Clostridioides difficile is a major cause of nosocomial infection worldwide causing antibiotic-associated diarrhea and some cases are leading to pseudomembranous colitis. The main virulence factors are toxin A and toxin B. Hypervirulent strains of C. difficile are linked to higher mortality rates and most of these strains produce additionally the C. difficile binary toxin (CDT) that possesses two subunits, CDTa and CDTb. The latter is responsible for binding and transfer of CDTa into the cytoplasm of target cells; CDTa is an ADP ribosyltransferase catalyzing the modification of actin fibers that disturbs the actin vs microtubule balance and induces microtubule-based protrusions of the cell membrane increasing the adherence of C. difficile. The underlying mechanisms remain elusive. Thus, we performed a screening experiment using MS-based proteomics and phosphoproteomics techniques. Epithelial Hep-2 cells were treated with CDTa and CDTb in a multiplexed study for 4 and 8 h. Phosphopeptide enrichment was performed using affinity chromatography with TiO2 and Fe-NTA; for quantification, a TMT-based approach and DDA measurements were used. More than 4,300 proteins and 5,600 phosphosites were identified and quantified at all time points. Although only moderate changes were observed on proteome level, the phosphorylation level of nearly 1,100 phosphosites responded to toxin treatment. The data suggested that CSNK2A1 might act as an effector kinase after treatment with CDT. Additionally, we confirmed ADP-ribosylation on Arg-177 of actin and the kinetic of this modification for the first time.

艰难梭菌是世界范围内导致抗生素相关性腹泻的医院感染的主要原因，并且一些病例导致伪膜性结肠炎。主要毒力因子是毒素 A 和毒素 B。艰难梭菌 与较高的死亡率有关，并且这些菌株中的大多数会额外产生 艰难梭菌二元毒素 (CDT) 具有两个亚基 CDTa 和 CDTb。后者负责将 CDTa 结合并转移到靶细胞的细胞质中；CDTa 是一种 ADP 核糖基转移酶，催化肌动蛋白纤维的修饰，扰乱肌动蛋白与微管的平衡，并诱导细胞膜基于微管的突起，增加肌动蛋白的粘附艰难梭菌. 潜在的机制仍然难以捉摸。因此，我们使用基于 MS 的蛋白质组学和磷酸蛋白质组学技术进行了筛选实验。在一项多重研究中，用 CDTa 和 CDTb 处理上皮 Hep-2 细胞 4 和 8 小时。使用 TiO2 和 Fe-NTA 亲和色谱法富集磷酸肽；为了量化，使用了基于 TMT 的方法和 DDA 测量。在所有时间点都鉴定并定量了超过 4,300 种蛋白质和 5,600 个磷酸位点。尽管在蛋白质组水平上仅观察到适度变化，但近 1,100 个磷酸化位点的磷酸化水平对毒素处理有反应。数据表明 CSNK2A1 在用 CDT 治疗后可能充当效应激酶。此外，