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

  • V. ENNES-VIDAL (a1), R. O. S. CASTRO (a2), C. BRITTO (a1), H. BARRABIN (a3), C. M. D'AVILA-LEVY (a1) and O. C. MOREIRA (a1) (a2)...


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.


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*Corresponding author: Instituto Oswaldo Cruz/FIOCRUZ, CEP: 21040-360, Rio de Janeiro, RJ, Brazil. Tel: +55 21 3865 8153. Fax: +55 21 2590-3495. E-mail:


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Barral, A., Pedral-Sampaio, D., Grimaldi Junior, G., Momen, H., McMahon-Pratt, D., Ribeiro de Jesus, A., Almeida, R., Badaro, M., Barral-Neto, M., Carvalho, E. M. and Johnson, W. D. Jr. (1991). Leishmaniasis in Bahia, Brazil: evidence that Leishmania amazonensis produces a wide spectrum of clinical disease. The American Journal of Tropical Medicine and Hygiene 44, 536546.
Barros, F. S., De Menezes, L. F., Pinheiro, A. A., Silva, E. F., Lopes, A. H., De Souza, W. and Meyer-Fernandes, J. R. (2000). Ectonucleotide diphosphohydrolase activities in Entamoeba histolytica. Archives of Biochemistry and Biophysics 375, 304314.
Bernardes, C. F., Meyer-Fernandes, J. R., Saad-Nehme, J., Vannier-Santos, M. A., Peres-Sampaio, C. E. and Vercesi, A. E. (2000). Effects of 4,49-diisothyocyanatostilbene-2,29-disulfonic acid on Trypanosoma cruzi proliferation and Ca(2+) homeostasis. International Journal of Biochemistry and Cell Biology 32, 519527.
Berrêdo-Pinho, M., Peres-Sampaio, C. E., Chrispim, P. P., Belmont-Firpo, R., Lemos, A. P., Martiny, A., Vannier-Santos, M. A. and Meyer-Fernandes, J. R. (2001). A Mg-dependent ecto-ATPase in Leishmania amazonensis and its possible role in adenosine acquisition and virulence. Archives of Biochemistry and Biophysics 391, 1624.
Bisaggio, D. F., Peres-Sampaio, C. E., Meyer-Fernandes, J. R. and Souto-Padron, T. (2003). Ecto-ATPase activity on the surface of Trypanosoma cruzi and its possible role in the parasite-host cell interaction. Parasitology Research 91, 273282.
Collopy-Junior, I., Esteves, F. F., Nimrichter, L., Rodrigues, M. L., Alviano, C. S. and Meyer-Fernandes, J. R. (2006). An ectophosphatase activity in Cryptococcus neoformans. FEMS Yeast Research 6, 10101017.
Convit, J., Pinardi, M. E. and Rondón, A. J. (1972). Diffuse cutaneous leishmaniasis: a disease due to an immunological defect in the host. Transactions of the Royal Society of Tropical Medicine and Hygiene 66, 603610.
De Jesus, J. B., de Sá Pinheiro, A. A., Lopes, A. H. and Meyer-Fernandes, J. R. (2002). An ectonucleotide ATP-diphosphohydrolase activity in Trichomonas vaginalis stimulated by galactose and its possible role in virulence. Zeitschrift der Naturforschung C 57, 890896.
De Pierre, J. W. and Karnovsky, M. L. (1973). Plasma membranes of mammalian cells: a review of methods for their characterization and isolation. Journal of Cell Biology 56, 275303.
De Pierre, J. W. and Karnovsky, M. L. (1974). Ecto-enzymes of the guinea pig polymorphonuclear leukocyte. Evidence for an ecto-adenosine monophosphatase, adenosine triphosphatase, and p-nitrophenyl phosphates. Journal of Biological Chemistry 249, 71117120.
