David L. Fouts

Cloning of a surface membrane glycoprotein specific for the infective form of Trypanosoma cruzi having adhesive properties to laminin

Trypomastigotes of Trypanosoma cruzi express a set of surface glycoproteins known, collectively, as Tc-85. A monoclonal antibody to these proteins, named H1A10, inhibits (50–90%) in vitro parasite interiorization into host cells, thus implicating these glycoproteins in the infection process. Two DNA inserts, a genomic DNA fragment and a full-length cDNA encoding the H1A10 epitope, have now been cloned and characterized. Results show that both have high sequence identity with all reported members of the gp85/trans-sialidase gene family, although the H1A10 epitope exists only in the Tc-85 subset of the family. The epitope has been mapped by competition of antibody binding to a Tc-85 recombinant protein with peptides having sequences predicted by the Tc-85 DNA sequence, which contains also putativeN-glycosylation sites and COOH-terminal glycosylphosphatidylinositol anchor insertion sites, as expected, since an N-glycan chain and a glycosylphosphatidylinositol anchor have been characterized previously in the Tc-85 subset. The protein encoded by the full-length cDNA insert binds to cells and in vitro to laminin, but not to gelatin or fibronectin, in a saturable manner. For the first time it was possible to assign a defined ligand to a sequenced glycoprotein belonging to the gp85 family. This fact, together with the reported binding of family members to cell surfaces, reinforces the hypothesis that this family encodes glycoproteins with similar sequences but differing enough as to bind to different ligands and thus forming a family of adhesion glycoproteins enabling the parasite to overcome the barriers interposed by cell membranes, extracellular matrices, and basal laminae.

Nucleotide sequence and transcription of a trypomastigote surface antigen gene of Trypanosoma cruzi

In previous studies we identified a 500-bp segment of the gene, TSA-1, which encodes an 85-kDa trypomastigote-specific surface antigen of the Peru strain of Trypanosoma cruzi. TSA-1 was shown to be located at a telomeric site and to contain a 27-bp tandem repeat unit within the coding region. This repeat unit defines a discrete subset of a multigene family and places the TSA-1 gene within this subset. In this study, we present the complete nucleotide sequence of the TSA-1 gene from the Peru strain. By homology matrix analysis, fragments of two other trypomastigote specific surface antigen genes, pTt34 and SA85-1.1, are shown to have extensive sequence homology with TSA-1 indicating that these genes are members of the same gene family as TSA-1. The TSA-1 subfamily was also found to be active in two other strains of T. cruzi, one of which contains multiple telomeric members and one of which contains a single non-telomeric member, suggesting that transcription is not necessarily dependent on the gene being located at a telomeric site. Also, while some of the sequences found in this gene family are present in 2 size classes of poly(A)+ RNA, others appear to be restricted to only 1 of the 2 RNA classes.

Evidence for Four Distinct Major Protein Components in the Paraflagellar Rod of Trypanosoma cruzi

The major structural proteins present in the paraflagellar rod of Trypanosoma cruzi migrate on SDS-polyacrylamide gels as two distinct electrophoretic bands. The gene encoding a protein present in the faster migrating band, designated PAR 2, has been identified previously. Here we report the isolation and partial characterization of three genes, designated par 1, par 3, and par 4, that encode proteins present in the two paraflagellar rod protein bands. Peptide-specific polyclonal antibodies and monoclonal antibodies against the four proteins encoded by these genes shows that PAR 1 and PAR 3 are present only in the slower migrating paraflagellar rod band, and that PAR 2 and PAR 4 are present only in the faster migrating band. Analysis of the nucleotide sequence of these genes and the amino acid sequence of the conceptual proteins encoded by them indicates that par 2 shares high sequence similarity with par 3 and both are members of a common gene family, of which par 1 may be a distant member. Analysis of gene copy number and steady-state RNA levels suggest that the close stoichiometric ratio of the four PAR proteins is likely maintained by homeostatic regulation of RNA levels rather than gene dosage.

Immunization with recombinant paraflagellar rod protein induces protective immunity against Trypanosoma cruzi infection

In the present study, we have produced recombinant paraflagellar rod proteins (PFR) and report their use for successful vaccination of mice against Trypanosoma cruzi. This protection is associated with a highly polarized type 1 cytokine production profile. Additionally, we have analyzed the gene sequence encoding PFR-2 to determine the degree of conservation among seven highly diverse strains of T. cruzi, and found it to be highly conserved. The results presented here indicate that the PFR antigens are highly conserved, and immunization with rPFR-1, PFR-2, or an equimolar mix of the PFR-1, -2, and -3 proteins provides protective immunity against Trypanosoma cruzi.

Paraflagellar rod protein‐specific CD8+ cytotoxic T lymphocytes target Trypanosoma cruzi‐infected host cells

Our previous studies show that in mice immunized with the paraflagellar rod (PFR) proteins of Trypanosoma cruzi protective immunity against this protozoan parasite requires MHC class I‐restricted T cell function. To determine whether PFR‐specific CD8+ T cell subsets are generated during T. cruzi infection, potential CTL targets in the PFR proteins were identified by scanning the amino acid sequences of the four PFR proteins for regions of 8–10 amino acids that conform to predicted MHC class I H‐2b binding motifs. A subset of the peptide sequences identified were synthesized and tested as target antigen in 51Cr‐release assays with effector cells from chronically infected T. cruzi mice. Short‐term cytotoxic T lymphocyte (CTL) lines specific for two of the peptides, PFR‐1164–171 and PFR‐3123–130, showed high levels of lytic activity against peptide‐pulsed target cells, secreted interferon (IFN)‐γ in response to parasite‐infected target cells, and were found to be CD8+, CD4−, CD3+, TCRαβ+ cells of the Tc1 subset. Challenge of PFR immunized CD8−/− and perforin‐deficient (PKO) mice confirmed that while CD8+ cells are required for survival of T. cruzi challenge infection, perforin activity is not required. Furthermore, while lytic activity of PFR‐specific CD8+ T cell lines derived from PKO mice was severely impaired, the IFN‐γ levels secreted by CTLs from PKO mice were equivalent to that of normal mice, suggesting that the critical role played by CD8+ T cells in immunity to the parasite may be secretion of type 1 cytokines rather than lysis of parasite infected host cells.