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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

Evaluation of role of LicNTPDases in the infection and adhesion of L. infantum chagasi to macrophages.(A) Adhesion of Leishmania promastigotes to macrophages. (B) Infection of macrophages by Leishmania promastigotes. (C) Treatment did not affect the amount of parasites in macrophages. In all assays (A, B and C): Macrophages treated with polyclonal antiserum anti-rLicNTPDase-2 prior to the infection or with the purified rLicNTPDase-2 before the contact with parasites are compared with macrophages treated with the parasites in the absence of intervention. Control  =  adhesion and infection assay without any intervention. Control with enzyme buffer  =  adhesion and infection assay in the presence of the buffer used to suspend rLicNTPDase-2. BSA was used as a non-related protein. The data reflect the mean + SE from three analyzed slides from each of three independent assays. The asterisks indicate significant differences (p<0.05) between the control and other samples.
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pntd-0003309-g008: Evaluation of role of LicNTPDases in the infection and adhesion of L. infantum chagasi to macrophages.(A) Adhesion of Leishmania promastigotes to macrophages. (B) Infection of macrophages by Leishmania promastigotes. (C) Treatment did not affect the amount of parasites in macrophages. In all assays (A, B and C): Macrophages treated with polyclonal antiserum anti-rLicNTPDase-2 prior to the infection or with the purified rLicNTPDase-2 before the contact with parasites are compared with macrophages treated with the parasites in the absence of intervention. Control  =  adhesion and infection assay without any intervention. Control with enzyme buffer  =  adhesion and infection assay in the presence of the buffer used to suspend rLicNTPDase-2. BSA was used as a non-related protein. The data reflect the mean + SE from three analyzed slides from each of three independent assays. The asterisks indicate significant differences (p<0.05) between the control and other samples.

Mentions: Previous work demonstrated a possible participation of the trypanosomatid ENTPDases in host infection. Here, we evaluated the influence of rLicNTPDase-2 and the polyclonal antibodies against this recombinant protein in the adhesion and infection of L. infantum chagasi in cultured macrophages. The non-related protein bovine serum albumin (BSA) and pre-immune serum were used as negative controls. When the macrophages were treated with the recombinant enzyme before the addition of parasites, there was a 48.3% reduction of adhesion and 43.91% reduction of infection (Figure 8 A–B). However, parasite proliferation (Figure 8C) was not influenced by any treatments, demonstrating that the effect observed in the adhesion step does not seem to influence the viability of internalized parasites. Taken together, these results suggest the possible existence of binding sites (e.g., receptors) for rLicNTPDase2 in the macrophages that may facilitate adherence and infection. Specifically, by binding to these receptors, the recombinant enzyme would most likely prevent the interaction of the parasite enzyme with the macrophage (Figure 8). However, we cannot exclude other possible explanations as discussed by Mariotini-Moura and co-workers [30]. Anti-rLicNTPDase-2 was also able to significantly reduce the adhesion (48.41% at 1∶100 dilution and 40.1% at 1∶50 dilution of the hyperimmune serum) and infection (45.4% at 1∶100 and 37.7% at 1∶50). We can speculate that the antibodies would bind to the parasite rLicNTPDases 1 and/or 2, preventing its interaction with a putative macrophage receptor. However, we cannot ignore other hypothesis such as the presence of unknown molecules that could interfere with the binding of rLicNTPDase to the host cells. Regardless of the mechanism, this area needs to be further investigated.


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)

Evaluation of role of LicNTPDases in the infection and adhesion of L. infantum chagasi to macrophages.(A) Adhesion of Leishmania promastigotes to macrophages. (B) Infection of macrophages by Leishmania promastigotes. (C) Treatment did not affect the amount of parasites in macrophages. In all assays (A, B and C): Macrophages treated with polyclonal antiserum anti-rLicNTPDase-2 prior to the infection or with the purified rLicNTPDase-2 before the contact with parasites are compared with macrophages treated with the parasites in the absence of intervention. Control  =  adhesion and infection assay without any intervention. Control with enzyme buffer  =  adhesion and infection assay in the presence of the buffer used to suspend rLicNTPDase-2. BSA was used as a non-related protein. The data reflect the mean + SE from three analyzed slides from each of three independent assays. The asterisks indicate significant differences (p<0.05) between the control and other samples.
© Copyright Policy
Related In: Results  -  Collection

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

pntd-0003309-g008: Evaluation of role of LicNTPDases in the infection and adhesion of L. infantum chagasi to macrophages.(A) Adhesion of Leishmania promastigotes to macrophages. (B) Infection of macrophages by Leishmania promastigotes. (C) Treatment did not affect the amount of parasites in macrophages. In all assays (A, B and C): Macrophages treated with polyclonal antiserum anti-rLicNTPDase-2 prior to the infection or with the purified rLicNTPDase-2 before the contact with parasites are compared with macrophages treated with the parasites in the absence of intervention. Control  =  adhesion and infection assay without any intervention. Control with enzyme buffer  =  adhesion and infection assay in the presence of the buffer used to suspend rLicNTPDase-2. BSA was used as a non-related protein. The data reflect the mean + SE from three analyzed slides from each of three independent assays. The asterisks indicate significant differences (p<0.05) between the control and other samples.
Mentions: Previous work demonstrated a possible participation of the trypanosomatid ENTPDases in host infection. Here, we evaluated the influence of rLicNTPDase-2 and the polyclonal antibodies against this recombinant protein in the adhesion and infection of L. infantum chagasi in cultured macrophages. The non-related protein bovine serum albumin (BSA) and pre-immune serum were used as negative controls. When the macrophages were treated with the recombinant enzyme before the addition of parasites, there was a 48.3% reduction of adhesion and 43.91% reduction of infection (Figure 8 A–B). However, parasite proliferation (Figure 8C) was not influenced by any treatments, demonstrating that the effect observed in the adhesion step does not seem to influence the viability of internalized parasites. Taken together, these results suggest the possible existence of binding sites (e.g., receptors) for rLicNTPDase2 in the macrophages that may facilitate adherence and infection. Specifically, by binding to these receptors, the recombinant enzyme would most likely prevent the interaction of the parasite enzyme with the macrophage (Figure 8). However, we cannot exclude other possible explanations as discussed by Mariotini-Moura and co-workers [30]. Anti-rLicNTPDase-2 was also able to significantly reduce the adhesion (48.41% at 1∶100 dilution and 40.1% at 1∶50 dilution of the hyperimmune serum) and infection (45.4% at 1∶100 and 37.7% at 1∶50). We can speculate that the antibodies would bind to the parasite rLicNTPDases 1 and/or 2, preventing its interaction with a putative macrophage receptor. However, we cannot ignore other hypothesis such as the presence of unknown molecules that could interfere with the binding of rLicNTPDase to the host cells. Regardless of the mechanism, this area needs to be further investigated.

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