Abstract
BackgroundVisceral 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 FindingsWe 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/SignificanceIn 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.
Highlights
Visceral leishmaniasis (VL) is a disease that, if not treated, is usually fatal
The bioinformatics studies shown the presence of two ENTPDase paralogs in kinetoplastids, except for T. cruzi, in which only one member of this family was observed [14]
In the context of the low levels of identity with mammalian ENTPDases and the previous knowledge of the T. cruzi NTPDase-1, we propose here a new nomenclature for the trypanosomatid ENTPDases: TpNTPDase1 for the trypanosomatid ENTPDases more similar to T. cruzi ENTPDase-1 (,70 kDa proteins) and TpNTPDase-2 for the lower molecular weight isoform (,40 kDa), which is absent from T. cruzi
Summary
Visceral leishmaniasis (VL) is a disease that, if not treated, is usually fatal. It affects thousands of people per year worldwide. The role of E-NTPDases in infectivity, virulence or purine acquisition of the pathogenic protozoan parasites has been investigated in Trypanosoma cruzi [8], Toxoplasma gondii [9] and the species of Leishmania that causes tegumental leishmaniasis [6,10,11,12] These studies suggested that the E-NTPDases have key roles in parasite infections through the subversion of extracellular nucleotide signaling pathways, those involving ATP and ADP. We investigated the subcellular localization of this protein and evaluated its involvement in macrophage infection and its natural expression in tissues from dogs with canine visceral leishmaniasis These approaches provided important steps towards the elucidation of the role of this protein in Leishmania infection and could open new fields for the rational design of new drugs or other biotechnological applications
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