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ROLE OF CATHEPSIN-LIKE CYSTEINE PROTEASES IN GIARDIA PATHOLOGY
A Bhargava1, J Cotton1, B Dixon2, R Yates3, P Beck4, J Ferraz4, A Buret11Biological Sciences, University of Calgary, Calgary, AB; 2Health Canada, Ottawa, ON; 3Faculty of Veterinary Medicine; 4Faculty of Medicine, University of Calgary, Calgary, AB
Aims: Giardia duodenalis, a non-invasive protozoan parasite of the upper small intestine, closely associates with the intestinal epithelial cells (IECs) and induces pathophysiological effects including heightened rates of IEC apoptosis, intestinal barrier dysfunction, and diffuse shortening of the brush border microvilli; this results in malabsorptive diarrhea. Giardia products that may initiate disease have yet to be identified. Cysteine proteases have been linked to pathology caused by other protozoan parasites, including Entamoeba and Leishmania. The Giardia genome contains several cathepsin B-, C-, and K/L-like cysteine protease genes of unknown function. The purpose of this study is to characterize cathepsin B- and L-like cysteine protease activity in Giardia infections, and determine if these play a role in the pathogenesis of giardiasis.
Methods: Human colonic (Caco-2) monolayers were co-incubated with G. duodenalis trophozoites (Assemblage A isolates NF, S2, WB or Assemblage B isolate GS/M)(MOI: 10:1 : 24 h). Giardia trophozoite and Caco-2 cell lysates and supernatants were incubated with cathepsin fluorogenic substrates to measure cathepsin activity. Experiments were performed in the presence or absence of a general cysteine protease inhibitor (E-64d), or a cathepsin B inhibitor (Ca-074) and processed for Western blotting to analyze for induction of apoptosis (caspase-3 activation) and intestinal barrier dysfunction (ZO-1).
Results: Giardia trophozoites secrete cathepsin L-like proteases in an isolate-independent manner in the presence and absence of Caco-2 monolayers. Supernatants from ex vivo human small intestinal biopsy tissues co-incubated with Giardia trophozoites showed a trend towards increased cathepsin B/L activity. Giardia trophozoites expressed cathepsin B activity in the presence or absence of Caco-2 cells in an isolate-dependent manner. Whole cell lysates collected from Caco-2 monolayers co-incubated with Giardia trophozoites showed an increase in intracellular cathepsin B activity in an isolate-independent manner. This required direct contact between Caco-2 cells and Giardia trophozoites. Inhibition of Giardia-induced increased intracellular cathepsin B activity with Ca-074 failed to block cleavage of ZO-1 and caspase-3.
Conclusions: Giardia trophozoites express and release cathepsin B- and L-like cysteine proteases, and induce epithelial cathepsin B-like activity when directly contacting enterocytes. Giardia-induced cathepsin B-like cysteine proteases do not appear to be responsible for activation of caspase-3 or disruption of ZO-1.