Expressão das NTPDases 1 e 2 de Leishmania infantum chagasi em sistema bacteriano e de célula de mamífero e estudo de suas influências na infecção de células raw 264.7 e na expressão de purino receptores P2

• Autor(a) principal: Bastos, Matheus Silva e
• Data de Publicação: 2014
• Tipo de documento: Tese
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/337
• topic: Leishmaniose visceral; Enzimas; NTPDases; Expressão heteróloga; Visceral leishmaniasis; Enzymes; NTPDase; Heterologous expression; CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA

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 NTPDase-1 and 2 from L. infantum chagasi (LicNTPDases) is the main etiologic agent of VL in Brazil. The LicNTPDase-1 and 2 are part of the E- NTPDase family, enzymes that are implicated as virulence factors of some parasites. They can participate in the purines salvage pathways and in the modulation of host immune response that dependent on extra cellular nucleotides. These enzymes have been indicated important as targets for the development of new drugs and effective vaccines for Leishmaniasis. In this work, were did comparative tests between different refolding protocols of the recombinant enzymes as well as comparison between activity of enzymes expressed in Escherichia coli and expressed in mammalian cells (COS-7). Moreover, many experiments in macrophages were done to determine if the recombinant enzymes can be involved in adhesion mechanisms of parasites and if these enzyme can modulate the P2 receptor expression. The data demonstrate that changing the refolding protocol was possible to increase the activity of LicNTPDase-2 , but the same effect was not observed for LicNTPDase-1, when these enzyme were expressed in bacterial. However, when the expression was done in COS-7 is possible to verify a activity increased of LicNTPDase-1. It was possible to see the same activity level between LicNTPDase-2 expressed in E. coli and expressed in COS-7. This result showed that LicNTPDase2 expressed in E. coli have a same shape than enzyme expressed in COS-7. This work was also possible to confirm the direct involvement of LicNTPDases in the parasites binding mechanism, showing a dependence of the concentration of recombinant. It was also possible to verify a possible relationship between the presence of these enzymes and modulation of expression of some xipurine receptors, macrophage treated with LicNTPDase-1 showed a up expression of mRNA of P2X3, P2X5, P2Y2 and a down expression for P2Y13, when the macrophage was treated with LicNTPDase-2 the P2X6 and P2X7 mRNA level decreased and when was checked the mRNA expression of infected macrophage was possible to verify that L. infantum chagasi can control the expression of P2X2, P2Y12 and P2Y13.