Mechanism of germination inhibition of Clostridioides difficile spores by an aniline substituted cholate derivative (CaPA)

Clostridioides difficile infection (CDI) is the major identifiable cause of antibiotic-associated diarrhea and has been declared an urgent threat by the CDC. C. difficile forms dormant and resistant spores that serve as infectious vehicles for CDI. To cause disease, C. difficile spores recognize taurocholate and glycine to trigger the germination process. In contrast to other sporulating bacteria, C. difficile spores are postulated to use a protease complex, CspABC, to recognize its germinants. Since spore germination is required for infection, we have developed anti-germination approaches for CDI prophylaxis. Previously, the bile salt analog CaPA (an aniline-substituted cholic acid) was shown to block spore germination and protect rodents from CDI caused by multiple C. difficile strains. In this study, we found that CaPA is an alternative substrate inhibitor of C. difficile spore germination. By competing with taurocholate for binding, CaPA delays C. difficile spore germination and reduces spore viability, thus diminishing the number of outgrowing vegetative bacteria. We hypothesize that the reduction of toxin-producing bacterial burden explains CaPAs protective activity against murine CDI. Previous data combined with our results suggests that CaPA binds tightly to C. difficile spores in a CspC-dependent manner and irreversibly trap spores in an alternative, time-delayed, and low yield germination pathway. Our results are also consistent with kinetic data suggesting the existence of at least two distinct bile salt binding sites in C. difficile spores.