The high identity of the Trypanosoma cruzi Group-I of trans-sialidases points them as promising vaccine immunogens.

Trans-sialidase (TS) superfamily of proteins comprises eight subgroups, being the proteins of Group-I (TS-GI) promising immunogens in vaccine approaches against Trypanosoma cruzi. Strikingly, TS-GI antigenic variability among parasite lineages and their influence on vaccine development has not been previously analyzed. Here, a search in GenBank detects 49 TS-GI indexed sequences, whereas the main infecting human different parasite discrete typing units (DTU) are represented. In silico comparison among these sequences indicate that they share an identity above 92%. Moreover, the antigenic regions (T-cell and B-cell epitopes) are conserved in most sequences or present amino acid substitutions that scarcely may alter the antigenicity. Additionally, since the generic term TS is usually used to refer to different immunogens of this broad family, a further in silico analysis of the TS-GI-derived fragments tested in preclinical vaccines was done to determine the coverage and identity among them, showing that overall amino acid identity of vaccine immunogens is high, but the segment coverage varies widely. Accordingly, strong H-2K, H-2I, and B-cell epitopes are dissimilarly represented among vaccine TS-derived fragments depending on the extension of the TG-GI sequence used. Moreover, bioinformatic analysis detected a set of 150 T-cell strong epitopes among the DTU-indexed sequences that strongly bind human HLA-I supertypes. In all currently reported experimental vaccines based on TS-GI fragments, mapping these 150 epitopes showed that they are moderately represented. However, despite vaccine epitopes do not present all the substitutions observed in the DTUs, these regions of the proteins are equally recognized by the same HLAs.  Interestingly, the predictions regarding global and South American population coverage estimated in these 150 epitopes are similar to the estimations in experimental vaccines when the complete sequence of TS-GI is used as an antigen. In silico prediction also shows that a number of these MHC-I restricted T-cell strong epitopes could be also cross-recognized by HLA-I supertypes and H-2Kb or H-2Kd backgrounds, indicating that these mice may be used to improve and facilitate the development of new TS-based vaccines and suggesting an immunogenic and protective potential in humans. Further molecular docking analyses were performed to strengthen these results. Taken together, different strategies that would cover more or eventually fully of these T-cell and also B-cell epitopes to reach a high level of coverage are considered.

Molecular Recognition of Surface Trans-Sialidases in Extracellular Vesicles of the Parasite Trypanosoma cruzi Using Atomic Force Microscopy (AFM).

Trans-sialidases (TS) are important constitutive macromolecules of the secretome present on the surface of Trypanosoma cruzi (T. cruzi) that play a central role as a virulence factor in Chagas disease. These enzymes have been related to infectivity, escape from immune surveillance and pathogenesis exhibited by this protozoan parasite. In this work, atomic force microscopy (AFM)-based single molecule-force spectroscopy is implemented as a suitable technique for the detection and location of functional TS on the surface of extracellular vesicles (EVs) released by tissue-culture cell-derived trypomastigotes (Ex-TcT). For that purpose, AFM cantilevers with functionalized tips bearing the anti-TS monoclonal antibody mAb 39 as a sense biomolecule are engineered using a covalent chemical ligation based on vinyl sulfonate click chemistry; a reliable, simple and efficient methodology for the molecular recognition of TS using the antibody-antigen interaction. Measurements of the breakdown forces between anti-TS mAb 39 antibodies and EVs performed to elucidate adhesion and forces involved in the recognition events demonstrate that EVs isolated from tissue-culture cell-derived trypomastigotes of T. cruzi are enriched in TS. Additionally, a mapping of the TS binding sites with submicrometer-scale resolution is provided. This work represents the first AFM-based molecular recognition study of Ex-TcT using an antibody-tethered AFM probe.

Anomeric 1,2,3-triazole-linked sialic acid derivatives show selective inhibition towards a bacterial neuraminidase over a trypanosome trans-sialidase.

