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Carlos A Buscaglia - One of the best experts on this subject based on the ideXlab platform.
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the Trypomastigote small surface antigen tssa regulates trypanosoma cruzi infectivity and differentiation
PLOS Neglected Tropical Diseases, 2017Co-Authors: Maria De Los Milagros Camara, Gaspar E Canepa, Andres B Lantos, Virginia Balouz, Hai Yu, Xi Chen, Oscar Campetella, Juan Mucci, Carlos A BuscagliaAbstract:Background TSSA (Trypomastigote Small Surface Antigen) is an antigenic, adhesion molecule displayed on the surface of Trypanosoma cruzi Trypomastigotes. TSSA displays substantial sequence identity to members of the TcMUC gene family, which code for the Trypomastigote mucins (tGPI-mucins). In addition, TSSA bears sequence polymorphisms among parasite strains; and two TSSA variants expressed as recombinant molecules (termed TSSA-CL and TSSA-Sy) were shown to exhibit contrasting features in their host cell binding and signaling properties. Methods/Principle findings Here we used a variety of approaches to get insights into TSSA structure/function. We show that at variance with tGPI-mucins, which rely on their extensive O-glycoslylation to achieve their protective function, TSSA seems to be displayed on the Trypomastigote coat as a hypo-glycosylated molecule. This has a functional correlate, as further deletion mapping experiments and cell binding assays indicated that exposition of at least two peptidic motifs is critical for the engagement of the ‘adhesive’ TSSA variant (TSSA-CL) with host cell surface receptor(s) prior to Trypomastigote internalization. These motifs are not conserved in the ‘non-adhesive’ TSSA-Sy variant. We next developed transgenic lines over-expressing either TSSA variant in different parasite backgrounds. In strict accordance to recombinant protein binding data, Trypomastigotes over-expressing TSSA-CL displayed improved adhesion and infectivity towards non-macrophagic cell lines as compared to those over-expressing TSSA-Sy or parental lines. These phenotypes could be specifically counteracted by exogenous addition of peptides spanning the TSSA-CL adhesion motifs. In addition, and irrespective of the TSSA variant, over-expression of this molecule leads to an enhanced Trypomastigote-to-amastigote conversion, indicating a possible role of TSSA also in parasite differentiation. Conclusion/Significance In this study we provided novel evidence indicating that TSSA plays an important role not only on the infectivity and differentiation of T. cruzi Trypomastigotes but also on the phenotypic variability displayed by parasite strains.
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sialic acid glycobiology unveils trypanosoma cruzi Trypomastigote membrane physiology
PLOS Pathogens, 2016Co-Authors: Andres B Lantos, Maria De Los Milagros Camara, Hai Yu, Xi Chen, Carlos A Buscaglia, Giannina Carlevaro, Beatriz Araoz, Pablo Ruiz Diaz, Mariano L Bossi, Carolyn R BertozziAbstract:Trypanosoma cruzi, the flagellate protozoan agent of Chagas disease or American trypanosomiasis, is unable to synthesize sialic acids de novo. Mucins and trans-sialidase (TS) are substrate and enzyme, respectively, of the glycobiological system that scavenges sialic acid from the host in a crucial interplay for T. cruzi life cycle. The acquisition of the sialyl residue allows the parasite to avoid lysis by serum factors and to interact with the host cell. A major drawback to studying the sialylation kinetics and turnover of the Trypomastigote glycoconjugates is the difficulty to identify and follow the recently acquired sialyl residues. To tackle this issue, we followed an unnatural sugar approach as bioorthogonal chemical reporters, where the use of azidosialyl residues allowed identifying the acquired sugar. Advanced microscopy techniques, together with biochemical methods, were used to study the Trypomastigote membrane from its glycobiological perspective. Main sialyl acceptors were identified as mucins by biochemical procedures and protein markers. Together with determining their shedding and turnover rates, we also report that several membrane proteins, including TS and its substrates, both glycosylphosphatidylinositol-anchored proteins, are separately distributed on parasite surface and contained in different and highly stable membrane microdomains. Notably, labeling for α(1,3)Galactosyl residues only partially colocalize with sialylated mucins, indicating that two species of glycosylated mucins do exist, which are segregated at the parasite surface. Moreover, sialylated mucins were included in lipid-raft-domains, whereas TS molecules are not. The location of the surface-anchored TS resulted too far off as to be capable to sialylate mucins, a role played by the shed TS instead. Phosphatidylinositol-phospholipase-C activity is actually not present in Trypomastigotes. Therefore, shedding of TS occurs via microvesicles instead of as a fully soluble form.
