Characterization of secreted Giardia intestinalis cysteine proteases

Sammanfattning: Giardia intestinalis, the causative agent of the diarrheal disease giardiasis, is a protozoan parasite that colonizes the upper small intestine of mammals, including humans. It can be divided into eight genotypes or assemblages (A through H) and only assemblage A and B are infective to humans. Giardiasis is a multi-factorial disease but few giardial virulence factors have been identified and characterized.In this thesis, we used proteomics to identify the major excretory-secretory products (ESPs) released by Giardia trophozoites of the WB and GS isolates during interaction with intestinal epithelial cells (IECs) in vitro (Paper I). To deepen our understanding of the role of ESPs in giardiasis, we focused on three specific secreted Giardia cysteine proteases (CPs; CP14019, CP16160 and CP16779). All the three CPs are capable of opening the apical junction complexes between IECs to degrade chemokines produced in response to Giardia (Paper II). This can partly explain the induction of symptoms and immunosuppression seen during giardiasis. We further studied the cleavage specificity of these CPs using substrate phage display and recombinant protein substrates. The preferred sequences were used to search potential human in vivo targets and a number of candidates were identified, including human immunoglobulins as well as defensins, that were subsequently shown to be efficiently cleaved by the CPs (Paper III). To investigate the involvement of CPs in mucus degradation, we tested the CPs on recombinant MUC2 constructs and full-length MUC2. MUC2 is the major component of the mucus layer in the small intestine. It was shown that CP14019 cleave MUC2 in the N-terminal, suggesting a mechanism that the parasite can use to disrupt/release the mucus gel network and get access to the intestinal epithelium of the host (Paper IV).In summary, this thesis has studied secreted Giardia CPs and their roles in Giardia infections, providing significant insights into the molecular pathogenesis of giardiasis.