Sialic acid is the natural substrate for sialidases and its chemical modification has been a useful approach to generate potent and selective inhibitors. Aiming at advancing the discovery of selective Trypanosoma cruzi trans-sialidase (TcTS) inhibitors, we have synthesised a small series of anomeric 1,2,3-triazole-linked sialic acid derivatives in good yields and high purity via copper-catalysed azide-alkyne cycloaddition (CuAAC, click chemistry) and evaluated their activity towards TcTS and neuraminidase. Surprisingly, the compounds showed practically no TcTS inhibition, whereas ca. 70% inhibition was observed for neuraminidase in relation to the analogues bearing hydrophobic substituents and ca. 5% for more polar substituents. These results suggest that polarity changes are less tolerated by neuraminidase due to the big difference in impact of hydrophobicity upon inhibition, thus indicating a simple approach to differentiate both enzymes. Moreover, such selectivity might be reasoned based on a possible steric hindrance caused by a bulky hydrophobic loop that sits over the TcTS active site and may prevent the hydrophobic inhibitors from binding. The present study is a step forward in exploiting subtle structural differences in sialidases that need to be addressed in order to achieve selective inhibition.

Biophysical and Biochemical Comparison of Extracellular Vesicles Produced by Infective and Non-Infective Stages of Trypanosoma cruzi.

Extracellular vesicles (EVs) are small lipid vesicles released by either any prokaryotic or eukaryotic cell, or both, with a biological role in cell-to-cell communication. In this work, we characterize the proteomes and nanomechanical properties of EVs released by tissue-culture cell-derived trypomastigotes (mammalian infective stage; (TCT)) and epimastigotes (insect stage; (E)) of Trypanosoma cruzi, the etiologic agent of Chagas disease. EVs of each stage were isolated by differential centrifugation and analyzed using liquid chromatography with tandem mass spectrometry (LC-MS/MS), dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), electron microscopy and atomic force microscopy (AFM). Measurements of zeta-potential were also included. Results show marked differences in the surface molecular cargos of EVs between both stages, with a noteworthy expansion of all groups of trans-sialidase proteins in trypomastigote's EVs. In contrast, chromosomal locations of trans-sialidases of EVs of epimastigotes were dramatically reduced and restricted to subtelomeric regions, indicating a possible regulatable expression of these proteins between both stages of the parasite. Regarding mechanical properties, EVs of trypomastigotes showed higher adhesion compared to the EVs of epimastigotes. These findings demonstrate the remarkable surface remodeling throughout the life cycle of T. cruzi, which shapes the physicochemical composition of the extracellular vesicles and could have an impact in the ability of these vesicles to participate in cell communication in completely different niches of infection.

Effect of the Tc13Tul antigen from Trypanosoma cruzi on splenocytes from naïve mice.

Trypanosoma cruzi, the etiological agent of Chagas disease, releases factors, including antigens from the trans-sialidase (TS) superfamily, which modulate the host immune responses. Tc13 antigens belong to group IV of TSs and are characterized by C-terminal EPKSA repeats. Here, we studied the effect of the Tc13 antigen from the Tulahuén strain, Tc13Tul, on primary cultures of splenocytes from naïve BALB/c mice. Recombinant Tc13Tul increased the percentage of viable cells and induced B (CD19+) lymphocyte proliferation. Tc13Tul stimulation also induced secretion of non-specific IgM and interferon-γ (IFN-γ). The same effects were induced by Tc13Tul on splenocytes from naïve C3H/HeJ mice. In vivo administration of Tc13Tul to naïve BALB/c mice increased non-specific IgG in sera. In addition, in vitro cultured splenocytes from Tc13Tul-inoculated mice secreted a higher basal level of non-specific IgM than controls and the in vitro Tc13Tul stimulation of these cells showed an enhanced effect on IgM and IFN-γ secretion. Our results indicate that Tc13Tul may participate in the early immunity in T. cruzi infection by favouring immune system evasion through B-cell activation and non-specific Ig secretion. In contrast, as IFN-γ is an important factor involved in T. cruzi resistance, this may be considered a Tc13Tul effect in favour of the host.

Sialic acid metabolism as a potential therapeutic target of atherosclerosis.

