Instead of receptor interaction or GT activity we focused on

Instead of receptor interaction or GT activity, we focused on the CPD and autoprocessing. The CPDs from the 2 toxins are highly homologous: each cysteine protease targets an intramolecular substrate and mediates InsP6-induced autoprocessing to release the GTD into host cytosol.40, 41 However, TcdB is more susceptible to InsP6-induced autocleavage than wild-type TcdA.7, 39 The cleaved GTD fragment from TcdB-intoxicated abnormal lipid metabolism is readily identifiable,6, 42, 43 whereas autocleavage of TcdA in host cells has not yet been identified. Therefore, we used TcdB in which CPD-mediated autoprocessing can be readily manipulated through mutagenesis or with chemical inhibitors. TcdB mutations at the CPD catalytic triad (TcdB-C698S) or autocleavage site (TcdB-L543A), or chemical inhibition of autoprocessing, blocked the release of the GTD and reduced the cytotoxicity of the toxin,6, 20, 22, 44 yet significantly enhanced the toxin’s ability to stimulate acute inflammatory responses in mouse intestine. The noncleavable mutant TcdB-L543A, with an intact functional CPD, showed significantly higher inflammatory activity than wild-type TcdB in both human intestinal tissues and immune cells, and also in animal intestines, showing that it is the autoprocessing, rather than the intrinsic cysteine protease activity of the toxins, that regulates the toxins’ proinflammatory activities. It also is possible that the products of autoprocessing regulate the inflammatory responses. The enhanced proinflammatory activity of TcdB-L543A is not likely owing to structural alteration of the toxin because a chemical inhibitor of the cysteine protease that blocks TcdB autoprocessing22, 45 converted wild-type TcdB into a potent enterotoxin in murine ileal loops, possibly keeping the full-length toxin bound to the endosome membrane. Thus, data presented in this work showed an unexpected function of CPD: regulation of the proinflammatory activity of C difficile glucosylating toxins. Because CPD activity clearly is associated with cytotoxicity,6, 7, 22, 46 CPD-mediated autoprocessing of the toxins may act as a fine tuner of their biological activities, in that it positively regulates toxins’ cytotoxicity, but negatively regulates their acute proinflammatory activity. Clinical studies analyzing various biomarkers of CDI patients show the strong relationships between intestinal inflammation and clinical disease severity and outcome.8, 47, 48, 49 Recently, Yacyshyn et al found that a proinflammatory PBMC phenotype in patients correlated with CDI recurrence, indicating a critical role of immune cells in response to C difficile infection. Our data showed that IL-8, a potent chemokine that recruits neutrophils, was the dominant cytokine produced by human colonic tissues in response to C difficile toxin treatment. The noncleavable mutant TcdB-L543A, when compared with wild-type TcdB, significantly up-regulated the production of several proinflammatory cytokines in human intestinal tissues as well as human PBMCs, suggesting that the autoprocessing of the toxins also may regulate the inflammatory response during CDI manifestation in human beings.