RESUMEN
Schistosomiasis, caused by Schistosoma mansoni trematode worm, affects more than 1.5 million people in Brazil. The current treatment consists in the administration of Praziquantel, the only medicine used for treatment for more than 40 years. Some of the limitations of this drug consist in its inactivity against schistosomula and parasite eggs, the appearance of resistant strains and non-prevention against reinfection. Thus, the objective of this study was to evaluate the effect of immunization with recombinant functional enzymes of the purine salvage pathway of S. mansoni, Nucleoside Diphosphate Kinase (NDPK) and Adenylosuccinate Lyase (ADSL), to evaluate the host immune response, as well as the parasite load after vaccination. For this, Balb/c mice were divided into 5 groups: control (uninfected and untreated), non-immunized/infected, NDPK infected, ADSL infected, and NDPK + ADSL infected. Immunized groups received three enzyme dosages, with a 15-day interval between each dose, and after 15 days of the last application the animals were infected with 80 cercariae of S. mansoni. On the 47th day after the infection, fecal eggs were counted and, on the 48th day after the infection, the evaluation of leukocyte response, parasite load, antibody production, cytokines quantification, and histopathological analysis were performed. The results showed that immunizations with NDPK, ADSL or NDPK + ADSL promoted a discreet reduction in eosinophil counts in lavage of peritoneal cavity. All immunized animals showed increased production and secretion of IgG1, IgG2a, and IgE antibodies. Increased production of IL-4 was observed in the group immunized with the combination of both enzymes (NDPK + ADSL). In addition, in all immunized groups there were reductions in egg counts in the liver and intestine, such as reductions in liver granulomas. Thus, we suggest that immunizations with these enzymes could contribute to the reduction of schistosomiasis transmission, besides being important in immunopathogenesis control of the disease.
Asunto(s)
Adenilosuccinato Liasa/inmunología , Antígenos Helmínticos/inmunología , Nucleósido-Difosfato Quinasa/inmunología , Schistosoma mansoni/enzimología , Schistosoma mansoni/inmunología , Esquistosomiasis mansoni/inmunología , Esquistosomiasis mansoni/parasitología , Animales , Antígenos Helmínticos/administración & dosificación , Biomarcadores , Citocinas/sangre , Eosinófilos , Femenino , Inmunización , Esquemas de Inmunización , Recuento de Leucocitos , Hígado/metabolismo , Hígado/parasitología , Hígado/patología , Ratones , Carga de Parásitos , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/inmunología , Esquistosomiasis mansoni/patología , Esquistosomiasis mansoni/prevención & controlRESUMEN
Nucleoside diphosphate kinase (NDPK) is a key enzyme in the control of cellular concentrations of nucleoside triphosphates, and has been shown to play important roles in many cellular processes. In this work we investigated the subcellular localization of the canonical NDPK1 from Trypanosoma cruzi (TcNDPK1), the etiological agent Chagas's Disease, and evaluated the effect of adding an additional weak protein-protein interaction domain from the green fluorescent protein (GFP). Immunofluorescence microscopy revealed that the enzyme from wild-type and TcNDPK1 overexpressing parasites has a cytosolic distribution, being the signal more intense around the nucleus. However, when TcNDPK1 was fused with dimeric GFP it relocalizes in non-membrane bounded granules also located adjacent to the nucleus. In addition, these granular structures were dependent on the quaternary structure of TcNDPK1 and GFP since mutations in residues involved in their oligomerization dramatically decrease the amount of granules. This phenomenon seems to be specific for TcNDPK1 since other cytosolic hexameric enzyme from T. cruzi, such as the NADP(+)-linked glutamate dehydrogenase, was not affected by the fusion with GFP. In addition, in parasites without GFP fusions granules could be observed in a subpopulation of epimastigotes under metacyclogenesis and metacyclic trypomastigotes. Organization into higher protein arrangements appears to be a singular feature of canonical NDPKs; however the physiological function of such structures requires further investigation.
Asunto(s)
Nucleósido-Difosfato Quinasa/metabolismo , Trypanosoma cruzi/enzimología , Animales , Anticuerpos Antiprotozoarios/inmunología , Western Blotting , Gránulos Citoplasmáticos/química , Citosol/enzimología , Digitonina , Regulación Enzimológica de la Expresión Génica , Proteínas Fluorescentes Verdes , Indicadores y Reactivos , Sustancias Luminiscentes , Ratones , Microscopía Fluorescente , Nucleósido-Difosfato Quinasa/química , Nucleósido-Difosfato Quinasa/inmunología , Estructura Cuaternaria de ProteínaRESUMEN
A soluble nucleoside diphosphate kinase (NDP kinase) was purified and characterized in epimastigote forms of Trypanosoma cruzi. The enzyme was purified by affinity chromatography on Blue-agarose and Q-Sepharose columns and by FPLC on a Superose 12 column. A membrane-associated NDP kinase was identified which accounts for 30% of total enzymatic activity. Western blot analysis of the soluble NDP kinase revealed a 16.5-kDa monomer recognized by polyclonal antibodies to NDP kinase from Dictyostelium discoideum, Candida albicans or human. Most of the T. cruzi NDP kinase is found in the cell as a hexamer composed of 16.5-kDa monomers. The Km values of the enzyme for ATP, GDP and dTDP were 0.2 +/- 0.008 mM, 0.125 +/- 0.012 mM and 0.4 +/- 0.009 mM, respectively. The parasite enzyme was stable, remained active at 65 degrees C and was found to tolerate up to 2.5 M urea. The 16.5-kDa subunit was phosphorylated with [gamma-32P]ATP or thiophosphorylated with [35S]GTP gamma S. The incubation of the 32P-labelled phosphoenzyme with unlabelled nucleoside 5'-diphosphates resulted in the formation of 32P-labelled nucleoside 5'-triphosphates without strict base specificity, indicating that the reaction mechanism of the T. cruzi enzyme is the same as reported for other NDP kinases. When the phosphoenzyme was incubated with a mixture of nucleoside 5'-diphosphates, GTP was preferentially formed.