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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-β. 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. [ABSTRACT FROM AUTHOR]

Chagas disease, caused by the parasitic protozoan Trypanosoma cruzi (T. cruzi), represents a worldwide public health issue. To date, there is no efficient treatment to combat this pathology, and the only drugs available are usually toxic to the patient. Through the enzyme trans-salidase, the parasite invades, infects, and multiplies intracellularly in the host cell. This protein has been considered an attractive target for developing or searching for compounds with potential trypanocidal activity. In this study, an in silico analysis was performed using a Food and Drug Administration-approved computational drug repositioning approach to identify compounds with anti-Chagas potential against two trans-sialidase proteins. Those compounds with potential inhibition were analyzed and selected through a molecular docking-based virtual screening. Forty-nine compounds were identified, of which forty-five are available on the market, and the rest were evaluated in silico. Our predicted results follow that these compounds are safe for human use and could be potential anti-trans-sialidase agents. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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-β. 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.

IntroductionOral 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.MethodsCell 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. ResultsTrypomastigote 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. DiscussionThen, 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.

Background: African animal trypanosomiasis hinders sustainable livestock productivity in sub-Saharan Africa. About 17 million infected cattle are treated with trypanocides annually but most of the drugs are associated with drawbacks, necessitating the search for a promising chemotherapeutic agent. Objectives: In this study, the effects of β-sitosterol on Trypanosoma congolense infection were investigated along with its effect on the trans-sialidase gene expressions. Results: Oral treatment with β-sitosterol at 15 and 30 mg/kg body weight (BW) for 14 days significantly (p < 0.05) reduced parasitemia and ameliorated the parasite-induced anemia. Also, the parasite-induced increase in serum urea level and renal histopathological damage scores in addition to renal hypertrophy was significantly (p < 0.05) reverted following treatment with 30 mg/kg BW β-sitosterol. The compound also significantly (p < 0.05) down-regulated the expression of TconTS1 but not TconTS2, TconTS3, and TconTS4. Correlation analysis between free serum sialic acid with the TconTS1 and TconTS2 gene variants revealed negative correlations in the β-sitosterol-treated groups although they were nonsignificant (p > 0.05) in the group treated with 15 mg/kg BW β-sitosterol. Similarly, a non-significant negative (p > 0.05) correlation between the biomolecule and the TconTS3 and TconTS4 gene variants was observed in the β-sitosterol-treated groups while positive correlations were observed in the infected untreated control group. Conclusion: The observed effect of β-sitosterol on T. congolense infection could make the compound a possible template for the design of novel trypanocides. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

BackgroundAfrican animal trypanosomiasis hinders sustainable livestock productivity in sub-Saharan Africa. About 17 million infected cattle are treated with trypanocides annually but most of the drugs are associated with drawbacks, necessitating the search for a promising chemotherapeutic agent.ObjectivesIn this study, the effects of β-sitosterol on Trypanosoma congolense infection were investigated along with its effect on the trans-sialidase gene expressions.ResultsOral treatment with β-sitosterol at 15 and 30 mg/kg body weight (BW) for 14 days significantly (p 0.05) in the group treated with 15 mg/kg BW β-sitosterol. Similarly, a non-significant negative (p > 0.05) correlation between the biomolecule and the TconTS3 and TconTS4 gene variants was observed in the β-sitosterol-treated groups while positive correlations were observed in the infected untreated control group.ConclusionThe observed effect of β-sitosterol on T. congolense infection could make the compound a possible template for the design of novel trypanocides.

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.

Trypanosoma congolense is a pathogenic African animal trypanosome species causing devastating conditions leading to death of an infected host. The drawbacks of the existing trypanocidal drugs have led to the search for new drug candidates. In this study, β‐ionone at 15 and 30 mg/kg body weight (BW) was orally administered to T. congolense infected rats for 14 days followed by an assessment of anemia, organ damages, and the expression of T. congolense trans‐sialidase gene variants. A significant decrease in parasitemia (p <.05) was observed in the animals treated with 15 mg/kg BW β‐ionone besides increased animal survival rate. A trypanosome‐induced decrease in packed cell volume (PCV) and histopathological changes across tissues was significantly (p <.05) ameliorated following treatment with both doses of β‐ionone. This is in addition to reversing the parasite‐induced upsurge in free serum sialic acid (FSA) and expression of T. congolense trans‐sialidase gene variants (TconTS1, TconTS3, and TconTS4). Correlation analysis revealed a positive correlation (p >.05) between FSA with the TconTS gene expressions. In addition, the compound inhibited partially purified T. congolense sialidase and phospholipase A2 via mixed inhibition pattern with inhibition binding constants of 25.325 and 4.550 µM, respectively, while molecular docking predicted binding energies of −5.6 kcal/mol for both enzymes. In conclusion, treatment with β‐ionone suppressed T. congolense proliferation and protected the animals against some of the parasite‐induced pathologies whilst the effect on anemia development might be due to inhibition of sialidase and PLA2 activities as well as the expression levels of TconTS3 and TconTS4. [ABSTRACT FROM AUTHOR]

