Occurrence of autophagy in basal stages from Echinococcus protoscoleces.

CAPARROS PEDRO A; LOOS, JULIA A.; NICOLAO MA. CELESTE; DENEGRI GUILLERMO M.; CUMINO ANDREA C.

Mar del Plata

Conferencia; IX Congreso de la Sociedad Argentina de Protozoología y Enfermedades Parasitarias; 2011

As an evolutionarily conserved and finely regulated process, autophagy plays a very important role in various biological events during the whole life of eukaryotes, including the cell survival or death decision. To preserve the maintenance of cellular homeostasis, cells utilize that degradative mechanism, whereas bulk cytoplasm is non-specifically sequestered within double‑membrane vesicles for subsequent degradation and recycling. Numerous genes that are essential for autophagy have been identified in higher eukaryotes, including C. elegans, Drosophila, mammals and plants. Previous reports in our laboratory have verified that, in a dose-dependent manner, rapalogs increased the lysosome-dependent cellular catabolic mechanism by accumulating acidic organelles in the cytoplasm, suggesting induction of autophagy as cellular death mechanism in pharmacological treatment protoscoleces. In this work were evidenced key autophagic related genes from platyhelminths, and others essential requirements for autophagosome formation in Echinococcus sp., pathogen causing cystic echinococcosis: the presence of proteins involved in the vesicle nucleation (phosphatidylinositol 3-kinase and Atg6); two enzymatic complexes (conjugation system -Atg5 and Atg12- and the lipidation system -Atg8, Atg3 and Atg7-) which mediate expansion and completion of the vesicles and the regulation system of autophagic induction by Tor kinase. Based on analyses of genomic data using tBLASTn from Echinococcus database, we have identified orthologs to all the members of these complexes in E. granulosus, since in principle these proteins retains all residues involved in the lipid-protein interactions in autophagosome structures, and we analyzed the expression of the key genes of these pathways. Also, we searched the EST database and found two isoforms of atg8 (I and II). We found that the transcription levels of two indicator genes Eg-atg8 and Eg-atg12 increased with the rapamycin treatment of the larval stage, and studies of confocal microscopy demonstrated the accumulation of acidic vesicular organelles using acridine orange. The expression of ATG8 protein (homologs to LC3 in mammals), a reliable marker of induction and progression of autophagy, was revealed with a heterologous antibody and immunohistochemistry localized in protoscoleces, indicating in addition, the formation of multimeric complexes. These findings suggest that the basal autophagy should play an important role in physiology of Echinococcus may be necessary for the maintenance of normal hipometabolism of parasitic larval stages.