Date Published: March 3, 2009
Publisher: Public Library of Science
Author(s): Ramon F. Santos, Marcela A. S. Pôssa, Matheus S. Bastos, Paulo M. M. Guedes, Márcia R. Almeida, Ricardo DeMarco, Sergio Verjovski-Almeida, Maria T. Bahia, Juliana L. R. Fietto, J. R. Meyer-Fernandes
Abstract: BackgroundThe protozoan Trypanosoma cruzi is the causative agent of Chagas disease. There are no vaccines or effective treatment, especially in the chronic phase when most patients are diagnosed. There is a clear necessity to develop new drugs and strategies for the control and treatment of Chagas disease. Recent papers have suggested the ecto-nucleotidases (from CD39 family) from pathogenic agents as important virulence factors. In this study we evaluated the influence of Ecto-Nucleoside-Triphosphate-Diphosphohydrolase (Ecto-NTPDase) activity on infectivity and virulence of T. cruzi using both in vivo and in vitro models.Methodology/Principal FindingsWe followed Ecto-NTPDase activities of Y strain infective forms (trypomastigotes) obtained during sequential sub-cultivation in mammalian cells. ATPase/ADPase activity ratios of cell-derived trypomastigotes decreased 3- to 6-fold and infectivity was substantially reduced during sequential sub-cultivation. Surprisingly, at third to fourth passages most of the cell-derived trypomastigotes could not penetrate mammalian cells and had differentiated into amastigote-like parasites that exhibited 3- to 4-fold lower levels of Ecto-NTPDase activities. To evidence the participation of T. cruzi Ecto-NTPDase1 in the infective process, we evaluated the effect of known Ecto-ATPDase inhibitors (ARL 67156, Gadolinium and Suramin), or anti-NTPDase-1 polyclonal antiserum on ATPase and ADPase hydrolytic activities in recombinant T. cruzi NTPDase-1 and in live trypomastigotes. All tests showed a partial inhibition of Ecto-ATPDase activities and a marked inhibition of trypomastigotes infectivity. Mice infections with Ecto-NTPDase-inhibited trypomastigotes produced lower levels of parasitemia and higher host survival than with non-inhibited control parasites.Conclusions/SignificanceOur results suggest that Ecto-ATPDases act as facilitators of infection and virulence in vitro and in vivo and emerge as target candidates in chemotherapy of Chagas disease.
Partial Text: Trypanosoma cruzi is the etiologic agent of Chagas disease, an endemic zoonosis present in some countries of South and Central Americas. WHO estimates suggested that 100 million people remain at risk of acquiring this infection . There are no vaccines or effective treatment for this disease, especially in the chronic phase . Many compounds are potential candidates to be used in the treatment for Chagas disease, such as TAK-187, D0870, albaconazole and allopurinol . In spite of these, there is a clear necessity to develop new drugs and strategies for the control and treatment of Chagas disease . From this point of view, virulence biomolecules, in particular those secreted or ecto-localized at the parasite’s plasma membrane seem to be good targets. The concentrations of extra cellular nucleotides and their derivative molecules, such as adenosine and inosine are linked to ecto-nucleotidase activities of cells ,. The role of ecto-nucleotidases as the major biomolecules involved in the control of purinergic signaling were demonstrated in various models, such as the dominant role of CD39 in the modulation of inflammation and immune response in the Langerhans cells  and in cardioprotection and protective responses to hypoxia/ischemia in murine model ,. ATP has been previously demonstrated as a “danger” extracellular signal induced by pathogen infection or injury, and it is able to trigger different cellular events such as proliferation, differentiation and chemotaxis, release of cytokines or lysosomal constituents, and generation of reactive oxygen or nitrogen species . Some authors believe that a high ecto-ATPase activity of pathogen is an adaptive parasitic behavior that made these organisms more virulent because they interfere with extracellular ATP signals –.
The present work shows evidence that continuing cultivation of trypomastigotes in VERO cells caused an infectivity decrease during cultivation and that only a small fraction of parasites was able to infect VERO cells and conclude another cellular cycle at the third to fourth passage. Parasites (trypomastigotes) that completed this passage presented 2.5-fold higher levels of ecto-ATPase activities when compared with amastigote-like parasites that could not penetrate into VERO cells. These data reinforce that a high ecto-ATPase activity is important for infectivity and suggest that the absence of host factors leads to loss of infectivity factors from T. cruzi. Similar suggestion was made by Bêrredo-Pinho et al.  when the ecto-ATPase activity was assayed on promastigotes from Leishmania amazonensis and showed that higher passages in acellular medium lead to low ecto-ATPase levels and avirulence . However, as we have used a single strain (Y) in our experiments, verification of correlation between infectivity and Ecto-ATPDase activity in other strains may provide further support to our hypothesis.