A TcdB-Targeting Antivirulence Compound Preserves Gut Homeostasis and Ameliorates Clostridioides difficile Infection

Clostridioides difficile (C. difficile) is a leading cause of antibiotic-associated diarrhea and colitis, with hypervirulent or resistant strains and high recurrence, limiting current therapies. Antivirulence therapy presents a promising alternative by neutralizing toxins without damaging the gut microbiota or promoting resistance. Here, we identified myricetin (MYR) and its derivatives, dihydromyricetin, and myricitrin (MYT), which effectively suppressed TcdB-mediated cell rounding. Mechanistically, MYR inhibits TcdB internalization, self-cleavage, and Rac1 glycosylation, thereby reducing the cytotoxicity. Microbiomics demonstrated that MYR promoted the expansion of beneficial bacteria such as Muribaculaceae and Lactobacillaceae while reducing the abundance of harmful bacteria, including Enterococcaceae and Enterobacteriaceae. Metabolomics further revealed that MYR elevated the levels of indole and its derivatives, including castanospermine. Castanospermine supplementation decreased the fecal bacterial load and ameliorated histopathological damage in infected mice. Collectively, MYR exerts dual functions by antagonizing virulence and sustaining microbiota homeostasis, thereby improving outcomes in C. difficile infections.