The EhCPADH112 Complex of Entamoeba histolytica Interacts with Tight Junction Proteins Occludin and Claudin-1 to Produce Epithelial Damage

Abstract

Entamoeba histolytica, the protozoan responsible for human amoebiasis, causes between 30,000 and 100,000 deaths per year worldwide. Amoebiasis is characterized by intestinal epithelial damage provoking severe diarrhea. However, the molecular mechanisms by which this protozoan causes epithelial damage are poorly understood. Here, we studied the initial molecular interactions between the E. histolytica EhCPADH112 virulence complex and epithelial MDCK and Caco-2 cells. By confocal microscopy, we discovered that after contact with trophozoites or trophozoite extracts (TE), EhCPADH112 and proteins forming this complex (EhCP112 and EhADH112) co-localize with occludin and claudin-1 at tight junctions (TJ). Immunoprecipitation assays revealed interaction between EhCPADH112 and occludin, claudin-1, ZO-1 and ZO-2. Overlay assays confirmed an interaction of EhCP112 and EhADH112 with occludin and claudin-1, whereas only EhADH112 interacted also with ZO-2. We observed degradation of all mentioned TJ proteins after incubation with TE. Importantly, inhibiting proteolytic activity or blocking the complex with a specific antibody not only prevented TJ protein degradation but also epithelial barrier disruption. Furthermore, we discovered that TE treatment induces autophagy and apoptosis in MDCK cells that could contribute to the observed barrier disruption. Our results suggest a model in which epithelial damage caused by E. histolytica is initiated by the interaction of EhCP112 and EhADH112 with TJ proteins followed by their degradation. Disruption of TJs then induces increased paracellular permeability, thus facilitating the entry of more proteases and other parasite molecules leading eventually to tissue destruction.

Adherens junctions and desmosomes are damaged byEntamoeba histolytica:Participation of EhCPADH complex and EhCP112 protease

“…Trophozoites adhere to the epithelium at the cell–cell contact (Li et al, ; Martinez‐Palomo et al, ), through molecules such as the EhCPADH complex (Arroyo & Orozco, ), the Gal‐Gal/NAc lectin (Leroy et al, ; Petri, Haque, & Mann, ), occludin‐like protein, and others (Goplen, Lejeune, Cornick, Moreau, & Chadee, ; Ravdin & Guerrant, ; Reed et al, ; Rosales‐Encina, Meza, Lopez‐De‐Leon, Talamas‐Rohana, & Rojkind, ). Furthermore, during epithelial penetration, other parasite mechanisms are involved, such as the insertion of amoebapores into the host cell membrane (Leippe, ), the dephosphorylation and degradation of host cell proteins (Betanzos et al, ; Leroy et al, ), the actin‐bundling protein villin proteolysis (Leroy et al, ), the activation of epithelial caspase 3‐like to induce host cell death (Huston, Houpt, Mann, Hahn, & Petri, ), the remodelling of the extracellular matrix with the cysteine proteinase A5 (CP‐A5) participation (Thibeaux et al, ), and the elaborated host inflammatory response (Begum, Quach, & Chadee, ). Of relevance is that during early contact with trophozoites, tight junctions (TJs) are disrupted and transepithelial electrical resistance (TEER) decreases (Betanzos et al, ; Lejeune, Moreau, & Chadee, ; Leroy et al, ; Li et al, ).…”

“…Furthermore, during epithelial penetration, other parasite mechanisms are involved, such as the insertion of amoebapores into the host cell membrane (Leippe, ), the dephosphorylation and degradation of host cell proteins (Betanzos et al, ; Leroy et al, ), the actin‐bundling protein villin proteolysis (Leroy et al, ), the activation of epithelial caspase 3‐like to induce host cell death (Huston, Houpt, Mann, Hahn, & Petri, ), the remodelling of the extracellular matrix with the cysteine proteinase A5 (CP‐A5) participation (Thibeaux et al, ), and the elaborated host inflammatory response (Begum, Quach, & Chadee, ). Of relevance is that during early contact with trophozoites, tight junctions (TJs) are disrupted and transepithelial electrical resistance (TEER) decreases (Betanzos et al, ; Lejeune, Moreau, & Chadee, ; Leroy et al, ; Li et al, ).…”

An In Vitro Model of the Blood–Brain Barrier: Naegleria fowleri Affects the Tight Junction Proteins and Activates the Microvascular Endothelial Cells

“…The results showed that the trophozoites and their CM produced a substantial cytopathic effect and an important decrease of TEER in the RBMEC culture. Previous reports showed that the cytopathic damage produced by A. castellanii, E. histolytica, Toxoplasma gondii, and Giardia duodenalis in HBMEC and MDCK cells, was the result of the action of their proteases (Betanzos et al 2013;Briceno et al 2016;Cuellar et al 2017;Halliez et al 2016;Khan and Siddiqui 2009;Shibayama et al 2013). To evaluate the damage produced by the amoebic proteases, we preincubated the trophozoites and their CM with E-64 or Aprotinin and observed that the cysteine proteases inhibitor but not the serine proteases exerted a protective effect on the endothelial monolayer.…”

“…Previous reports showed that the cytopathic damage produced by A. castellanii , E. histolytica, Toxoplasma gondii, and Giardia duodenalis in HBMEC and MDCK cells, was the result of the action of their proteases (Betanzos et al. ; Briceno et al. ; Cuellar et al.…”

Entamoeba histolytica: Host parasite interactions at the colonic epithelium

“…This leads to the rapid loss of TER and increases paracellular permeability. 47 Additionally, Eh contains an occludin-like protein which possibly functions to displace the host TJ occludin resulting in a decrease in TER. 48 The expression of TJ proteins has been studied in the context of a mouse colonic loop model using control animals and also mice lacking a bonafide mucus layer.…”