Trypanosoma congolense is a pathogenic African animal trypanosome species causing devastating conditions leading to death of an infected host. The drawbacks of the existing trypanocidal drugs have led to the search for new drug candidates. In this study, β‐ionone at 15 and 30 mg/kg body weight (BW) was orally administered to T. congolense infected rats for 14 days followed by an assessment of anemia, organ damages, and the expression of T. congolense trans‐sialidase gene variants. A significant decrease in parasitemia (p <.05) was observed in the animals treated with 15 mg/kg BW β‐ionone besides increased animal survival rate. A trypanosome‐induced decrease in packed cell volume (PCV) and histopathological changes across tissues was significantly (p <.05) ameliorated following treatment with both doses of β‐ionone. This is in addition to reversing the parasite‐induced upsurge in free serum sialic acid (FSA) and expression of T. congolense trans‐sialidase gene variants (TconTS1, TconTS3, and TconTS4). Correlation analysis revealed a positive correlation (p >.05) between FSA with the TconTS gene expressions. In addition, the compound inhibited partially purified T. congolense sialidase and phospholipase A2 via mixed inhibition pattern with inhibition binding constants of 25.325 and 4.550 µM, respectively, while molecular docking predicted binding energies of −5.6 kcal/mol for both enzymes. In conclusion, treatment with β‐ionone suppressed T. congolense proliferation and protected the animals against some of the parasite‐induced pathologies whilst the effect on anemia development might be due to inhibition of sialidase and PLA2 activities as well as the expression levels of TconTS3 and TconTS4. [ABSTRACT FROM AUTHOR]

Atherosclerosis is one of the leading causes of death in developed and developing countries. The atherogenicity phenomenon cannot be separated from the role of modified low-density lipoproteins (LDL) in atherosclerosis development. Among the multiple modifications of LDL, desialylation deserves to be discussed separately, since its atherogenic effects and contribution to atherogenicity are often underestimated or, simply, forgotten. Vladimir Tertov is linked to the origin of the research related to desialylated lipoproteins, including the association of modified LDL with atherogenicity, autoimmune nature of atherosclerosis, and discovery of sialidase activity in blood plasma. The review will briefly discuss all the above-mentioned information, with a description of the current situation in the research. [ABSTRACT FROM AUTHOR]

• Mucosal vaccine strategy can protect against T. cruzi infection. • TS antigen plus c-di-AMP trigger systemic and mucosal TS-specific antibodies. • TS antigen plus c-di-AMP display a Th1-biased response. • A nasal vaccine based in TS antigen plus c-di-AMP control oral T. cruzi infection. The new generation of vaccines for Chagas disease, are focused to induce both humoral and cellular response to effectively control Trypanosoma cruzi parasites. The administration of vaccine formulations intranasally has the advantage over parenteral routes that can induce a specific response at mucosal and systemic levels. This study aimed to evaluate and compare the immunogenicity and prophylactic effectiveness of two Trans-sialidase (TS)-based mucosal vaccines against T. cruzi administered intranasally. Vaccines consisted of a recombinant fragment of TS expressed in Lactococcus lactis formulated in two different adjuvants. The first, was an immunostimulant particle (ISPA, an ISCOMATRIX-like adjuvant), while the second was the dinucleotide c-di-AMP, which have shown immunostimulant properties at the mucosal level. BALB/c mice were immunized intranasally (3 doses, one every two weeks) with each formulation (TS + ISPA or TS + c-di-AMP) and with TS alone or vehicle (saline solution) as controls. Fifteen days after the last immunization, both TS + ISPA or TS + c-di-AMP induced an evident systemic humoral and cellular response, as judged by the increased plasma anti-TS IgG2a titers and IgG2a/IgG1 ratio and enhanced cellular response against TS. Plasma derived antibodies from TS + c-di-AMP also inhibit in vitro the invasion capacity of T. cruzi. Furthermore, specific secretory IgA was more enhanced in TS + c-di-AMP group. Protective efficacy was proved in vaccinated animals by an oral T. cruzi -challenge. Parasitemia control was only achieved by animals vaccinated with TS + c-di-AMP, despite all vaccinates groups showed enhanced CD8+IFN-γ+ T cell numbers. In addition, it was reflected during the acute phase in a significant reduction of tissue parasite load, clinical manifestations and diminished tissue damage. The better prophylactic capacity elicited by TS + c-di-AMP was related to the induction of neutralizing plasma antibodies and augmented levels of mucosal IgA since TS + ISPA and TS + c-di-AMP groups displayed similar immunogenicity and CD8+IFN-γ+ T-cell response. Therefore, TS + c-di-AMP formulation appears as a promising strategy for prophylaxis against T. cruzi. [ABSTRACT FROM AUTHOR]

ABSTRACT Trans-sialidases (TS) are unusual enzymes present on the surface of Trypanosoma cruzi, the causative agent of Chagas disease. Encoded by the largest gene family in the T. cruzi genome, only few members of the TS family have catalytic activity. Active trans-sialidases (aTS) are responsible for transferring sialic acid from host glycoconjugates to mucins, also present on the parasite surface. The existence of several copies of TS genes has impaired the use of reverse genetics to study this highly polymorphic gene family. Using CRISPR-Cas9, we generated aTS knockout cell lines displaying undetectable levels of TS activity, as shown by sialylation assays and labeling with antibodies that recognize sialic acid-containing mucins. In vitro infection assays showed that disruption of aTS genes does not affect the parasite’s capacity to invade cells or to escape from the parasitophorous vacuole but resulted in impaired differentiation of amastigotes into trypomastigotes and parasite egress from the cell. When inoculated into mice, aTS mutants were unable to establish infection even in the highly susceptible gamma interferon (IFN-γ) knockout mice. Mice immunized with aTS mutants were fully protected against a challenge infection with the virulent T. cruzi Y strain. Altogether, our results confirmed the role of aTS as a T. cruzi virulence factor and indicated that aTS play a major role during the late stages of intracellular development and parasite egress. Notably, mutants lacking TS activity are completely avirulent in animal models of infection and may be used as a live attenuated vaccine against Chagas disease. IMPORTANCE Trypanosoma cruzi is the causative agent of Chagas disease, a neglected tropical disease that affects approximately 6 to 8 million people and for which there is no effective treatment or vaccine. The parasite expresses a family of surface proteins, named trans-sialidases, responsible for transferring sialic acid from host glycoconjugates to parasite mucins. Although recognized as a main virulence factor, the multiple roles of these proteins during infection have not yet been fully characterized, mainly because the presence of several copies of aTS genes has impaired their study using reverse genetics. By applying CRISPR-Cas9, we generated aTS knockout parasites and showed that, although aTS parasite mutants were able to infect cells in vitro, they have an impaired capacity to egress from the infected cell. Importantly, aTS mutants lost the ability to cause infection in vivo but provided full protection against a challenge infection with a virulent strain.

