Strain-Dependent Induction of Enterocyte Apoptosis byGiardia lambliaDisrupts Epithelial Barrier Function in a Caspase-3-Dependent Manner

Abstract

We recently demonstrated that Giardia lamblia rearranges cytoskeletal proteins and reduces transepithelial electrical resistance. The effect of G. lamblia on enterocyte apoptosis is unknown, and a possible link between microbially induced enterocyte apoptosis and altered epithelial permeability has yet to be established. The aim of this study was to assess whether G. lamblia induces enterocyte apoptosis in duodenal epithelial monolayers and whether this effect increases epithelial permeability. Monolayers of nontransformed human duodenal epithelial cells were incubated with sonicated or live G. lamblia trophozoites (NF, S2, WB, or PB strains) for 8, 24, and 48 h. Cell cultures were assessed for apoptosis by Hoechst fluorescence staining, enzyme-linked immunosorbent assay for apoptotic nucleosomes, and electron microscopy. In separate experiments, monolayers were pretreated with or without 120 M caspase-3 inhibitor (Z-DEVD-FMK) for 1 h and were assessed for production of apoptotic nucleosomes, tight junctional integrity (with fluorescent ZO-1 staining followed by confocal laser microscopy), and transepithelial permeability for fluorescein isothiocyanate-dextran. G. lamblia strains NF and S2, but not strains WB or PB, induced enterocyte apoptosis within the monolayers, and this effect was inhibited by Z-DEVD-FMK pretreatment. Using the G. lamblia NF isolate, additional experiments investigated the possible link between enterocyte apoptosis and altered epithelial permeability. G. lamblia NF disrupted tight junctional ZO-1 and increased epithelial permeability, but these effects were also prevented by pretreatment with the caspase-3 inhibitor. These findings indicate that strain-dependent induction of enterocyte apoptosis may contribute to the pathogenesis of giardiasis. This effect is responsible for a loss of epithelial barrier function by disrupting tight junctional ZO-1 and increasing permeability in a caspase-3-dependent manner.

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

“…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.…”

SGLT-1-mediated glucose uptake protects human intestinal epithelial cells against Giardia duodenalis-induced apoptosis

“…Giardia duodenalis strain NF was obtained from an epidemic outbreak of human giardiasis in Newfoundland, Canada (Chin et al, 2002). Giardia duodenalis trophozoites were grown axenically at 37 ° C in Diamond's TYI-S33 media supplemented with piperacillin (Piprecil; Wyeth-Ayerst Canada Inc., Montreal, Que., Canada) in 15 ml polystyrene centrifuge tubes (Falcon, Becton-Dickinson and Co., Franklin Lakes, NJ) and sub-cultured every 7 days to maintain the line.…”

“…High concentrations of bacterial lipopolysaccharide (LPS) may also increase epithelial apoptosis and intestinal permeability ( [Yu et al, 2005], [Yu et al, 2006] and [Chin et al, 2006]). While it has been established that G. duodenalis-induced intestinal permeability defects require caspase-3 activation and appear to be strain-dependent ( and [Chin et al, 2002]), the mechanisms responsible for G. duodenalis-induced epithelial apoptosis remain incompletely understood.…”

“…Physiological extrusion of senescent apoptotic enterocytes does not compromise intestinal barrier function ( [Madara, 1990] and [Watson et al, 2005]). In contrast, exposure to enteric pathogens such as G. duodenalis, Escherichia coli, Salmonella enteritica or Helicobacter pylori induces excessive enterocytic apoptosis, which may adversely affect epithelial tight junctional integrity ( [Jones et al, 2000], [Le'Negrate et al, 2001], [Chin et al, 2002], [Paesold et al, 2002], , [Yu et al, 2005], [Troeger et al, 2007] and [Panaro et al, 2007]). High concentrations of bacterial lipopolysaccharide (LPS) may also increase epithelial apoptosis and intestinal permeability ( [Yu et al, 2005], [Yu et al, 2006] and [Chin et al, 2006]).…”

The role of epithelial malfunction in the pathogenesis of enteropathogenic E. coli-induced diarrhea

“…50 It should be noted that certain gastrointestinal pathogens, such as the parasite Giardia lambia, are known to modulate epithelial permeability through the induction of apoptosis. 51,52 As mentioned before, EPEC induces apoptosis in infected IECs in a caspase-dependent manner. 53,54 Nevertheless, recent studies concluded that the increase in epithelial permeability caused by EPEC is caspase-independent, as the inhibition of different initiator and executioner caspases did not restore the epithelial barrier properties.…”