Protein SUMOylation is crucial for phagocytosis in Entamoeba histolytica trophozoites

During phagocytosis, a key event in the virulence of the protozoan Entamoeba histolytica, several molecules in concert contact the target, generate pseudopodia, and internalize and digest the ingested prey. Posttranslational modifications provide proteins the timing and signaling to intervene in these processes. SUMOylation is a posttranslational modification that in several systems grants a fine tuning for protein functions, protein interactions and cellular location, but it has not been studied in E. histolytica. In this paper, we characterized the E. histolytica SUMO gene and its product (EhSUMO) and elucidated the EhSUMO 3D-structure. Furthermore, here we studied the relevance of SUMOylation in phagocytosis, particularly in its association with EhADH (an ALIX family protein) and EhVps32 (a protein of the ESCRT-III complex), both involved in phagocytosis. Our results indicated that EhSUMO has an extended N-terminus that differentiates other SUMO from ubiquitin. It also presents the GG residues at the C-terminus and the {Psi}KXE/D binding motif, both involved in target protein contact. Additionally, E. histolytica genome possesses the enzymes belonging to the SUMOylation-deSUMOylation machineries. Confocal microscopy assays, using -EhSUMO antibodies disclosed a remarkable membrane activity with convoluted and changing structures in trophozoites during erythrophagocytosis. SUMOylated proteins appeared in pseudopodia, phagocytic channels, and around the adhered and ingested erythrocytes. Docking analysis predicted interaction of EhSUMO with EhADH, and immunoprecipitation and immunofluorescence assays revealed that the EhADH-EhSUMO association increased during phagocytosis, whereas the EhVps32-EhSUMO interaction appeared stronger since basal conditions. In EhSUMO knocked down trophozoites, the bizarre membranous structures disappeared, and EhSUMO interaction with EhADH and EhVps32 diminished. Our results evidenced the presence of a SUMO gene in E. histolytica and the SUMOylation relevance during phagocytosis. Authors AbstractPhagocytosis is one of the main functions that Entamoeba histolyitica trophozoites carry out during the invasion to the host. Many proteins are involved in this fascinating event, in which the plasmatic membrane undergoes to multiple and speedy changes. Posttraductional modifications activate proteins in the precise time that they must get involved. SUMOylation, that consists in the non-covalent binding of SUMO protein with target molecules, is one of the main changes suffered by proteins in order to enable them to participate in cellular functions. SUMOylation had not been studied in E. histolytica nor in phagocytosis, and our working hypothesis is that this event is deeply engaged in the ingestion of target molecules and cells. The results of this paper prove the presence of an intronless bona fide EhSUMO gene encoding for a predicted 12.6 kDa protein that is actively involved in phagocytosis. Silencing of the EhSUMO gene affected the rate of phagocytosis and interfered with the EhADH and EhVps32 function, two proteins involved in phagocytosis, strongly supporting the importance of SUMOylation in this event.