Summary TcdB, an intracellular bacterial toxin that inactivates small GTPases, is a major Clostridium difficile virulence factor. Recent studies have found that TcdB produced by emerging/hypervirulent strains of C. difficile is more potent than TcdB from historical strains, and in the current work, studies were performed to investigate the underlying mechanisms for this change in TcdB toxicity. Using a series of biochemical analyses we found that TcdB from a hypervirulent strain (TcdBHV) was more efficient at autoprocessing than TcdB from a historical strain (TcdBHIST). TcdBHV and TcdBHIST were activated by similar concentrations of IP6; however, the overall efficiency of processing was 20% higher for TcdBHV. Using an activity‐based fluorescent probe (AWP19) an intermediate, activated but uncleaved, form of TcdBHIST was identified, while only a processed form of TcdBHV could be detected under the same conditions. Using a much higher concentration (200 µM) of the probe revealed an activated uncleaved form of TcdBHV, indicating a preferential and more efficient engagement of intramolecular substrate than TcdBHIST. Furthermore, a peptide‐based inhibitor (Ac‐GSL‐AOMK) was found to block the cytotoxicity of TcdBHIST at a lower concentration than required to inhibit TcdBHV. These findings suggest that TcdBHV may cause increased cytotoxicity due to more efficient autoprocessing.

The production of cAMP from Bacillus anthracis edema toxin (ET) activates gene expression in macrophages through a complex array of signaling pathways, most of which remain poorly defined. In this study, the tumor suppressor protein adenomatous polyposis coli (APC) was found to be important for the up-regulation of previously defined ET-stimulated genes (Vegfa, Ptgs2, Arg2, Cxcl2, Sdc1, and Cebpb). A reduction in the expression of these genes after ET exposure was observed when APC was disrupted in macrophages using siRNA or in bone marrow-derived macrophages obtained from C57BL/6J-Apc Min mice, which are heterozygous for a truncated form of APC. In line with this observation, ET increased the expression of APC at the transcriptional level, leading to increased amounts of APC in the nucleus. The mechanism utilized by APC to increase ETinduced gene expression was determined to depend on the ability of APC to interact with C/EBP ␤, which is a transcription factor activated by cAMP. Coimmunoprecipitation experiments found that APC associated with C/EBP ␤ and that levels of this complex increase after ET exposure. A further connection was uncovered when silencing APC was determined to reduce the ET-induced phosphorylation of C/EBP ␤ at Thr-188. This ET-mediated phosphorylation of C/EBP ␤ was blocked by glycogen synthase kinase 3 (GSK-3) inhibitors, suggesting that GSK-3 is involved in the activation of C/EBP ␤ and supporting the idea of APC helping direct interactions between GSK-3 and C/EBP ␤. These results indicate that ET stimulates gene expression by promoting the formation of an inducible protein complex consisting of APC and C/EBP ␤.