Chagas’ disease is caused by the protozoan Trypanosoma cruzi, described in the early 20th century by the Brazilian physician Dr. Carlos Chagas. There was a great amount of research devoted to diagnosis, treatment and prevention of the disease. One of the most important discoveries made since then, impacting the understanding of how the parasite interacts with the host’s immune system, was the description of trans-sialidase. It is an unique enzyme, capable of masking the parasite’s presence from the host, while at the same time dampening the activation of CD8+ T cells, the most important components of the immune response. 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. The importance of the trans-sialidase enzyme brought life to many studies for the design of diagnostic tests, drugs and vaccines. While many groups have been prolific, such efforts have encountered problems, among them: the fact that while T. cruzi have many genes that are unique to the parasite, it relies on multiple copies of them and the difficulty in providing epitopes that result in effective and robust immune responses. In this review, we aim to convey the importance of trans-sialidase as well as to provide a history, including the initial failures and the most promising successes in the chasing of a working vaccine for a disease that is endemic in many tropical countries, including Brazil.

Abstract 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.

BACKGROUND Trypanosoma cruzi is an important human pathogen in Latin America with nearly seven million people infected. It has a large degree of genetic diversity, classified into six discrete typing units (DTUs), which probably influences its physiological behavior and clinical manifestations. Several genotyping methods are available, with distinct performance on easiness, cost, resolution and applicability; no method excels in all parameters. OBJECTIVES AND METHODS To devise a molecular method for T. cruzi genotyping, based on polymerase chain reaction (PCR) amplification of a single target with multiple copies in the nuclear genome by large scale sequencing. We have applied this method to 29 T. cruzi isolates, comprising all described DTUs. FINDINGS We were able to classify all samples into sub DTU level with high robustness. Evolutionary relationship between DTUs were ascertained, suggesting that TcIII and TcIV DTUs are non-hybrid, and DTU IV is more similar to the common ancestral. CONCLUSION As the TS-LSS method is based on a single PCR reaction, comprising several copies of the target, it is probably useful for clinical samples, when the amount of DNA is a limiting factor. As large scale sequencing systems become more common, the TS-LSS method can be increasingly applied for T. cruzi genotyping.

Trypanosoma cruzi (T. cruzi) is a hemoflagellate protozoan parasite that causes Chagas disease, a neglected tropical disease that affects more than 6 million people around the world, mostly in Latin America. Despite intensive research, there is no vaccine available; therefore, new approaches are needed to further improve vaccine efficacy. It is well established that experimental T. cruzi infection induces a marked immunosuppressed state, which includes notably increases of CD11b+ GR-1+ myeloid-derived suppressor cells (MDSCs) in the spleen, liver and heart of infected mice. We previously showed that a trans-sialidase based vaccine (TSf-ISPA) is able to confer protection against a virulent T. cruzi strain, stimulating the effector immune response and decreasing CD11b+ GR-1+ splenocytes significantly. Here, we show that even in the immunological context elicited by the TSf-ISPA vaccine, the remaining MDSCs are still able to influence several immune populations. Depletion of MDSCs with 5 fluorouracil (5FU) at day 15 post-infection notably reshaped the immune response, as evidenced by flow cytometry of spleen cells obtained from mice after 21 days post-infection. After infection, TSf-ISPA-vaccinated and 5FU-treated mice showed a marked increase of the CD8 response, which included an increased expression of CD107a and CD44 markers in CD8+ cultured splenocytes. In addition, vaccinated and MDSC depleted mice showed an increase in the percentage and number of CD4+ Foxp3+ regulatory T cells (Tregs) as well as in the expression of Foxp3+ in CD4+ splenocytes. Furthermore, depletion of MDSCs also caused changes in the percentage and number of CD11chigh CD8α+ dendritic cells as well as in activation/maturation markers such as CD80, CD40 and MHCII. Thus, the obtained results suggest that MDSCs not only play a role suppressing the effector response during T. cruzi infection, but also strongly modulate the immune response in vaccinated mice, even when the vaccine formulation has a significant protective capacity. Although MDSC depletion at day 15 post-infection did not ameliorated survival or parasitemia levels, depletion of MDSCs during the first week of infection caused a beneficial trend in parasitemia and mice survival of vaccinated mice, supporting the possibility to target MDSCs from different approaches to enhance vaccine efficacy. Finally, since we previously showed that TSf-ISPA immunization causes a slight but significant increase of CD11b+ GR-1+ splenocytes, here we also targeted those cells at the stage of immunization, prior to T. cruzi challenge. Notably, 5FU administration before each dose of TSf-ISPA vaccine was able to significantly ameliorate survival and decrease parasitemia levels of TSf-ISPA-vaccinated and infected mice. Overall, this work supports that targeting MDSCs may be a valuable tool during vaccine design against T. cruzi, and likely for other pathologies that are characterized by the subversion of the immune system.