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mapping antigenic motifs in the Trypomastigote small surface antigen from trypanosoma cruzi
Clinical and Vaccine Immunology, 2015Co-Authors: Virginia Balouz, Maria De Los Milagros Camara, Gaspar E Canepa, Fernan Aguero, Santiago J Carmona, Romina Volcovich, Nicolas Gonzalez, Jaime Altcheh, Carlos A BuscagliaAbstract:The Trypomastigote small surface antigen (TSSA) is a mucin-like molecule from Trypanosoma cruzi, the etiological agent of Chagas disease, which displays amino acid polymorphisms in parasite isolates. TSSA expression is restricted to the surface of infective cell-derived Trypomastigotes, where it functions as an adhesin and engages surface receptors on the host cell as a prerequisite for parasite internalization. Previous results have established TSSA-CL, the isoform encoded by the CL Brener clone, as an appealing candidate for use in serology-based diagnostics for Chagas disease. Here, we used a combination of peptide- and recombinant protein-based tools to map the antigenic structure of TSSA-CL at maximal resolution. Our results indicate the presence of different partially overlapping B-cell epitopes clustering in the central portion of TSSA-CL, which contains most of the polymorphisms found in parasite isolates. Based on these results, we assessed the serodiagnostic performance of a 21-amino-acid-long peptide that spans TSSA-CL major antigenic determinants, which was similar to the performance of the previously validated glutathione S-transferase (GST)-TSSA-CL fusion molecule. Furthermore, the tools developed for the antigenic characterization of the TSSA antigen were also used to explore other potential diagnostic applications of the anti-TSSA humoral response in Chagasic patients. Overall, our present results provide additional insights into the antigenic structure of TSSA-CL and support this molecule as an excellent target for molecular intervention in Chagas disease.
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involvement of tssa Trypomastigote small surface antigen in trypanosoma cruzi invasion of mammalian cells
Biochemical Journal, 2012Co-Authors: Gaspar E Canepa, Maria Sol Degese, Alexandre Budu, Celia R S Garcia, Carlos A BuscagliaAbstract:TSSA (Trypomastigote small surface antigen) is a polymorphic mucin-like molecule displayed on the surface of Trypanosoma cruzi Trypomastigote forms. To evaluate its functional properties, we undertook comparative biochemical and genetic approaches on isoforms present in parasite stocks from extant evolutionary lineages (CL Brener and Sylvio X-10). We show that CL Brener TSSA, but not the Sylvio X-10 counterpart, exhibits dose-dependent and saturable binding towards non-macrophagic cell lines. This binding triggers Ca2+-based signalling responses in the target cell while providing an anchor for the invading parasite. Accordingly, exogenous addition of either TSSA-derived peptides or specific antibodies significantly inhibits invasion of CL Brener, but not Sylvio X-10, Trypomastigotes. Non-infective epimastigote forms, which do not express detectable levels of TSSA, were stably transfected with TSSA cDNA from either parasite stock. Although both transfectants produced a surface-associated mucin-like TSSA product, epimastigotes expressing CL Brener TSSA showed a ~2-fold increase in their attachment to mammalian cells. Overall, these findings indicate that CL Brener TSSA functions as a parasite adhesin, engaging surface receptor(s) and inducing signalling pathways on the host cell as a prerequisite for parasite internalization. More importantly, the contrasting functional features of TSSA isoforms provide one appealing mechanism underlying the differential infectivity of T . cruzi stocks. Abbreviations: Ab, antibody; DAPI, 4′,6-diamidino-2-phenylindole; DMEM, Dulbecco's modified Eagle's medium; ERK, extracellular-signal-regulated kinase; FBS, fetal bovine serum; gp, glycoprotein; GPI, glycosylphosphatidylinositol; GST, glutathione transferase; HBSS, Hanks balanced salt solution; hEGF, human endothelial growth factor; HEK, human embryonic kidney; HRP, horseradish peroxidase; IFA, indirect immunofluorescence assay; mAb, monoclonal Ab; MAPK, mitogen-activated protein kinase; MEM, minimal essential medium; PI-PLC, phosphatidylinositol-specific phospholipase C; RT, reverse transcription; SP, signal peptide; TS, trans-sialidase; TSSA, Trypomastigote small surface antigen; UTR, untranslated region
Mario Steindel - One of the best experts on this subject based on the ideXlab platform.
