In-depth characterization of the Clostridioides difficile phosphoproteome to identify Ser/Thr kinases substrates

Clostridioides difficile is the leading cause of intestinal nosocomial post-antibiotic infections in adults. During infection, the bacterium must rapidly respond and adapt to the host environment by using survival strategies. Protein phosphorylation is a reversible post-translational modification employed ubiquitously for signal transduction and cellular regulation. Recently, Hanks-type Serine/Threonine kinases (STKs) and phosphatases (STPs), have emerged as important players in bacterial cell signaling and pathogenicity. However, characterization of STKs targets in prokaryotes remained a difficult challenge. Here, we applied and adapted the TiO2 phosphopeptide enrichment approach to determine the phosphoproteome of C. difficile. We have identified and quantified 2497 proteins representing 63,1% of the theoretical proteome. Among these proteins, 649 contained phosphorylated peptides with a localization probability >0,75. This pathogen encodes two STKs (PrkC and CD2148) and one associated phosphatase (STP), PrkC was shown to be crucial in the regulation of cell wall homeostasis, antibiotic resistance and cell division. Comparative phosphoproteomics of wild-type, prkC, CD2148, stp and double prkC CD2148 strains were performed in order to determinate STKs targets. 104 proteins were identified as specific substrates of PrkC, 46 of CD2148 and 94 of both kinases. Using a combination of in vivo and in vitro approaches, FtsK and Spo0A were validated as a direct target of PrkC. This study provides a detailed mapping of kinase-substrate relationships which may aid in the identification of new biomarkers and therapeutic targets.

艰难梭菌（Clostridioides difficile）是成人医院获得性抗生素相关性肠道感染的首要致病菌。在感染过程中，该细菌需借助生存策略快速响应并适配宿主环境。蛋白质磷酸化是一种广泛用于信号转导与细胞调控的可逆性翻译后修饰。近年来，Hanks型丝氨酸/苏氨酸激酶（STKs）与磷酸酶（STPs）已被证实是细菌细胞信号转导与致病过程中的关键调控因子。然而，原核生物中丝氨酸/苏氨酸激酶底物的鉴定仍是一项极具挑战性的难题。本研究采用并优化了二氧化钛（TiO2）磷酸肽富集策略，以解析艰难梭菌的磷酸化蛋白质组。本研究共鉴定并定量到2497种蛋白质，占该菌理论蛋白质组的63.1%。其中649种蛋白质含有点位定位概率大于0.75的磷酸化肽段。该致病菌编码两种丝氨酸/苏氨酸激酶（PrkC与CD2148）以及一种关联磷酸酶（STP）；其中PrkC已被证实对细胞壁稳态、抗生素抗性与细胞分裂的调控至关重要。为鉴定丝氨酸/苏氨酸激酶的底物，本研究对野生型、prkC缺失株、CD2148缺失株、stp缺失株以及prkC与CD2148双缺失株开展了比较磷酸化蛋白质组学分析。研究共鉴定出104种PrkC的特异性底物、46种CD2148的特异性底物，以及94种可被两种激酶共同磷酸化的底物。通过结合体内与体外实验手段，本研究验证了FtsK与Spo0A是PrkC的直接磷酸化底物。本研究详细绘制了激酶-底物调控关系图谱，可为新型生物标志物与治疗靶点的发掘提供重要参考。