CONICET | Buscador de Institutos y Recursos Humanos

Autophagy in Echinococcus: searching for its physiological and pharmacological triggersCumino Andrea C., Loos Julia A., Nicolao M. Celeste, Dávila Valeria A., Caparrós Pedro A., Rodriguez R. ChristianFacultad de Ciencias Exactas y Naturales, Universidad Nacional de Mar del Plata, Conicet, Email: acumino@gmail.comAutophagy is a fundamental catabolic pathway conserved from yeast to mammals, but which remains unknown in cestoda. The best-characterized functions of autophagy include maintenance of cellular quality control and provision of an alternate source of energy during starvation, a physiological condition of Echinococcus granulosus larval stages (permanent nutritional dependence on the hosts and low cellular energy generation in the parasite life cycle -Zheng et al., 2013), which produces cystic echinococcosis disease or hydatidosis in humans. In this project, the autophagy has been cellular and molecularly analyzed under basal conditions in the larval stages (metacestode and protoscolex forms) as well as under the pharmacological induction of rapamycin (positive control) and metformin, arsenic trioxide, bortezomib and octreotide (study cases). Metacestode sensitivity to rapamycin (Rm) and metformin (Met) as well as TORC1 and AMPK expression in protoscoleces and metacestodes have been demonstrated (Cumino et al., 2010; Loos & Cumino 2015). Genes coding for key autophagy-related proteins were also identified in the Echinococcus genome. These genes were involved in autophagosome formation and transcriptional over-expression of Eg-atg5, Eg-atg6, Eg-atg8, Eg-atg12, Eg-atg16 and Eg-atg18 in the drug treatments. A single FoxO transcription factor was identified in E. granulosus (Eg-FoxO), the first transcription factor that is necessary and sufficient to induce autophagy in the larvae of Drosophila melanogaster and Caenorhabditis elegans (Klionsky et al., 2012). Eg-FoxOs share similar DNA binding specificity, with the core binding motif being defined as TTGTTTAC (Furuyama et al., 2000). We thus analyzed Eg-FoxO transcriptional target candidates predicted by the 1 kb upstream survey of each Echinococcus atg gene and we detected that the conserved binding motif described for FoxO-activated genes was present in upstream sequences from Eg-atg8 and Eg-atg12 putative promoters. Thus, Echinococcus autophagy could be regulated by non-transcriptional inhibition through TOR and by transcription-dependent up-regulation via FoxO-like protein.The presence of Eg-Atg8 punctate images and the high gene expression level of atg genes from control parasites suggest the occurrence of basal autophagy in the larval stages (Loos et al., 2014). Our experiments also showed an Eg-Atg8 level increment proportional to drug concentration, and a punctate pattern was detected in the syncytial tegument and in several parenchymal cells of the soma in treated parasites. Another important observation was the presence of Eg-Atg8 punctate dot formations in the terminal vesicle system and vesicularised protoscoleces, which suggests that autophagy has a particular role in development towards vesicular differentiation. On the other hand, SEM/EDAX images and high Eg-Atg8 polypeptide levels within the free cytoplasmic matrix of biomineralized cells as the calcareous corpuscles were observed, which owe their origin and cellular activity to the occurrence of autophagic events in cestode tissues (Loos et al., 2014) leading to reconsideration of the theory proposed by McCullough and Fairweather (1987) on corpuscle autophagic development. The presence of multilamellar structures and focal deposition of excessive amounts of calcium into calcareous corpuscles could be a reason for autophagy after endoplasmic reticulum stress, being different from those cells deprived of nutrients (Ogata et al., 2006). In addittion, ultrastructural studies showed that Rm treated parasites had an isolation membrane, autophagosomes and autolysosomes, all of which evidenced the autophagic flux. As the endoplasmic reticulum is frequently observed in close proximity to autophagosomes, it has been proposed as the source of membrane for the autophagosome or else as the plataform.Actually we are interested in describing the lipophagy and glycophagy as homeostatic processes that could occur in the parasite, which could also be pharmacologically inferred by Met. Since starvation and acute lipid stimulus increase autophagic sequestration of lipid droplets (LDs), we find that providing a physiological fatty acid such as oleic acid (OA), the number and total area of LDs detected by bodipy 493/503 (a lipid droplet marker) were significantly higher compared to the control. Given the differences in organelle size between autophagosme-lysosome and LDs, it is likely that autophagosome assembly occurs at the surface of the lipid droplet. In this line evidence, the proximity and colocalization of LDs with Eg-Atg8 were determined by confocal microscopy. In support of this notion, other proteins that upstream regulator of autophagy as Atg5, Atg6 and Atg7 could be analyzed on the LD surface in order to study the lipophagy in the parasite. Additionally, it has been revealed that starvation-induced activation of the transcription factor FoxO modulates LDs by upregulation of lysosomal acid lipase-mediated autophagy via TFEB, other master regulator of autophagy and lysosoma biogenesis also identified in the parasite. Thus, in the following experiment we analyzed the functional activity of FoxO and TFBE in Echinococcus sp. regarding in autophagic degradation of neutral lipids and glycogen. The detection of the autophagic machinery in this parasite represents a basic starting point to unravel the role of autophagy under both physiological and stress conditions which will allow to identificate new strategies for drug discovery against neglected parasitic diseases caused by cestoda.

enviar mensaje