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

rLic-NTPDase-2: refolding, substrate preference and pH dependence.(A) Refolding assay- the enzymatic activity was measured just after the refolding (zero point) and up to six days later using ADP as the substrate. (B) The preference for different substrates was assessed in the presence of the cofactors calcium (grey bars) or magnesium (black bars). Inset shows the hydrolysis values with SD. (C) ATP (open circles) and ADP (black circles) were used to evaluate enzymatic activity as a function of pH. The pH-dependence test was performed in buffer containing 50 mM MES, 50 mM Tris, 50 mM HEPES, 3 mM MgCl2, 116 mm NaCl, 5.4 mM KCl and 2.5 mM nucleotide. The SDs represent the those from the average of three independent experiments performed in triplicate. The free phosphate released was measured using the malachite green method.
© Copyright Policy
Related In: Results  -  Collection

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

pntd-0003309-g004: rLic-NTPDase-2: refolding, substrate preference and pH dependence.(A) Refolding assay- the enzymatic activity was measured just after the refolding (zero point) and up to six days later using ADP as the substrate. (B) The preference for different substrates was assessed in the presence of the cofactors calcium (grey bars) or magnesium (black bars). Inset shows the hydrolysis values with SD. (C) ATP (open circles) and ADP (black circles) were used to evaluate enzymatic activity as a function of pH. The pH-dependence test was performed in buffer containing 50 mM MES, 50 mM Tris, 50 mM HEPES, 3 mM MgCl2, 116 mm NaCl, 5.4 mM KCl and 2.5 mM nucleotide. The SDs represent the those from the average of three independent experiments performed in triplicate. The free phosphate released was measured using the malachite green method.

Mentions: Two of the main characteristics of ENTPDases are the ability to use a broad range of nucleotides as substrates and the dependence on divalent cations, mainly calcium or magnesium, as cofactors [33],[34]. The first step of the rLicNTPDase-2 biochemical characterization was analysis of the substrate specificity using calcium or magnesium as co-factors. The rLicNTPDase-2 nucleotidase activity was determined using the substrates ATP, ADP, GTP, GDP, UTP and UDP. To rule out the possibility that the protein preparation contained any phosphatases, an assay performed using p-nitrophenylphosphate (pNPP), a substrate of acid and alkaline phosphatases, showed no detectable activity. rLicNTPDase-2 was capable of hydrolyzing all tested triphosphate and diphosphate nucleotides. Under the tested conditions (excess substrate), we observed the highest activity for GTP and the least activity for ATP. The enzyme showed maximum activity between the first and second day after refolding (Figure 4A). Additionally, as shown in Figure 4B, the enzyme did not discriminate between calcium or magnesium ions as cofactors. To assess whether this promiscuity would depend only on a divalent cation regardless of the ion, we tested other divalent cations as cofactors (nickel and zinc), and both abolished the ATPase activity.


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)

rLic-NTPDase-2: refolding, substrate preference and pH dependence.(A) Refolding assay- the enzymatic activity was measured just after the refolding (zero point) and up to six days later using ADP as the substrate. (B) The preference for different substrates was assessed in the presence of the cofactors calcium (grey bars) or magnesium (black bars). Inset shows the hydrolysis values with SD. (C) ATP (open circles) and ADP (black circles) were used to evaluate enzymatic activity as a function of pH. The pH-dependence test was performed in buffer containing 50 mM MES, 50 mM Tris, 50 mM HEPES, 3 mM MgCl2, 116 mm NaCl, 5.4 mM KCl and 2.5 mM nucleotide. The SDs represent the those from the average of three independent experiments performed in triplicate. The free phosphate released was measured using the malachite green method.
© Copyright Policy
Related In: Results  -  Collection

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

pntd-0003309-g004: rLic-NTPDase-2: refolding, substrate preference and pH dependence.(A) Refolding assay- the enzymatic activity was measured just after the refolding (zero point) and up to six days later using ADP as the substrate. (B) The preference for different substrates was assessed in the presence of the cofactors calcium (grey bars) or magnesium (black bars). Inset shows the hydrolysis values with SD. (C) ATP (open circles) and ADP (black circles) were used to evaluate enzymatic activity as a function of pH. The pH-dependence test was performed in buffer containing 50 mM MES, 50 mM Tris, 50 mM HEPES, 3 mM MgCl2, 116 mm NaCl, 5.4 mM KCl and 2.5 mM nucleotide. The SDs represent the those from the average of three independent experiments performed in triplicate. The free phosphate released was measured using the malachite green method.
Mentions: Two of the main characteristics of ENTPDases are the ability to use a broad range of nucleotides as substrates and the dependence on divalent cations, mainly calcium or magnesium, as cofactors [33],[34]. The first step of the rLicNTPDase-2 biochemical characterization was analysis of the substrate specificity using calcium or magnesium as co-factors. The rLicNTPDase-2 nucleotidase activity was determined using the substrates ATP, ADP, GTP, GDP, UTP and UDP. To rule out the possibility that the protein preparation contained any phosphatases, an assay performed using p-nitrophenylphosphate (pNPP), a substrate of acid and alkaline phosphatases, showed no detectable activity. rLicNTPDase-2 was capable of hydrolyzing all tested triphosphate and diphosphate nucleotides. Under the tested conditions (excess substrate), we observed the highest activity for GTP and the least activity for ATP. The enzyme showed maximum activity between the first and second day after refolding (Figure 4A). Additionally, as shown in Figure 4B, the enzyme did not discriminate between calcium or magnesium ions as cofactors. To assess whether this promiscuity would depend only on a divalent cation regardless of the ion, we tested other divalent cations as cofactors (nickel and zinc), and both abolished the ATPase activity.

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