Abstract
Here, we investigated the role of EhVps32 protein (a member of the endosomal-sorting complex required for transport) in endocytosis of Entamoeba histolytica, a professional phagocyte. Confocal microscopy, TEM and cell fractionation revealed EhVps32 in cytoplasmic vesicles and also located adjacent to the plasma membrane. Between 5 to 30 min of phagocytosis, EhVps32 was detected on some erythrocytes-containing phagosomes of acidic nature, and at 60 min it returned to cytoplasmic vesicles and also appeared adjacent to the plasma membrane. TEM images revealed it in membranous structures in the vicinity of ingested erythrocytes. EhVps32, EhADH (an ALIX family member), Gal/GalNac lectin and actin co-localized in the phagocytic cup and in some erythrocytes-containing phagosomes, but EhVps32 was scarcely detected in late phagosomes. During dextran uptake, EhVps32, EhADH and Gal/GalNac lectin, but not actin, co-localized in pinosomes. EhVps32 recombinant protein formed oligomers composed by rings and filaments. Antibodies against EhVps32 monomers stained cytoplasmic vesicles but not erythrocytes-containing phagosomes, suggesting that in vivo oligomers are formed on phagosome membranes. The involvement of EhVps32 in phagocytosis was further study in pNeoEhvps32-HA-transfected trophozoites, which augmented almost twice their rate of erythrophagocytosis as well as the membranous concentric arrays built by filaments, spirals and tunnel-like structures. Some of these structures apparently connected phagosomes with the phagocytic cup. In concordance, the EhVps32-silenced G3 trophozoites ingested 80% less erythrocytes than the G3 strain. Our results suggest that EhVps32 participates in E. histolytica phagocytosis and pinocytosis. It forms oligomers on erythrocytes-containing phagosomes, probably as a part of the scission machinery involved in membrane invagination and intraluminal vesicles formation.
Highlights
Entamoeba histolytica is the protozoan responsible for human amoebiasis, considered the third cause of death in the world due to parasitic diseases [1]
Trophozoites of E. histolytica represent an excellent model to study endosomal-sorting complex required for transport components due to their high endocytic activity and vesicle trafficking
We discovered the presence of membranous concentric helicoidally and tunnel-like structures constituted by EhVps32 and EhADH that may have a dynamic role in membrane remodeling and in the generation of intraluminal vesicles in the phagosomes
Summary
Entamoeba histolytica is the protozoan responsible for human amoebiasis, considered the third cause of death in the world due to parasitic diseases [1]. Phagocytosis is a key factor in the parasite virulence and several proteins involved in this event have been already unveiled [2,3,4,5,6,7,8,9], among them the Gal/GalNac lectin [10], EhC2PK, EhCaBP1, EhAK1 [4,11,12] and the EhCPADH complex, formed by a protease (EhCP112) and an adhesin (EhADH) [2], which is a member of the ALIX (apoptosis-linked gene 2-interacting protein X) family [13]. EhADH interacts with EhVps32 [16], a protein described in mammals as a member of the endosomal sorting complex required for transport (ESCRT). ESCRT participates in a number of cellular events such as cell division and autophagy, among others [18,19,20]
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