Molecular and antigenic characterization of Trypanosoma cruzi TolT proteins

Background TolT was originally described as a Trypanosoma cruzi molecule that accumulated on the trypomastigote flagellum bearing similarity to bacterial TolA colicins receptors. Preliminary biochemical studies indicated that TolT resolved in SDS-PAGE as ~3–5 different bands with sizes between 34 and 45 kDa, and that this heterogeneity could be ascribed to differences in polypeptide glycosylation. However, the recurrent identification of TolT-deduced peptides, and variations thereof, in trypomastigote proteomic surveys suggested an intrinsic TolT complexity, and prompted us to undertake a thorough reassessment of this antigen. Methods/Principle findings Genome mining exercises showed that TolT constitutes a larger-than-expected family of genes, with at least 12 polymorphic members in the T. cruzi CL Brener reference strain and homologs in different trypanosomes. According to structural features, TolT deduced proteins could be split into three robust groups, termed TolT-A, TolT-B, and TolT-C, all of them showing marginal sequence similarity to bacterial TolA proteins and canonical signatures of surface localization/membrane association, most of which were herein experimentally validated. Further biochemical and microscopy-based characterizations indicated that this grouping may have a functional correlate, as TolT-A, TolT-B and TolT-C molecules showed differences in their expression profile, sub-cellular distribution, post-translational modification(s) and antigenic structure. We finally used a recently developed fluorescence magnetic beads immunoassay to validate a recombinant protein spanning the central and mature region of a TolT-B deduced molecule for Chagas disease serodiagnosis. Conclusion/Significance This study unveiled an unexpected genetic and biochemical complexity within the TolT family, which could be exploited for the development of novel T. cruzi biomarkers with diagnostic/therapeutic applications.
Author summary Chagas disease, caused by the protozoan Trypanosoma cruzi, is a lifelong and debilitating neglected illness of major significance in Latin America, for which no vaccine or adequate drugs are yet available. Identification of novel biomarkers able to transcend the current limits of diagnostic and/or therapeutic assessment methods hence surfaces as a main priority in Chagas disease applied research. In this framework, we herein undertook a thorough biochemical and antigenic characterization of T. cruzi TolT surface antigens. Our results unveil an unexpected complexity within this family, with at least 12 polymorphic TolT genes in the T. cruzi CL Brener reference strain genome. According to structural features, TolT deduced molecules could be split into three robust groups that show differences in their structural features, expression profile, sub-cellular distribution, post-translational modification(s) and antigenic structure. Overall, we show that TolT molecules are conspicuously expressed by both major mammal-dwelling stages of the parasite, and that they are differentially recognized by the immune system in Chagasic patients and in T. cruzi-infected mammals. Our findings are discussed in terms of the evolution and possible structural/functional roles of TolT molecules, as well as in terms of their applicability in Chagas disease serodiagnosis.