Giardia duodenalis Surface Cysteine Proteases Induce Cleavage of the Intestinal Epithelial Cytoskeletal Protein Villin via Myosin Light Chain Kinase

Abstract

Giardia duodenalis infections are among the most common causes of waterborne diarrhoeal disease worldwide. At the height of infection, G. duodenalis trophozoites induce multiple pathophysiological processes within intestinal epithelial cells that contribute to the development of diarrhoeal disease. To date, our understanding of pathophysiological processes in giardiasis remains incompletely understood. The present study reveals a previously unappreciated role for G. duodenalis cathepsin cysteine proteases in intestinal epithelial pathophysiological processes that occur during giardiasis. Experiments first established that Giardia trophozoites indeed produce cathepsin B and L in strain-dependent fashion. Co-incubation of G. duodenalis with human enterocytes enhanced cathepsin production by Assemblage A (NF and S2 isolates) trophozoites, but not when epithelial cells were exposed to Assemblage B (GSM isolate) trophozoites. Direct contact between G. duodenalis parasites and human intestinal epithelial monolayers resulted in the degradation and redistribution of the intestinal epithelial cytoskeletal protein villin; these effects were abolished when parasite cathepsin cysteine proteases were inhibited. Interestingly, inhibition of parasite proteases did not prevent degradation of the intestinal tight junction-associated protein zonula occludens 1 (ZO-1), suggesting that G. duodenalis induces multiple pathophysiological processes within intestinal epithelial cells. Finally, this study demonstrates that G. duodenalis-mediated disruption of villin is, at least, in part dependent on activation of myosin light chain kinase (MLCK). Taken together, this study indicates a novel role for parasite cathepsin cysteine proteases in the pathophysiology of G. duodenalis infections.

Whole genome sequencing of dog specific assemblages C and D of Giardia duodenalis from single and pooled cysts indicates host associated genes

“…The formation of the 6 paralogs of genome GSB within clades 7, 8 and 9 can also be caused by selection, because it is known that substrate specificity varies between the paralogs and orthologs of the cathepsins of Giardia . Recombinants of 3 paralogs of cathepsins from GL50803 (Liu et al 2018), but also orthologs of GL50803 and GL50581 (Bhargava et al 2015, Cotton et al 2014) showed differences in substrate specificity. Therefore, the presence of more cathepsin variants within an assemblage, might help to make a less restricted host repertoire possible.…”

“…The most abundant proteins in both isolates were variant-specific surface proteins (VSPs), cathepsins, high cysteine membrane proteins and tenacins. Differences in proteolytic activity and specificity of the cathepsin paralogs and orthologs of different isolates/assemblages have been described (Bhargava et al 2015; Lui et al 2018). So far, nothing is known about the cathepsins of the assemblages of the dog.…”

Interactions ofGiardia sp.with the intestinal barrier: Epithelium, mucus, and microbiota

“…24,25 Second, Giardia cathepsin B-like proteases induce myosin light chain kinase (MLCK)-mediated villin breakdown and contribute to Giardia-induced microbiota toxicity. 26,27 Findings from other reports suggest that soluble factors secreted by colonic epithelial cell lines are sufficient to up-regulate trophozoite membranous and secreted proteins, such as cathepsin-B precursors, cystatin and VSPs, independent of direct parasite attachment. 20,26 Despite very few virulence factors have been identified and characterized in Giardia to date, an unknown 58-kDa product released by Giardia trophozoites has also been reported to induce physiologic changes at mucosal surface.…”

“…26,27 Findings from other reports suggest that soluble factors secreted by colonic epithelial cell lines are sufficient to up-regulate trophozoite membranous and secreted proteins, such as cathepsin-B precursors, cystatin and VSPs, independent of direct parasite attachment. 20,26 Despite very few virulence factors have been identified and characterized in Giardia to date, an unknown 58-kDa product released by Giardia trophozoites has also been reported to induce physiologic changes at mucosal surface. In particular, this factor activates signal transduction pathways in enterocytes leading to anion hypersecretion and intestinal fluid accumulation.…”

“…56,62 In contrast, cleavage of villin induced by Giardia cathepsin-like protease is independent of caspase-3 activity. 26 Therefore, in giardiasis, some of the epithelial pathophysiology is apoptosis-dependent, while some is not.…”