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Penetration of the salivary glands of Rhodnius domesticus Neiva & Pinto, 1923 (Hemiptera: Reduviidae) by Trypanosoma rangeli Tejera, 1920 (Protozoa: Kinetoplastida)
Parasitology Research, 2005Co-Authors: Rosane M. S. Meirelles, Andrea Henriques-pons, Maurilio J. Soares, Mario SteindelAbstract:Penetration of the heteroxenous protozoan Trypanosoma rangeli into the salivary glands of its invertebrate host Rhodnius domesticus has been investigated here using different approaches. Electron microscopy showed that epimastigotes coming from the insect hemocoel cross the basal lamina that surrounds the salivary glands and penetrate through the gland cells cytoplasm. After reaching the gland lumen, epimastigote forms remain adhered to the gland cell microvilli by their flagella, while metacyclic Trypomastigotes are found swimming free in the saliva. Analysis by flow cytometry, western blotting and hemolytic activity allowed to demonstrate the presence in T. rangeli of a hemolytic molecule with antigenic cross-reactivity with murine perforin, which could be used by the parasites to reach the salivary gland lumen. This molecule, which we named as rangelysin, has 120 kDa molecular weight, is able to induce hemolysis only in acidic pH, and is produced by both Trypomastigote and epimastigote forms.
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penetration of the salivary glands of rhodnius domesticus neiva pinto 1923 hemiptera reduviidae by trypanosoma rangeli tejera 1920 protozoa kinetoplastida
Parasitology Research, 2005Co-Authors: Rosane M. S. Meirelles, Andrea Henriquespons, Maurilio J. Soares, Mario SteindelAbstract:Penetration of the heteroxenous protozoan Trypanosoma rangeli into the salivary glands of its invertebrate host Rhodnius domesticus has been investigated here using different approaches. Electron microscopy showed that epimastigotes coming from the insect hemocoel cross the basal lamina that surrounds the salivary glands and penetrate through the gland cells cytoplasm. After reaching the gland lumen, epimastigote forms remain adhered to the gland cell microvilli by their flagella, while metacyclic Trypomastigotes are found swimming free in the saliva. Analysis by flow cytometry, western blotting and hemolytic activity allowed to demonstrate the presence in T. rangeli of a hemolytic molecule with antigenic cross-reactivity with murine perforin, which could be used by the parasites to reach the salivary gland lumen. This molecule, which we named as rangelysin, has 120 kDa molecular weight, is able to induce hemolysis only in acidic pH, and is produced by both Trypomastigote and epimastigote forms.
