Occurrence of autophagy in basal stages from Echinococcus protoscoleces

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

Mar del Plata

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

Sociedad Argentina de Protozoología

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 among other mechanisms that degradative pathway. As autophagosomes (double-membrane vesicles) engulf a portion of cytoplasm for subsequent degradation and recycling, autophagy is generally thought to be a nonselective degradation system. Numerous genes that are essential for autophagy have been identified in higher eukaryotes, including C. elegans, Drosophila, mammals and plants. Previous rapamycin assays in our laboratory suggested that autophagy could explain cell death observed as a possible mechanism in pharmacological treatment of 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 retain all residues involved in the lipid-protein and protein-protein interactions in autophagosome structures, so we analyzed the key gene expression 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 accumulation of acidic vesicular organelles using acridine orange. The expression of ATG8 protein (homologs to LC3 in mammals), a reliable marker 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.