Sialic acid (Sia), the acylated derivative of the nine-carbon sugar neuraminic acid, is a terminal component of the oligosaccharide chains of many glycoproteins and glycolipids. In light of its important biological and pathological functions, the relationship between Sia and coronary artery disease (CAD) has been drawing great attentions recently. Large-scale epidemiological surveys have uncovered a positive correlation between plasma total Sia and CAD risk. Further research demonstrated that N-Acetyl-Neuraminic Acid, acting as a signaling molecule, triggered myocardial injury via activation of Rho/ROCK-JNK/ERK signaling pathway both in vitro and in vivo. Moreover, there were some evidences showing that the aberrant sialylation of low-density lipoprotein, low-density lipoprotein receptor and blood cells was involved in the pathological process of atherosclerosis. Significantly, the Sia regulates immune response by binding to sialic acid-binding immunoglobulin-like lectin (Siglecs). The Sia-Siglecs axis is involved in the immune inflammation of atherosclerosis. The generation of Sia and sialylation of glycoconjugate both depend on many enzymes, such as sialidase, sialyltransferase and trans-sialidase. Abnormal activation or level of these enzymes associated with atherosclerosis, and inhibitors of them might be new CAD treatments. In this review, we focus on summarizing current understanding of Sia metabolism and of its relevance to atherosclerosis.

The Atherogenic Role of Circulating Modified Lipids in Atherosclerosis.

Lipid accumulation in the arterial wall is a crucial event in the development of atherosclerotic lesions. Circulating low-density lipoprotein (LDL) is the major source of lipids that accumulate in the atherosclerotic plaques. It was discovered that not all LDL is atherogenic. In the blood plasma of atherosclerotic patients, LDL particles are the subject of multiple enzymatic and non-enzymatic modifications that determine their atherogenicity. Desialylation is the primary and the most important atherogenic LDL modification followed by a cascade of other modifications that also increase blood atherogenicity. The enzyme trans-sialidase is responsible for the desialylation of LDL, therefore, its activity plays an important role in atherosclerosis development. Moreover, circulating modified LDL is associated with immune complexes that also have a strong atherogenic potential. Moreover, it was shown that antibodies to modified LDL are also atherogenic. The properties of modified LDL were described, and the strong evidence indicating that it is capable of inducing intracellular accumulation of lipids was presented. The accumulated evidence indicated that the molecular properties of modified LDL, including LDL-containing immune complexes can serve as the prognostic/diagnostic biomarkers and molecular targets for the development of anti-atherosclerotic drugs.

Binding of triazole-linked galactosyl arylsulfonamides to galectin-3 affects Trypanosoma cruzi cell invasion.

The synthesis of the O-3 triazole-linked galactosyl arylsulfonamides 1-7 as potential inhibitors of Trypanosoma cruzi cell invasion is described. These target compounds were synthesized by Cu(I)-catalysed azide-alkyne cycloaddition reaction ('click chemistry') between different azide arylsulfonamides and the alkyne-based sugar 3-O-propynyl-ßGalOMe. Inhibition assays of T. cruzi cell invasion with compounds 1-7 showed reduced values of infection index (∼20) for compounds 3 and 5, bearing the corresponding 5-methylisoxazole and 2,4-dimethoxypyrimidine groups, which also presented higher binding affinities to galectin-3 (EC50 17-18 µM) in Corning Epic label-free assays. In agreement with experimental results, the assessment of the theoretical binding of compounds 1-7 to galectin-3 by MM/PBSA method displayed higher affinities for compounds 3 (-9.7 kcal/mol) and 5 (-11.1 kcal/mol). Overall, these achievements highlight compounds 3 and 5 as potential T. cruzi cell invasion blockers by means of a galectin-3 binding-related mechanism, revealing galectin-3 as an important host target for design of novel anti-trypanosomal agents.

Benzoic Acid Derivatives with Trypanocidal Activity: Enzymatic Analysis and Molecular Docking Studies toward Trans-Sialidase.

