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.

In SituImmunolocalization and Stage-Dependent Expression of a Secretory Serine Protease inLeishmania donovaniand Its Role as a Vaccine Candidate

“…Differentiation of procyclic promastigotes into metacyclic promastigotes (metacyclogenesis) is known to be crucial for infectivity (3,9). Infective-stage promastigotes are generated in vitro as organisms approach the stationary phase of growth (2,29). To correlate these changes in the expression of the 115-kDa protease with metacyclogenesis, the expression of the protease was studied in promastigotes derived from 0 to 96 h of culture.…”

“…The coverslips were kept at 37°C under a humidified 5% CO 2 atmosphere. After sequential incubation for 24 h, 48 h, and 72 h, the cells were fixed in methanol and were stained with Giemsa stain for the determination of intracellular parasite numbers (2,22). In a separate set of experiments, parasites pretreated with the anti-115-kDa antibody were incubated with the macrophages, which had previously been incubated only with the purified 115-kDa protease (1 g/ml) (4), and the experiments proceeded as described above.…”

Differential Expression and Characterization of a Member of the Mucin-Associated Surface Protein Family Secreted by Trypanosoma cruzi

“…The TSs transfer sialic acid from the host cell membrane to the parasite's glycoproteins and bind the parasite to the host cell during the invasion process (27,45). gp82 is another glycoprotein known to be involved in the invasion process; it forms an integral part of the surface of the parasite, where it plays a role in freeing calcium from the intracellular deposits and the disassembly of F-actin (4,15,21,25,47).…”

“…To obtain the hydrophilic fraction of the M membrane, we used the method described by Atayde et al (4).…”

The gp82 Surface Molecule of Trypanosoma cruzi Metacyclic Forms

“…On the other hand, consistent with the sequence/function relationship, a gp82 family member C03 that displays considerable differences in P4 and P8 sequences (Fig. 6.1 ) exhibits reduced cell adhesion capacity (Atayde et al 2007 ). C03 protein is not recognized by mAb 3F6 and its cellular localization varies depending on the parasite strain.…”