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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.

Trypanosoma cruzi, the flagellate protozoan agent of Chagas disease or American trypanosomiasis, is unable to synthesize sialic acids de novo. Mucins and trans-sialidase (TS) are substrate and enzyme, respectively, of the glycobiological system that scavenges sialic acid from the host in a crucial interplay for T. cruzi life cycle. The acquisition of the sialyl residue allows the parasite to avoid lysis by serum factors and to interact with the host cell. A major drawback to studying the sialylation kinetics and turnover of the trypomastigote glycoconjugates is the difficulty to identify and follow the recently acquired sialyl residues. To tackle this issue, we followed an unnatural sugar approach as bioorthogonal chemical reporters, where the use of azidosialyl residues allowed identifying the acquired sugar. Advanced microscopy techniques, together with biochemical methods, were used to study the trypomastigote membrane from its glycobiological perspective. Main sialyl acceptors were identified as mucins by biochemical procedures and protein markers. Together with determining their shedding and turnover rates, we also report that several membrane proteins, including TS and its substrates, both glycosylphosphatidylinositol-anchored proteins, are separately distributed on parasite surface and contained in different and highly stable membrane microdomains. Notably, labeling for α(1,3)Galactosyl residues only partially colocalize with sialylated mucins, indicating that two species of glycosylated mucins do exist, which are segregated at the parasite surface. Moreover, sialylated mucins were included in lipid-raft-domains, whereas TS molecules are not. The location of the surface-anchored TS resulted too far off as to be capable to sialylate mucins, a role played by the shed TS instead. Phosphatidylinositol-phospholipase-C activity is actually not present in trypomastigotes. Therefore, shedding of TS occurs via microvesicles instead of as a fully soluble form.

Trypanosoma cruzi is the etiologic agent of Chagas disease. Although this is not a free-living organism it has conserved a contractile vacuole complex (CVC) to regulate its osmolarity. This obligate intracellular pathogen is, in addition, dependent on surface proteins to invade its hosts. Here we used a combination of genetic and biochemical approaches to delineate the contribution of the CVC to the traffic of glycosylphosphatidylinositol (GPI)-anchored proteins to the plasma membrane of the parasite and promote host invasion. While T. cruzi Rab11 (GFP-TcRab11) localized to the CVC, a dominant negative (DN) mutant tagged with GFP (GFP-TcRab11DN) localized to the cytosol, and epimastigotes expressing this mutant were less responsive to hyposmotic and hyperosmotic stress. Mutant parasites were still able to differentiate into metacyclic forms and infect host cells. GPI-anchored trans-sialidase (TcTS), mucins of the 60-200 KDa family, and trypomastigote small surface antigen (TcTSSA II) co-localized with GFP-TcRab11 to the CVC during transformation of intracellular amastigotes into trypomastigotes. Mucins of the gp35/50 family also co-localized with the CVC during metacyclogenesis. Parasites expressing GFP-TcRab11DN prevented TcTS, but not other membrane proteins, from reaching the plasma membrane, and were less infective as compared to wild type cells. Incubation of these mutants in the presence of exogenous recombinant active, but not inactive, TcTS, and a sialic acid donor, before infecting host cells, partially rescued infectivity of trypomastigotes. Taking together these results reveal roles of TcRab11 in osmoregulation and trafficking of trans-sialidase to the plasma membrane, the role of trans-sialidase in promoting infection, and a novel unconventional mechanism of GPI-anchored protein secretion.