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differentiation of trypanosoma rangeli high production of infective Trypomastigote forms in vitro
Parasitology Research, 2002Co-Authors: Leonardo B Koerich, Priscilla Emmanuellemachado, Kelly De O Santos, Edmundo C Grisard, Mario SteindelAbstract:In the present study, we report a simple method to induce high Trypanosoma rangeli differentiation in vitro, producing a large number of infective Trypomastigote forms. Parasites from SC-58 (Brazil) and Choachi (Colombia) strains were cultivated at 27 °C in TC-100, Grace and DMEM media, each supplemented with 5% fetal bovine serum and prepared at three distinct pHs (6.0, 7.0, 8.0). Differentiation was microscopically evaluated at 0, 3 and 6 days after cultivation in each medium by determining the percentage of Trypomastigotes in Giemsa-stained smears. Our data revealed similar results for both T. rangeli strains, showing (after 6 days of cultivation in DMEM medium, pH 8.0) the presence of about 80% of Trypomastigotes. These culture-derived Trypomastigotes proved to be infective to both Balb-C mice and Rhodnius spp, reaching the triatomine's salivary glands. Our results describe a new and easy method to induce high T. rangeli differentiation in vitro, allowing further studies on the antigenic constitution of Trypomastigotes.
Maria De Los Milagros Camara - One of the best experts on this subject based on the ideXlab platform.
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the Trypomastigote small surface antigen tssa regulates trypanosoma cruzi infectivity and differentiation
PLOS Neglected Tropical Diseases, 2017Co-Authors: Maria De Los Milagros Camara, Gaspar E Canepa, Andres B Lantos, Virginia Balouz, Hai Yu, Xi Chen, Oscar Campetella, Juan Mucci, Carlos A BuscagliaAbstract:Background TSSA (Trypomastigote Small Surface Antigen) is an antigenic, adhesion molecule displayed on the surface of Trypanosoma cruzi Trypomastigotes. TSSA displays substantial sequence identity to members of the TcMUC gene family, which code for the Trypomastigote mucins (tGPI-mucins). In addition, TSSA bears sequence polymorphisms among parasite strains; and two TSSA variants expressed as recombinant molecules (termed TSSA-CL and TSSA-Sy) were shown to exhibit contrasting features in their host cell binding and signaling properties. Methods/Principle findings Here we used a variety of approaches to get insights into TSSA structure/function. We show that at variance with tGPI-mucins, which rely on their extensive O-glycoslylation to achieve their protective function, TSSA seems to be displayed on the Trypomastigote coat as a hypo-glycosylated molecule. This has a functional correlate, as further deletion mapping experiments and cell binding assays indicated that exposition of at least two peptidic motifs is critical for the engagement of the ‘adhesive’ TSSA variant (TSSA-CL) with host cell surface receptor(s) prior to Trypomastigote internalization. These motifs are not conserved in the ‘non-adhesive’ TSSA-Sy variant. We next developed transgenic lines over-expressing either TSSA variant in different parasite backgrounds. In strict accordance to recombinant protein binding data, Trypomastigotes over-expressing TSSA-CL displayed improved adhesion and infectivity towards non-macrophagic cell lines as compared to those over-expressing TSSA-Sy or parental lines. These phenotypes could be specifically counteracted by exogenous addition of peptides spanning the TSSA-CL adhesion motifs. In addition, and irrespective of the TSSA variant, over-expression of this molecule leads to an enhanced Trypomastigote-to-amastigote conversion, indicating a possible role of TSSA also in parasite differentiation. Conclusion/Significance In this study we provided novel evidence indicating that TSSA plays an important role not only on the infectivity and differentiation of T. cruzi Trypomastigotes but also on the phenotypic variability displayed by parasite strains.
