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

Ausencio Galindo,Cecilia Bañuelos,Sarita Montaño,Bibiana Chávez-Munguía,Guillermina García-Rivera,Rosario Javier-Reyna,Lizbeth Salazar-Villatoro,Jaime Ortega-Lopez,Esther Orozco

doi:10.3389/fcimb.2021.770759

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.

Highlights

The plasma and internal membranes of the trophozoites of Entamoeba histolytica, the protozoan responsible of human amoebiasis, (WHO, 1997), are constantly producing invaginations, forming vesicles that fuse and split, and generating endosomes, multivesicular bodies (MVBs), tubes and pseudopodia; events necessary to capture, ingest and digest the prey, as well as for the selection of proteins that will be recycled or secreted
endosomal sorting complex required for transport (ESCRT)-I is an elongated heterotetrameric complex (1:1:1:1) that interacts by one side with the HRS/STAM (ESCRT-0), and by the other with the Vps36 protein (ESCRT-II)
In E. histolytica, the ESCRT-III proteins have been studied, but little is known on the other complexes of the ESCRT machinery

Summary

Introduction

The plasma and internal membranes of the trophozoites of Entamoeba histolytica, the protozoan responsible of human amoebiasis, (WHO, 1997), are constantly producing invaginations, forming vesicles that fuse and split, and generating endosomes, multivesicular bodies (MVBs), tubes and pseudopodia; events necessary to capture, ingest and digest the prey, as well as for the selection of proteins that will be recycled or secreted. The endosomal sorting complex required for transport (ESCRT), formed by the ESCRT-0, ESCRT-I, ESCRT-II, ESCRT-III complexes, and accessory proteins, is one of the main players in vesicle trafficking. In E. histolytica, ESCRT machinery is involved in phagocytosis (Lopez-Reyes et al, 2010; AvalosPadilla et al, 2015; Avalos-Padilla et al, 2018), a virulence landmark of the parasite (Garcıá -Rivera et al, 1999). This protozoan is an excellent model to study the ESCRT machinery and its role in events involving vesicular trafficking, such as phagocytosis and tissue invasion (Alfred and Vaccari, 2016; Liu et al, 2019; Vietri et al, 2020)

Methods

Results

Conclusion