Limits...
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

L. infantum chagasi promastigote ecto-nucleotidase activity and surface ENTPDase localization using anti-rLicNTPDase-2.(A) The promastigote ecto-nucleotidase activity assays were performed in live total promastigotes from log phase growth using different substrates and Mg2+ as cofactor. The activities of recombinant enzyme (rLicNTPDase-2) are shown in the inset box. The SDs represent those of the average of three independent experiments performed in triplicate. The free phosphate released was measured using the malachite green method. (B) Western blotting using anti-rLicNTPDase-2 recognizes both recombinant isoforms rLicNTPDase-1 and rLicNTPDase-2. Purified rLicNTPDase-1 and rLicNTPDase-2 were run in 10% SDS-PAGE and blotted onto a nitrocellulose membrane. The membrane was incubated with purified rabbit polyclonal antibodies to anti-rLicNTPDase-2 (1∶100) as the primary antibody and with anti-rabbit IgG conjugated with FITC (1∶10,000) as the secondary antibody. (C) Distribution of ENTPDases at the surface of L. infantum chagasi promastigotes. Non-permeabilized cells fixed with paraformaldehyde were incubated with anti- rLicNTPDase-2 (1∶50) as primary antibody and with Alexa 488-conjugated goat anti-rabbit IgG (1∶400) as secondary antibody. The glass slides were mounted with Prolong Gold Antifade Reagent (Molecular Probes) and examined by confocal microscopy (Leica, SP5). Bar scale  = 2 µm.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4230930&req=5

pntd-0003309-g006: L. infantum chagasi promastigote ecto-nucleotidase activity and surface ENTPDase localization using anti-rLicNTPDase-2.(A) The promastigote ecto-nucleotidase activity assays were performed in live total promastigotes from log phase growth using different substrates and Mg2+ as cofactor. The activities of recombinant enzyme (rLicNTPDase-2) are shown in the inset box. The SDs represent those of the average of three independent experiments performed in triplicate. The free phosphate released was measured using the malachite green method. (B) Western blotting using anti-rLicNTPDase-2 recognizes both recombinant isoforms rLicNTPDase-1 and rLicNTPDase-2. Purified rLicNTPDase-1 and rLicNTPDase-2 were run in 10% SDS-PAGE and blotted onto a nitrocellulose membrane. The membrane was incubated with purified rabbit polyclonal antibodies to anti-rLicNTPDase-2 (1∶100) as the primary antibody and with anti-rabbit IgG conjugated with FITC (1∶10,000) as the secondary antibody. (C) Distribution of ENTPDases at the surface of L. infantum chagasi promastigotes. Non-permeabilized cells fixed with paraformaldehyde were incubated with anti- rLicNTPDase-2 (1∶50) as primary antibody and with Alexa 488-conjugated goat anti-rabbit IgG (1∶400) as secondary antibody. The glass slides were mounted with Prolong Gold Antifade Reagent (Molecular Probes) and examined by confocal microscopy (Leica, SP5). Bar scale  = 2 µm.

Mentions: To evaluate the presence of ecto-nucleotidase activity in L. infantum chagasi, we analyzed general ecto-nucleotidase activities directly on live promastigotes. As shown in Figure 6A, the parasites were able to hydrolyze all tested nucleotides (ATP, ADP, AMP, GTP, GDP, UTP and UDP). The hydrolysis of ATP was similar to ADP, GTP, GDP and UDP. UTPase activity was the lowest ecto-nucleotidase activity observed using this approach. These results indicate that this parasite possesses broad ecto-nucleotidase activity on its surface because no permeabilization was made in this assay. Using another L. infantum strain (BH46) and a reaction medium with a different composition, Maia et al. [42] found similar results indicating a broad ecto-nucleotidase activity in L. infantum promastigotes.


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)

L. infantum chagasi promastigote ecto-nucleotidase activity and surface ENTPDase localization using anti-rLicNTPDase-2.(A) The promastigote ecto-nucleotidase activity assays were performed in live total promastigotes from log phase growth using different substrates and Mg2+ as cofactor. The activities of recombinant enzyme (rLicNTPDase-2) are shown in the inset box. The SDs represent those of the average of three independent experiments performed in triplicate. The free phosphate released was measured using the malachite green method. (B) Western blotting using anti-rLicNTPDase-2 recognizes both recombinant isoforms rLicNTPDase-1 and rLicNTPDase-2. Purified rLicNTPDase-1 and rLicNTPDase-2 were run in 10% SDS-PAGE and blotted onto a nitrocellulose membrane. The membrane was incubated with purified rabbit polyclonal antibodies to anti-rLicNTPDase-2 (1∶100) as the primary antibody and with anti-rabbit IgG conjugated with FITC (1∶10,000) as the secondary antibody. (C) Distribution of ENTPDases at the surface of L. infantum chagasi promastigotes. Non-permeabilized cells fixed with paraformaldehyde were incubated with anti- rLicNTPDase-2 (1∶50) as primary antibody and with Alexa 488-conjugated goat anti-rabbit IgG (1∶400) as secondary antibody. The glass slides were mounted with Prolong Gold Antifade Reagent (Molecular Probes) and examined by confocal microscopy (Leica, SP5). Bar scale  = 2 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pntd-0003309-g006: L. infantum chagasi promastigote ecto-nucleotidase activity and surface ENTPDase localization using anti-rLicNTPDase-2.(A) The promastigote ecto-nucleotidase activity assays were performed in live total promastigotes from log phase growth using different substrates and Mg2+ as cofactor. The activities of recombinant enzyme (rLicNTPDase-2) are shown in the inset box. The SDs represent those of the average of three independent experiments performed in triplicate. The free phosphate released was measured using the malachite green method. (B) Western blotting using anti-rLicNTPDase-2 recognizes both recombinant isoforms rLicNTPDase-1 and rLicNTPDase-2. Purified rLicNTPDase-1 and rLicNTPDase-2 were run in 10% SDS-PAGE and blotted onto a nitrocellulose membrane. The membrane was incubated with purified rabbit polyclonal antibodies to anti-rLicNTPDase-2 (1∶100) as the primary antibody and with anti-rabbit IgG conjugated with FITC (1∶10,000) as the secondary antibody. (C) Distribution of ENTPDases at the surface of L. infantum chagasi promastigotes. Non-permeabilized cells fixed with paraformaldehyde were incubated with anti- rLicNTPDase-2 (1∶50) as primary antibody and with Alexa 488-conjugated goat anti-rabbit IgG (1∶400) as secondary antibody. The glass slides were mounted with Prolong Gold Antifade Reagent (Molecular Probes) and examined by confocal microscopy (Leica, SP5). Bar scale  = 2 µm.
Mentions: To evaluate the presence of ecto-nucleotidase activity in L. infantum chagasi, we analyzed general ecto-nucleotidase activities directly on live promastigotes. As shown in Figure 6A, the parasites were able to hydrolyze all tested nucleotides (ATP, ADP, AMP, GTP, GDP, UTP and UDP). The hydrolysis of ATP was similar to ADP, GTP, GDP and UDP. UTPase activity was the lowest ecto-nucleotidase activity observed using this approach. These results indicate that this parasite possesses broad ecto-nucleotidase activity on its surface because no permeabilization was made in this assay. Using another L. infantum strain (BH46) and a reaction medium with a different composition, Maia et al. [42] found similar results indicating a broad ecto-nucleotidase activity in L. infantum promastigotes.

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