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sialic acid glycobiology unveils trypanosoma cruzi Trypomastigote membrane physiology
PLOS Pathogens, 2016Co-Authors: Andres B Lantos, Maria De Los Milagros Camara, Hai Yu, Xi Chen, Carlos A Buscaglia, Giannina Carlevaro, Beatriz Araoz, Pablo Ruiz Diaz, Mariano L Bossi, Carolyn R BertozziAbstract:Trypanosoma cruzi, the flagellate protozoan agent of Chagas disease or American trypanosomiasis, is unable to synthesize sialic acids de novo. Mucins and trans-sialidase (TS) are substrate and enzyme, respectively, of the glycobiological system that scavenges sialic acid from the host in a crucial interplay for T. cruzi life cycle. The acquisition of the sialyl residue allows the parasite to avoid lysis by serum factors and to interact with the host cell. A major drawback to studying the sialylation kinetics and turnover of the Trypomastigote glycoconjugates is the difficulty to identify and follow the recently acquired sialyl residues. To tackle this issue, we followed an unnatural sugar approach as bioorthogonal chemical reporters, where the use of azidosialyl residues allowed identifying the acquired sugar. Advanced microscopy techniques, together with biochemical methods, were used to study the Trypomastigote membrane from its glycobiological perspective. Main sialyl acceptors were identified as mucins by biochemical procedures and protein markers. Together with determining their shedding and turnover rates, we also report that several membrane proteins, including TS and its substrates, both glycosylphosphatidylinositol-anchored proteins, are separately distributed on parasite surface and contained in different and highly stable membrane microdomains. Notably, labeling for α(1,3)Galactosyl residues only partially colocalize with sialylated mucins, indicating that two species of glycosylated mucins do exist, which are segregated at the parasite surface. Moreover, sialylated mucins were included in lipid-raft-domains, whereas TS molecules are not. The location of the surface-anchored TS resulted too far off as to be capable to sialylate mucins, a role played by the shed TS instead. Phosphatidylinositol-phospholipase-C activity is actually not present in Trypomastigotes. Therefore, shedding of TS occurs via microvesicles instead of as a fully soluble form.
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mapping antigenic motifs in the Trypomastigote small surface antigen from trypanosoma cruzi
Clinical and Vaccine Immunology, 2015Co-Authors: Virginia Balouz, Maria De Los Milagros Camara, Gaspar E Canepa, Fernan Aguero, Santiago J Carmona, Romina Volcovich, Nicolas Gonzalez, Jaime Altcheh, Carlos A BuscagliaAbstract:The Trypomastigote small surface antigen (TSSA) is a mucin-like molecule from Trypanosoma cruzi, the etiological agent of Chagas disease, which displays amino acid polymorphisms in parasite isolates. TSSA expression is restricted to the surface of infective cell-derived Trypomastigotes, where it functions as an adhesin and engages surface receptors on the host cell as a prerequisite for parasite internalization. Previous results have established TSSA-CL, the isoform encoded by the CL Brener clone, as an appealing candidate for use in serology-based diagnostics for Chagas disease. Here, we used a combination of peptide- and recombinant protein-based tools to map the antigenic structure of TSSA-CL at maximal resolution. Our results indicate the presence of different partially overlapping B-cell epitopes clustering in the central portion of TSSA-CL, which contains most of the polymorphisms found in parasite isolates. Based on these results, we assessed the serodiagnostic performance of a 21-amino-acid-long peptide that spans TSSA-CL major antigenic determinants, which was similar to the performance of the previously validated glutathione S-transferase (GST)-TSSA-CL fusion molecule. Furthermore, the tools developed for the antigenic characterization of the TSSA antigen were also used to explore other potential diagnostic applications of the anti-TSSA humoral response in Chagasic patients. Overall, our present results provide additional insights into the antigenic structure of TSSA-CL and support this molecule as an excellent target for molecular intervention in Chagas disease.
Valeria Tekiel - One of the best experts on this subject based on the ideXlab platform.
