Epithelial Cells Expressing EhADH, An Entamoeba histolytica Adhesin, Exhibit Increased Tight Junction Proteins.

Abigail Betanzos,Elizabeth Hernández-Nava,Dxinegueela Zanatta,Cecilia Bañuelos,Patricia Cuellar,Esther Orozco

doi:10.3389/fcimb.2018.00340

Abstract

In Entamoeba histolytica, the EhADH adhesin together with the EhCP112 cysteine protease, form a 124 kDa complex named EhCPADH. This complex participates in trophozoite adherence, phagocytosis and cytolysis of target cells. EhCPADH and EhCP112 are both involved on epithelium damage, by opening tight junctions (TJ) and reaching other intercellular junctions. EhADH is a scaffold protein belonging to the ALIX family that contains a Bro1 domain, expresses at plasma membrane, endosomes and cytoplasm of trophozoites, and is also secreted to the medium. Contribution of EhADH to TJ opening still remains unknown. In this paper, to elucidate the role of EhADH on epithelium injury, we followed two strategies: producing a recombinant protein (rEhADH) and transfecting the ehadh gene in MDCK cells. Results from the first strategy revealed that rEhADH reached the intercellular space of epithelial cells and co-localized with claudin-1 and occludin at TJ region; later, rEhADH was mainly internalized by clathrin-coated vesicles. In the second strategy, MDCK cells expressing EhADH (MDCK-EhADH) showed the adhesin at plasma membrane. In addition, MDCK-EHADH cells exhibited adhesive features, producing epithelial aggregation and adherence to erythrocytes, as described in trophozoites. Surprisingly, the adhesin expression produced an increase of claudin-1, occludin, ZO-1 and ZO-2 at TJ, and also the transepithelial electric resistance (TEER), which is a measure of TJ gate function. Moreover, MDCK-EhADH cells resulted more susceptible to trophozoites attack, as showed by TEER and cytopathic experiments. Overall, our results indicated that EhADH disturbed TJ from the extracellular space and also intracellularly, suggesting that EhADH affects by itself TJ proteins, and possibly synergizes the action of other parasite molecules during epithelial invasion.

Highlights

Entamoeba histolytica is the protozoan responsible for human amoebiasis that infects 50 million people and kills between 30 and 100 thousand individuals around the world (Singh et al, 2016)
Confocal microscopy images showed that immediately after Recombinant EhADH (rEhADH) addition, the protein located at cellular borders, it appeared co-localizing with occludin and claudin-1 at the tight junctions (TJ) region (Figures 2, 3). xz-planes images revealed that rEhADH was posed firstly on the apical surface of cells
Confocal images revealed that after cells contact with rEhADH (1 and 5 min), occludin and claudin-1 were delocalized from the cellular borders and eventually, both proteins were internalized to the cytoplasm (15 min)

Summary

Introduction

Entamoeba histolytica is the protozoan responsible for human amoebiasis that infects 50 million people and kills between 30 and 100 thousand individuals around the world (Singh et al, 2016). Amoebiasis is characterized by acute diarrhea due to the substantial damage of the colonic epithelium produced by E. histolytica trophozoites (Cornick and Chadee, 2017). Trophozoites attach to and displace over the epithelium, contacting the epithelial cell surface. They open the intercellular spaces by gradual separation of adjacent cells. Several molecules are involved in this process, such as Gal/GalNAc lectin, amoebapores, cysteine and serine proteases, prostaglandin E2 (PGE2), the EhCPADH complex, among others (Chadee et al, 1987; Leippe, 1997; García-Rivera et al, 1999; Meléndez-López et al, 2007; Lejeune et al, 2011; Cornick et al, 2016)

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