Biliverdin targeting TcdB-DRBD inhibits Clostridioides difficile virulence and restores gut microbiota in Mongolian gerbils (Meriones unguiculatus).

The incidence of Clostridioides difficile infection (CDI) has been rising globally in recent years. Treating CDI is complicated by antibiotic-induced disruption of the normal gut microbiota, which promotes CDI recurrence and increases the risk of therapeutic failure. We used an AI-assisted approach to screen small molecule inhibitors targeting the receptor binding domain of toxin B (TcdB). Biliverdin (BV) had strong binding affinities with all TcdB variants. In vitro results showed that BV exhibited no cytotoxic effects on cells and didn't affect growth of C. difficile, yet markedly suppressed cytotoxic effects induced by TcdB1-4. Encapsulating BV in intestinal epithelial cell-derived extracellular vesicles (I-EVs) significantly recovered body weight, enhanced survival rate, reduced TcdB load, and alleviated intestinal lesions in treated gerbils. Notably, BV treatment not only restored the abundance of gut microbiota but also significantly increased the quantity of gut-beneficial Firmicutes. BV also exerted its anti-CDI effect by restoring the short-chain fatty acid metabolic network. Our findings indicate that BV shows promise as a natural small-molecule therapeutic that attenuates broad-spectrum TcdB-induced injuries, highlighting its potential for clinical translation in CDI treatment.