Molecular characterization of serine-, alanine-, and proline-rich proteins of Trypanosoma cruzi and their possible role in host cell infection.

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 Ca2+ 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.

Mapping of B-cell epitopes in a Trypanosoma cruzi immunodominant antigen expressed in natural infections.

Tc40 is an immunodominant antigen present in natural Trypanosoma cruzi infections. This immunogen was thoroughly mapped by using overlapping amino acid sequences identified by gene cloning and chemical peptide synthesis. To map continuous epitopes of the Tc40 antigen, an epitope expression library was constructed and screened with sera from human chagasic patients. A major, linear B-cell epitope spanning residues 403 to 426 (PAKAAAPPAA) was identified in the central domain of Tc40. A synthetic peptide spanning this region reacted strongly with 89.8% of the serum samples from T. cruzi-infected individuals. This indicates that the main antigenic site is defined by the linear sequence of the peptide rather than a conformation-dependent structure. The major B-cell epitope of Tc40 shares a high degree of sequence identity with T. cruzi ribosomal and RNA binding proteins, suggesting the existence of cross-reactivity among these molecules.