DePamphilis, M. L. and Cleland, W. W. (1973). Preparation and properties of chromium(III)-nucleotide complexes for use in the study of enzyme mechanisms. Biochemistry 12, 37143724.
De Souza, M. C., De Assis, E. A., Gomes, R. S., Marques da Silva, E. A., Melo, M. N., Fietto, J. L. and Afonso, L. C. (2010). The influence of ecto-nucleotidases on Leishmania amazonensis infection and immune response in C57B/6 mice. Acta Tropica 115, 262269.
Dutra, P. M., Dias, F. A., Santos, M. A., Rodrigues, C. O., Romeiro, A., Attias, M., De Souza, W., Lopes, A. H. and Meyer-Fernandes, J. R. (2001). Secreted phosphatase activities in trypanosomatid parasites of plants modulated by platelet-activating factor. Phytopathology 91, 408414.
Einholm, A. P., Vilsen, B. and Andersen, J. P. (2004). Importance of transmembrane segment M1 of the sarcoplasmic reticulum Ca2+-ATPase in Ca2+ occlusion and phosphoenzyme processing. Journal of Biological Chemistry 279, 1588815896.
Furuya, T., Zhong, L., Meyer-Fernandes, J. R., Lu, H. G., Moreno, S. N. and Docampo, R. (1998). Ecto-protein tyrosine phosphatase activity in Trypanosoma cruzi infective stages. Molecular and Biochemical Parasitology 92, 339348.
Hopfe, M. and Henrich, B. (2008). OppA, the ecto-ATPase of Mycoplasma hominis induces ATP release and cell death in HeLa cells. BMC Microbiology 4, 855.
Jesus, J. B., Lopes, A. H. and Meyer-Fernandes, J. R. (2002). Characterization of an ecto-ATPase of Tritrichomonas foetus. Veterinary Parasitology 103, 2942.
Kiffer-Moreira, T., Fernandes Sampaio, M. E., Alviano, D. S., Axelband, F., Cesar, G. V., Cosentino-Gomes, D., Rodrigues, M. L., Nimrichter, L., Vieyra, A., Alviano, C. S. and Meyer-Fernandes, J. R. (2010). Biochemical characterization of an ecto-ATP diphosphohydrolase activity in Candida parapsilosis and its possible role in adenosine acquisition and pathogenesis. FEMS Yeast Research 10, 735746.
la Sala, A., Ferrari, D., Di Virgilio, F., Idzko, M., Norgauer, J. and Girolomoni, G. (2003). Alerting and tuning the immune response by extracellular nucleotides. Journal of Leukocyte Biology 73, 339343.
Linnertz, H., Thonges, D. and Schoner, W. (1995). Na+/K(+)-ATPase with a blocked E1ATP site still allows backdoor phosphorylation of the E2ATP site. European Journal of Biochemistry 232, 420424.
Menna Barreto, R. F. S., Corrêa, J. R., Pinto, A. V., Soares, M. J. and de Castro, S. L. (2007). Mitochondrial disruption and DNA fragmentation in Trypanosoma cruzi induced by naphthoimidazoles synthesized from beta-lapachone. Parasitology Research 101, 895905.
Meyer-Fernandes, J. R. (2002). Ecto-ATPases in protozoa parasites: looking for a function. Parasitology International 51, 299303.
Mizumoto, N., Kumamoto, T., Robson, S. C., Sévigny, J., Matsue, H., Enjyoji, K. and Takashima, A. (2002). CD39 is the dominant Langerhans cell-associated ecto-NTPDase: modulatory roles in inflammation and immune responsiveness. Nature Medicine 8, 358365.
Moreira, O. C., Rios, P. F. and Barrabin, H. (2005). Inhibition of plasma membrane Ca(2+)-ATPase by CrATP. LaATP but not CrATP stabilizes the Ca(2+)-occluded state. Biochimica et Biophysica Acta 1708, 411419.
Moreira, O. C., Rios, P. F., Esteves, F. F., Meyer-Fernandes, J. R. and Barrabin, H. (2009). CrATP as a new inhibitor of ecto-ATPases of trypanosomatids. Parasitology 136, 544.
