IDEHU   05542
INSTITUTO DE ESTUDIOS DE LA INMUNIDAD HUMORAL PROF. RICARDO A. MARGNI
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Cloning and immunological characterization of the cistein proteinase B of L. brasiliensis
Autor/es:
BIVONA, AUGUSTO E.; CERNY, NATACHA; SANCHEZ-ALBERTI, ANDRÉS; MATOS, MARINA; MALCHIODI, EMILIO L.; CAZORLA, SILVIA I.
Lugar:
Ouro Preto
Reunión:
Simposio; Keystone Symposium on (E1) The Innate Immune Response in the Pathogenesis of Infectious Disease; 2013
Institución organizadora:
CNPq ? the National Council for Scientific and Technological Development, Brazil, and FAPEMIG
Resumen:
Leishmaniosis is one of the neglected tropical diseases. Depending on the virulence factor of the parasites and the immune response established by the host, a wide spectrum of diseases can appear. The most prevalent and widespread in South America is cutaneous leishmaiosis caused mainly by Leishmania braziliensis. The parasite contains a cistein proteinase B (CPB), which gene is organized as: pre-region, propeptide, catalytic domain, and C-terminal extension. The exact roles of the CP and its domains in Leishmania pathogenesis have yet to be defined. The aim of this work is to analyse if the CPB or any of its domains, play a role in the modulation of the host immunoresponse elicited by the parasite. From the synthetized gen of the CPB of L. brasiliensis, we synthetized by PCR the sequences of 954, 657 and 297 bp corresponding to the entire CPB and its N- and C-terminal domains, respectively. The expressed proteins in E. coli were purified by affinity chromatography using Ni-NTA resin and refolded by exhaustive dialysis in glycerol-PBS. We evaluated by Western blot the identity of the proteins with serum of Leishmania infected patients. We also find that specific antisera against the whole CBP was able to recognize the two domains. The binding ability of the recombinant CPB on different Pattern Recognition Receptors, using HEK-blue cells was analysed. CPB was able to bind TLR2 in a doses dependent manner. By contrast, TLR9 and TLR8 were not activated by CPB. Peritoneal macrophages from Balb-c mice were cultured with CPB and its domains, observing an increased in the NO production in the presence of the CPB (14.9 µM) that was abolished with denatured protein (6.6 µM) or in protein absence (3.8 µM). Surprisingly, macrophages incubated with N-terminal domains in presence of polimixin B increased NO production (13.3 µM), which was not observed with the C-terminal domains (6.8 µM). These results suggest that macrophages, the major target cells for Leishmania infection will be activated in the production of NO by the CPB and its N-terminal domain. Macrophages activation will be mediated, at least in part, by the interaction of the L.brasiliensis CPB with TLR2.