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Updated: Mar 3, 2026
Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291
Published on: December 10, 2016
Clostridium difficile toxin glucosyltransferase domains in complex with a non-hydrolyzable UDP-glucose analogue
Joseph W Alvin1, D Borden Lacy2
1Chemical and Physical Biology Program, Vanderbilt University, Nashville, TN 37232, USA.
Abstract:
Clostridium difficile is the leading cause of hospital-acquired diarrhea and pseudomembranous colitis worldwide. The organism produces two homologous toxins, TcdA and TcdB, which enter and disrupt host cell function by glucosylating and thereby inactivating key signalling molecules within the host. As a toxin-mediated disease, there has been a significant interest in identifying small molecule inhibitors of the toxins' glucosyltransferase activities. This study was initiated as part of an effort to identify the mode of inhibition for a small molecule inhibitor of glucosyltransferase activity called apigenin. In the course of trying to get co-crystals with this inhibitor, we determined five different structures of the TcdA and TcdB glucosyltransferase domains and made use of a non-hydrolyzable UDP-glucose substrate. While we were able to visualize apigenin bound in one of our structures, the site was a crystal packing interface and not likely to explain the mode of inhibition. Nevertheless, the structure allowed us to capture an apo-state (one without the sugar nucleotide substrate) of the TcdB glycosyltransferase domain that had not been previously observed. Comparison of this structure with structures obtained in the presence of a non-hydrolyzable UDP-glucose analogue have allowed us to document multiple conformations of a C-terminal loop important for catalysis. We present our analysis of these five new structures with the hope that it will advance inhibitor design efforts for this important class of biological toxins.
Insights
Researchers studied Clostridium difficile toxins A and B (TcdA/TcdB) to understand how inhibitors work. New crystal structures reveal toxin conformations, aiding the design of drugs targeting these hospital-acquired infection agents.
Area of Science:
- Biochemistry
- Structural Biology
- Microbiology
Background:
- Clostridium difficile infections (CDI) are a major cause of hospital-acquired diarrhea and colitis.
- The disease is mediated by two toxins, TcdA and TcdB, which inactivate host cell signaling molecules via glucosyltransferase activity.
- Developing small molecule inhibitors of TcdA and TcdB glucosyltransferase activity is a key therapeutic strategy.
Purpose of the Study:
- To investigate the mode of inhibition of a glucosyltransferase inhibitor, apigenin.
- To determine the structural basis for TcdA and TcdB glucosyltransferase activity.
- To provide structural insights for the design of novel CDI therapeutics.
Main Methods:
- X-ray crystallography was used to determine five distinct structures of TcdA and TcdB glucosyltransferase domains.
- A non-hydrolyzable UDP-glucose analog was employed as a substrate.
- Structural comparisons were made between apo-state and substrate-bound states.
Main Results:
- Five new crystal structures of TcdA and TcdB glucosyltransferase domains were determined.
- Apigenin was observed bound at a crystal packing interface, not a likely inhibitory site.
- A novel apo-state structure of the TcdB glucosyltransferase domain was captured.
- Multiple conformations of a catalytically important C-terminal loop were identified.
Conclusions:
- The determined structures provide valuable insights into the conformational dynamics of TcdB glucosyltransferase.
- Understanding these conformations is crucial for rational drug design targeting TcdA and TcdB.
- These findings may accelerate the development of effective inhibitors against Clostridium difficile toxins.
