Autophagy regulates survival through transcription dependent mechanisms in Echinococcus granulosus

LOOS JULIA A; NICOLAO MA. CELESTE; CUMINO ANDREA C

Distrito Federal

Congreso; 13th Congreso International of Parasitology; 2014

Sociedad Internacional de Parasitología

Autophagy regulates survival through transcription dependent mechanisms in Echinococcus granulosus Loos Julia A.1*, Nicolao M. Celeste1*, Cumino Andrea C1. 1Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Mar del Plata, Funes 3350, MDP, Argentina. CONICET. *Authors contributed equally BACKGROUND: Autophagy participates in cell survival and intervenes actively in the larval development in helminths. TORC1 pharmacological inhibition, through rapamycin or metformin (agonist drug of AMP activated protein kinase -AMPK-), induce autophagy. AMPK, an eukaryotic metabolism master regulator, is activated when intracellular ATP levels fall, regulating growth, development and autophagy. In this work, we studied how the autophagy that occurs in Echinococcus granulosus larval stages is induced by AMPK activation and different Ca2+ mobilizing agents. METHODS: Protoscoleces from hydatid cysts of infected cattle and metacestodes from CF1 mice with secondary hydatidosis were maintained in in vitro culture. SEM and TEM were used to investigate autophagosome progress. Expression of autophagy (atg1-atg18), ER stress and FoxO genes were analyzed by RT-PCR/qPCR from drug-treated parasites (metformin, cyclosporine-A, TFP, BAPTA and bortezomib). In toto immunofluorescence and immunoassays to determine the Eg-Atg8/LC3 expression and localization were performed. Eg-AMPK-α (total and AMPK-PThr176) and Eg-calcineurin-A expression by confocal microscope were evaluated. Finally, a single CREB homologue was identified. Data were compared using the student?s t test. RESULTS: Eg-AMPK is activated in basal condition and in metformin treatment, inducing autophagy in the cestode. Metformin treated parasites demonstrated autophagic structures from ultrastructural images, upregulated Eg-Atg8/LC3II expression and Eg-atg6-8-12-16-18 transcription, reduced glycogen level and Eg-pepck and Eg-g6pase (genes coordinated by CREB via) in both larval stages. Eg-Atg8/LC3, detected in the tegument, excretory system and calcareous corpuscles, co-localized with Eg-AMPK-α in these calcium-stored cells. CONCLUSIONS: Echinococcus autophagy could be regulated by non-transcriptional inhibition through TOR, transcription-dependent up-regulation via FoxO and AMPK and calcineurin via CREB to promote survival in Echinococcus lifespan.