Chagas, or American trypanosomiasis, remains an important public health problem in developing countries. In the last decade, trans-sialidase has become a pharmacological target for new anti-Chagas drugs. In this work, the aims were to design and find a new series of benzoic acid derivatives as trans-sialidase (TS) inhibitors and anti-trypanosomal agents. Three compounds (14, 18, and 19) sharing a para-aminobenzoic acid moiety showed more potent trypanocidal activity than the commercially available drugs nifurtimox and benznidazole in both strains: the lysis concentration of 50% of the population (LC50) was <0.15 µM on the NINOA strain, and LC50 < 0.22 µM on the INC-5 strain. Additionally, compound 18 showed a moderate inhibition (47%) on the trans-sialidase enzyme and a binding model similar to DANA (pattern A).

Mechanisms of the sialidase and trans-sialidase activities of bacterial sialyltransferases from glycosyltransferase family 80.

Many important biological functions are mediated by complex glycan structures containing the nine-carbon sugar sialic acid (Sia) at terminal, non-reducing positions. Sia are introduced onto glycan structures by enzymes known as sialyltransferases (STs). Bacterial STs from the glycosyltransferase family GT80 are a group of well-studied enzymes used for the synthesis of sialylated glycan structures. While highly efficient at sialyl transfer, these enzymes also demonstrate sialidase and trans-sialidase activities for which there is some debate surrounding the corresponding enzymatic mechanisms. Here we propose a mechanism for STs from the glycosyltransferase family GT80 in which sialidase and trans-sialidase activities occur through reverse sialylation of CMP. The resulting CMP-Sia is then enzymatically hydrolyzed or used as a donor in subsequent ST reactions resulting in sialidase and trans-sialidase activities, respectively. We provide evidence for this mechanism by demonstrating that CMP is required for sialidase and trans-sialidase activities and that its removal with phosphatase ablates activity. We also confirm the formation of CMP-Sia using a coupled enzyme assay. A clear understanding of the sialidase and trans-sialidase mechanisms for this class of enzymes allows for more effective use of these enzymes in the synthesis of glycoconjugates.

Recombination-driven generation of the largest pathogen repository of antigen variants in the protozoan Trypanosoma cruzi.

BACKGROUND: The protozoan parasite Trypanosoma cruzi, causative agent of Chagas disease, depends upon a cell surface-expressed trans-sialidase (ts) to avoid activation of complement-mediated lysis and to enhance intracellular invasion. However these functions alone fail to account for the size of this gene family in T. cruzi, especially considering that most of these genes encode proteins lacking ts enzyme activity. Previous whole genome sequencing of the CL Brener clone of T. cruzi identified ~1400 ts variants, but left many partially assembled sequences unannotated. RESULTS: In the current study we reevaluated the trans-sialidase-like sequences in this reference strain, identifying an additional 1779 full-length and partial ts genes with their important features annotated, and confirming the expression of previously annotated "pseudogenes" and newly annotated ts family members. Multiple EM for Motif Elicitation (MEME) analysis allowed us to generate a model T. cruzi ts (TcTS) based upon the most conserved motif patterns and demonstrated that a common motif order is highly conserved among ts family members. Using a newly developed pipeline for the analysis of recombination within large gene families, we further demonstrate that TcTS family members are undergoing frequent recombination, generating new variants from the thousands of functional and non-functional ts gene segments but retaining the overall structure of the core TcTS family members. CONCLUSIONS: The number and variety as well as high recombination frequency of TcTS family members supports strong evolutionary pressure, probably exerted by immune selection, for continued variation in ts sequences in T. cruzi, and thus for a unique immune evasion mechanism for the large ts gene family.

Multivalent sialylation of ß-thio-glycoclusters by Trypanosoma cruzi trans sialidase and analysis by high performance anion exchange chromatography.

