Leishmania amazonensis: Biological and biochemical characterization of ecto-nucleoside triphosphate diphosphohydrolase activities

Abstract

The presence of Leishmania amazonensis ecto-nucleoside triphosphate triphosphohydrolase activities was demonstrated using antibodies against different NTPDase members by Western blotting, flow cytometry, and immunoelectron microscopy analysis. Living promastigote cells sequentially hydrolyzed the ATP molecule generating ADP, AMP, and adenosine, indicating that this surface enzyme may play a role in the salvage of purines from the extracellular medium. The L. amazonensis ecto-NTPDase activities were insensitive to Triton X-100, but they were enhanced by divalent cations, such as Mg(2+). In addition, the ecto-NTPDase activities decreased with time for 96 h when promastigotes were grown in vitro. On the other hand, these activities increased considerably when measured in living amastigote forms. Furthermore, the treatment with adenosine, a mediator of several relevant biological phenomena, induced a decrease in the reactivity with anti-CD39 antibody, raised against mammalian E-NTPDase, probably because of down regulation in the L. amazonensis ecto-NTPDase expression. Also, adenosine and anti-NTPDase antibodies induced a significant diminishing in the interaction between promastigotes of L. amazonensis and mouse peritoneal macrophages.

Immune and inflammatory responses to Leishmania amazonensis isolated from different clinical forms of human leishmaniasis in CBA mice

“…Analysis of nucleotide and adenosine concentrations was performed as previously described (Pinheiro et al 2006).…”

Leishmania-released nucleoside diphosphate kinase prevents ATP-mediated cytolysis of macrophages

“…Of particular interest is extracellular ATP (eATP), which is expected to accumulate at the site of inflammation [12][13][14], important in immunity against microbial infection. The released enzymes include adenylate kinase, 5′-nucleotidase (and/or ATPase/phosphatase), ecto-ATPase [15], ecto-nucleoside triphosphate diphosphohydrolase (NTPDase) [16] and NdK. The last enzyme utilizes eATP as its principal substrate [9,10,[17][18][19][20].…”

The Leishmania-Macrophage Interactions: Role of E-NTPDases and Purinergic Signaling

“…Furthermore, they showed higher expression and activity in amastigote than in promastigote forms and that adenosine down-regulated the expression of this enzyme. Additionally, they showed that adenosine and anti-E-NTPDase antibodies decreased the adhesion of promastigotes to mouse peritoneal macrophages [74] .…”

“…They showed for the first time that virulent L. amazonensis presented higher ecto-ATPase activity than the avirulent promastigotes. In 2006, Pinheiro and co-workers demonstrated that the surface E-NTPDase from L. amazonensis is a protein from the CD39 family and that this enzyme may play a role in the purine salvage pathway [74] . Summary overview of purinergic signaling in the context of the immune system during homeostasis, dangerous stimuli or in the face of Leishmania infection.…”

“…A summary of the general view of ecto-nucleotidases, ecto-nucleosides and mammalian immune system homeostasis control is shown in Figure 1. Additionally, E-NTPDases had been found in several protozoan parasites including the following: Toxoplasma gondii [72] Trichomonas vaginalis [73] Trypanosoma cruzi [40] , Leishmania amazonensis [74] , Leishmania braziliensis [75] and other organisms [46] . In Trypanosomatids, these enzymes are important components of the pathway of the salvation of purines [40] because these parasites are unable to perform de novo synthesis of purines [76] .…”