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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

Immunohistochemistry using anti-rLicNTPDase-2 in the lymph nodes of naturally infected dogs.(A) Lymph nodes from 48 Leishmania-positive dogs were evaluated by immunohistochemistry (IHC) using anti-rLicNTPDase-2. The results of the IHC are compared with ELISA data of the same samples using a Biomanguinhos Kit. (B) An example of IHC result using polyclonal anti-rLicNTPDase-2. (C) The zoom of section C is from image B.
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pntd-0003309-g009: Immunohistochemistry using anti-rLicNTPDase-2 in the lymph nodes of naturally infected dogs.(A) Lymph nodes from 48 Leishmania-positive dogs were evaluated by immunohistochemistry (IHC) using anti-rLicNTPDase-2. The results of the IHC are compared with ELISA data of the same samples using a Biomanguinhos Kit. (B) An example of IHC result using polyclonal anti-rLicNTPDase-2. (C) The zoom of section C is from image B.

Mentions: In our previous work, we demonstrated that the recombinant Lic-NTPDase-2 is a good novel antigen for immunological diagnosis of canine visceral leishmaniasis [21]. That work suggested the presence of active Leishmania in dogs but only via an indirect method because we used the recombinant protein as a target to measure the levels of specific antibodies in samples of serum from dogs. Here, we directly investigated the presence of amastigotes in naturally infected dogs. Leishmania can infect many different organs of mammalian hosts. In this work, we used immunohistochemistry with antibodies against rLic-NTPDase-2 to directly evaluate the expression of LicNTPDases in the lymph nodes of naturally infected dogs (Figure 9). In this approach, 45 samples (95.7%) showed immunoperoxidase staining, directly demonstrating the presence of the recognized antigen in the tissue. These data show for the first time the expression of LicNTPDases in tissues from naturally infected dogs and corroborate previous data from our group that demonstrated the potential application of rLicNTPDase-2 in the diagnosis of canine Leishmaniasis [21].


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)

Immunohistochemistry using anti-rLicNTPDase-2 in the lymph nodes of naturally infected dogs.(A) Lymph nodes from 48 Leishmania-positive dogs were evaluated by immunohistochemistry (IHC) using anti-rLicNTPDase-2. The results of the IHC are compared with ELISA data of the same samples using a Biomanguinhos Kit. (B) An example of IHC result using polyclonal anti-rLicNTPDase-2. (C) The zoom of section C is from image B.
© Copyright Policy
Related In: Results  -  Collection

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

pntd-0003309-g009: Immunohistochemistry using anti-rLicNTPDase-2 in the lymph nodes of naturally infected dogs.(A) Lymph nodes from 48 Leishmania-positive dogs were evaluated by immunohistochemistry (IHC) using anti-rLicNTPDase-2. The results of the IHC are compared with ELISA data of the same samples using a Biomanguinhos Kit. (B) An example of IHC result using polyclonal anti-rLicNTPDase-2. (C) The zoom of section C is from image B.
Mentions: In our previous work, we demonstrated that the recombinant Lic-NTPDase-2 is a good novel antigen for immunological diagnosis of canine visceral leishmaniasis [21]. That work suggested the presence of active Leishmania in dogs but only via an indirect method because we used the recombinant protein as a target to measure the levels of specific antibodies in samples of serum from dogs. Here, we directly investigated the presence of amastigotes in naturally infected dogs. Leishmania can infect many different organs of mammalian hosts. In this work, we used immunohistochemistry with antibodies against rLic-NTPDase-2 to directly evaluate the expression of LicNTPDases in the lymph nodes of naturally infected dogs (Figure 9). In this approach, 45 samples (95.7%) showed immunoperoxidase staining, directly demonstrating the presence of the recognized antigen in the tissue. These data show for the first time the expression of LicNTPDases in tissues from naturally infected dogs and corroborate previous data from our group that demonstrated the potential application of rLicNTPDase-2 in the diagnosis of canine Leishmaniasis [21].

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