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tctasv c a protein family in trypanosoma cruzi that is predominantly Trypomastigote stage specific and secreted to the medium
PLOS ONE, 2013Co-Authors: Guillermo Bernabo, Daniel O Sanchez, Maria Ziliani, Gabriela V Levy, Lucas D Caeiro, Valeria TekielAbstract:Among the several multigene families codified by the genome of T. cruzi, the TcTASV family was the latest discovered. The TcTASV (Trypomastigote, Alanine, Serine, Valine) family is composed of ∼40 members, with conserved carboxi- and amino-termini but with a variable central core. According to the length and sequence of the central region the family is split into 3 subfamilies. The TcTASV family is conserved in the genomes of – at least – lineages TcI and TcVI and has no orthologues in other trypanosomatids. In the present work we focus on the study of the TcTASV-C subfamily, composed by 16 genes in the CL Brener strain. We determined that TcTASV-C is preferentially expressed in Trypomastigotes, but it is not a major component of the parasite. Both immunoflourescence and flow cytometry experiments indicated that TcTASV-C has a clonal expression, i.e. it is not expressed by all the parasites of a certain population at the same time. We also determined that TcTASV-C is phosphorylated and glycosylated. TASV-C is attached to the parasite surface by a GPI anchor and is shed spontaneously into the medium. About 30% of sera from infected hosts reacted with TcTASV-C, confirming its exposition to the immune system. Its superficial localization and secretory nature suggest a possible role in host-parasite interactions.
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tctasv a novel protein family in trypanosoma cruzi identified from a subtractive Trypomastigote cdna library
PLOS Neglected Tropical Diseases, 2010Co-Authors: Elizabeth A Garcia, Daniel O Sanchez, Maria Ziliani, Fernan Aguero, Guillermo Bernabo, Valeria TekielAbstract:BACKGROUND: The identification and characterization of antigens expressed in Trypanosoma cruzi stages that parasitize mammals are essential steps for the development of new vaccines and diagnostics. Genes that are preferentially expressed in Trypomastigotes may be involved in key processes that define the biology of Trypomastigotes, like cell invasion and immune system evasion. METHODOLOGY/PRINCIPAL FINDINGS: With the initial aim of identifying Trypomastigote-specific expressed tags, we constructed and sequenced an epimastigote-subtracted Trypomastigote cDNA library (library TcT-E). More than 45% of the sequenced clones of the library could not be mapped to previously annotated mRNAs or proteins. We validated the presence of these transcripts by reverse northern blot and northern blot experiments, therefore providing novel information about the mRNA expression of these genes in Trypomastigotes. A 280-bp consensus element (TcT-E element, TcT-Eelem) located at the 3' untranslated region (3' UTR) of many different open reading frames (ORFs) was identified after clustering the TcT-E dataset. Using an RT-PCR approach, we were able to amplify different mature mRNAs containing the same TcT-Eelem in the 3' UTR. The proteins encoded by these ORFs are members of a novel surface protein family in T. cruzi, (which we named TcTASV for T. cruzi Trypomastigote, Alanine, Serine and Valine rich proteins). All members of the TcTASV family have conserved coding amino- and carboxy-termini, and a central variable core that allows partitioning of TcTASV proteins into three subfamilies. Analysis of the T. cruzi genome database resulted in the identification of 38 genes/ORFs for the whole TcTASV family in the reference CL-Brener strain (lineage II). Because this protein family was not found in other trypanosomatids, we also looked for the presence of TcTASV genes in other evolutionary lineages of T. cruzi, sequencing 48 and 28 TcTASVs members from the RA (lineage II) and Dm28 (lineage I) T. cruzi strains respectively. Detailed phylogenetic analyses of TcTASV gene products show that this gene family is different from previously characterized mucin (TcMUCII), mucin-like, and MASP protein families. CONCLUSIONS/SIGNIFICANCE: We identified TcTASV, a new gene family of surface proteins in T. cruzi.
