Quantitative Phosphoproteome Analysis of Clostridioides difficile Toxin B Treated Human Epithelial Cells

The large clostridial glucosylating toxin B (TcdB) is a major virulence factor of the nosocomial pathogen Clostridioides difficile. TcdB inhibits small GTPases by glucosylation leading to impaired downstream signaling. TcdB also possesses a glucosyltransferase independent effect described as pyknosis. To elucidate the impact of TcdB and its glucosylation-inactive mutant TcdB(NXN) on the kinome of human cells, SILAC labeled HEp-2 cells were treated with 2 nM TcdB for 8 h. Phosphopeptides were enriched using SCX chromatography, IMAC and TiO(2) followed shotgun mass spectrometry analysis. Overall 4,197 phosphopeptides were identified; more than 1,200 phosphosites responded to treatment with TcdB or TcdB(NXN). The data suggested that predominantly stress-activated MAPK-dependent signaling pathways were triggered by toxin B treatment.