Leishmania infantum ecto-nucleoside triphosphate diphosphohydrolase-2 is an apyrase involved in macrophage infection and expressed in infected dogs

PLoS Negl Trop Dis. 2014 Nov 13;8(11):e3309. doi: 10.1371/journal.pntd.0003309. eCollection 2014 Nov.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Apyrase / chemistry
  • Apyrase / genetics
  • Apyrase / metabolism*
  • Cell Line
  • Dogs
  • Female
  • Leishmania infantum / chemistry
  • Leishmania infantum / cytology
  • Leishmania infantum / enzymology*
  • Leishmania infantum / metabolism
  • Leishmaniasis, Visceral / parasitology*
  • Lymph Nodes / parasitology
  • Macrophages / parasitology*
  • Mice
  • Molecular Sequence Data
  • Phylogeny
  • Protozoan Proteins / chemistry
  • Protozoan Proteins / genetics
  • Protozoan Proteins / metabolism*
  • Rabbits
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Sequence Alignment

Substances

  • Protozoan Proteins
  • Recombinant Proteins
  • Apyrase

Grants and funding

The authors gratefully acknowledge the Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for financial support from grant number CNPq/CAPES 552459/2011-9 URL http://www.cnpq.br/ and http://www.capes.gov.br/ and FAPEMIG grant number APQ-00754-11 and APQ-02437-10 http://www.fapemig.br/. We thank FAPEMIG for FFdC, LBP fellowships; CNPq for JLRF, MRA, LCCA, MSeB and ACAdS, and CMM fellowships; and CAPES for CMdO, RDSV and RFdS fellowships. MMAB received financial support from the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) URL www.fapesp.br/. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.