Artigos
URI permanente para esta coleçãohttps://locus.ufv.br/handle/123456789/11847
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Item Immunogenomic screening approach to identify new antigens for the serological diagnosis of chronic Chagas’ disease(Applied Microbiology and Biotechnology, 2018-05-07) Elisei, Rutyanne Maria Tonelli; Matos, Christiane Santos; Carvalho, Ana Maria Ravena Severino; Chaves, Ana Thereza; Medeiros, Fernanda Alvarenga Cardoso; Barbosa, Ronaldo; Marcelino, Andreza Pain; Emidio, Kenia dos Santos; Coelho, Eduardo Antonio Ferraz; Duarte, Mariana Costa; Mendes, Tiago Antônio de Oliveira; Rocha, Manoel Otávio da Costa; Menezes-Souza, DanielSerological tests are preferentially used for the diagnosis of Chagas’ disease (CD) during the chronic phase because of the low parasitemia and high anti-Trypanosoma cruzi antibody titers. However, the current methods showed several disadvantages, as contradictory or inconclusive results, mainly related to the characteristics of the antigens used, in general, crude or whole parasites, but also due to antigen production protocol and the experimental conditions used in serological tests. Thus, better-quality serological assays are urgently needed. Here, we performed a wide immunogenomic screen strategy to identify conserved linear B-cell epitopes in the predicted proteome based on genome sequence from T. cruzi strains to will be applied as synthetic peptides in the serodiagnosis of the chronic CD. Three B-cell epitopes derived from mucin-associated surface protein (MASP) family, expressed in both infective parasite stages, trypomastigote and amastigotes, conserved in T. cruzi strains, and highly divergent as compared with Leishmania spp. proteome, were selected for this study. The results demonstrated that synthetic peptide 2 and a mixture of peptides (Mix II: peptides 2 and 3) were able to identify all chronic CD cases, indeterminate or Chagas cardiomyopathy clinical presentation, and simultaneously able to discriminate infections caused by Leishmania parasites, with high accuracy (98.37 and 100.00%, respectively) and agreement (kappa index = 0.967 and 1.000, respectively) with direct methods as compared to current diagnostic pipeline performed by reference laboratories in Brazil. This study represents an interesting strategy for the discovery of new antigens applied to serologic diagnosis of infectious diseases and for the technological development of platforms for large-scale production of diagnostic tests.Item Immobilization of NTPDase-1 from Trypanosoma cruzi and development of an online label-free assay(Journal of Analytical Methods in Chemistry, 2016-12-14) Calil, Felipe Antunes; Lima, Juliana Maria; Oliveira, Arthur Henrique Cavalcante de; Mariotini-Moura, Christiane; Fietto, Juliana Lopes Rangel; Cardoso, Carmen LuciaThe use of IMERs (Immobilized Enzyme Reactors) as a stationary phase coupled to high performance chromatographic systems is an interesting approach in the screening of new ligands. In addition, IMERs offer many advantages over techniques that employ enzymes in solution. The enzyme nucleoside triphosphate diphosphohydrolase (NTPDase-1) from Trypanosoma cruzi acts as a pathogen infection facilitator, so it is a good target in the search for inhibitors. In this paper, immobilization of NTPDase-1 afforded ICERs (Immobilized Capillary Enzyme Reactors). A liquid chromatography method was developed and validated to monitor the ICER activity. The conditions for the application of these bioreactors were investigated, and excellent results were obtained. The enzyme was successfully immobilized, as attested by the catalytic activity detected in the TcNTPDase-1-ICER chromatographic system. Kinetic studies on the substrate ATP gave K M of 0.317 ± 0.044 mmol·L−1, which still presented high affinity compared to in solution. Besides that, the ICER was stable for 32 days, enough time to investigate samples of possible inhibitors, including especially the compound Suramin, that inhibited 51% the enzyme activity at 100 µmol·L−1, which is in accordance with the data for the enzyme in solution.Item Trypanosoma cruzi nucleoside triphosphate diphosphohydrolase 1 (TcNTPDase-1) biochemical characterization, immunolocalization and possible role in host cell adhesion(Acta Tropica, 2013-11-19) Mariotini-Moura, Christiane; Bastos, Matheus Silva e; Castro, Felipe Freitas de; Trindade, Mellina Lanna; Vasconcellos, Raphael de Souza; Neves-do-Valle, Myrian Augusta Araújo; Moreira, Bernardo Pereira; Santos, Ramon de Freitas; Oliveira, Claudia Miranda de; Cunha, Luana Celina Seraphim; Souto, Xênia Macedo; Bressan, Gustavo Costa; Silva-Júnior, Abelardo; Baqui, Munira Muhammad Abdel; Bahia, Maria Terezinha; Almeida, Márcia Rogéria de; Meyer-Fernandes, José Roberto; Fietto, Juliana Lopes RangelPrevious work has suggested that Trypanosoma cruzi diphosphohydrolase 1 (TcNTPDase-1) may be involved in the infection of mammalian cells and serve as a potential target for rational drug design. In this work, we produced recombinant TcNTPDase-1 and evaluated its nucleotidase activity, cellular localization and role in parasite adhesion to mammalian host cells. TcNTPDase-1 was able to utilize a broad range of triphosphate and diphosphate nucleosides. The enzyme's Km for ATP (0.096 mM) suggested a capability to influence the host's ATP-dependent purinergic signaling. The use of specific polyclonal antibodies allowed us to confirm the presence of TcNTPDase-1 at the surface of parasites by confocal and electron microscopy. In addition, electron microscopy revealed that TcNTPDase-1 was also found in the flagellum, flagellum insertion region, kinetoplast, nucleus and intracellular vesicles. The presence of this enzyme in the flagellum insertion region and vesicles suggests that it may have a role in nutrient acquisition, and the widespread distribution of TcNTPDase-1 within the parasite suggests that it may be involved in other biological process. Adhesion assays using anti-TcNTPDase-1 polyclonal antibodies as a blocker or purified recombinant TcNTPDase-1 as a competitor revealed that the enzyme has a role in parasite–host cell adhesion. These data open new frontiers to future studies on this specific parasite–host interaction and other unknown functions of TcNTPDase-1 related to its ubiquitous localization.