Trypanosoma cruzi (T. cruzi) is a hemoflagellate protozoan parasite that causes Chagas disease, a neglected tropical disease that affects more than 6 million people around the world, mostly in Latin America. Despite intensive research, there is no vaccine available; therefore, new approaches are needed to further improve vaccine efficacy. It is well established that experimental T. cruzi infection induces a marked immunosuppressed state, which includes notably increases of CD11b+ GR-1+ myeloid-derived suppressor cells (MDSCs) in the spleen, liver and heart of infected mice. We previously showed that a trans-sialidase based vaccine (TSf-ISPA) is able to confer protection against a virulent T. cruzi strain, stimulating the effector immune response and decreasing CD11b+ GR-1+ splenocytes significantly. Here, we show that even in the immunological context elicited by the TSf-ISPA vaccine, the remaining MDSCs are still able to influence several immune populations. Depletion of MDSCs with 5 fluorouracil (5FU) at day 15 post-infection notably reshaped the immune response, as evidenced by flow cytometry of spleen cells obtained from mice after 21 days post-infection. After infection, TSf-ISPA-vaccinated and 5FU-treated mice showed a marked increase of the CD8 response, which included an increased expression of CD107a and CD44 markers in CD8+ cultured splenocytes. In addition, vaccinated and MDSC depleted mice showed an increase in the percentage and number of CD4+ Foxp3+ regulatory T cells (Tregs) as well as in the expression of Foxp3+ in CD4+ splenocytes. Furthermore, depletion of MDSCs also caused changes in the percentage and number of CD11chigh CD8α+ dendritic cells as well as in activation/maturation markers such as CD80, CD40 and MHCII. Thus, the obtained results suggest that MDSCs not only play a role suppressing the effector response during T. cruzi infection, but also strongly modulate the immune response in vaccinated mice, even when the vaccine formulation has a significant protective capacity. Although MDSC depletion at day 15 post-infection did not ameliorated survival or parasitemia levels, depletion of MDSCs during the first week of infection caused a beneficial trend in parasitemia and mice survival of vaccinated mice, supporting the possibility to target MDSCs from different approaches to enhance vaccine efficacy. Finally, since we previously showed that TSf-ISPA immunization causes a slight but significant increase of CD11b+ GR-1+ splenocytes, here we also targeted those cells at the stage of immunization, prior to T. cruzi challenge. Notably, 5FU administration before each dose of TSf-ISPA vaccine was able to significantly ameliorate survival and decrease parasitemia levels of TSf-ISPA-vaccinated and infected mice. Overall, this work supports that targeting MDSCs may be a valuable tool during vaccine design against T. cruzi , and likely for other pathologies that are characterized by the subversion of the immune system. [ABSTRACT FROM AUTHOR]

Atherosclerosis is one of the leading causes of death in developed and developing countries. The atherogenicity phenomenon cannot be separated from the role of modified low-density lipoproteins (LDL) in atherosclerosis development. Among the multiple modifications of LDL, desialylation deserves to be discussed separately, since its atherogenic effects and contribution to atherogenicity are often underestimated or, simply, forgotten. Vladimir Tertov is linked to the origin of the research related to desialylated lipoproteins, including the association of modified LDL with atherogenicity, autoimmune nature of atherosclerosis, and discovery of sialidase activity in blood plasma. The review will briefly discuss all the above-mentioned information, with a description of the current situation in the research.

The knowledge that the immune system is composed of a regulatory/suppressor arm added a new point of view to better understand the nature of several pathologies including cancer, transplants, infections and autoimmune diseases. The striking discoveries concerning molecules and cells involved in this kind of regulation were followed by the elucidation of equally notable mechanisms used by several pathogens to manipulate the host immune system. Vaccines against pathogens are an invaluable tool developed to help the immune system cope with a potential infection or prevent disease pathology. Nowadays, there is accumulated evidence indicating that the powerful stimulation capacity of vaccines influences not only the effector arm of the immune system but also cells with regulatory/suppressor capacity, such as myeloid derived suppressor cells (MDSCs) and Foxp3+ regulatory T cells (Tregs). Trypanosoma cruzi (T. cruzi) is a protozoan parasite with a complex life cycle that has evolved several strategies to influence the regulatory immune response. Although diverse vaccine formulations have been able to stimulate the effector response, achieving non-sterilizing protection against T. cruzi , the influence of the vaccine candidates on the regulatory machinery has scarcely been assessed. This fact may not only reveal important information concerning how vaccines may influence cells with regulatory/suppressor capacity but also open the possibility to analyze whether vaccines are able to disrupt the mechanisms used by some pathogens to manipulate the host regulatory circuits. The aim of this review is to summarize and discuss available data related to the role of cellular components, like MDSCs and Foxp3+ Tregs, during T. cruzi infection, and the potential utility of those populations as additional targets for the rational design of vaccines. [ABSTRACT FROM AUTHOR]

Trans-sialidase of Trypanosoma cruzi (TcTS) is a key enzyme in the infection process from parasite to host; therefore, it has been considered an important target for developing new anti-Chagas drugs. Different compounds with trypanocidal activity and/or inhibition of TcTS have been reported; however, some benzoic acid derivatives have shown high enzymatic inhibition but low trypanocidal activity and viceversa. These results show that each compound may possess a different mechanism of action. Based on the above, the compound 4-amino-3-nitrobenzoic acid (16), a potent TcTS inhibitor (77% inhibition in enzymatic assays) was selected to evaluate its effects on the expression level of the TS gene in T. cruzi epimastigotes and determine its involvement in the mechanism of action. Results showed an increase in the expression level of the TcTS gene, which confirmed that compound 16, has a direct effect on TcTS. [ABSTRACT FROM AUTHOR]

