(C) Densitometry analysis of rings in (B)

(C) Densitometry analysis of rings in (B). To explore the molecular mechanisms underlying this, we studied and the damage produced by the recombinant cysteine protease (rEhCP112) on TJ functions and proteins. rEhCP112 reduced TEER in Caco-2 cells in a dose- and time-dependent manner; and EhCP112-overexpressing trophozoites provoked major epithelial injury compared to control trophozoites. rEhCP112 penetrated through the intercellular space, and consequently the ion flux increased and the TJs fence function was disturbed. However, macromolecular flux was not altered. Functional assays revealed specific association of rEhCP112 with claudin-1 and claudin-2, that are both involved TMOD2 in regulating ion flux and fence function. Of note, rEhCP112 did not interact with occludin that is responsible for regulating macromolecular flux. Moreover, rEhCP112 degraded and delocalized claudin-1, thus affecting interepithelial adhesion. Concomitantly, expression of the leaky claudin-2 at TJ, first increased and then it was degraded. CGP77675 is the causative agent of amoebiasis, responsible for up to 100,000 deaths worldwide per year (Mortimer and Chadee, 2010). Trophozoites colonize the large intestine producing watery and bloody diarrhea (Espinosa-Cantellano and Martnez-Palomo, 2000; Haque et al., 2003). The intestinal epithelium forms a barrier that prevents pathogens entrance and regulates nutrient acquisition (Hodges and Gill, 2010). This barrier is constituted by a highly-organized CGP77675 monolayer of polarized epithelial cells that are bound mainly through tight junctions (TJs), adherens junctions (AJs), and desmosomes (DSMs) (Sousa et al., 2005). TJs are localized at the most apical region of the intercellular space and are the first barrier for pathogens (Sousa et al., 2005). TJs are formed by transmembrane proteins such as occludin and claudins, and cytoplasmatic plaque proteins such as ZO-1 and ZO-2, which bind to the actin cytoskeleton (Liang and Weber, 2014). Occludin participates in the regulation of the macromolecules flux, while claudins mediate ion flux control. Such paracellular flux is considered CGP77675 as a TJ gate function. Moreover, claudins also restrict proteins and lipids diffusion within membranes, thus contributing to epithelial polarization. This function is known as TJs fence function (Lingaraju et al., 2015). During intestinal invasion and colonization, pathogens destabilize TJs by different mechanisms. In rotavirus, VP8 protein alters the localization of occludin and ZO-1 (Nava et al., 2004). secretes an enterotoxin which binds to and disintegrates claudins (Sonoda et al., 1999); whereas, enteropathogenic activates the epithelial RhoA kinase, contracting actin perijunctional ring and opening TJs (Matsuzawa et al., 2005); sp. activate the epidermal growth factor receptor pathway and elevate the expression of claudin-2 in the colon (Zhang et al., 2013). The protozoan affects the distribution of claudin-1 and ZO-1 and decreases intestinal transepithelial electrical resistance (TEER) (Maia-Brigagao et al., 2012). trophozoites damage the intestinal epithelium and drop TEER in cultured epithelial cells, such as MDCK (Martinez-Palomo et al., 1985; Betanzos et al., 2013), T84 (Leroy et al., 2000; Lejeune et al., 2011), and Caco-2 (Li et al., 1994) cell monolayers. Prostaglandin E2 (PGE2) (Lejeune et al., 2011) and the EhCPADH complex (Betanzos et al., 2013) drop TEER. The concerted action of these and other molecules allows the trophozoites invasion to the intestinal epithelium. has 50 putative cystein proteases (CPs), some of them are secreted and involved in the damage to epithelium (Serrano-Luna et al., 2013). Among CPs, EhCP1 cleaves key components of the host immune system, C3 complement factor, immunoglobulin G, and pro-interleukin-18 (Melndez-Lpez et al., 2007); EhCP2 cleaves the chemokines CCL2, CCL13, and CXCL8, and the resulting proteolysis products modulate the chemotaxis of leukocytes (Pertuz Belloso et al., 2004; Irmer et al., 2009); EhCP5 elicits the fast release of mucin by goblet cells (Cornick et al., 2016); whereas EhCP112 degrades collagen type I, gelatin, fibronectin, and hemoglobin and damages epithelial cells (Arroyo and Orozco, 1987; Garcia-Rivera et al., 1997; Banuelos et al., 2005). EhCP112 together with EhADH (an ALIX family protein) forms the EhCPADH virulence complex. EhCP112 has a canonical catalytic domain and an RGD sequence that in other organisms interacts with integrins (Ruoslahti, 1996; Bruchhaus et al., 2003). However, at molecular level, its contribution to the epithelial damage remains unknown. It is known that pre-treatment of trophozoites with protease inhibitors or an -EhCPADH antibody prevents injury (Betanzos et al., 2013), suggesting that EhCP112 indeed participates in TJs disruption. Here, we studied and the molecular mechanisms that EhCP112 follows to.