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  • During host cell interactions G intestinalis trophozoites up


    During host-cell interactions, G. intestinalis trophozoites up-regulate several cell-associated proteins, including high-cysteine membrane proteins (HCMPs) and variant surface proteins (VSPs) [8]. The parasite also releases around 200 proteins upon host-cell contact, including many proteases [9,10]. Since Giardia is a non-invasive parasite, the secreted proteins have been suggested to be involved in the pathogenesis of the disease [1]. Among the secreted proteins, cysteine protease (CP) activities have emerged as important virulence factors in the pathogenesis of G. intestinalis. This stems mainly from the observation of their association with attenuated immune cell chemotaxis by cleaving chemokines [11,12], intestinal epithelial cell (IEC) villin breakdown [13], disruption the cellular junctions and increase of the intestinal permeability [[14], [15], [16], [17]], effects on the intestinal normal flora and biofilm formation [18] and inhibition of growth of intestinal bacterial pathogens [19]. The intestinal mucus layer acts as both a physical and immunological barrier, providing protection of the luminal surface of the gastrointestinal tract [20]. It is important in order to prevent normal flora bacteria and enteric pathogens from accessing the IECs and the underlying tissue. It was recently demonstrated that CP activities in Giardia play a role in degrading the intestinal mucus layer but the specific CPs were never identified [21]. There are numerous other antimicrobial proteins embedded in the mucus layer, e.g. defensins, lysozyme and secretory GTP-Binding Protein Fragment, G alpha synthesis [20], and they have all been suggested to be important for protection against Giardia infections [[22], [23], [24]]. The current assembly of the G. intestinalis WB genome ( contains 26 CP genes [25]. Among these 9 members belong to the cathepsin B-like subfamily [9,26,27], including CP10217, CP14019, CP16160, CP16468, CP16779 and CP17516 which are the major CPs secreted from G. intestinalis during the interaction with IECs [9,10]. The proteolytic activities of some of the CPs (CP14019, CP16160 and CP16779) and their roles in the cleavage of apical junctional complexes and chemokines and induction of apoptosis have recently been studied [11,28]. The most highly expressed CP in trophozoites, CP14019, was given the name giardiapain-1 due to its papain-like features and putative role in virulence [28]. It has also recently been shown that certain VSPs have CP activities, further increasing their importance for pathogenesis [29]. However, the roles of most CPs in the molecular pathogenesis of G. intestinalis remain unclear. One way to address this question is to first determine the substrate specificity of the main secreted Giardia CPs and then perform a search for targets according to their substrate specificity. To date, little research has been done on the substrate specificity of G. intestinalis CPs. In the only published study, DuBois et al. investigated the substrate specificity of CP14019, or CP2 as it was called, using a positional scanning synthetic combinatorial library [26]. They showed that CP14019 has a preference for K in the P1 position, L, M, V or F in the P2 position, P in the P3 position and no major specificity in the P4 position [26]. The aim of this study was to further characterize three of the secreted Giardia CPs that are phylogenetically related (CP14019, CP16160 and CP16779) based on their cleavage specificity in order to identify new targets and to determine if they have different cleavage specificity. Substrate phage display was used to determine the substrate specificity and model substrates, with motifs predicted to be cleaved by the CPs, were tested in vitro. It showed that the three related, secreted CPs are broad specificity proteases with slightly different specificities and it identified human IgG and IgA antibodies and defensins as potential targets of the secreted CPs.
    Materials and methods