CrATP interferes in the promastigote-macrophage interaction in Leishmania amazonensis infection

Recent have shown the relationship between Ecto-Nucleoside-Triphosphate-Diphosphohydrolases (Ecto-NTPDases or ecto-nucleotidases) and virulence and infectivity in trypanosomatids. In this work, the inhibition of the ecto-ATPase activities and promastigote growth of Leishmania amazonensis by CrATP was characterized. Furthermore, this compound was used to investigate the role of ecto-nucleotidase in the interaction of L. amazonensis with resident peritoneal macrophages obtained from BALB/c mice. CrATP partially inhibits the ecto-ATPase activity, presenting Ki values of 575·7±199·1 and 383·5±79·0 μm, in the presence or absence of 5 mm MgCl2, respectively. The apparent Kms for ATP (2·9±0·5 mm to Mg2+-dependent ecto-ATPase and 0·4±0·2 mm to Mg2+-independent ecto-ATPase activities) are not significantly altered by CrATP, suggesting a reversible non-competitive inhibition of both enzymes. When CrATP was added to the cultivation medium at 500 μm, it drastically inhibited the cellular growth. The interaction of promastigote forms of L. amazonensis with BALB/c peritoneal macrophages is strongly affected by CrATP. When the parasites were treated with 500 μm CrATP before interacting with macrophages, the adhesion and endocytic indices were strongly reduced to 53·0±14·8% and 39·8±1·1%, respectively. These results indicate that ecto-nucleotidase plays an important role in the infection process caused by Leishmania amazonensis.