EhVps23: A Component of ESCRT-I That Participates in Vesicular Trafficking and Phagocytosis of Entamoeba histolytica

Abstract

The endosomal sorting complex required for transport (ESCRT) is formed by ESCRT-0, ESCRT-I, ESCRT-II, ESCRT-III complexes, and accessory proteins. It conducts vesicular trafficking in eukaryotes through the formation of vesicles and membrane fission and fusion events. The trophozoites of Entamoeba histolytica, the protozoan responsible for human amoebiasis, presents an active membrane movement in basal state that increases during phagocytosis and tissue invasion. ESCRT-III complex has a pivotal role during these events, but ESCRT-0, ESCRT-I and ESCRT-II have been poorly studied. Here, we unveiled the E. histolytica ESCRT-I complex and its implication in vesicular trafficking and phagocytosis, as well as the molecular relationships with other phagocytosis-involved molecules. We found a gene encoding for a putative EhVps23 protein with the ubiquitin-binding and Vps23 core domains. In basal state, it was in the plasma membrane, cytoplasmic vesicles and multivesicular bodies, whereas during phagocytosis it was extensively ubiquitinated and detected in phagosomes and connected vesicles. Docking analysis, immunoprecipitation assays and microscopy studies evidenced its interaction with EhUbiquitin, EhADH, EhVps32 proteins, and the lysobisphosphatidic acid phospholipid. The knocking down of the Ehvps23 gene resulted in lower rates of phagocytosis. Our results disclosed the concert of finely regulated molecules and vesicular structures participating in vesicular trafficking-related events with a pivotal role of EhVps23.

EhVps23, an ESCRT-I Member, Is a Key Factor in Secretion, Motility, Phagocytosis and Tissue Invasion by Entamoeba histolytica

“…This protozoan causes human amoebiasis, affecting 50 million ( Bercu et al., 2007 ) and killing 100,000 individuals around the world each year ( World Health Organization, 1997 ). In our efforts to understand the molecular basis of the aggressive mechanism of the trophozoites, we have studied proteins of the endosomal sorting complexes required for transport (ESCRT) ( Avalos-Padilla et al., 2015 ; Avalos-Padilla et al., 2018 ; Galindo et al., 2021 ), and others associated that cooperate in molecular trafficking and virulence events of this parasite ( Lopez-Reyes et al., 2010 ; Bañuelos et al., 2012 ).…”

“…We know that the E. histolytica ESCRT machinery possesses at least 19 genes ( Lopez-Reyes et al., 2010 ), whose products act in several steps of endosomal sorting, vesicular trafficking and phagocytosis ( Lopez-Reyes et al., 2010 ; Bañuelos et al., 2012 ; Avalos-Padilla et al., 2015 ; Avalos-Padilla et al., 2018 ; Galindo et al., 2021 ). So far, we have found that the ESCRT-III complex ( Avalos-Padilla et al., 2015 ; Avalos-Padilla et al., 2018 ) proteins work together and with the EhADH protein, an ALIX family member ( Bañuelos et al., 2012 ), and the EhVps4 ATPase ( Lopez-Reyes et al., 2010 ), the enzyme required to maintain the cycle of binding and release of ESCRT-III proteins from the endosomal membranes.…”

“…So far, we have found that the ESCRT-III complex ( Avalos-Padilla et al., 2015 ; Avalos-Padilla et al., 2018 ) proteins work together and with the EhADH protein, an ALIX family member ( Bañuelos et al., 2012 ), and the EhVps4 ATPase ( Lopez-Reyes et al., 2010 ), the enzyme required to maintain the cycle of binding and release of ESCRT-III proteins from the endosomal membranes. Meanwhile, EhVps23 (ESCRT-I) interacts with EhADH, the lysobisphosphatidic acid (LBPA) and EhUbiquitin ( Galindo et al., 2021 ).…”

“…Yeast Vps23 and mammalian TSG101 proteins form part of the tetrameric conserved ESCRT-I complex in both systems ( Kostelansky et al., 2007 ; Flower et al., 2020 ). In contrast, EhVps23, the E. histolytica orthologue is the unique member of this complex characterized until now in the parasite ( Galindo et al., 2021 ). It is predominantly a cytosolic protein, but when the trophozoites undergo phagocytosis, EhVps23 is ubiquitinated and appears close to the erythrocytes and in the endosomes, phagosomes and multivesicular bodies (MVBs).…”

“…It is predominantly a cytosolic protein, but when the trophozoites undergo phagocytosis, EhVps23 is ubiquitinated and appears close to the erythrocytes and in the endosomes, phagosomes and multivesicular bodies (MVBs). Co-immunoprecipitation and immunofluorescence microscopy experiments have shown that it interacts directly or indirectly with EhADH ( Galindo et al., 2021 ). In addition, structural and docking analysis predicted that the contact of EhVps23 with EhADH, EhUbiquitin, and EhVps32 proteins is through the amino terminus, whereas it contacts the LBPA by the C-terminal region, possibly generating a multiprotein complex.…”