Localização ultraestrutural da Nucleosídeo Trifosfato Dfosfohidrolase 1 (NTPDase 1) antigênica de Leishmania (Viannia) braziliensis

Abstract

An NTPDase activity was previously characterized in L. (V.) braziliensis promastigote forms (Lb). In this work, its localization ultrastructural was obtained by cytochemical techniques, using a JEM-1011 electron microscope. Enzyme activity was found distributed as an electron dense cerium phosphate deposit at the surface of the plasma membrane, at mitochondrial membrane, kinetoplast, flagellar pocket and flagellum of Lb when they were incubated in standard reaction medium with ATP, ADP or GDP, and in the presence of classical ATPase, phosphatase and nucleotidase inhibitors. A polypeptide belonging to the conserved domain B from the potato apyrase, homologue to the domain B from the Lb NTPDase 1 isoform, was then obtained as a 6xHis tag polypeptide (r-potDomain B), which was inoculated in BALB/c mice. Polyclonal anti-r-potDomínio B antibodies, when immobilized on Protein A-Sepharose, immunoprecipitated 43-46% of the ATPase and ADPase activities from the parasite preparation. By Western blots, the immune serum recognized band of 48 kDa in both parasite preparations and immunoprecipitated complex, confirming the NTPDase 1 identity. Serum samples of American cutaneous leishmaniasis (ACL) patients were tested in ELISA using r-potDomain B as coating antigen. The IgG1 (0.077 0.059) or IgG3 (0.020 0.024) antibody reactivity of ACL patients was significantly (P < 0.05) higher when compared to the control (IgG1, 0.047 0.029, cut-off, 0.105; IgG3, 0.010 0.009, cut-off, 0.029), and 9-10 (30-33%) of the 30 patients were seropositive for r-potDomain B. The IgG2 (0.054 0.064; 17% seropositivity, 5/30) or IgG4 (0.077 0.031; 23% seropositivity, 7/30) antibody reactivity of ACL patients was similar to the control (IgG2, 0.050 0.021, cut-off, 0.092; IgG4, 0.059 0.023, cut-off, 0.105). These results suggested that the domain B (r82-121) from the L. (V.) braziliensis NTPDase 1 is potentially involved in the host immune response. In ultrastructural and immunocytochemical study, polyclonal anti-r-potDomain B antibodies identified the NTPDase 1 isoform on the parasite surface and, also, at mitochondrial membrane, kinetoplast, flagellar pocket and flagellum. These results suggested that phosphohydrolytic activity from the parasite NTPDase 1 is possibly associated with nutrient recuperation and/or as a protective mechanism against the host organism under conditions that involve nucleotides. Additionally, polyclonal anti-r-potDomain B antibodies (A) inhibited 22-72% of the ATPase and ADPase activities from the total Lb preparation, and (B) after complement system inactivation, and used in MTT cell proliferation assays, significantly inhibited 42% of the promastigote growth after 24 h of incubation, when compared to the cell growth in medium alone, or in the presence of serum from healthy mouse or rabbit. The domain B (r82-121) from the L. (V.) braziliensis NTPDase 1 is a new therapeutic target. The recombinant r-potDomain B polypeptide and antibodies produced against it could be useful in studies of this parasite protein and American cutaneous leishmaniasis.