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identification of novel vaccine candidates for chagas disease by immunization with sequential fractions of a Trypomastigote cdna expression library
Vaccine, 2009Co-Authors: Valeria Tekiel, Catalina D Albasoto, Stella Gonzalez M Cappa, Miriam Postan, Daniel O SanchezAbstract:The protozoan Trypanosoma cruzi is the etiological agent of Chagas’ disease, a major chronic infection in Latin America. Currently, there are neither effective drugs nor vaccines for the treatment or prevention of the disease. Several T. cruzi surface antigens are being tested as vaccines but none of them proved to be completely protective, probably because they represent only a limited repertoire of all the possible T. cruzi target molecules. Taking into account that the Trypomastigote stage of the parasite must express genes that allow the parasite to disseminate into the tissues and invade cells, we reasoned that genes preferentially expressed in Trypomastigotes represent potential targets for immunization. Here we screened an epimastigote-subtracted Trypomastigote cDNA expression library by genetic immunization, in order to find new vaccine candidates for Chagas’ disease. After two rounds of immunization and challenge with Trypomastigotes, this approach led to the identification of a pool of 28 gene fragments that improved in vivo protection. Sequence analysis of these putative candidates revealed that 19 out of 28 (67.85%) of the genes were hypothetical proteins or unannotated T. cruzi open reading frames, which certainly would not
Gaspar E Canepa - One of the best experts on this subject based on the ideXlab platform.
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the Trypomastigote small surface antigen tssa regulates trypanosoma cruzi infectivity and differentiation
PLOS Neglected Tropical Diseases, 2017Co-Authors: Maria De Los Milagros Camara, Gaspar E Canepa, Andres B Lantos, Virginia Balouz, Hai Yu, Xi Chen, Oscar Campetella, Juan Mucci, Carlos A BuscagliaAbstract:Background TSSA (Trypomastigote Small Surface Antigen) is an antigenic, adhesion molecule displayed on the surface of Trypanosoma cruzi Trypomastigotes. TSSA displays substantial sequence identity to members of the TcMUC gene family, which code for the Trypomastigote mucins (tGPI-mucins). In addition, TSSA bears sequence polymorphisms among parasite strains; and two TSSA variants expressed as recombinant molecules (termed TSSA-CL and TSSA-Sy) were shown to exhibit contrasting features in their host cell binding and signaling properties. Methods/Principle findings Here we used a variety of approaches to get insights into TSSA structure/function. We show that at variance with tGPI-mucins, which rely on their extensive O-glycoslylation to achieve their protective function, TSSA seems to be displayed on the Trypomastigote coat as a hypo-glycosylated molecule. This has a functional correlate, as further deletion mapping experiments and cell binding assays indicated that exposition of at least two peptidic motifs is critical for the engagement of the ‘adhesive’ TSSA variant (TSSA-CL) with host cell surface receptor(s) prior to Trypomastigote internalization. These motifs are not conserved in the ‘non-adhesive’ TSSA-Sy variant. We next developed transgenic lines over-expressing either TSSA variant in different parasite backgrounds. In strict accordance to recombinant protein binding data, Trypomastigotes over-expressing TSSA-CL displayed improved adhesion and infectivity towards non-macrophagic cell lines as compared to those over-expressing TSSA-Sy or parental lines. These phenotypes could be specifically counteracted by exogenous addition of peptides spanning the TSSA-CL adhesion motifs. In addition, and irrespective of the TSSA variant, over-expression of this molecule leads to an enhanced Trypomastigote-to-amastigote conversion, indicating a possible role of TSSA also in parasite differentiation. Conclusion/Significance In this study we provided novel evidence indicating that TSSA plays an important role not only on the infectivity and differentiation of T. cruzi Trypomastigotes but also on the phenotypic variability displayed by parasite strains.
