Giardia Cysteine Proteases: The Teeth behind the Smile.

Giardia duodenalis is one of the most prevalent human enteropathogens and a major cause of diarrheal disease worldwide. Cysteine proteases (CPs) have been identified as major virulence factors in protozoan parasites, playing important roles in disease pathogenesis and in parasitic life cycles. G. duodenalis exhibits high proteolytic activity, and CPs play significant roles in giardiasis. Giardia CPs are directly involved in intestinal epithelial junctional complex disruption, intestinal epithelial cell apoptosis, and degradation of host immune factors, including chemokines and immunoglobulins. Giardia CPs have also been implicated in mucus depletion and microbiota dysbiosis induced by the parasite. This review discusses the most recent advances in characterization of Giardia Assemblage A and B CPs, including cathepsin B (catB)-like proteases.

Allain T ，Fekete E ，Buret AG 《-》

Secreted Giardia intestinalis cysteine proteases disrupt intestinal epithelial cell junctional complexes and degrade chemokines.

Liu J ，Ma'ayeh S ，Peirasmaki D ，Lundström-Stadelmann B ，Hellman L ，Svärd SG ... -  《-》

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.

Liu J ，Fu Z ，Hellman L ，Svärd SG ... -  《-》

Giardia duodenalis induces pathogenic dysbiosis of human intestinal microbiota biofilms.

Giardia duodenalis is a prevalent cause of acute diarrheal disease worldwide. However, recent outbreaks in Italy and Norway have revealed a link between giardiasis and the subsequent development of chronic post-infectious irritable bowel syndrome. While the mechanisms underlying the causation of post-infectious irritable bowel syndrome remain obscure, recent findings suggest that alterations in gut microbiota communities are linked to the pathophysiology of irritable bowel syndrome. In the present study, we use a laboratory biofilm system to culture and enrich mucosal microbiota from human intestinal biopsies. Subsequently, we show that co-culture with Giardia induces disturbances in biofilm species composition and biofilm structure resulting in microbiota communities that are intrinsically dysbiotic - even after the clearance of Giardia. These microbiota abnormalities were mediated in part by secretory-excretory Giardia cysteine proteases. Using in vitro cell culture and germ-free murine infection models, we show that Giardia-induced disruptions of microbiota promote bacterial invasion, resulting in epithelial apoptosis, tight junctional disruption, and bacterial translocation across an intestinal epithelial barrier. Additionally, these dysbiotic microbiota communities resulted in increased activation of the Toll-like receptor 4 signalling pathway, and overproduction of the pro-inflammatory cytokine IL-1beta in humanized germ-free mice. Previous studies that have sought explanations and risk factors for the development of post-infectious irritable bowel syndrome have focused on features of enteropathogens and attributes of the infected host. We propose that polymicrobial interactions involving Giardia and gut microbiota may cause persistent dysbiosis, offering a new interpretation of the reasons why those afflicted with giardiasis are predisposed to gastrointestinal disorders post-infection.

Beatty JK ，Akierman SV ，Motta JP ，Muise S ，Workentine ML ，Harrison JJ ，Bhargava A ，Beck PL ，Rioux KP ，McKnight GW ，Wallace JL ，Buret AG ... -  《-》

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

Bhargava A ，Cotton JA ，Dixon BR ，Gedamu L ，Yates RM ，Buret AG ... -  《PLoS One》