Cleavage specificity of recombinant Giardia intestinalis cysteine proteases: Degradation of immunoglobulins and defensins.

Giardia intestinalis is a protozoan parasite and the causative agent of giardiasis, a common diarrheal disease. Cysteine protease (CP) activities have been suggested to be involved in Giardia's pathogenesis and we have recently identified and characterized three secreted Giardia CPs; CP14019, CP16160 and CP16779. Here we have studied the cleavage specificity of these CPs using substrate phage display and recombinant protein substrates. The phage display analyses showed that CP16160 has both chymase and tryptase activity and a broad substrate specificity. This was verified using recombinant protein substrates containing different variants of the cleavage sites. Phage display analyses of CP14019 and CP16779 failed but the substrate specificity of CP14019 and CP16779 was tested using the recombinant substrates generated for CP16160. CP16160 and CP14019 showed similar substrate specificity, while CP16779 has a slightly different substrate specificity. The consensus sequence for cleavage by CP16160, obtained from phage display analyses, was used in an in silico screen of the human intestinal proteome for detection of potential targets. Immunoglobulins, including IgA and IgG and defensins (α-HD6 and β-HD1) were predicted to be targets and they were shown to be cleaved by the recombinant CPs in vitro. Our results suggest that the secreted Giardia CPs are key players in the interaction with host cells during Giardia infections since they can cleave several components of the human mucosal defense machinery.