Structure-based targeting of the C. difficile toxin (CDT) from hypervirulent bacterial strains

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Abstract – Targeting Clostridium difficile infection (CDI) is challenging because treatments are limited to a small number of antibiotics, colonoscopy, and experimental methods (i.e. fecal transplant), and there are unacceptably high recurrence rates, particularly with hypervirulent strains such as NAP1/P1/027. One serious problem with hypervirulent strains is that they have a binary toxin termed the C. difficile toxin (CDT), in addition to the enterotoxins TsdA and TsdB. The CDT binary toxin has an enzymatic component, termed CDTa, and a pore-forming or delivery subunit termed CDTb. The cytotoxic response to host cells is the result of a catalytic transfer of the ADP-ribose moiety of NAD to host G-actin, its physiological substrate, via CDTa. Delivery of CDTa to the cytoplasm is via the pore-forming component of the binary toxin, CDTb. While treatment options exist for TcdA/TcdB, there is no therapy to target CDT and the mechanism of action for the CDTa/CDTb binary toxin associated with the hypervirulent strains of CDI is not well understood. In our labs, a structural biology approach is underway via cryoEM to characterize the CDTa/CDTb binary complex using active and fully processed full-length constructs of both CDTa and CDTb (Aim 1). This will be followed by more detailed structural and dynamic studies involving specific domains of the binary toxins using NMR and X-ray crystallography (Aim 1). In Aim 2, studies of complexes relevant to host cell engagement are planned to include those of CDTb and the CDTa/CDTb complex in membrane-like media and bound to constructs derived from host cell receptors (i.e. CD44, LSR). Together the structures will be used to define regions on CDT important for engaging the host cell and provide information necessary to block this process, as a therapeutic approach. Because other infectious bacteria make use of similar “Type III” systems for delivering ADP- ribosylating toxins (i.e. Corynebacterium diphtheriae, diphtheria toxin, Pseudomonas aeruginosa, and others), it is anticipated that the research completed here for CDI could benefit treatment strategies for hypervirulent C. difficile as well as for other dangerous bacterial infections.