The last decades have produced a plethora of evidence on the role of glycans, from cell adhesion to signaling pathways. Much of that information pertains to their role on the immune system and their importance on the surface of many human pathogens. A clear example of this is the flagellated protozoan Trypanosoma cruzi, which displays on its surface a great variety of glycoconjugates, including O-glycosylated mucin-like glycoproteins, as well as multiple glycan-binding proteins belonging to the trans-sialidase (TS) family. Among the latter, different and concurrently expressed molecules may present or not TS activity, and are accordingly known as active (aTS) and inactive (iTS) members. Over the last thirty years, it has been well described that T. cruzi is unable to synthesize sialic acid (SIA) on its own, making use of aTS to steal the host's SIA. Although iTS did not show enzymatic activity, it retains a substrate specificity similar to aTS (α-2,3 SIA-containing glycotopes), displaying lectinic properties. It is accepted that aTS members act as virulence factors in mammals coursing the acute phase of the T. cruzi infection. However, recent findings have demonstrated that iTS may also play a pathogenic role during T. cruzi infection, since it modulates events related to adhesion and invasion of the parasite into the host cells. Since both aTS and iTS proteins share structural substrate specificity, it might be plausible to speculate that iTS proteins are able to assuage and/or attenuate biological phenomena depending on the catalytic activity displayed by aTS members. Since SIA-containing glycotopes modulate the host immune system, it should not come as any surprise that changes in the sialylation of parasite's mucin-like molecules, as well as host cell glycoconjugates might disrupt critical physiological events, such as the building of effective immune responses. This review aims to discuss the importance of mucin-like glycoproteins and both aTS and iTS for T. cruzi biology, as well as to present a snapshot of how disturbances in both parasite and host cell sialoglycophenotypes may facilitate the persistence of T. cruzi in the infected mammalian host.

Lactococcus lactis is a promising candidate for the development of mucosal vaccines. More than 20 years of experimental research supports this immunization approach. In addition, 3′ 5′- cyclic di-adenosine monophosphate (c-di-AMP) is a bacterial second messenger that plays a key role in the regulation of diverse physiological functions (potassium and cellular wall homeostasis, among others). Moreover, recent studies showed that c-di-AMP has a strong mucosal adjuvant activity that promotes both humoral and cellular immune responses. In this study, we report the development of a novel mucosal vaccine prototype based on a genetically engineered L. lactis strain. First, we demonstrate that homologous expression of cdaA gen in L. lactis is able to increase c-di-AMP levels. Thus, we hypothesized that in vivo synthesis of the adjuvant can be combined with production of an antigen of interest in a separate form or jointly in the same strain. Therefore, a specifically designed fragment of the trans-sialidase (TScf) enzyme from the Trypanosoma cruzi parasite, the etiological agent of Chagas disease, was selected to evaluate as proof of concept the immune response triggered by our vaccine prototypes. Consequently, we found that oral administration of a L. lactis strain expressing antigenic TScf combined with another L. lactis strain producing the adjuvant c-di-AMP could elicit a TS-specific immune response. Also, an additional L. lactis strain containing a single plasmid with both cdaA and tscf genes under the Pcit and Pnis promoters, respectively, was also able to elicit a specific immune response. Thus, the current report is the first one to describe an engineered L. lactis strain that simultaneously synthesizes the adjuvant c-di-AMP as well as a heterologous antigen in order to develop a simple and economical system for the formulation of vaccine prototypes using a food grade lactic acid bacterium.

Trypanosoma brucei is the etiological agent of African trypanosomiasis responsible for human and animal infections. T. brucei is transmitted by infected tsetse flies. There is no vaccine for the disease and drugs available for treatment are inefficient and high toxicity. In this context, it is a priority to find antigenic targets suitable for the development of new diagnostic tools, drugs and vaccines. In this work, we report that mice infected with T. b. brucei produce antibodies against trans-sialidase recombinant protein (TS). In addition, we also demonstrate that bloodstream T. b. brucei express messenger RNA related to the TS gene. Collectively, our data strongly suggest that bloodstream forms of T. b. brucei also express the TS gene, that to date was described only in the procyclic forms of the T. b. brucei. In conclusion, these results highlight the importance of TS protein as a possible antigen target during infection caused by T. b. brucei. [ABSTRACT FROM AUTHOR]

Abstract The trans -sialidase protein expressed by Trypanosoma cruzi is an important enzyme in the life cycle of this human pathogenic parasite and is considered a promising target for the development of new drug treatments against Chagas' disease. Here we describe α-amino phosphonates as a novel class of inhibitor of T. cruzi trans -sialidase. Molecular modelling studies were initially used to predict the active-site binding affinities for a series of amino phosphonates, which were subsequently synthesised and their IC 50 s determined in vitro. The measured inhibitory activities show some correlation with the predictions from molecular modelling, with 1-napthyl derivatives found to be the most potent inhibitors having IC 50 s in the low micromolar range. Interestingly, kinetic analysis of the mode of inhibition demonstrated that the α-aminophosphonates tested here operate in a non-competitive manner. Graphical abstract Image 1 Highlights • Aryl α-aminophosphonates are effective inhibitors of T. cruzi trans -sialidase. • Inhibition of trans -sialidase is improved using substituted aryl α-aminophosphonates. • Aryl α-aminophosphonates inhibit trans -sialidase in a non-competitive manner. [ABSTRACT FROM AUTHOR]

