Signal transduction induced in Trypanosoma cruzi metacyclic trypomastigotes during the invasion of mammalian cells.

Penetration of Trypanosoma cruzi into mammalian cells depends on the activation of the parasite's protein tyrosine kinase and on the increase in cytosolic Ca2+ concentration. We used metacyclic trypomastigotes, the T. cruzi developmental forms that initiate infection in mammalian hosts, to investigate the association of these two events and to identify the various components of the parasite signal transduction pathway involved in host cell invasion. We have found that i) both the protein tyrosine kinase activation, as measured by phosphorylation of a 175-kDa protein (p175), and Ca2+ mobilization were induced in the metacyclic forms by the HeLa cell extract but not by the extract of T. cruzi-resistant K562 cells; ii) treatment of parasites with the tyrosine kinase inhibitor genistein blocked both p175 phosphorylation and the increase in cytosolic Ca2+ concentration; iii) the recombinant protein J18, which contains the full-length sequence of gp82, a metacyclic stage surface glycoprotein involved in target cell invasion, interfered with tyrosine kinase and Ca2+ responses, whereas the monoclonal antibody 3F6 directed at gp82 induced parasite p175 phosphorylation and Ca2+ mobilization; iv) treatment of metacyclic forms with phospholipase C inhibitor U73122 blocked Ca2+ signaling and impaired the ability of the parasites to enter HeLa cells, and v) drugs such as heparin, a competitive IP3-receptor blocker, caffeine, which affects Ca2+ release from IP3-sensitive stores, in addition to thapsigargin, which depletes intracellular Ca2+ compartments and lithium ion, reduced the parasite infectivity. Taken together, these data suggest that protein tyrosine kinase, phospholipase C and IP3 are involved in the signaling cascade that is initiated on the parasite cell surface by gp82 and leads to Ca2+ mobilization required for target cell invasion.

Signal transduction induced in Trypanosoma cruzi metacyclic trypomastigotes during the invasion of mammalian cells

Penetration of Trypanosoma cruzi into mammalian cells depends on the activation of the parasite's protein tyrosine kinase and on the increase in cytosolic Ca2+ concentration. We used metacyclic trypomastigotes, the T. cruzi developmental forms that initiate infection in mammalian hosts, to investigate the association of these two events and to identify the various components of the parasite signal transduction pathway involved in host cell invasion. We have found that i) both the protein tyrosine kinase activation, as measured by phosphorylation of a 175-kDa protein (p175), and Ca2+ mobilization were induced in the metacyclic forms by the HeLa cell extract but not by the extract of T. cruzi-resistant K562 cells; ii) treatment of parasites with the tyrosine kinase inhibitor genistein blocked both p175 phosphorylation and the increase in cytosolic Ca2+ concentration; iii) the recombinant protein J18, which contains the full-length sequence of gp82, a metacyclic stage surface glycoprotein involved in target cell invasion, interfered with tyrosine kinase and Ca2+ responses, whereas the monoclonal antibody 3F6 directed at gp82 induced parasite p175 phosphorylation and Ca2+ mobilization; iv) treatment of metacyclic forms with phospholipase C inhibitor U73122 blocked Ca2+ signaling and impaired the ability of the parasites to enter HeLa cells, and v) drugs such as heparin, a competitive IP3-receptor blocker, caffeine, which affects Ca2+ release from IP3-sensitive stores, in addition to thapsigargin, which depletes intracellular Ca2+ compartments and lithium ion, reduced the parasite infectivity. Taken together, these data suggest that protein tyrosine kinase, phospholipase C and IP3 are involved in the signaling cascade that is initiated on the parasite cell surface by gp82 and leads to Ca2+ mobilization required for target cell invasion.

Characterization of the cell adhesion site of Trypanosoma cruzi metacyclic stage surface glycoprotein gp82.

The surface glycoprotein gp82, expressed in the insect-stage metacyclic trypomastigotes of Trypanosoma cruzi, has been implicated in mammalian cell invasion. Here we have characterized the cell adhesion site of gp82 by using recombinant proteins and synthetic peptides based on gp82. The recombinant protein Del-4/8, lacking 65 amino acids of gp82 central domain (at positions 257 to 321), was virtually devoid of cell-binding activity and lacked the ability to inhibit parasite invasion, in contrast to J18, the construct containing the full-length gp82 sequence (amino acids 1 to 516). Constructs with shorter deletions, i.e., Del-4 (deleted from 257 to 271) and Del-8 (deleted from 293 to 321), bound to target cells to a significantly lesser degree than did J18. The sites deleted in recombinant proteins Del-4 and Del-8 contained acidic amino acids critical for cell adhesion. Thus, the cell-binding capacity of protein Del-E/D, lacking the glutamic acid (259/260) and aspartic acid (303/304) pairs, was negligible, as was its capacity to inhibit parasite internalization. Of a set of synthetic peptides spanning the gp82 central domain, a 22-mer hybrid peptide, p4/8, formed by two noncontiguous sequences (at positions 257 to 273 and 302 to 306) and containing the four acidic residues, competed with the binding of J18 protein to target cells and significantly inhibited ( approximately 60%) the penetration of parasites. This peptide, generated by the juxtaposition of sequences that are separated by a hydrophobic stretch in the linear molecule, appears to be mimicking a conformation-dependent cell-binding site of gp82. Experiments of antibody competition with a set of 20-mer overlapping peptides mapped the epitope for 3F6, a monoclonal antibody directed to gp82 that inhibits parasite invasion, to the sequence represented by peptide p3 (244 to 263), which has a partial overlap with the cell adhesion site.

Infectivity of Trypanosoma cruzi strains is associated with differential expression of surface glycoproteins with differential Ca2+ signalling activity.

Mammalian cell invasion assays, using metacyclic trypomastigotes of Trypanosoma cruzi G and CL strains, showed that the CL strain enters target cells in several-fold higher numbers as compared with the G strain. Analysis of expression of surface glycoproteins in metacyclic forms of the two strains by iodination, immunoprecipitation and FACS, revealed that gp90, undetectable in the CL strain, is one of the major surface molecules in the G strain, that expression of gp82 is comparable in both strains and that gp35/50 is expressed at lower levels in the CL strain. Purified gp90 and gp35/50 bound more efficiently than gp82 to cultured HeLa cells. However, the intensity of the Ca2+ response triggered in HeLa cells by gp82 was significantly higher than that induced by gp35/50 or gp90. Most of the Ca2+ signalling activity of the metacyclic extract towards HeLa cells was due to gp82 and was inhibitable by gp82-specific monoclonal antibody 3F6. Ca2+ mobilization was also triggered in metacyclic trypomastigotes by host-cell components; it was mainly gp82-mediated and more intense in the CL than in the G strain. We propose that expression of gp90 and gp35/50 at high levels impairs binding of metacyclic forms to host cells through productive gp82-mediated interaction, which leads to the target-cell and parasite Ca2+ mobilization required for invasion. Analysis of metacyclic forms of eight additional T. cruzi strains corroborated the inverse correlation between infectivity and expression of gp90 and gp35/50.