Protein SUMOylation is crucial for phagocytosis inEntamoeba histolyticatrophozoites
Authors: Díaz-Hernández Mitzi , Reyna Rosario Javier , Sotto-Ortega Izaid , García-Rivera Guillermina , Montaño Maricela Sarita , Betanzos Abigail , Orozco Esther 
Published: February 2021

AbstractDuring phagocytosis, a key event in the virulence of the protozoanEntamoeba 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 inE. histolytica. In this paper, we characterized theE. histolytica SUMOgene 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 ΨKXE/D binding motif, both involved in target protein contact. Additionally,E. histolyticagenome possesses the enzymes belonging to the SUMOylation-deSUMOylation machineries. Confocal microscopy assays, using α−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. InEhSUMOknocked down trophozoites, the bizarre membranous structures disappeared, and EhSUMO interaction with EhADH and EhVps32 diminished. Our results evidenced the presence of aSUMOgene inE. histolyticaand the SUMOylation relevance during phagocytosis.Author’s AbstractPhagocytosis is one of the main functions thatEntamoeba histolyiticatrophozoites 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 inE. histolyticanor 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 intronlessbona fide EhSUMOgene encoding for a predicted 12.6 kDa protein that is actively involved in phagocytosis. Silencing of theEhSUMOgene 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.