TcdB from hypervirulent Clostridium difficile exhibits increased efficiency of autoprocessing

Abstract

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 (TcdB HV) was more efficient at autoprocessing than TcdB from a historical strain (TcdB HIST). TcdB HV and TcdB HIST were activated by similar concentrations of IP6; however, the overall efficiency of processing was 20% higher for TcdB HV. Using an activity-based fluorescent probe (AWP19) an intermediate, activated but uncleaved, form of TcdB HIST was identified, while only a processed form of TcdB HV could be detected under the same conditions. Using a much higher concentration (200μM) of the probe revealed an activated uncleaved form of TcdB HV, indicating a preferential and more efficient engagement of intramolecular substrate than TcdB HIST. Furthermore, a peptide-based inhibitor (Ac-GSL-AOMK) was found to block the cytotoxicity of TcdB HIST at a lower concentration than required to inhibit TcdB HV. These findings suggest that TcdB HV may cause increased cytotoxicity due to more efficient autoprocessing. © 2012 Blackwell Publishing Ltd.