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

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

doi:10.3389/fcimb.2022.835654

Abstract

The EhVps23 protein, an orthologue of the yeast Vps23 and the mammalian TSG101 proteins, is the single member of the ESCRT-I complex of Entamoeba histolytica identified and characterized until now. EhVps23 actively participates in vesicular trafficking and phagocytosis, which influence several cellular events. In this paper, we investigated the role of EhVps23 in virulence-related functions, including the invasive capacity of trophozoites, using transfected trophozoites. Trophozoites overexpressing the EhVps23 protein (Neo-EhVps23) presented helical arrangements in the cytoplasm, similar to the ones formed by EhVps32 for scission of vesicles. By confocal and transmission electron microscopy, EhVps23 was detected in multivesicular bodies, vesicles, and the extracellular space. It was secreted in vesicles together with other proteins, including the EhADH adhesin. Probably, these vesicles carry molecules that participate in the prey capture or in cell-cell communication. Mass spectrometry of precipitates obtained using α-EhVps23 antibodies, evidenced the presence of proteins involved in motility, phagocytosis, vesicular trafficking and secretion. The study of cellular functions, revealed that Neo-EhVps23 trophozoites exhibit characteristics similar to those described for mammalian transformed cells: they grew 50% faster than the control; presented a significant higher rate of phagocytosis, and migrated five-fold faster than the control, in concordance with the low rate of migration exhibited by Ehvps23-knocked down trophozoites. In addition, Neo-EhVps23 trophozoites produced dramatic liver abscesses in experimental animals. In conclusion, our results showed that EhVps23 overexpression gave to the trophozoites characteristics that resemble cancer cells, such as increased cell proliferation, migration, and invasion. The mutant that overexpresses EhVps23 can be a good study model to explore different events related to the transformation of malignant cells.

Highlights

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)
EhVps23 is a protein actively involved in the E. histolytica vesicular trafficking through the interaction with proteins of the ESCRT machinery and other molecules such as EhADH and
Extracts of NeoEhVps23 trophozoites grown with 40 μg/ml of gentamicin (Hamann et al, 1995) showed the band 50% stronger than the one developed in trophozoites transfected with the empty vector, used as control (Figures 1C, D)

Summary

Introduction

Several studies have identified the genes and proteins required for endosomal sorting in mammalian cells. Even when these are fundamental for nutrition, and capturing and internalization of the prey, they remain poorly known in Entamoeba histolytica. 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). EhVps (ESCRT-I) interacts with EhADH, the lysobisphosphatidic acid (LBPA) and EhUbiquitin (Galindo et al, 2021)

Methods

Results

Conclusion