Abstract Rational drug design creates innovative therapeutics based on knowledge of the biological target to provide more effective and responsible therapeutics. Chagas disease, endemic throughout Latin America, is caused by Trypanosoma cruzi, a protozoan parasite. Current therapeutics are problematic with widespread calls for new approaches. Researchers are using rational drug design for Chagas disease and one target receiving considerable attention is the T. cruzi trans-sialidase protein (TcTS). In T. cruzi, trans-sialidase catalyzes the transfer of sialic acid from a mammalian host to coat the parasite surface membrane and avoid immuno-detection. However, the role of TcTS in pathology variance among and within genetic variants of the parasite is not well understood despite numerous studies. Previous studies reported the crystalline structure of TcTS and the TS protein structure in other trypanosomes where the enzyme is often inactive. However, no study has examined the role of natural selection in genetic variation in TcTS. To understand the role of natural selection in TcTS DNA sequence and protein variation, we examined a 471 bp portion of the TcTS gene from 48 T. cruzi samples isolated from insect vectors. Because there may be multiple parasite genotypes infecting one insect and there are multiple copies of TcTS per parasite genome, all 48 sequences had multiple polymorphic bases. To resolve these polymorphisms, we examined cloned sequences from two insect vectors. The data are analyzed to understand the role of natural selection in shaping genetic variation in TcTS and interpreted in light of the possible role of TcTS as a drug target. The analysis highlights negative or purifying selection on three amino acids previously shown to be important in TcTS transfer activity. One amino acid in particular, Tyr342, is a strong candidate for a drug target because it is under negative selection and amino acid substitutions inactivate TcTS transfer activity. Author summary Chagas disease is caused by the protozoan parasite Trypanosoma cruzi and transmitted to humans and other mammals primarily by Triatomine insects. Being endemic in many South and Central American countries and affecting millions of people the need for new more effective and safe therapies is evident. Here, we examine genetic variation and natural selection on DNA (471 bp) and amino acid (157 aa) sequence data of the T. cruzi trans-sialdiase (TcTS) protein, often suggested as a candidate for rational drug design. In our surveyed region of the protein there were five amino acid residues that have been shown to be integral to the function of TcTS. We found that three were under strong negative selection making them ideal candidates for drug design; however, one was under balancing selection and should be avoided as a drug target. Our study provides new information into identifying potential targets for a new Chagas drug. Highlights • Trypanosoma cruzi trans-sialidase has been suggested as a target for rational drug design. • We found genetic variation in a 498 nucleotide portion of this gene within and between genomes. • Selection signatures in this portion vary between the catalytic and non-catalytic regions. • There is strong purifying selection at three of the five amino acids involved in catalysis. • Drug design is discussed with respect to variation and selection. [ABSTRACT FROM AUTHOR]

Trans-sialidase from Trypanosoma cruzi (Tc-TS) belongs to a superfamily of proteins that may have enzymatic activity. While enzymatically active members (Tc-aTS) are able to transfer sialic acid from the host cell sialyl-glycoconjugates onto the parasite or to other molecules on the host cell surface, the inactive members (Tc-iTS) are characterized by their lectinic properties. Over the last 10 years, several papers demonstrated that, individually, Tc-aTS or Tc-iTS is able to modulate several biological events. Since the genes encoding Tc-iTS and Tc-aTS are present in the same copy number, and both proteins portray similar substrate-specificities as well, it would be plausible to speculate that such molecules may compete for the same sialyl-glycan structures and govern numerous immunobiological phenomena. However, their combined effect has never been evaluated in the course of an acute infection. In this study, we investigated the ability of both proteins to modulate the production of inflammatory signals, as well as the homing of T cells to the cardiac tissue of infected mice, events that usually occur during the acute phase of T. cruzi infection. The results showed that the intravenous administration of Tc-iTS, but not Tc-aTS protected the cardiac tissue from injury caused by reduced traffic of inflammatory cells. In addition, the ability of Tc-aTS to modulate the production of inflammatory cytokines was attenuated and/or compromised when Tc-iTS was co-injected in the same proportions. These results suggest that although both proteins present structural similarities and compete for the same sialyl-glycan epitopes, they might present distinct immunomodulatory properties on T cells following T. cruzi infection.

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). [ABSTRACT FROM AUTHOR]

Trypanosoma cruzi , the etiologic agent of Chagas disease, is covered by a dense glycocalix mainly composed by glycoproteins called mucins which are also the acceptors of sialic acid in a reaction catalyzed by a trans-sialidase (TcTS). Sialylation of trypomastigote mucins protects the parasite from lysis by the anti α-Gal p antibodies from serum. The TcTS is essential for the infection process since T. cruzi is unable to biosynthesize sialic acid. The enzyme specifically transfers it from a terminal β- d -Gal p unit in the host glycoconjugate to terminal β- d -Gal p units in the parasite mucins to construct the d -NeuNAc(α2→3)β- d -Gal p motif. On the other hand, although galactose is the most abundant sugar in mucins of both, the infective trypomastigotes and the insect stage epimastigotes, α- d -Gal p is only present in the infective stage whereas β- d -Gal f is characteristic of the epimastigote stage of the less virulent strains. Neither α- d -Gal p nor d -Gal f is acceptor of sialic acid. In the mucins, some of the oligosaccharides are branched with terminal β- d -Gal p units to be able to accept sialic acid in the TcTS reaction. Based on previous reports showing that anti α-Gal p antibodies only partially colocalize with sialic acid, we have undertaken the synthesis of the trisaccharide α- d -Gal p (1→3)-[β- d -Gal p (1→6)]- d -Gal p , the smallest structure containing both, the antigenic d -Gal p (α1→3)- d -Gal p unit and the sialic acid-acceptor β- d -Gal p unit. The trisaccharide was obtained as the 6-aminohexyl glycoside to facilitate further conjugation for biochemical studies. The synthetic approach involved the α-galactosylation at O-4 of a suitable precursor of the reducing end, followed by β-galactosylation at O-6 of the same precursor and introduction of the 6-aminohexyl aglycone. The fully deprotected trisaccharide was successfully sialylated by TcTS using either 3′-sialyllactose or fetuin as donors. The product, 6-aminohexyl α- d -NeuNAc(2→3)-β- d -Gal p (1→6)-[α- d -Gal p (1→3)]-β- d -Gal p , was purified and characterized. [ABSTRACT FROM AUTHOR]