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mapping antigenic motifs in the Trypomastigote small surface antigen from trypanosoma cruzi
Clinical and Vaccine Immunology, 2015Co-Authors: Virginia Balouz, Maria De Los Milagros Camara, Gaspar E Canepa, Fernan Aguero, Santiago J Carmona, Romina Volcovich, Nicolas Gonzalez, Jaime Altcheh, Carlos A BuscagliaAbstract:The Trypomastigote small surface antigen (TSSA) is a mucin-like molecule from Trypanosoma cruzi, the etiological agent of Chagas disease, which displays amino acid polymorphisms in parasite isolates. TSSA expression is restricted to the surface of infective cell-derived Trypomastigotes, where it functions as an adhesin and engages surface receptors on the host cell as a prerequisite for parasite internalization. Previous results have established TSSA-CL, the isoform encoded by the CL Brener clone, as an appealing candidate for use in serology-based diagnostics for Chagas disease. Here, we used a combination of peptide- and recombinant protein-based tools to map the antigenic structure of TSSA-CL at maximal resolution. Our results indicate the presence of different partially overlapping B-cell epitopes clustering in the central portion of TSSA-CL, which contains most of the polymorphisms found in parasite isolates. Based on these results, we assessed the serodiagnostic performance of a 21-amino-acid-long peptide that spans TSSA-CL major antigenic determinants, which was similar to the performance of the previously validated glutathione S-transferase (GST)-TSSA-CL fusion molecule. Furthermore, the tools developed for the antigenic characterization of the TSSA antigen were also used to explore other potential diagnostic applications of the anti-TSSA humoral response in Chagasic patients. Overall, our present results provide additional insights into the antigenic structure of TSSA-CL and support this molecule as an excellent target for molecular intervention in Chagas disease.
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involvement of tssa Trypomastigote small surface antigen in trypanosoma cruzi invasion of mammalian cells
Biochemical Journal, 2012Co-Authors: Gaspar E Canepa, Maria Sol Degese, Alexandre Budu, Celia R S Garcia, Carlos A BuscagliaAbstract:TSSA (Trypomastigote small surface antigen) is a polymorphic mucin-like molecule displayed on the surface of Trypanosoma cruzi Trypomastigote forms. To evaluate its functional properties, we undertook comparative biochemical and genetic approaches on isoforms present in parasite stocks from extant evolutionary lineages (CL Brener and Sylvio X-10). We show that CL Brener TSSA, but not the Sylvio X-10 counterpart, exhibits dose-dependent and saturable binding towards non-macrophagic cell lines. This binding triggers Ca2+-based signalling responses in the target cell while providing an anchor for the invading parasite. Accordingly, exogenous addition of either TSSA-derived peptides or specific antibodies significantly inhibits invasion of CL Brener, but not Sylvio X-10, Trypomastigotes. Non-infective epimastigote forms, which do not express detectable levels of TSSA, were stably transfected with TSSA cDNA from either parasite stock. Although both transfectants produced a surface-associated mucin-like TSSA product, epimastigotes expressing CL Brener TSSA showed a ~2-fold increase in their attachment to mammalian cells. Overall, these findings indicate that CL Brener TSSA functions as a parasite adhesin, engaging surface receptor(s) and inducing signalling pathways on the host cell as a prerequisite for parasite internalization. More importantly, the contrasting functional features of TSSA isoforms provide one appealing mechanism underlying the differential infectivity of T . cruzi stocks. Abbreviations: Ab, antibody; DAPI, 4′,6-diamidino-2-phenylindole; DMEM, Dulbecco's modified Eagle's medium; ERK, extracellular-signal-regulated kinase; FBS, fetal bovine serum; gp, glycoprotein; GPI, glycosylphosphatidylinositol; GST, glutathione transferase; HBSS, Hanks balanced salt solution; hEGF, human endothelial growth factor; HEK, human embryonic kidney; HRP, horseradish peroxidase; IFA, indirect immunofluorescence assay; mAb, monoclonal Ab; MAPK, mitogen-activated protein kinase; MEM, minimal essential medium; PI-PLC, phosphatidylinositol-specific phospholipase C; RT, reverse transcription; SP, signal peptide; TS, trans-sialidase; TSSA, Trypomastigote small surface antigen; UTR, untranslated region
