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

Vasconcellos Rde S, Mariotini-Moura C, Gomes RS, Serafim TD, Firmino Rde C, Silva E Bastos M, Castro FF, Oliveira CM, Borges-Pereira L, de Souza AC, de Souza RF, Gómez GA, Pinheiro Ada C, Maciel TE, Silva-Júnior A, Bressan GC, Almeida MR, Baqui MM, Afonso LC, Fietto JL - PLoS Negl Trop Dis (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Biologia Geral, Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil; Instituto Nacional de Biotecnologia Estrutural e Química Medicinal em Doenças Infecciosas (INBEQMeDI), São Carlos, São Paulo, Brazil.

ABSTRACT

Background: 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.

Methodology/principal findings: 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.

Conclusions/significance: 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.

No MeSH data available.


Related in: MedlinePlus

Analyses of expression and purification of rLicNTPDase-2.(A) Protein extract from E. coli carrying the empty vector pET21b. (B) Protein extract from E. coli carrying the vector pET21b plus rLicNTPDase-2. (C) Purified rLicNTPDase-2 (3 µg) stained by the silver method. SDS lanes indicate samples analyzed after SDS (10%)-PAGE stained with Coomassie blue. WB lanes indicate the same SDS-PAGE samples analyzed by Western blot using anti-His produced in rabbit as primary antibody (1∶4000) and anti-rabbit-IgG conjugated with FITC as secondary antibody (1∶6000). The nitrocellulose membrane was analyzed using an FLA 5100 (Fujifilm) instrument at 475 nm, with a blue filter.
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pntd-0003309-g003: Analyses of expression and purification of rLicNTPDase-2.(A) Protein extract from E. coli carrying the empty vector pET21b. (B) Protein extract from E. coli carrying the vector pET21b plus rLicNTPDase-2. (C) Purified rLicNTPDase-2 (3 µg) stained by the silver method. SDS lanes indicate samples analyzed after SDS (10%)-PAGE stained with Coomassie blue. WB lanes indicate the same SDS-PAGE samples analyzed by Western blot using anti-His produced in rabbit as primary antibody (1∶4000) and anti-rabbit-IgG conjugated with FITC as secondary antibody (1∶6000). The nitrocellulose membrane was analyzed using an FLA 5100 (Fujifilm) instrument at 475 nm, with a blue filter.

Mentions: The soluble/ecto domain of rLicNTPDase-2 (L41 to E425) was cloned into the bacterial expression vector pET21b (+). The expression was performed in E. coli BL21 (DE3) RIL cells. This construct displayed a satisfactory overexpression of the recombinant protein (Figure 3). The recombinant protein was refolded and purified by affinity chromatography, resulting in a unique band protein with the expected molecular weight of 43.96 kDa as visualized by silver staining (Figure 3C). The expression of rLicNTPDase-2 was confirmed by Western blot (Figure 3B and C).


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

Vasconcellos Rde S, Mariotini-Moura C, Gomes RS, Serafim TD, Firmino Rde C, Silva E Bastos M, Castro FF, Oliveira CM, Borges-Pereira L, de Souza AC, de Souza RF, Gómez GA, Pinheiro Ada C, Maciel TE, Silva-Júnior A, Bressan GC, Almeida MR, Baqui MM, Afonso LC, Fietto JL - PLoS Negl Trop Dis (2014)

Analyses of expression and purification of rLicNTPDase-2.(A) Protein extract from E. coli carrying the empty vector pET21b. (B) Protein extract from E. coli carrying the vector pET21b plus rLicNTPDase-2. (C) Purified rLicNTPDase-2 (3 µg) stained by the silver method. SDS lanes indicate samples analyzed after SDS (10%)-PAGE stained with Coomassie blue. WB lanes indicate the same SDS-PAGE samples analyzed by Western blot using anti-His produced in rabbit as primary antibody (1∶4000) and anti-rabbit-IgG conjugated with FITC as secondary antibody (1∶6000). The nitrocellulose membrane was analyzed using an FLA 5100 (Fujifilm) instrument at 475 nm, with a blue filter.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4230930&req=5

pntd-0003309-g003: Analyses of expression and purification of rLicNTPDase-2.(A) Protein extract from E. coli carrying the empty vector pET21b. (B) Protein extract from E. coli carrying the vector pET21b plus rLicNTPDase-2. (C) Purified rLicNTPDase-2 (3 µg) stained by the silver method. SDS lanes indicate samples analyzed after SDS (10%)-PAGE stained with Coomassie blue. WB lanes indicate the same SDS-PAGE samples analyzed by Western blot using anti-His produced in rabbit as primary antibody (1∶4000) and anti-rabbit-IgG conjugated with FITC as secondary antibody (1∶6000). The nitrocellulose membrane was analyzed using an FLA 5100 (Fujifilm) instrument at 475 nm, with a blue filter.
Mentions: The soluble/ecto domain of rLicNTPDase-2 (L41 to E425) was cloned into the bacterial expression vector pET21b (+). The expression was performed in E. coli BL21 (DE3) RIL cells. This construct displayed a satisfactory overexpression of the recombinant protein (Figure 3). The recombinant protein was refolded and purified by affinity chromatography, resulting in a unique band protein with the expected molecular weight of 43.96 kDa as visualized by silver staining (Figure 3C). The expression of rLicNTPDase-2 was confirmed by Western blot (Figure 3B and C).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Biologia Geral, Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil; Instituto Nacional de Biotecnologia Estrutural e Química Medicinal em Doenças Infecciosas (INBEQMeDI), São Carlos, São Paulo, Brazil.

ABSTRACT

Background: 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.

Methodology/principal findings: 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.

Conclusions/significance: 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.

No MeSH data available.


Related in: MedlinePlus