Trans-sialidase from Trypanosoma cruzi (Tc-TS) belongs to a superfamily of proteins that may have enzymatic activity. While enzymatically active members (Tc-aTS) are able to transfer sialic acid from the host cell sialyl-glycoconjugates onto the parasite or to other molecules on the host cell surface, the inactive members (Tc-iTS) are characterized by their lectinic properties. Over the last 10 years, several papers demonstrated that, individually, Tc-aTS or Tc-iTS is able to modulate several biological events. Since the genes encoding Tc-iTS and Tc-aTS are present in the same copy number, and both proteins portray similar substrate-specificities as well, it would be plausible to speculate that such molecules may compete for the same sialyl-glycan structures and govern numerous immunobiological phenomena. However, their combined effect has never been evaluated in the course of an acute infection. In this study, we investigated the ability of both proteins to modulate the production of inflammatory signals, as well as the homing of T cells to the cardiac tissue of infected mice, events that usually occur during the acute phase of T. cruzi infection. The results showed that the intravenous administration of Tc-iTS, but not Tc-aTS protected the cardiac tissue from injury caused by reduced traffic of inflammatory cells. In addition, the ability of Tc-aTS to modulate the production of inflammatory cytokines was attenuated and/or compromised when Tc-iTS was co-injected in the same proportions. These results suggest that although both proteins present structural similarities and compete for the same sialyl-glycan epitopes, they might present distinct immunomodulatory properties on T cells following T. cruzi infection. [ABSTRACT FROM AUTHOR]

Chagas is a lethal chronic disease that currently affects 8–10 million people worldwide, primarily in South and Central America. Trypanosoma cruzi trans-sialidase is an enzyme that is of vital importance for the survival of the parasite due to its key role in the transfer of sialic acid from the host to the parasite surface and it also helps the parasite combat the host's immune system. This enzyme has no equivalent human enzyme; thus, it has become an interesting target for the development of inhibitors that combat the parasite. In this review, we summarize three classes of inhibitors (acceptor, donor and unrelated) with their inhibition values and their mode of action against this enzyme. Based on molecular docking, molecular dynamics and structure-activity relationship studies, it has been discovered that the molecules with –NH2, –OH and –COOH groups on an aromatic ring could be used as a scaffold for the development of new and potent trans-sialidase inhibitors due to their key interaction with active enzyme sites. In particular, carboxylic acid derivatives have importance over the sugar moiety due to their ease of synthesis and unique structure-activity relationship. [ABSTRACT FROM AUTHOR]

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-2K b or H-2K d 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., (© 2023 Wiley Periodicals LLC.)

Trypanosoma cruzi, the etiological agent of Chagas disease exhibits multiple mechanisms to guarantee its establishment and persistence in the infected host. It has been well demonstrated that T. cruzi is not able to synthesize sialic acids (Sia). To acquire the monosaccharide, the parasite makes use of a multifunctional enzyme called trans-sialidase (Tc-TS). Since this enzyme has no analogous in the vertebrate host, it has been used as a target in drug therapy development. Tc-TS preferentially catalyzes the transfer of Sia from the host glycoconjugates to the terminal β-galactopyranosyl residues of mucin-like molecules present on the parasite's cell surface. Alternatively, the enzyme can sialylate/re-sialylate glycoconjugates expressed on the surface of host cells. Since its discovery, several studies have shown that T. cruzi employs the Tc-TS activity to modulate the host cell sialoglycophenotype, thus favoring its perpetuation in the infected vertebrate. In this review, we summarize the dynamic of host/parasite sialylglycophenotype modulation, highlighting its role in the subversion of host immune response in order to promote the establishment of persistent chronic infection.

Chagas disease is caused by the flagellate protozoan Trypanosoma cruzi, affecting millions of people throughout Latin America. The parasite dampens host immune response causing modifications in diverse lymphoid compartments, including the thymus. T. cruzi trans-sialidase (TS) seems to play a fundamental role in such immunopathological events. This unusual enzyme catalysed the transference of sialic acid molecules from host glycoconjugates to acceptor molecules placed on the parasite surface. TS activity mediates several biological effects leading to the subversion of the host immune system, favouring both parasite survival and the establishment of chronic infection. This review summarizes current findings on the role of TS on the immune response during T. cruzi infection.

Trypanosoma cruzi , the agent of Chagas disease, can infect through conjunctive or oral mucosas. Therefore, the induction of mucosal immunity by vaccination is relevant not only to trigger local protection but also to stimulate both humoral and cell-mediated responses in systemic sites to control parasite dissemination. In a previous study, we demonstrated that a nasal vaccine based on a Trans-sialidase (TS) fragment plus the mucosal STING agonist c-di-AMP, was highly immunogenic and elicited prophylactic capacity. However, the immune profile induced by TS-based nasal vaccines at the nasopharyngeal-associated lymphoid tissue (NALT), the target site of nasal immunization, remains unknown. Hence, we analyzed the NALT cytokine expression generated by a TS-based vaccine plus c-di-AMP (TSdA+c-di-AMP) and their association with mucosal and systemic immunogenicity. The vaccine was administered intranasally, in 3 doses separated by 15 days each other. Control groups received TSdA, c-di-AMP, or the vehicle in a similar schedule. We demonstrated that female BALB/c mice immunized intranasally with TSdA+c-di-AMP boosted NALT expression of IFN-γ and IL-6, as well as IFN-β and TGF-β. TSdA+c-di-AMP increased TSdA-specific IgA secretion in the nasal passages and also in the distal intestinal mucosa. Moreover, T and B-lymphocytes from NALT-draining cervical lymph nodes and spleen showed an intense proliferation after ex-vivo stimulation with TSdA. Intranasal administration of TSdA+c-di-AMP provokes an enhancement of TSdA-specific IgG 2a and IgG 1 plasma antibodies, accompanied by an increase IgG 2a /IgG 1 ratio, indicative of a Th1-biased profile. In addition, immune plasma derived from TSdA+c-di-AMP vaccinated mice exhibit in-vivo and ex-vivo protective capacity. Lastly, TSdA+c-di-AMP nasal vaccine also promotes intense footpad swelling after local TSdA challenge. Our data support that TSdA+c-di-AMP nasal vaccine triggers a NALT mixed pattern of cytokines that were clearly associated with an evident mucosal and systemic immunogenicity. These data are useful for further understanding the immune responses elicited by the NALT following intranasal immunization and the rational design of TS-based vaccination strategies for prophylaxis against T. cruzi. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Trypanosoma cruzi, the etiological agent of Chagas disease exhibits multiple mechanisms to guarantee its establishment and persistence in the infected host. It has been well demonstrated that T. cruzi is not able to synthesize sialic acids (Sia). To acquire the monosaccharide, the parasite makes use of a multifunctional enzyme called trans-sialidase (Tc-TS). Since this enzyme has no analogous in the vertebrate host, it has been used as a target in drug therapy development. Tc-TS preferentially catalyzes the transfer of Sia from the host glycoconjugates to the terminal β-galactopyranosyl residues of mucin-like molecules present on the parasite's cell surface. Alternatively, the enzyme can sialylate/re-sialylate glycoconjugates expressed on the surface of host cells. Since its discovery, several studies have shown that T. cruzi employs the Tc-TS activity to modulate the host cell sialoglycophenotype, thus favoring its perpetuation in the infected vertebrate. In this review, we summarize the dynamic of host/parasite sialoglycophenotype modulation, highlighting its role in the subversion of host immune response in order to promote the establishment of persistent chronic infection. [ABSTRACT FROM AUTHOR]

