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  • In summary our data show


    In summary, our data show a novel mechanism: CPD-mediated autoprocessing of C difficile toxins regulates their proinflammatory activities. Thus, our study provides a new understanding of the molecular mechanisms of the pathogenesis of C difficile toxins and insights into designing new therapeutics against CDI.
    Introduction Colorado potato beetle (Leptinotarsa decemlineata Say, CPB) is the most detrimental potato insect pest. It causes large economic losses in potato production. It also demonstrates a great ability to adapt to different classes of insecticides used [1]. It is therefore of great interest to find new chemical classes of insecticides, preferably from natural origin. Plants respond to herbivore attack by an array of defenses, including induced synthesis of protease inhibitors that inhibit the digestive proteases of the feeding herbivores and reduce their growth, development and fertility. Concurrently, herbivorous insects adapt to plant defenses and compensate for the decreased proteolytic activity [2], [3], [4], [5]. CPB larvae utilize predominantly cysteine proteases, intestains, for protein digestion. In response to dietary protease inhibitors they overexpress these cysteine proteases, express insensitive cysteine proteases, proteases of other catalytic classes and proteases that inactivate ingested protease inhibitors [5], [6], [7], [8], [9], [10]. Several types of protease inhibitors have been tested as potential agents for plant protection against CPB in the form of dietary additives or transgenic plants, but with varying success [6], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20]. Rice and barley cystatins expressed in potato caused lower weight gain in feeding assays with CPB larvae. The effect was concentration dependent for rice cystatin [16] and Glutathione (GSH/GSSG/Total) Fluorometric Assay Kit synthesis at the level of digestive protease composition was observed for barley cystatin [11]. A delay in development and a change in digestive protease composition was observed in CPB larvae fed with transgenic potato expressing an inhibitor of aspartic proteases from tomato [6]. Experiments using protease inhibitors of animal origin expressed heterologously in potato were less promising. Serine protease inhibitors from locust caused a slight delay in development [14] and, for the cysteine protease inhibitor from sea anemone, equistatin, insufficient levels of expression were achieved for protection against CPB larvae, resulting from protein degradation by Arg/Lys and legumain-type Asn-specific cysteine proteases [18]. An interesting effect was observed with cysteine protease inhibitors of fungal origin, macrocypins, which caused decreased larval weight gain and delay in development but, unlike any other dietary protease inhibitor investigated, no effect was observed in CPB guts on gene expression of known adaptation-related digestive enzymes [21]. Macrocypins from parasol mushroom constitute family I85 in the MEROPS classification and belong to the mycocypin superfamily of cysteine protease inhibitors unique to basidiomycetes [22], [23]. Clitocypin (Clt) is a cysteine protease inhibitor from clouded agaric (Clitocybe nebularis) that belongs to family I48 of the MEROPS classification [24], [25], [26], [27] and, together with macrocypins, forms the mycocypin superfamily. They possess the β-trefoil fold that affords them resistance to proteolytic degradation and tolerance to extreme pH and temperatures [28], [29]. Clitocypin is a strong inhibitor of papain-like cysteine proteases (family C1), especially human endopeptidases cathepsin L, V and K, but not exopeptidases cathepsins B and H. Clitocypin also inhibits asparaginyl endopeptidase (legumain, family C13) through a different inhibitory reactive site [25], [26], [29]. Mushrooms are a rich source of proteins with insecticidal activity [30]. A crude protein extract of C. nebularis fruiting bodies exhibited a strong antinutritional activity against CPB larvae, which has been partially attributed to lectins [31]. Since macrocypins were shown to be effective inhibitors of larval growth, we investigated the antinutritional potential of clitocypin from C. nebularis against CPB. Here, we report the mode of action of clitocypin as a protective agent against CPB larvae and its consequent potential as an insecticide.