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Adenosine-to-inosine conversion at position 34 (A34-to-I) of certain tRNAs is essential for expanding their decoding capacity. This reaction is catalyzed by the adenosine deaminase acting on tRNA (ADAT) complex, which in Eukarya is formed by two subunits: ADAT2 and ADAT3. We herein identified and thoroughly characterized the ADAT molecules from the protozoan pathogen Trypanosoma cruzi, the causative agent of Chagas Disease. TcADAT2 and TcADAT3 spontaneously form a catalytically active complex, as shown by expression in engineered bacteria and/or by the increased ex vivo tRNA A-to-I deamination activity of T. cruzi epimastigotes overexpressing TcADAT subunits. Importantly, enhanced TcADAT2/3 activity in transgenic parasites caused a shift in their in vivo tRNAThrAGU signature, which correlated with significant changes in the expression of the Thr-rich TcSMUG proteins. To our knowledge, this is the first evidence indicating that T. cruzi tRNA editing can be modulated in vivo, in turn post-transcriptionally changing the expression of specific genes. Our findings suggest tRNA editing/availability as a forcible step in controlling gene expression and driving codon adaptation in T. cruzi. Moreover, we unveil certain differences between parasite and mammalian host tRNA editing and processing, such as cytosine-to-uridine conversion at position 32 of tRNAThrAGU in T. cruzi, that may be exploited for the identification of novel druggable targets of intervention., (© 2022 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

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 (Galfβ1-4[Galpβ1-6]GlcNAcα) from their O-linked glycans were identified as main adhesion determinants for these molecules. Interestingly, exogenous addition of a synthetic Galfβ1-4[Galpβ1-6]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., Competing Interests: The authors have declared that no competing interests exist

The immunodominant epitope α-d-Galp-(1 → 3)-β-d-Galp-(1 → 4)-d-GlcNAc, expressed in the mucins of the infective trypomastigote stage of Trypanosoma cruzi has been proposed for multiple clinical applications, from serodiagnosis of protozoan caused diseases to xenotransplantation or cancer vaccinology. It was previously shown that the analogue trisaccharide, with glucose in the reducing end instead of GlcNAc, was as efficient as the natural trisaccharide for recognition of chagasic antibodies. Here we describe the synthesis of α-d-Galp-(1 → 3)-β-d-Galp-(1 → 4)-d-Glcp functionalized as the 6-aminohexyl glycoside and its conjugation to BSA using the squarate method. The conjugate of 6-aminohexyl α-d-Galp-(1 → 3)-β-d-Galp was also prepared. Both neoglycoconjugates were recognized by serum samples of Trypanosoma cruzi-infected individuals and thus, are promising tools for the improvement of Chagas disease diagnostic applications., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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., Competing Interests: The authors have declared that no competing interests exist.

Background: TSSA (Trypomastigote Small Surface Antigen) is an antigenic, adhesion molecule displayed on the surface of Trypanosoma cruzi trypomastigotes. TSSA displays substantial sequence identity to members of the TcMUC gene family, which code for the trypomastigote mucins (tGPI-mucins). In addition, TSSA bears sequence polymorphisms among parasite strains; and two TSSA variants expressed as recombinant molecules (termed TSSA-CL and TSSA-Sy) were shown to exhibit contrasting features in their host cell binding and signaling properties., Methods/principle Findings: Here we used a variety of approaches to get insights into TSSA structure/function. We show that at variance with tGPI-mucins, which rely on their extensive O-glycoslylation to achieve their protective function, TSSA seems to be displayed on the trypomastigote coat as a hypo-glycosylated molecule. This has a functional correlate, as further deletion mapping experiments and cell binding assays indicated that exposition of at least two peptidic motifs is critical for the engagement of the 'adhesive' TSSA variant (TSSA-CL) with host cell surface receptor(s) prior to trypomastigote internalization. These motifs are not conserved in the 'non-adhesive' TSSA-Sy variant. We next developed transgenic lines over-expressing either TSSA variant in different parasite backgrounds. In strict accordance to recombinant protein binding data, trypomastigotes over-expressing TSSA-CL displayed improved adhesion and infectivity towards non-macrophagic cell lines as compared to those over-expressing TSSA-Sy or parental lines. These phenotypes could be specifically counteracted by exogenous addition of peptides spanning the TSSA-CL adhesion motifs. In addition, and irrespective of the TSSA variant, over-expression of this molecule leads to an enhanced trypomastigote-to-amastigote conversion, indicating a possible role of TSSA also in parasite differentiation., Conclusion/significance: In this study we provided novel evidence indicating that TSSA plays an important role not only on the infectivity and differentiation of T. cruzi trypomastigotes but also on the phenotypic variability displayed by parasite strains.