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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

rLicNTPDase-2 ATPase activity in the presence of known partial inhibitors of ENTPDases and a 5′nucleotidase inhibitor.Purified rLicNTPDase-2 was assayed in the absence of inhibitors (control column) or in the presence of inhibitors: ARL 67156 (ARL) 300 µM, gadolinium chloride 300 µM, suramin 100 µM, sodium azide 2 mM and ammonium molybdate 3 µM (a 5′nucleotidase inhibitor). The SDs represent those from the average of three independent experiments performed in triplicate. The free phosphate released was measured using the malachite green method. Statistical analyses were performed using ANOVA, and the significant differences between the control and inhibitors assays are shown with asterisks (p<0.05).
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pntd-0003309-g005: rLicNTPDase-2 ATPase activity in the presence of known partial inhibitors of ENTPDases and a 5′nucleotidase inhibitor.Purified rLicNTPDase-2 was assayed in the absence of inhibitors (control column) or in the presence of inhibitors: ARL 67156 (ARL) 300 µM, gadolinium chloride 300 µM, suramin 100 µM, sodium azide 2 mM and ammonium molybdate 3 µM (a 5′nucleotidase inhibitor). The SDs represent those from the average of three independent experiments performed in triplicate. The free phosphate released was measured using the malachite green method. Statistical analyses were performed using ANOVA, and the significant differences between the control and inhibitors assays are shown with asterisks (p<0.05).

Mentions: To prove that rLicNTPDase is a genuine apyrase, we tested various partial inhibitors of E-NTPDases described in previous works. We used the following agents: ARL 67156 (6-N, N-Diethyl-bc-dibromomethylene-D-adenosine-5-triphosphate), considered to be a selective inhibitor of ecto-ATPase and able to partially inhibit the NTPDase-1 of T. cruzi[8], [38]; gadolinium, a lanthanide capable of inhibiting the ecto-NTPDase of the electric organ of Torpedo[39]; suramin, a naphthylurea polysulfone compound that has been shown to be a partial inhibitor of the ecto-ATPDase and the NTPDase-1 of T. cruzi[8], [40]; sodium azide, a mitochondrial ATPase inhibitor that is also able to partially inhibit the ecto-ATPase of T. cruzi[14] and ammonium molybdate, an inhibitor of 5'-nucleotidase and acid phosphatase [41]. ARL 67156 and suramin partially inhibited the enzyme activity, approximately 38.5% and 46.83%, respectively (Figure 5). No significant inhibition was observed using the other tested compounds. On the other hand, a previous study using live promastigotes from L. infantum demonstrated a significant inhibition of ecto-ADPase (80%) and ecto-ATPase activity (24%) by sodium azide [42]. This conflicting result may be explained by an action of azide on ecto-nucleotidases or NTPDases other than the isoform named here as LicNTPDase-2. Another possibility is that under the conditions assayed in live parasites, this isoform could be partially inhibited by azide. Regarding the recombinant enzyme NTPDase-1 of T. cruzi, the compound that showed the greatest inhibition was 100 µM suramin (∼50%), while 300 µM gadolinium inhibited only approximately 20% of the activity [8]. These results indicate that despite belonging to the same family and having the five ACR conserved regions, there are differences in the sensitivity of the ENTPDases from different parasites to inhibitors.


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)

rLicNTPDase-2 ATPase activity in the presence of known partial inhibitors of ENTPDases and a 5′nucleotidase inhibitor.Purified rLicNTPDase-2 was assayed in the absence of inhibitors (control column) or in the presence of inhibitors: ARL 67156 (ARL) 300 µM, gadolinium chloride 300 µM, suramin 100 µM, sodium azide 2 mM and ammonium molybdate 3 µM (a 5′nucleotidase inhibitor). The SDs represent those from the average of three independent experiments performed in triplicate. The free phosphate released was measured using the malachite green method. Statistical analyses were performed using ANOVA, and the significant differences between the control and inhibitors assays are shown with asterisks (p<0.05).
© Copyright Policy
Related In: Results  -  Collection

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

pntd-0003309-g005: rLicNTPDase-2 ATPase activity in the presence of known partial inhibitors of ENTPDases and a 5′nucleotidase inhibitor.Purified rLicNTPDase-2 was assayed in the absence of inhibitors (control column) or in the presence of inhibitors: ARL 67156 (ARL) 300 µM, gadolinium chloride 300 µM, suramin 100 µM, sodium azide 2 mM and ammonium molybdate 3 µM (a 5′nucleotidase inhibitor). The SDs represent those from the average of three independent experiments performed in triplicate. The free phosphate released was measured using the malachite green method. Statistical analyses were performed using ANOVA, and the significant differences between the control and inhibitors assays are shown with asterisks (p<0.05).
Mentions: To prove that rLicNTPDase is a genuine apyrase, we tested various partial inhibitors of E-NTPDases described in previous works. We used the following agents: ARL 67156 (6-N, N-Diethyl-bc-dibromomethylene-D-adenosine-5-triphosphate), considered to be a selective inhibitor of ecto-ATPase and able to partially inhibit the NTPDase-1 of T. cruzi[8], [38]; gadolinium, a lanthanide capable of inhibiting the ecto-NTPDase of the electric organ of Torpedo[39]; suramin, a naphthylurea polysulfone compound that has been shown to be a partial inhibitor of the ecto-ATPDase and the NTPDase-1 of T. cruzi[8], [40]; sodium azide, a mitochondrial ATPase inhibitor that is also able to partially inhibit the ecto-ATPase of T. cruzi[14] and ammonium molybdate, an inhibitor of 5'-nucleotidase and acid phosphatase [41]. ARL 67156 and suramin partially inhibited the enzyme activity, approximately 38.5% and 46.83%, respectively (Figure 5). No significant inhibition was observed using the other tested compounds. On the other hand, a previous study using live promastigotes from L. infantum demonstrated a significant inhibition of ecto-ADPase (80%) and ecto-ATPase activity (24%) by sodium azide [42]. This conflicting result may be explained by an action of azide on ecto-nucleotidases or NTPDases other than the isoform named here as LicNTPDase-2. Another possibility is that under the conditions assayed in live parasites, this isoform could be partially inhibited by azide. Regarding the recombinant enzyme NTPDase-1 of T. cruzi, the compound that showed the greatest inhibition was 100 µM suramin (∼50%), while 300 µM gadolinium inhibited only approximately 20% of the activity [8]. These results indicate that despite belonging to the same family and having the five ACR conserved regions, there are differences in the sensitivity of the ENTPDases from different parasites to inhibitors.

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