Development of a non-radiolabeled glucosyltransferase activity assay for C. difficile toxin A and B using ultra performance liquid chromatography

- A novel non-radioactive glucosyltransferase assay was developed for C. difficile toxin A and toxin B.
- Reverse phase UPLC was used to measure RhoA protein in this activity assay.
- Four different forms of RhoA can be separated with this UPLC method.
- The Km and kcat are determined for the first time for full length toxin A and toxin B.
- EM images of genetically mutated toxins are identical to native toxins.

Clostridium difficile infection (CDI) is the leading cause of gastroenteritis-associated death in the United States. The major virulent factors of C. difficile are toxin A (TcdA) and toxin B (TcdB). Toxicity is mediated by the glucosyltransferase domains on TcdA and TcdB wherein a glucose is transferred from UDP-glucose to Ras homolog family member A (RhoA) receptor. This modification results in disruption of critical cell signaling events. Vaccination against these toxins is considered the best way to combat the CDI. In order to produce non-toxic TcdA and TcdB antigens, their glucosyltransferase domains were genetically mutated to inactivate the toxin activity. We have developed a reverse phase ultra performance liquid chromatographic (RP-UPLC) method to measure this glucosyltransferase activity by separating RhoA and glucosylated RhoA. Glucosylated RhoA and RhoA have a retention time (RT) of 31.25 and 31.95 min. We determine for the first time the glucosyltransferase kinetics (Km and kcat) of both full length TcdA and TcdB to RhoA and demonstrate that the genetically mutated TcdA and TcdB show no glucosyltransferase activity. Furthermore, two-dimensional electron microscopy (2D EM) data demonstrates that the overall global structures of mutated toxins do not change compared to native toxins.