Atg8 is involved in endosomal and phagosomal acidification in the parasitic protist Entamoeba histolytica.

Autophagy is one of two major bulk protein degradation systems and is conserved throughout eukaryotes. The protozoan Entamoeba histolytica, which is a human intestinal parasite, possesses a restricted set of autophagy-related (Atg) proteins compared with other eukaryotes and thus represents a suitable model organism for studying the minimal essential components and ancestral functions of autophagy. E. histolytica possesses two conjugation systems: Atg8 and Atg5/12, although a gene encoding Atg12 is missing in the genome. Atg8 is considered to be the central and authentic marker of autophagosomes, but recent studies have demonstrated that Atg8 is not exclusively involved in autophagy per se, but other fundamental mechanisms of vesicular traffic. To investigate this question in E. histolytica, we studied on Atg8 during the proliferative stage. Atg8 was constitutively expressed in both laboratory-maintained and recently established clinical isolates and appeared to be lipid-modified in logarithmic growth phase, suggesting a role of Atg8 in non-stress and proliferative conditions. These findings are in contrast to those for Entamoeba invadens, in which autophagy is markedly induced during an early phase of differentiation from the trophozoite into the cyst. The repression of Atg8 gene expression in En. histolytica by antisense small RNA-mediated transcriptional gene silencing resulted in growth retardation, delayed endocytosis and reduced acidification of endosomes and phagosomes. Taken together, these results suggest that Atg8 and the Atg8 conjugation pathway have some roles in the biogenesis of endosomes and phagosomes in this primitive eukaryote.

Analysis of autophagy in the enteric protozoan parasite Entamoeba.

Entamoeba histolytica is the enteric protozoan parasite that causes human amoebiasis. We have previously shown that autophagy is involved in proliferation and differentiation in the related species Entamoeba invadens, which infects reptiles and develops similar clinical manifestations. Because this group of protists possesses only a limited set of genes known to participate in autophagy in other eukaryotes, it potentially represents a useful model for studying the core system of autophagy and provides tools to elucidate the evolution of eukaryotes and their organelles. Here we describe the methods to study autophagy in Entamoeba.

Autophagy during proliferation and encystation in the protozoan parasite Entamoeba invadens.

Autophagy is one of the three systems responsible for the degradation of cytosolic proteins and organelles. Autophagy has been implicated in the stress response to starvation, antigen cross-presentation, the defense against invading bacteria and viruses, differentiation, and development. Saccharomyces cerevisiae Atg8 and its mammalian ortholog, LC3, play an essential role in autophagy. The intestinal protozoan parasite Entamoeba histolytica and a related reptilian species, Entamoeba invadens, possess the Atg8 conjugation system, consisting of Atg8, Atg4, Atg3, and Atg7, but lack the Atg5-to-Atg12 conjugation system. Immunofluorescence imaging revealed that polymorphic Atg8-associated structures emerged in the logarithmic growth phase and decreased in the stationary phase and also increased in the early phase of encystation in E. invadens. Immunoblot analysis showed that the increase in phosphatidylethanolamine-conjugated membrane-associated Atg8 was also accompanied by the emergence of Atg8-associated structures during the proliferation and differentiation mentioned above. Specific inhibitors of class I and III phosphatidylinositol 3-kinases simultaneously inhibited both the growth of trophozoites and autophagy and also both encystation and autophagy in E. invadens. These results suggest that the core machinery for autophagy is conserved and plays an important role during proliferation and differentiation in Entamoeba.

Entamoeba histolytica: identification of EhGPCR-1, a novel putative G protein-coupled receptor that binds to EhRabB.

EhRabB is an Entamoeba histolytica protein involved in phagocytosis. However, proteins that regulate the EhRabB activity are unknown. Here, we report the identification of a putative G protein-coupled receptor of E. histolytica (EhGPCR-1) that binds to EhRabB. By two-hybrid screening, we found a 372-bp cDNA fragment that encodes the C-terminus of EhGPCR-1. The cloning and sequence of the full-length cDNA revealed that it predicts a polypeptide with two tyrosine-based sorting signals for endocytosis and seven transmembranal domains. These results suggest that EhGPCR-1 could be a GPCR involved in phagocytosis. EhGPCR-1 could be a member of the Rhodopsin family, characterized by a short N-terminus without cysteine residues.