Giardia duodenalis: analysis of secreted proteases upon trophozoite-epithelial cell interaction in vitro

Abstract

Protease secretion by Giardia duodenalis trophozoites upon interaction with epithelial cells and its association with the parasite adhesion was studied in co-cultures of parasites with IEC6 epithelial cell monolayers in the presence or absence of protease inhibitors. Proteolytic activity in supernatants from trophozoites was enhanced when they were co-cultured with IEC6 cells. This activity was strongly inhibited by pre-incubation of live trophozoites with E-64 and TPCK and a concomitant inhibition of parasite adhesion to IEC6 cells was observed. These data suggest that trophozoites secrete cysteine-type proteases that play a role in the adhesion of G. duodenalis to epithelial cells.Key words: Giardia duodenalis -protease secretion -adhesion Giardiasis caused by the flagellated, intestinal protozoan parasite Giardia duodenalis (syn. G. lamblia, G. intestinalis) manifests as acute and chronic diarrhoea in communities of both developing and developed countries. It is also a leading cause of defined waterborne diarrhoea worldwide (Adam 1991). Infection results from ingestion of cysts, most commonly from faecal contamination of water and food whereas community-wide outbreaks result from contaminated water supplies. After excystation in the upper small intestine, adhesion of trophozoites to epithelial intestinal cells represents the first step in the pathogenesis of the disease. The use of IEC6, an epithelial cell line derived from rat small intestine, is a well validated model in which the inhibitory effect of both actin inhibitors and chelating agents on the attachment of G. duodenalis trophozoites has been previously demonstrated (McCabe et al. 1991). It is also well recognized that the adhesion process is a multi-factorial event that involves a suction force of the adhesive disk (Knaippe 1990), a mechanical process related to contractile proteins of the adhesive disk and the ventrolateral flange (Chávez et al. 1995) and surface molecules that allow the binding of trophozoites to receptors to host epithelial cells (Pegado & Souza 1994). It has been postulated that proteases may be involved in this process. Several studies have demonstrated protease activity in total extracts of Giardia trophozoites or in parasite vacuoles (Feely & Dyer 1987, Hare et al. 1989) including the Portland 1 (P1) strain cultured in vitro, with the predominance of cysteine, serine, and aspartic protease activities (Williams & Coombs 1995). It has also been shown that cysteine proteases of P1 strain degraded protein substrates such as gelatin, collagen, albumin, and azocasein while serine proteases were mainly active against haemoglobin (Coradi & Guimarães 2006). In addition, the presence of cysteine protease activities in excretory/secretory (E/S) products of P1 strain has also been reported (Jiménez et al. 2000). These E/S products are associated with mucosal injuries observed in murine giardiasis (Jiménez et al. 2004). However the possible relation between protease secretion and the adhesion of G. duodenalis to epithelial cells has not b...

Release of metabolic enzymes by Giardia in response to interaction with intestinal epithelial cells

“…It is possible that these fragments interfered with the identification of the 58 kDa protein. Moreover, several unknown proteins with masses ranging from 15 to 225 kDa as well as unidentified proteins with cysteine-type protease activity have been reported in supernatants of Giardia-host cell co-cultures [15,33,34]. This is the first time giardial ADI, OCT and enolase have been shown to be present in Giardia culture supernatants.…”

“…In vitro interaction models for G. lamblia and intestinal cells are well established [7][8][9][10][11], as are rodent models [12][13][14] and this has increased the knowledge of the infection [15][16][17].…”

“…Recently several cysteine-type proteases of G. lamblia origin were detected after co-incubations with rat small intestine epithelial cells [15], and other host cell-Giardia interaction experiments and giardiasis patient data have shown that Giardia reduces the epithelial barrier function [16,17] and induces apoptosis [14,[21][22][23], but the effector protein(s) have never been identified. To improve our understanding of host-parasite interactions during infection, we asked whether exposure of G. lamblia to human intestinal epithelial cells (IEC) might lead to release of trophozoite proteins into the medium.…”

Giardia duodenalis: New Research Developments in Pathophysiology, Pathogenesis, and Virulence Factors

“…In addition, these proteases are further subdivided into cathepsin B or cathepsin L superfamilies (reviewed in [104,106]). Cysteine protease activity has been reported in G. duodenalis cultures [51,107]. However, the functions of these proteases are mostly unknown, and their role in giardiasis remains obscure.…”

“…Disruptions of epithelial tight junctional proteins by Giardia are caspase-3-dependent, similarly to other enteric disorders [38•, 46, 47••, 48, 49]. However, recent findings also point to a direct role for cysteine proteases released by the parasite in the proteolytic disruption of epithelial villin, an important constituent of brush border microvilli [50,51]. Remodeling of intestinal epithelial villin has also been observed during late stages of G. duodenalis infection in vivo where it occurs via CD4+ and CD8+ immune responses [52•].…”