Leishmania infantum ecto-nucleoside triphosphate diphosphohydrolase-2 is an apyrase involved in macrophage infection and expressed in infected dogs.

Autor: Raphael De Souza Vasconcellos, Christiane Mariotini-Moura, Rodrigo Saar Gomes, Tiago Donatelli Serafim, Rafaela de Cássia Firmino, Matheus Silva E Bastos, Felipe Freitas de Castro, Claudia Miranda de Oliveira, Lucas Borges-Pereira, Anna Cláudia Alves de Souza, Ronny Francisco de Souza, Gabriel Andres Tafur Gómez, Aimara da Costa Pinheiro, Talles Eduardo Ferreira Maciel, Abelardo Silva-Júnior, Gustavo Costa Bressan, Márcia Rogéria Almeida, Munira Muhammad Abdel Baqui, Luís Carlos Crocco Afonso, Juliana Lopes Rangel Fietto
Jazyk: angličtina
Rok vydání: 2014
Předmět:
Zdroj: PLoS Neglected Tropical Diseases, Vol 8, Iss 11, p e3309 (2014)
Druh dokumentu: article
ISSN: 1935-2727
1935-2735
DOI: 10.1371/journal.pntd.0003309
Popis: Visceral leishmaniasis is an important tropical disease, and Leishmania infantum chagasi (synonym of Leishmania infantum) is the main pathogenic agent of visceral leishmaniasis in the New World. Recently, ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) were identified as enablers of infection and virulence factors in many pathogens. Two putative E-NTPDases (∼70 kDa and ∼45 kDa) have been found in the L. infantum genome. Here, we studied the ∼45 kDa E-NTPDase from L. infantum chagasi to describe its natural occurrence, biochemical characteristics and influence on macrophage infection.We used live L. infantum chagasi to demonstrate its natural ecto-nucleotidase activity. We then isolated, cloned and expressed recombinant rLicNTPDase-2 in bacterial system. The recombinant rLicNTPDase-2 hydrolyzed a wide variety of triphosphate and diphosphate nucleotides (GTP> GDP = UDP> ADP> UTP = ATP) in the presence of calcium or magnesium. In addition, rLicNTPDase-2 showed stable activity over a pH range of 6.0 to 9.0 and was partially inhibited by ARL67156 and suramin. Microscopic analyses revealed the presence of this protein on cell surfaces, vesicles, flagellae, flagellar pockets, kinetoplasts, mitochondria and nuclei. The blockade of E-NTPDases using antibodies and competition led to lower levels of parasite adhesion and infection of macrophages. Furthermore, immunohistochemistry showed the expression of E-NTPDases in amastigotes in the lymph nodes of naturally infected dogs from an area of endemic visceral leishmaniasis.In this work, we cloned, expressed and characterized the NTPDase-2 from L. infantum chagasi and demonstrated that it functions as a genuine enzyme from the E-NTPDase/CD39 family. We showed that E-NTPDases are present on the surface of promastigotes and in other intracellular locations. We showed, for the first time, the broad expression of LicNTPDases in naturally infected dogs. Additionally, the blockade of NTPDases led to lower levels of in vitro adhesion and infection, suggesting that these proteins are possible targets for rational drug design.
Databáze: Directory of Open Access Journals
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