Trypanosoma cruzi surface mucins are involved in the attachment to the Triatoma infestans rectal ampoule.

BACKGROUND: Trypanosoma cruzi, the agent of Chagas disease, is a protozoan parasite transmitted to humans by blood-sucking triatomine vectors. However, and despite its utmost biological and epidemiological relevance, T. cruzi development inside the digestive tract of the insect remains a poorly understood process. METHODS/PRINCIPLE FINDINGS: Here we showed that Gp35/50 kDa mucins, the major surface glycoproteins from T. cruzi insect-dwelling forms, are involved in parasite attachment to the internal cuticle of the triatomine rectal ampoule, a critical step leading to its differentiation into mammal-infective forms. Experimental evidence supporting this conclusion could be summarized as follows: i) native and recombinant Gp35/50 kDa mucins directly interacted with hindgut tissues from Triatoma infestans, as assessed by indirect immunofluorescence assays; ii) transgenic epimastigotes over-expressing Gp35/50 kDa mucins on their surface coat exhibited improved attachment rates (~2-3 fold) to such tissues as compared to appropriate transgenic controls and/or wild-type counterparts; and iii) certain chemically synthesized compounds derived from Gp35/50 kDa mucins were able to specifically interfere with epimastigote attachment to the inner lining of T. infestans rectal ampoules in ex vivo binding assays, most likely by competing with or directly blocking insect receptor(s). A solvent-exposed peptide (smugS peptide) from the Gp35/50 kDa mucins protein scaffolds and a branched, Galf-containing trisaccharide (Galfß1-4[Galpß1-6]GlcNAcα) from their O-linked glycans were identified as main adhesion determinants for these molecules. Interestingly, exogenous addition of a synthetic Galfß1-4[Galpß1-6]GlcNAcα derivative or of oligosaccharides containing this structure impaired the attachment of Dm28c but not of CL Brener epimastigotes to triatomine hindgut tissues; which correlates with the presence of Galf residues on the Gp35/50 kDa mucins' O-glycans on the former but not the latter parasite clone. CONCLUSION/SIGNIFICANCE: These results provide novel insights into the mechanisms underlying T. cruzi-triatomine interplay, and indicate that inter-strain variations in the O-glycosylation of Gp35/50 kDa mucins may lead to differences in parasite differentiation and hence, in parasite transmissibility to the mammalian host. Most importantly, our findings point to Gp35/50 kDa mucins and/or the Galf biosynthetic pathway, which is absent in mammals and insects, as appealing targets for the development of T. cruzi transmission-blocking strategies.

Homologous Expression of Glycosylphosphatidylinositol-Anchored Glycoproteins in Trypanosoma cruzi.

The surface coat of Trypanosoma cruzi is covered with glycosylphosphatidylinositol (GPI)-anchored glycoproteins (GAGPs) that contribute to parasite protection and to the establishment of a persistent infection in both the insect vector and the mammalian host. Multiple GAGPs that vary by amino acid sequence and/or posttranslational modifications are co-expressed on the parasite surface coat, hence curtailing structural/functional analyses on these molecules. Studies in our lab have indicated that GAGP-tagged variants expressed by transfected parasites undergo analogous posttranslational processing than endogenous ones and therefore constitute suitable tools to overcome these limitations. In this chapter, we detail the entire methodological pipeline for the efficient homologous expression of GAGPs in T. cruzi: from a simple strategy for the simultaneously cloning and tagging of the gene of interest to the biochemical validation of the parasite-expressed product.

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.