Platelets contribute to vessel formation through the release of angiogenesis-modulating factors stored in their α-granules. Galectins, a family of lectins that bind β-galactoside residues, are up-regulated in inflammatory and cancerous tissues, trigger platelet activation and mediate vascularization processes. Here we aimed to elucidate whether the release of platelet-derived proangiogenic molecules could represent an alternative mechanism through which galectins promote neovascularization. We show that different members of the galectin family can selectively regulate the release of angiogenic molecules by human platelets. Whereas Galectin (Gal)-1, -3, and -8 triggered vascular endothelial growth factor (VEGF) release, only Gal-8 induced endostatin secretion. Release of VEGF induced by Gal-8 was partially prevented by COX-1, PKC, p38 and Src kinases inhibitors, whereas Gal-1-induced VEGF secretion was inhibited by PKC and ERK blockade, and Gal-3 triggered VEGF release selectively through a PKC-dependent pathway. Regarding endostatin, Gal-8 failed to stimulate its release in the presence of PKC, Src and ERK inhibitors, whereas aspirin or p38 inhibitor had no effect on endostatin release. Despite VEGF or endostatin secretion, platelet releasates generated by stimulation with each galectin stimulated angiogenic responses in vitro including endothelial cell proliferation and tubulogenesis. The platelet angiogenic activity was independent of VEGF and was attributed to the concerted action of other proangiogenic molecules distinctly released by each galectin. Thus, secretion of platelet-derived angiogenic molecules may represent an alternative mechanism by which galectins promote angiogenic responses and its selective blockade may lead to the development of therapeutic strategies for angiogenesis-related diseases.

Trypanosoma cruzi the agent of Chagas disease is a monophyletic but heterogeneous group conformed by several Discrete Typing Units (DTUs) named TcI to TcVI characterized by genetic markers. The trans-sialidase (TS) is a virulence factor involved in cell invasion and pathogenesis that is differentially expressed in aggressive and less virulent parasite stocks. Genes encoding TS-related proteins are included in a large family divided in several groups but only one of them contains TS genes. Two closely related genes differing in a T/C transition encode the enzymatically active TS (aTS) and a lectin-like TS (iTS). We quantified the aTS/iTS genes from TcII and TcVI aggressive and TcI low virulent strains and found variable aTS number (1-32) per haploid genome. In spite of being low TS enzyme-expressers, TcI strains carry 28-32 aTS gene copies. The intriguing absence of iTS genes in TcI strains together with the presence of aTS/iTS in TcII and TcVI strains (virulent) were observed. Moreover, after sequencing aTS/iTS from 38 isolates collected along the Americas encompassing all DTUs, the persistent absence of the iTS gene in TcI, TcIII and TcIV was found. In addition, the sequence clustering together with T/C transition analysis correlated to DTUs of T. cruzi. The consistence of TS results with both evolutionary genome models proposed for T. cruzi, namely the "Two Hybridization" and the "Three Ancestor" was discussed and reviewed to fit present findings. Parasite stocks to attempt genetic KO or to assay the involvement of iTS in parasite biology and virulence are finally available.

Trans-sialidase (TS), a virulence factor from Trypanosoma cruzi, is an enzyme playing key roles in the biology of this protozoan parasite. Absent from the mammalian host, it constitutes a potential target for the development of novel chemotherapeutic drugs, an urgent need to combat Chagas' disease. TS is involved in host cell invasion and parasite survival in the bloodstream. However, TS is also actively shed by the parasite to the bloodstream, inducing systemic effects readily detected during the acute phase of the disease, in particular, hematological alterations and triggering of immune cells apoptosis, until specific neutralizing antibodies are elicited. These antibodies constitute the only known submicromolar inhibitor of TS's catalytic activity. We now report the identification and detailed characterization of a neutralizing mouse monoclonal antibody (mAb 13G9), recognizing T. cruzi TS with high specificity and subnanomolar affinity. This mAb displays undetectable association with the T. cruzi superfamily of TS-like proteins or yet with the TS-related enzymes from Trypanosoma brucei or Trypanosoma rangeli. In immunofluorescence assays, mAb 13G9 labeled 100% of the parasites from the infective trypomastigote stage. This mAb also reduces parasite invasion of cultured cells and strongly inhibits parasite surface sialylation. The crystal structure of the mAb 13G9 antigen-binding fragment in complex with the globular region of T. cruzi TS was determined, revealing detailed molecular insights of the inhibition mechanism. Not occluding the enzyme's catalytic site, the antibody performs a subtle action by inhibiting the movement of an assisting tyrosine (Y₁₁₉), whose mobility is known to play a key role in the trans-glycosidase mechanism. As an example of enzymatic inhibition involving non-catalytic residues that occupy sites distal from the substrate-binding pocket, this first near atomic characterization of a high affinity inhibitory molecule for TS provides a rational framework for novel strategies in the design of chemotherapeutic compounds.