The synthesis of multivalent sialylated glycoclusters is herein addressed by a chemoenzymatic approach using the trans-sialidase of Trypanosoma cruzi (TcTS). Multivalent ß-thio-galactopyranosides and ß-thio-lactosides were used as acceptor substrates and 3'-sialyllactose as the sialic acid donor. High performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) was shown to be an excellent technique for the analysis of the reaction products. Different eluting conditions were optimized to allow the simultaneous resolution of the sialylated species, as well as their neutral precursors. The TcTS efficiently transferred sialyl residues to di, tri, tetra and octa ß-thiogalactosides. In the case of an octavalent thiolactoside, up to six polysialylated compounds could be resolved. Preparative sialylation reactions were performed using the tetravalent and octavalent acceptor substrates. The main sialylated derivatives could be unequivocally assigned by MALDI mass spectrometry. Inhibition of the transfer to the natural substrate, N-acetyllactosamine, was also studied. The octalactoside caused 82 % inhibition of sialic acid transfer when we used equimolar concentrations of donor, acceptor and inhibitor.

Evidence of ternary complex formation in Trypanosoma cruzi trans-sialidase catalysis.

Trypanosoma cruzi trans-sialidase (TcTS) is a key target protein for Chagas disease chemotherapy. In this study, we investigated the implications of active site flexibility on the biochemical mechanism of TcTS. Molecular dynamics studies revealed remarkable plasticity in the TcTS catalytic site, demonstrating, for the first time, how donor substrate engagement with the enzyme induces an acceptor binding site in the catalytic pocket that was not previously captured in crystal structures. Furthermore, NMR data showed cooperative binding between donor and acceptor substrates, supporting theoretical results. In summary, our data put forward a coherent dynamic framework to understand how a glycosidase evolved its highly efficient trans-glycosidase activity.

The trans-sialidase, the major Trypanosoma cruzi virulence factor: Three decades of studies.

Chagas' disease is a potentially life-threatening disease caused by the protozoan parasite Trypanosoma cruzi. Since the description of Chagas'disease in 1909 extensive research has identified important events in the disease in order to understand the biochemical mechanism that modulates T. cruzi-host cell interactions and the ability of the parasite to ensure its survival in the infected host. Exactly 30 years ago, we presented evidence for the first time of a trans-sialidase activity in T. cruzi (T. cruzi-TS). This enzyme transfers sialic acid from the host glycoconjugates to the terminal ß-galactopyranosyl residues of mucin-like molecules on the parasite's cell surface. Thenceforth, many articles have provided convincing data showing that T. cruzi-TS is able to govern relevant mechanisms involved in the parasite's survival in the mammalian host, such as invasion, escape from the phagolysosomal vacuole, differentiation, down-modulation of host immune responses, among others. The aim of this review is to cover the history of the discovery of T. cruzi-TS, as well as some well-documented biological effects encompassed by this parasite's virulence factor, an enzyme with potential attributes to become a drug target against Chagas disease.

Automated N-glycan profiling of a mutant Trypanosoma rangeli sialidase expressed in Pichia pastoris, using tandem mass spectrometry and bioinformatics.

A mutant Trypanosoma rangeli sialidase, Tr7, expressed in Pichia pastoris, exhibits significant trans-sialidase activity, and has been used for analytical-scale production of sialylated human milk oligosaccharides. Mass spectrometry-based site-specific N-glycoprofiling of Tr7 showed that heterogeneous high-mannose type N-glycans were present at all the five potential N-linked glycosites. N-linked glycans in Tr7 were predominantly neutral oligosaccharides with compositions Man(8-16)GlcNA(c2), but also mono- and di-phosphorylated oligosaccharides in the forms of Man(9-15)P(1)GlcNA(c2) and Man(9-14)P(2)GlcNA(c2), respectively. Some phosphorylated N-linked glycans further contained an additional HexNAc, which has not previously been reported in P. pastoris-expressed proteins. We compiled a method pipeline that combined hydrophilic interaction liquid chromatography enrichment of glycopeptides, high accuracy mass spectrometry and automated interpretation of the mass spectra with in-house developed "MassAI" software, which proved efficient in glycan site microheterogeneity analysis. Functional analysis showed that the deglycosylated Tr7 retained more than 90% of both the sialidase and trans-sialidase activities relative to the glycosylated Tr7.

Synthesis of the O-linked hexasaccharide containing ß-D-Galp-(1→2)-D-Galf in Trypanosoma cruzi mucins. Differences on sialylation by trans-sialidase of the two constituent hexasaccharides.

