Fuctional characterization and expression of Echinococcus granulosus Calcineurin

NICOLAO, MARÍA CELESTE; LOOS, JULIA A.; CUMINO, ANDREA C.

Guayaquil

Congreso; XXI Congreso Latinoamericano de Parasitología (FLAP); 2013

Sociedad Latinoamericana de Medicina Tropical y Parasitología

Introduction. Calcineurin is a Ca2+/calmodulin-activated serine/threonine-phosphatase (PP2B) with critical role in the coupling of calcium signals to cellular responses. It is a heterodimeric protein consisting of a catalytic (CaN-A) and a regulatory calcium-binding subunit (CaN-B), and is inhibited by cyclophilin-cyclosporin A and FKBP-FK506 complexes. Its inactivation blocks different cellular processes resulting in cell death. In this study we focused on the functional characterization of the catalytic subunit of calcineurin in the Echinococcus granulosus larval stages.Materials and methods. E. granulosus can-A, can-B and chp genes were identified in protoscoleces and cysts by RT-PCR and sequencing (GenBank KC146906, HQ454284 and KC146907, respectively). Bioinformatic analysis revealed that their predicted proteins possess all residues implicated in the binding of pharmacological ligands. Also, we performed in toto immunofluorescence assays in protoscoleces to determine CaN-A localization and its association in exocytic processes using the FM4-64 fluorescent dye. We determined the CaN-A PP2B activity in basal conditions, with activators (Cl2Ni) and inhibitors (CsA) from protoscolex and metacestode protein extracts in presence of imidazol and phosphatases inhibitors. Results. We demonstrated in protoscoleces that in low calcium conditions (BAPTA/ClLa) Eg-can-B and Eg-chp expressions were down-regulated respect to controls. Eg-CaN-A represents an active protein phosphatase, since PP2B was the 60% of the total serine/threonine phosphatase activity. Eg-CaN-A was mainly immunolocalized in tegument, suckers and excretory bladder. Eg-CaN-A expression was higher in CsA-treated protoscoleces and lower in BAPTA/ClLa-treated protoscoleces compared with controls. Finally, we examined the unloading kinetics of FM4-64 after stimulation with KCl and HCl, demonstrating that the exocytosis was significantly reduced in CsA-treated larvae, in agreement with other invertebrate species.Conclusion. We have carried out a functional analysis of the E. granulosus calcineurin which coordinate multiple calcium-dependent dephosphorylation processes in metazoa. These findings contribute to elucidate conserved elements of signal transduction cascades and define novel drug targets.