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Successful infection by Trypanosoma cruzi , the agent of Chagas' disease, is critically dependent on host cell invasion by metacyclic trypomastigote (MT) forms. Two main metacyclic stage-specific surface molecules, gp82 and gp90, play determinant roles in target cell invasion in vitro and in oral T. cruzi infection in mice. The structure and properties of gp82, which is highly conserved among T. cruzi strains, are well known. Information on gp90 is still rather sparse. Here, we attempted to fill that gap. gp90, purified from poorly invasive G strain MT and expressing gp90 at high levels, inhibited HeLa cell lysosome spreading and the gp82-mediated internalization of a highly invasive CL strain MT expressing low levels of a diverse gp90 molecule. A recombinant protein containing the conserved C-terminal domain of gp90 exhibited the same properties as the native G strain gp90: it counteracted the host cell lysosome spreading induced by recombinant gp82 and exhibited an inhibitory effect on HeLa cell invasion by CL strain MT. Assays to identify the gp90 sequence associated with the property of downregulating MT invasion, using synthetic peptides spanning the gp90 C-terminal domain, revealed the sequence GVLYTADKEW. These data, plus the findings that lysosome spreading was induced upon HeLa cell interaction with CL strain MT, but not with G strain MT, and that in mixed infection CL strain MT internalization was inhibited by G strain MT, suggest that the inhibition of target cell lysosome spreading is the mechanism by which the gp90 molecule exerts its downregulatory role.

We previously reported the isolation of a novel protein gene family, termed SAP (serine-, alanine-, and proline-rich protein), from Trypanosoma cruzi . Aided by the availability of the completed genome sequence of T. cruzi , we have now identified 39 full-length sequences of SAP, six pseudogenes and four partial genes. SAPs share a central domain of about 55 amino acids and can be divided into four groups based on their amino (N)- and carboxy (C)-terminal sequences. Some SAPs have conserved N- and C-terminal domains encoding a signal peptide and a glycosylphosphatidylinositol anchor addition site, respectively. Analysis of the expression of SAPs in metacyclic trypomastigotes by two-dimensional electrophoresis and immunoblotting revealed that they are likely to be posttranslationally modified in vivo. We have also demonstrated that some SAPs are shed into the extracellular medium. The recombinant SAP exhibited an adhesive capacity toward mammalian cells, where binding was dose dependent and saturable, indicating a possible ligand-receptor interaction. SAP triggered the host cell Ca 2+ response required for parasite internalization. A cell invasion assay performed in the presence of SAP showed inhibition of internalization of the metacyclic forms of the CL strain. Taken together, these results show that SAP is involved in the invasion of mammalian cells by metacyclic trypomastigotes, and they confirm the hypothesis that infective trypomastigotes exploit an arsenal of surface glycoproteins and shed proteins to induce signaling events required for their internalization.

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 Ca2+ mobilization from thapsigargin-sensitive stores. Like gp82, gp30 triggered the host cell Ca2+ response required for parasite internalization. Purified gp30 and the recombinant gp82 inhibited HeLa cell invasion of metacyclic forms of isolates 569 and 588 by similar to90 and similar to70%, 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. Universidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Immunol & Parasitol, BR-04023062 São Paulo, Brazil Univ Fed Goias, Lab Doenca Chagas, Hosp Clin, Fac Med, Goiania, Go, Brazil Universidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Immunol & Parasitol, BR-04023062 São Paulo, Brazil Web of Science

Trypanosoma cruzi , the agent of Chagas disease, binds to diverse extracellular matrix proteins. Such an ability prevails in the parasite forms that circulate in the bloodstream and contributes to host cell invasion. Whether this also applies to the insect-stage metacyclic trypomastigotes, the developmental forms that initiate infection in the mammalian host, is not clear. Using T. cruzi CL strain metacyclic forms, we investigated whether fibronectin bound to the parasites and affected target cell invasion. Fibronectin present in cell culture medium bound to metacyclic forms and was digested by cruzipain, the major T. cruzi cysteine proteinase. G strain, with negligible cruzipain activity, displayed a minimal fibronectin-degrading effect. Binding to fibronectin was mediated by gp82, the metacyclic stage-specific surface molecule implicated in parasite internalization. When exogenous fibronectin was present at concentrations higher than cruzipain can properly digest, or fibronectin expression was stimulated by treatment of epithelial HeLa cells with transforming growth factor beta, the parasite invasion was reduced. Treatment of HeLa cells with purified recombinant cruzipain increased parasite internalization, whereas the treatment of parasites with cysteine proteinase inhibitor had the opposite effect. Metacyclic trypomastigote entry into HeLa cells was not affected by anti-β1 integrin antibody but was inhibited by anti-fibronectin antibody. Overall, our results have indicated that the cysteine proteinase of T. cruzi metacyclic forms, through its fibronectin-degrading activity, is implicated in host cell invasion.

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 (∼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.