The hexasaccharide ß-D-Galp-(1→2)-[ß-D-Galp-(1→3)]-ß-D-Galp-(1→6)-[ß-D-Galp(1→2)-ß-D-Galf(1→4)]-D-GlcNAc (10) and its ß-D-Galf-(1→2)-ß-D-Galf containing isomer (7) are the largest carbohydrates in mucins of some strains of Trypanosoma cruzi. The terminal ß-D-Galp units are sites of sialylation by the parasite trans-sialidase. Hexasaccharide 10 was chemically synthesized for the first time by a [3+3] nitrilium based convergent approach, using the trichloroacetimidate method of glycosylation. The (1)H NMR spectrum of its alditol was identical to the spectrum of the product released by ß-elimination from the parasite mucin. The trans-sialylation reaction studied on the benzyl glycoside of 10 showed two monosialylated products whose relative abundance changed with time. On the other hand, only one product was produced by sialylation of the benzyl glycoside of 7. A preparative synthesis of the latter and spectroscopic analysis of the product unequivocally established the sialylation site at the less hindered (1→3)-linked galactopyranose.

Design, synthesis and enzymatic evaluation of 3-O-substituted aryl ß-D-galactopyranosides as inhibitors of Trypanosoma cruzi trans-sialidase.

The trans-sialidase of Trypanosoma cruzi (TcTS) is a surface enzyme that modifies the parasite glycocalyx covering it with sialic acid. This process is essential to adhesion and invasion mechanisms in life cycle of the protozoan in the human host, making TcTS a very attractive molecular target for drug design. Using the TcTS substrate 3'-sialyllactose as prototype, D-galactose-derived potential inhibitors of TcTS were designed using strategies of molecular modification. Ten new aryl galactosides modified at carbon-3 were synthesized employing classical carbohydrate chemistry and dibutyltin oxide method for regioselective 3-O-alkylations and evaluated against TcTS by spectrofluorimetry. The 4-methoxycarbonyl-2-nitrophenyl 3-O-carboxymethyl-ß-D-galactopyranoside was the most active compound inhibiting 21% of TcTS enzymatic activity at 1 mM.

Synthesis of divalent ligands of ß-thio- and ß-N-galactopyranosides and related lactosides and their evaluation as substrates and inhibitors of Trypanosoma cruzi trans-sialidase.

In this work we describe the synthesis of mono- and divalent ß-N- and ß-S-galactopyranosides and related lactosides built on sugar scaffolds and their evaluation as substrates and inhibitors of the Trypanosoma cruzi trans-sialidase (TcTS). This enzyme catalyzes the transfer of sialic acid from an oligosaccharidic donor in the host, to parasite ßGalp terminal units and it has been demonstrated that it plays an important role in the infection. Herein, the enzyme was also tested as a tool for the chemoenzymatic synthesis of sialic acid containing glycoclusters. The transfer reaction of sialic acid was performed using a recombinant TcTS and 3'-sialyllactose as sialic acid donor, in the presence of the acceptor having ßGalp non reducing ends. The products were analyzed by high performance anion exchange chromatography with pulse amperometric detection (HPAEC-PAD). The ability of the different S-linked and N-linked glycosides to inhibit the sialic acid transfer reaction from 3'-sialyllactose to the natural substrate N-acetyllactosamine, was also studied. Most of the substrates behaved as good acceptors and moderate competitive inhibitors. A di-N-lactoside showed to be the strongest competitive inhibitor among the compounds tested (70% inhibition at equimolar concentration). The usefulness of the enzymatic trans-sialylation for the preparation of sialylated ligands was assessed by performing a preparative sialylation of a divalent substrate, which afforded the monosialylated compound as main product, together with the disialylated glycocluster.

Oral infectivity through carnivorism in murine model of Trypanosoma cruzi infection.

