CD44 is a macrophage receptor for TcdB from <i>Clostridioides difficile</i> that <i>via</i> its lysine-158 succinylation contributes to inflammation

Toxin B (TcdB) is a critical virulence factor in <i>Clostridioides difficile</i>-associated disease (CDAD), which activates macrophages to promote inflammation and epithelial damage. However, the mechanism by which TcdB targets inflammation-related receptors on the macrophage surface and the underlying molecular mechanisms remain unknown. The frizzled-binding domain of TcdB (TcdB-FBD) is a promising target of TcdB. Here, FBD was found to trigger macrophage inflammation, similar to TcdB, but did not induce cytotoxicity. Thus, using FBD as a bait protein, macrophage CD44 was identified as an inflammation-related receptor for TcdB/FBD. The role of CD44 was confirmed by CRISPR/Cas9-mediated gene knockout in macrophages and CD44 knockout mice. Using 4-D label-free succinylation quantitative modification proteomics, we demonstrated that TcdB/FBD binds to CD44 in macrophages, promotes CD44 K158 succinylation <i>via</i> SUCLG2 suppression, and enhances NF-κB translocation/transcriptional activity, thereby driving inflammation. Finally, blocking the binding of TcdB to CD44 was demonstrated as a favorable strategy for inhibiting TcdB-mediated macrophage inflammation. This study not only provides a new therapeutic target for the prevention and treatment of CDAD but also elucidates a new molecular mechanism underlying the inflammatory effect of TcdB <i>via</i> the TcdB/FBD-CD44 axis.