We have cloned and sequenced a cDNA for a metacyclic trypomastigote-specific glycoprotein with a molecular mass of 90 kDa, termed MTS-gp90. By immunoblotting, antibodies to the MTS-gp90 recombinant protein reacted exclusively with a 90-kDa antigen of metacyclic trypomastigotes. The insert of the MTS-gp90 cDNA clone strongly hybridized with a single 3.0-kb mRNA of metacyclic forms, whereas the hybridization signal with epimastigote mRNA was weak and those with RNAs from other developmental stages were negative, indicating that transcription of the MTS-gp90 gene is developmentally regulated. A series of experiments showed that the MTS-gp90 gene is present in multiple copies in the Trypanosoma cruzi genome, arranged in a nontandem manner, and that there are at least 40 copies of the gene per haploid genome. Sequence analysis of recombinant MTS-gp90 revealed 40 to 60% identity at the amino acid level with members of a family of mammalian stage-specific, 85-kDa surface antigens of T. cruzi. However, there are considerable differences in the amino acid compositions outside the homology region.

In this study, we examined the immunochemical properties of the 35- and 50-kDa (35/50-kDa) surface glycoconjugates expressed on the surface of metacyclic trypomastigotes of Trypanosoma cruzi using three different monoclonal antibodies directed to this component. The 35/50-kDa surface antigen was expressed by metacyclic trypomastigotes of 11 different strains of T. cruzi and displayed a significant degree of molecular polymorphism. Results of immunoblotting and complement-mediated lysis were consistent with such diversity. Different monoclonal antibodies reacted with distinct epitopes on the 35/50-kDa antigen, which is resistant to proteases and behaves as an amphiphilic component.

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.

Upon oral infection, Trypanosoma cruzi metacyclic trypomastigotes invade and replicate in the gastric mucosal epithelium, being apparently uniquely specialized for adhesion to mucin and mucosal invasion. Here we investigated the involvement of gp82, the metacyclic-stage-specific surface glycoprotein implicated in host cell entry, in both adhesion to gastric mucin and invasion of the mucosal epithelium upon oral challenge. Metacyclic forms, preincubated with a control monoclonal antibody (MAb) or with MAb 3F6 directed to gp82, were administered orally to BALB/c mice, and parasitemia was monitored. Mice that received parasites treated with MAb 3F6 had greatly reduced parasitemia, displaying at the peak a mean number of blood parasites more than 100-fold lower than that of the control group. MAbs directed to other T. cruzi surface glycoproteins had no such effect. gp82, as either a native or a recombinant molecule, but not the metacyclic trypomastigote surface molecule gp90 or gp35/50, bound to gastric mucin in enzyme-linked immunosorbent assays. MAb 3F6 significantly inhibited the penetration of cultured epithelial HeLa cells by metacyclic forms in the absence or in the presence of gastric mucin. Mucin alone did not affect parasite internalization. Parasite infectivity was not altered by treatment of metacyclic forms with pepsin, to which gp82 was resistant, or with proteinase K, which removed the N-terminal portion of gp82 but preserved its host cell binding site. Taken together, these findings suggest that gp82 mediates the interaction of metacyclic trypomastigotes with gastric mucin and the subsequent penetration of underlying epithelial cells.

Peptides derived from the surface glycoprotein gp82 of Trypanosoma cruzi , previously implicated in the parasite’s invasion of host cells, were expressed as fusions to the protein LamB of Escherichia coli in a region known to be exposed on the cell surface. Bacteria expressing these proteins adhered to HeLa cells in a manner that mimics the pattern of parasite invasion of mammalian cells. Purified LamB fusion proteins were shown to bind to HeLa cells and to inhibit infection by T. cruzi , supporting the notion that these gp82-derived peptides can mediate interaction of the parasite with its host.

It was shown in this work that the infectivity of metacyclic forms of Trypanosoma cruzi was affected upon interaction with the monoclonal antibody (10D8), which reacts with a carbohydrate epitope of the 35- and 50-kilodalton (kDa) surface glycoconjugates. The invasion of Vero cells by metacyclic forms of strains Tulahuen and G was inhibited 50 to 67% in the presence of 10D8 (10 micrograms/ml), whereas a nonrelated monoclonal antibody to Plasmodium berghei had no such effect. In mice that were inoculated with metacyclic forms preincubated with 10D8 or that had passively received 10D8 before challenge with metacyclic forms, a considerable decrease in the parasitemia levels was observed. The 35- and 50-kDa antigens were detectable by the galactose oxidase and sodium boro[3H]hydride procedure but not by surface iodination or metabolic labeling with [35S]methionine, suggesting that they may be of glycolipid nature. The finding that the 35- and 50-kDa antigens are major bands recognized by sera of mice immunized with killed metacyclic forms and protected against acute infection, in addition to the results with 10D8, indicate that these glycoconjugates may play an important role in the metacyclic form-host cell association that initiates T. cruzi infection.

The surface antigen makeup of metacyclic trypomastigote forms of strain G of Trypanosoma cruzi, which produce a subpatent infection in mice, differed from those of the virulent strains Y and CL. A 100,000-molecular-weight protein, barely detectable on the Y or CL cell surface, appeared as the main surface antigen of the G metacyclic trypomastigotes. In addition, the G metacyclic forms differed from those of the virulent strains in their susceptibility to complement-mediated immunolysis.