TcdB of Clostridioides difficile mediates necrosis in epithelial cells and is depend on RAS

A Clostridioides difficile infection (CDI) is the most common nosocomial infection worldwide. The main virulence factors are TcdA and TcdB, which inhibit small Rho-GTPases. Inhibition of small Rho-GTPases leads to the so-called cytopathic effect, a reorganization of the actin cytoskeleton, an impairment of the colon epithelium barrier function, and inflammation. Additionally, TcdB induces a necrotic cell death termed pyknosis in vitro independently from its glucosyltransferas-es characterized by chromatin condensation and ROS production. To understand the underlying mechanism of this pyknotic effect, we conducted a large-scale phosphoproteomic study. We in-cluded the analysis of alterations in the phosphoproteome after treatment with TcdA, that was investigated for the first time. TcdA exhibit no glucosyltransferase-independent effect and was, thus, a good control to elucidate the underlying mechanism of the glucosyltransferase inde-pendent effect of TcdB. We found RAS to be a central upstream regulator for the glucosyltrans-ferase independent effect of TcdB. Inhibition of RAS leads to a 68% reduction in necrosis. Further analysis revealed apolipoprotein C-III (APOC3) as a possible crucial factor of CDI induced in-flammation in vivo.