Characterization of the Autophagy Marker Protein Atg8 Reveals Atypical Features of Autophagy in Plasmodium falciparum

Abstract

Conventional autophagy is a lysosome-dependent degradation process that has crucial homeostatic and regulatory functions in eukaryotic organisms. As malaria parasites must dispose a number of self and host cellular contents, we investigated if autophagy in malaria parasites is similar to the conventional autophagy. Genome wide analysis revealed a partial autophagy repertoire in Plasmodium, as homologs for only 15 of the 33 yeast autophagy proteins could be identified, including the autophagy marker Atg8. To gain insights into autophagy in malaria parasites, we investigated Plasmodium falciparum Atg8 (PfAtg8) employing techniques and conditions that are routinely used to study autophagy. Atg8 was similarly expressed and showed punctate localization throughout the parasite in both asexual and sexual stages; it was exclusively found in the pellet fraction as an integral membrane protein, which is in contrast to the yeast or mammalian Atg8 that is distributed among cytosolic and membrane fractions, and suggests for a constitutive autophagy. Starvation, the best known autophagy inducer, decreased PfAtg8 level by almost 3-fold compared to the normally growing parasites. Neither the Atg8-associated puncta nor the Atg8 expression level was significantly altered by treatment of parasites with routinely used autophagy inhibitors (cysteine (E64) and aspartic (pepstatin) protease inhibitors, the kinase inhibitor 3-methyladenine, and the lysosomotropic agent chloroquine), indicating an atypical feature of autophagy. Furthermore, prolonged inhibition of the major food vacuole protease activity by E64 and pepstatin did not cause accumulation of the Atg8-associated puncta in the food vacuole, suggesting that autophagy is primarily not meant for degradative function in malaria parasites. Atg8 showed partial colocalization with the apicoplast; doxycycline treatment, which disrupts apicoplast, did not affect Atg8 localization, suggesting a role, but not exclusive, in apicoplast biogenesis. Collectively, our results reveal several atypical features of autophagy in malaria parasites, which may be largely associated with non-degradative processes.

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

“…1410,1411 Note that although the lysosomal protease inhibitors E64 and pepstatin block lysosomal degradative activity in Plasmodium, these inhibitors do not affect ATG8 levels and associated structures, suggesting a need for alternate methodologies to investigate autophagy in this model system. 1412 In conventional autophagy, the final destination of autophagosomes is their fusion with lysosomes for intracellular degradation. However, T. gondii and certain stages of Plasmodium (insect and hepatic) lack degradative lysosomes, which makes questionable the presence of canonical autophagosomes and a process of autophagy in these parasites.…”

Protein Degradation Systems as Antimalarial Therapeutic Targets

“…Using the ubiquitin-like protein ATG8 (PF3D7_1019900) as a marker, researchers have demonstrated punctate staining and partial co-localization with the apicoplast marker acyl carrier protein (ACP, PF3D7_0208500). In these studies, ART treatment decreased PfATG8 levels as evidenced by a decrease in intensity in immunofluorescence assays and Western blot, while the number of vesicles remained the same [71, 72]. However, treatment with the lysosomal Na + /H + pump inhibitor Bafilomycin A1 or lysosomal cysteine protease inhibitor E64d did not lead to accumulation of ATG8.…”

Virtual Screening and Experimental Validation Identify Novel Inhibitors of the Plasmodium falciparum Atg8–Atg3 Protein–Protein Interaction

“…Autophagy is a catabolic process performed by eukaryotic cells in order to maintain homeostasis and to degrade unwanted or toxic cellular content, such as misfolded proteins . The autophagy pathway has been demonstrated to be important for various functions in yeast and humans, but studies in the past five years have also shown that autophagy is necessary in human pathogens, such as Plasmodium …”

“…Our PTA compounds were designed to interfere with the Atg8–Atg3 protein–protein interaction and thereby prevent the lipidation of Atg8. Only this lipidated form of Atg8 is active: it participates in elongation of the autophagosomal membrane in higher eukaryotes, and is the predominant species in Plasmodium …”