Many important biological functions are mediated by complex glycan structures containing the ninecarbon 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 transsialidase 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. [ABSTRACT FROM AUTHOR]

Chagas disease is caused by the flagellate protozoan Trypanosoma cruzi, affecting millions of people throughout Latin America. The parasite dampens host immune response causing modifications in diverse lymphoid compartments, including the thymus. T. cruzi trans-sialidase (TS) seems to play a fundamental role in such immunopathological events. This unusual enzyme catalyses the transference of sialic acid molecules from host glycoconjugates to acceptor molecules placed on the parasite surface. TS activity mediates several biological effects leading to the subversion of host immune system, hence favoring both parasite survival and the establishment of chronic infection. This review summarizes current findings on the roles of TS in the immune response during T. cruzi infection. [ABSTRACT FROM AUTHOR]

Cloning of the T-cell receptor genes is a critical step when generating T-cell receptor transgenic mice. Because T-cell receptor molecules are clonotypical, isolation of their genes requires reverse transcriptase-assisted PCR using primers specific for each different Valpha or Vß genes or by the screening of cDNA libraries generated from RNA obtained from each individual T-cell clone. Although feasible, these approaches are laborious and costly. The aim of the present study was to test the application of the non-palindromic adaptor-PCR method as an alternative to isolate the genes encoding the T-cell receptor of an antigen-specific T-cell hybridoma. For this purpose, we established hybridomas specific for trans-sialidase, an immunodominant Trypanosoma cruzi antigen. These T-cell hybridomas were characterized with regard to their ability to secrete interferon-gamma, IL-4, and IL-10 after stimulation with the antigen. A CD3+, CD4+, CD8- interferon-gamma-producing hybridoma was selected for the identification of the variable regions of the T-cell receptor by the non-palindromic adaptor-PCR method. Using this methodology, we were able to rapidly and efficiently determine the variable regions of both T-cell receptor chains. The results obtained by the non-palindromic adaptor-PCR method were confirmed by the isolation and sequencing of the complete cDNA genes and by the recognition with a specific antibody against the T-cell receptor variable ß chain. We conclude that the non-palindromic adaptor-PCR method can be a valuable tool for the identification of the T-cell receptor transcripts of T-cell hybridomas and may facilitate the generation of T-cell receptor transgenic mice.

Trans-sialidase (TS) is a polymorphic protein superfamily described in members of the protozoan genus Trypanosoma . Of the eight TS groups recently described, TS group I proteins (some of which have catalytic activity) are present in the distantly related Trypanosoma brucei and Trypanosoma cruzi phylogenetic clades, whereas other TS groups have only been described in some species belonging to the T. cruzi clade. In the present study we analyzed the repertoire, distribution and phylogenetic relationships of TS genes among species of the T. cruzi clade based on sequence similarity, multiple sequence alignment and tree-reconstruction approaches using TS sequences obtained with the aid of PCR-based strategies or retrieved from genome databases. We included the following representative isolates of the T. cruzi clade from South America: T. cruzi , T. cruzi Tcbat, Trypanosoma cruzi marinkellei , Trypanosoma dionisii , Trypanosoma rangeli and Trypanosoma conorhini . The cloned sequences encoded conserved TS protein motifs Asp-box and VTVxNVxLYNR but lacked the FRIP motif (conserved in TS group I). The T. conorhini sequences were the most divergent. The hybridization patterns of TS probes with chromosomal bands confirmed the abundance of these sequences in species in the T. cruzi clade. Divergence and relationship analysis placed most of the TS sequences in the groups defined in T. cruzi. Further examination of members of TS group II, which includes T. cruzi surface glycoproteins implicated in host cell attachment and invasion, showed that sequences of T. cruzi Tcbat grouped with those of T. cruzi genotype TcI. Our analysis indicates that different members of the T. cruzi clade, with different vertebrate hosts, vectors and pathogenicity, share the extensive expansion and sequence diversification of the TS gene family. Altogether, our results are congruent with the evolutionary history of the T. cruzi clade and represent a contribution to the understanding of the molecular evolution and role of TS proteins in trypanosomes. [ABSTRACT FROM AUTHOR]

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 fromthe 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 aimof 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. [ABSTRACT FROM AUTHOR]