Entamoeba histolytica: EhADH, an Alix Protein, Participates in Several Virulence Events through Its Different Domains

Author:

Zanatta Dxinegueela1,Betanzos Abigail1ORCID,Azuara-Liceaga Elisa2ORCID,Montaño Sarita3ORCID,Orozco Esther1ORCID

Affiliation:

1. Department of Infectomics and Molecular Pathogenesis, Center for Research and Advanced Studies of National Polytechnic Institute, Mexico City 07360, Mexico

2. Postgraduate in Genomic Sciences, Autonomous University of Mexico City, Mexico City 03100, Mexico

3. Laboratory of Bioinformatics and Molecular Simulation, Faculty of Biological Chemistry Sciences, Autonomous University of Sinaloa, Sinaloa 80030, Mexico

Abstract

Entamoeba histolytica is the protozoan causative of human amoebiasis. The EhADH adhesin (687 aa) is a protein involved in tissue invasion, phagocytosis and host-cell lysis. EhADH adheres to the prey and follows its arrival to the multivesicular bodies. It is an accessory protein of the endosomal sorting complexes required for transport (ESCRT) machinery. Here, to study the role of different parts of EhADH during virulence events, we produced trophozoites overexpressing the three domains of EhADH, Bro1 (1–400 aa), Linker (246–446 aa) and Adh (444–687 aa) to evaluate their role in virulence. The TrophozBro11–400 slightly increased adherence and phagocytosis, but these trophozoites showed a higher ability to destroy cell monolayers, augment the permeability of cultured epithelial cells and mouse colon, and produce more damage to hamster livers. The TrophozLinker226–446 also increased the virulence properties, but with lower effect than the TrophozBro11–400. In addition, this fragment participates in cholesterol transport and GTPase binding. Interestingly, the TrophozAdh444–687 produced the highest effect on adherence and phagocytosis, but it poorly influenced the monolayers destruction; nevertheless, they augmented the colon and liver damage. To identify the protein partners of each domain, we used recombinant peptides. Pull-down assays and mass spectrometry showed that Bro1 domain interplays with EhADH, Gal/GalNAc lectin, EhCPs, ESCRT machinery components and cytoskeleton proteins. While EhADH, ubiquitin, EhRabB, EhNPC1 and EhHSP70 were associated to the Linker domain, and EhADH, EhHSP70, EhPrx and metabolic enzymes interacted to the Adh domain. The diverse protein association confirms that EhADH is a versatile molecule with multiple functions probably given by its capacity to form distinct molecular complexes.

Funder

National Council for Science and Technology

Publisher

MDPI AG

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