Mosmann, T. (1983). Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of Immunological Methods 65, 5563.
Nakaar, V., Beckers, C. J., Polotsky, V. and Joiner, K. A. (1998). Basis for substrate specificity of the Toxoplasma gondii nucleoside triphosphate hydrolase. Molecular and Biochemical Parasitology 97, 209220.
Pauls, H., BredenBröcker, B. and Schoner, W. (1980). Inactivation of (Na+, K+)-ATPase by Chromium (III) complexes of nucleotide triphosphate. European Journal of Biochemistry 109, 523533.
Peres-Sampaio, C. E., de Almeida-Amaral, E. E., Giarola, N. L. and Meyer-Fernandes, J. R. (2008). Leishmania amazonensis: effects of heat shock on ecto-ATPase activity. Experimental Parasitology 119, 135143.
Plesner, L. (1995). Ecto-ATPases: identities and functions. International Review of Cytology 158, 141142.
Pinheiro, C. M., Martins-Duarte, E. S., Ferraro, R. B., Fonseca De Souza, A. L., Gomes, M. T., Lopes, A. H., Vannier-Santos, M. A., Santos, A. L. and Meyer-Fernandes, J. R. (2006). Leishmania amazonensis: biological and biochemical characterization of ecto-nucleoside triphosphate diphosphohydrolase activities. Experimental Parasitology 114, 1625.
Sansom, F. M., Riedmaier, P., Newton, H. J., Dunstone, M. A., Müller, C. E., Stephan, H., Byres, E., Beddoe, T., Rossjohn, J., Cowan, P. J., d'Apice, A. J., Robson, S. C. and Hartland, E. L. (2008). Enzymatic properties of an ecto-nucleoside triphosphate diphosphohydrolase from Legionella pneumophila: substrate specificity and requirement for virulence. Journal of Biological Chemistry 283, 1290912918.
Santos, R. F., Pôssa, M. A., Bastos, M. S., Guedes, P. M., Almeida, M. R., Demarco, R., Verjovsky-Almeida, S., Bahia, M. T. and Fietto, J. L. (2009). Influence of Ecto-Nucleoside Triphosphate Diphosphohydrolase Activity on Trypanosoma cruzi Infectivity and Virulence. PloS Neglected Tropical Diseases 3:e387.
Souza, V. L., Veras, P. S., Welby-Borges, M., Silva, T. M., Leite, B. R., Ferraro, R. B., Meyer-Fernandes, J. R., Barral, A., Costa, J. M. and Freitas, L. A. (2011). Immune and inflammatory responses to Leishmania amazonensis isolated from different clinical forms of human leishmaniasis in CBA mice. Memorias Instituto Oswaldo Cruz 106, 2331.
Vilsen, B. and Andersen, J. P. (1992 a). CrATP-induced Ca2+ occlusion in mutants of the Ca2+-ATPase of sarcoplasmic reticulum. Journal of Biological Chemistry 267, 2573925743.
Vilsen, B. and Andersen, J. P. (1992 b). Interdependence of Ca2+ occlusion sites in the unphosphorylated sarcoplasmic reticulum Ca2+-ATPase Complex with CrATP. Journal of Biological Chemistry 267, 35393550.
Walseth, T. F. and Johnson, R. A. (1979). The enzymatic preparation of alpha-[32P]nucleosides triphosphates, cyclic [32P]AMP and cyclic [32P]GMP. Biochimica et Biophysica Acta 562, 1131.
World Health Organization (2009). Leishmaniasis: magnitude of the problem. World Health Organization. (July 2009, last date accessed).
Zimmermann, H. (1999). Two novel families of ectonucleotidases: molecular structures, catalytic properties and a search for a function. Trends in Pharmacological Sciences 20, 231236.



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