-dimensional gel electrophoresis and immunoblotting, molecules of 82 to 86 kDa, focusing at pH 4.6 to 5.4, and molecules of 72 to 88 kDa, focusing at pH 4.9 to 5.7, were visualized in CL and G strains, respectively. Flow cytometry and microscopic analysis revealed in both strains similar expression of MAb 3F6-reactive gp82 in live and permeabilized parasites, indicating its surface localization. The reaction of live parasites of both strains with anti-J18 antibodies was weaker than with MAb 3F6 and was increased by permeabilization. Anti-C03 antibodies bound predominantly to flagellar components in permeabilized G strain parasites, but in the CL strain the flagellum was not the preferential target for these antibodies. Host cell invasion of metacyclic forms was inhibited by J18 protein, as well as by MAb 3F6 and anti-J18 antibodies, but not by C03 protein or anti-C03 antibodies.

FIG. 1.

Sequences of proteins of T. cruzi metacyclic trypomastigote gp82 family members. Shown are the proteins C03 (CL strain), R31 (CL strain), and J18 (G strain). In proteins R31 and J18 are the sequences (indicated by lines above the sequences) identified as the epitope for MAb 3F6 (P3) and as the conformational cell binding site (P4 and P8). The alternative initiator methionines in the C03 are indicated by asterisks. The conserved motif VTV, characteristic of all members of the gp85/TS family, and the potential glycosylphosphatidylinositol (GPI) anchor consensus sequence are indicated (lines above sequences). Points represent residues that are conserved in the three proteins, nonconserved amino acids are indicated, and dashes represent residues that are lacking in the indicated sequence. Note the high degree of identity between protein R31 and J18, as opposed to the much lower sequence identity of these proteins in relation to protein C03.

FIG. 2.

Properties of recombinant proteins J18 and C03. (A) The sequences identified as the epitope for MAb 3F6 (P3) and as the conformational cell binding site (P4 and P8) are aligned to show the differences between J18 and C03 proteins, with asterisks indicating the changed residues. (B) Immunoblot of purified J18 and C03 proteins using MAb 3F6. Note the lack of reactivity of C03 protein. (C) Binding of proteins J18 and C03 to HeLa cells, assayed as described in Materials and Methods. The reaction was revealed by sequential incubation with polyclonal antiserum directed to J18 or to C03 and anti-mouse IgG conjugated to peroxidase. OD₄₉₂, optical density at 492 nm.

FIG. 3.

Cross-reactivity between anti-J18 and anti-C03 antibodies. Purified recombinant proteins J18 and C03, as well as the metacyclic trypomastigote extract of T. cruzi strains G and CL, were analyzed by immunoblotting using polyclonal antibodies to J18 or C03 protein.

FIG. 4.

Cellular localization of proteins of the gp82 family in metacyclic forms of the T. cruzi CL strain. Live or permeabilized parasites were reacted with MAb 3F6 or polyclonal antibodies to J18 or C03 and processed for analysis by flow cytometry or for visualization by fluorescence microscopy. DAPI staining of parasite kinetoplast and nucleus appears in blue. Note the similar expression patterns of MAb 3F6-reactive molecules in live and permeabilized parasites, whereas the molecules recognized by anti-J18 and anti-C03 antibodies were in part localized intracellularly, as indicated by the high fluorescence intensity of permeabilized parasites compared to their live counterparts.

FIG. 5.

Cellular localization of proteins of the gp82 family in metacyclic forms of the T. cruzi G strain. Live or permeabilized parasites were reacted with MAb 3F6 or polyclonal antibodies to J18 or C03 and processed for analysis by flow cytometry or for visualization by fluorescence microscopy. Note the similar expression patterns of MAb 3F6-reactive molecules in live and permeabilized parasites, whereas the molecules recognized by anti-J18 antibodies were in part located intracellularly, and those reacting with anti-C03 antibodies were associated with the flagellum and did not react with live parasites.

FIG. 6.

Effect of different proteins and antibodies on host cell invasion by T. cruzi metacyclic trypomastigotes (CL strain). (A) HeLa cells were incubated with parasites in the absence or presence of the indicated protein at 40 μg/ml. (B) Parasites were incubated with the indicated antibodies for 30 min. After antibodies were washed out, the parasites were incubated with HeLa cells. In both panels, the incubation time was 1 h, the cells were stained with Giemsa, and the numbers of intracellular parasites were counted in at least 500 cells. Values in panel A are the means ± standard deviation of three independent assays performed in duplicate. The difference between the control and the cells incubated with J18 protein was significant (P < 0.0005). In panel B the values are the means ± standard deviation of triplicates of one representative experiment. A significant difference between the control and the parasites incubated with MAb 3F6 (P < 0.0005) or with anti-J18 antibodies (P < 0.005) was found.