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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

Sub-cellular localization of ENTPDases in L. infantum chagasi promastigotes.Electron micrographs using polyclonal antibodies to rLicNTPDase-2 anti-IgG conjugated to 10 nm colloidal gold (A–H). Letters and symbols indicate different localizations: nucleus (N) (white arrow), mitochondria and kinetoplasts (K) (white arrowhead), internal vesicles (black arrowhead), flagellum (F) and flagellar pocket (black arrow) and cell surface (dashed black arrow). No staining was observed in the control (H). Bars: A, C, F, G = 0.1 µm and B = 0.3 µm, C, D and E, H 0.2 µm.
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pntd-0003309-g007: Sub-cellular localization of ENTPDases in L. infantum chagasi promastigotes.Electron micrographs using polyclonal antibodies to rLicNTPDase-2 anti-IgG conjugated to 10 nm colloidal gold (A–H). Letters and symbols indicate different localizations: nucleus (N) (white arrow), mitochondria and kinetoplasts (K) (white arrowhead), internal vesicles (black arrowhead), flagellum (F) and flagellar pocket (black arrow) and cell surface (dashed black arrow). No staining was observed in the control (H). Bars: A, C, F, G = 0.1 µm and B = 0.3 µm, C, D and E, H 0.2 µm.

Mentions: The next step in this work was a detailed investigation of the localization of the TpNTPDases by electron microscopy to confirm the presence of ENTPDases on the cell surface, as observed by the ecto-nucleotidase activity (Figure 6A) and confocal data (Figure 6C). Figure 7A–G shows the presence of the protein on the cell surface, nucleus, flagellum and flagellar pocket region. Additionally, the gold particles stained the kinetoplast, mitochondria and internal vesicles. No staining was observed in the control assay (Figure 7H). The intracellular localization profile is similar to recent studies of L. braziliensis and L.amazonensis promastigotes [43], [47] and T. cruzi[30] epimastigotes and reinforces the ubiquitous localization of these proteins and the requirement for further investigations in this area.


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)

Sub-cellular localization of ENTPDases in L. infantum chagasi promastigotes.Electron micrographs using polyclonal antibodies to rLicNTPDase-2 anti-IgG conjugated to 10 nm colloidal gold (A–H). Letters and symbols indicate different localizations: nucleus (N) (white arrow), mitochondria and kinetoplasts (K) (white arrowhead), internal vesicles (black arrowhead), flagellum (F) and flagellar pocket (black arrow) and cell surface (dashed black arrow). No staining was observed in the control (H). Bars: A, C, F, G = 0.1 µm and B = 0.3 µm, C, D and E, H 0.2 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pntd-0003309-g007: Sub-cellular localization of ENTPDases in L. infantum chagasi promastigotes.Electron micrographs using polyclonal antibodies to rLicNTPDase-2 anti-IgG conjugated to 10 nm colloidal gold (A–H). Letters and symbols indicate different localizations: nucleus (N) (white arrow), mitochondria and kinetoplasts (K) (white arrowhead), internal vesicles (black arrowhead), flagellum (F) and flagellar pocket (black arrow) and cell surface (dashed black arrow). No staining was observed in the control (H). Bars: A, C, F, G = 0.1 µm and B = 0.3 µm, C, D and E, H 0.2 µm.
Mentions: The next step in this work was a detailed investigation of the localization of the TpNTPDases by electron microscopy to confirm the presence of ENTPDases on the cell surface, as observed by the ecto-nucleotidase activity (Figure 6A) and confocal data (Figure 6C). Figure 7A–G shows the presence of the protein on the cell surface, nucleus, flagellum and flagellar pocket region. Additionally, the gold particles stained the kinetoplast, mitochondria and internal vesicles. No staining was observed in the control assay (Figure 7H). The intracellular localization profile is similar to recent studies of L. braziliensis and L.amazonensis promastigotes [43], [47] and T. cruzi[30] epimastigotes and reinforces the ubiquitous localization of these proteins and the requirement for further investigations in this area.

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