Introduction: Oral transmission of T. cruzi is probably the most frequent transmission mechanism in wild animals. This observation led to the hypothesis that consuming raw or undercooked meat from animals infected with T. cruzi may be responsible for transmitting the infection. Therefore, the general objective of this study was to investigate host-pathogen interactions between the parasite and gastric mucosa and the role of meat consumption from infected animals in the oral transmission of T. cruzi. Methods: Cell infectivity assays were performed on AGS cells in the presence or absence of mucin, and the roles of pepsin and acidic pH were determined. Moreover, groups of five female Balb/c mice were fed with muscle tissue obtained from mice in the acute phase of infection by the clone H510 C8C3hvir of T. cruzi, and the infection of the fed mice was monitored by a parasitemia curve. Similarly, we assessed the infective capacity of T. cruzi trypomastigotes and amastigotes by infecting groups of five mice Balb/c females, which were infected orally using a nasogastric probe, and the infection was monitored by a parasitemia curve. Finally, different trypomastigote and amastigote inoculums were used to determine their infective capacities. Adhesion assays of T. cruzi proteins to AGS stomach cells were performed, and the adhered proteins were detected by western blotting using monoclonal or polyclonal antibodies and by LC-MS/MS and bioinformatics analysis. Results: Trypomastigote migration in the presence of mucin was reduced by approximately 30%, whereas in the presence of mucin and pepsin at pH 3.5, only a small proportion of parasites were able to migrate (∼6%). Similarly, the ability of TCTs to infect AGS cells in the presence of mucin is reduced by approximately 20%. In all cases, 60-100% of the animals were fed meat from mice infected in the acute phase or infected with trypomastigotes or amastigotes developed high parasitemia, and 80% died around day 40 post-infection. The adhesion assay showed that cruzipain is a molecule of trypomastigotes and amastigotes that binds to AGS cells. LC-MS/MS and bioinformatics analysis, also confirmed that transialidase, cysteine proteinases, and gp63 may be involved in TCTs attachment or invasion of human stomach cells because they can potentially interact with different proteins in the human stomach mucosa. In addition, several human gastric mucins have cysteine protease cleavage sites. Discussion: Then, under our experimental conditions, consuming meat from infected animals in the acute phase allows the T. cruzi infection. Similarly, trypomastigotes and amastigotes could infect mice when administered orally, whereas cysteinyl proteinases and trans-sialidase appear to be relevant molecules in this infective process.

Intranasal Trans-Sialidase Vaccine Mitigates Acute and Chronic Pathology in a Preclinical Oral Chagas Disease Model.

Chagas disease, caused by Trypanosoma cruzi, leads to severe complications in 30% of infected individuals, including acute myocarditis and chronic fibrosing cardiomyopathy. Despite the significant burden of this disease, there is currently no licensed vaccine available to prevent it. This study aimed to evaluate the mucosal and systemic immunogenicity as well as the prophylactic efficacy of a mucosal vaccine candidate and its impact on both acute and chronic cardiomyopathy. The results showed that the nasal administration of trans-sialidase (TS) plus c-di-AMP (TS+A) vaccine elicited a NALT expression of IFN-γ, IL-17a and IL-4 mRNA as well as a nasal-specific production of IgA. An in vivo challenge with TS also triggered increased proliferation of lymphocytes from the NALT, sentinel cervical lymph node, and spleen. TS+A immunization increased the plasma levels of Th1/Th2/Th17 cytokines and elicited an evident cellular response by which to judge enhanced delayed-type hypersensitivity responses following a TS footpad challenge. After oral infection, TS+A-vaccinated mice showed significantly reduced parasitemia and parasite load in the heart, muscles and intestines, while markers of hepatic and muscle damage as well as clinical manifestations of acute infection were strongly diminished. TS+A also attenuated acute myocarditis and the expression of inflammatory markers in the heart. The protection conferred by TS+A extended into the chronic phase, where it resulted in a clear reduction in chronic myocarditis, fibrosis and functional electrocardiographic abnormalities, associated with a decreased expression of the pro-fibrotic TGF-ß. These results revealed that it is possible to develop a mucosal vaccine against T. cruzi based on TS and c-di-AMP that is capable of reducing the development of Chagas cardiomyopathy, the hallmark of Chagas disease.