Expressão heteróloga e caracterização bioquímica e biológica da NTPDase-1 de Leishmania infantum chagasi

• Ano de defesa: 2011
• Autor(a) principal: Bastos, Matheus Silva e
• Orientador(a): Fietto, Juliana Lopes Rangel
• Banca de defesa: Fernandes, José Roberto Meyer , Bressan, Gustavo Costa , Silva, Eduardo de Almeida Marques da
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de Viçosa
• Programa de Pós-Graduação: Mestrado em Bioquímica Agrícola
• Departamento: Bioquímica e Biologia molecular de plantas; Bioquímica e Biologia molecular animal
• País: BR
• Palavras-chave em Português: NTPDase-1; Leishmania; Expressão heteróloga
• Palavras-chave em Inglês: NTPDase-1; Leishmania; Heterologous expression
• Área do conhecimento CNPq: CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOLOGIA MOLECULAR
• Link de acesso: http://locus.ufv.br/handle/123456789/2435

The Visceral Leishmaniasis (VL) represents an important global health problem in many countries including Brazil and it has seen a growing number of cases in humans and dogs. The dogs are being considered the main domestic reservoir responsible for maintaining the disease in the outskirts of large cities. The aim of this work is the study of Leishmania infantum chagasi NTPDase-1. L. infantum chagasi is the main etiologic agent of VL in Brazil. The NTPDase-1 is part of the E-NTPDase family, enzymes that are implicated as virulence factors of some parasites. They participate in the purines salvage pathways and in the modulation of host immune response that dependend on extra cellular nucleotides. These enzymes are important targets for the development of new drugs and effective vaccines for Leishmaniasis. In this work were made bioinformatics analysis of E-NTPDase from L. braziliensis, L. infantum, L. major, Trypanosoma brucei and T. cruzi. Moreover, was made the cloning, expression in bacterial system, purification and characterization of soluble portion of the enzyme NTPDase 1 from L. infantum chagasi. The purified enzyme was also used for production of polyclonal antiserum. In addition were evaluated the role of recombinant NTPDase-1 in the biology of f L. braziliensis. The results of the bioinformatics analysis showed the existence of two groups of Trypanosomatide NTPDases named here as NTPDase-1 and NTPDase-2. These groups presented similar predicted glycosilation pattern, transmembrane domain, disulphide bonds and substrate specifity. Comparing the T. cruzi NTPDase-1 and L. infantum chagasi NTPDase-1 (L.i. NTPDase-1),characterized in this work, we observe that the pH of maximum activity is different: the enzyme from T. cruzi has a higher activity at alkaline pH (7.5 to 8) and the NTPDase-1 from L. infantum chagasi in acidic pH (5.5 to 6.0). L.i. NTPDase-1 showed specificity for GTP while the other enzymes of Trypanosomatids hydrolyze various tri-and diphosphate nucleotides. In the analysis of biological effect of addition of L.i. NTPDase-1 in axenic culture of promastigotes of L. braziliensis, there was a possible influence on in vitro metacyclogenesis. All data are discussed in terms of possible roles of L.i. NTPDase-1 in the biology of L. infantum chagasi and the infection vector and host, opening new prospects for studies in this area. The data suggests that this enzyme may influence the mechanism of metacyclogenesis by some process not yet known.