Laboratoire Pathogénese des Bactéries Anaérobies, UMR CNRS 6047, Institut Pasteur, Université Paris Cité, Paris, France.
Plateforme Protéomique, Unité de Technologie et Service Spectrométrie de Masse pour la biologie, CNRS USR 2000, Institut Pasteur, Université Paris Cité, Paris, France.
Mol Cell Proteomics. 2022 Nov;21(11):100428. doi: 10.1016/j.mcpro.2022.100428. Epub 2022 Oct 14.
Clostridioides difficile is the leading cause of postantibiotic diarrhea in adults. During infection, the bacterium must rapidly 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. Hanks-type serine/threonine kinases (STKs) and serine/threonine phosphatases have emerged as important players in bacterial cell signaling and pathogenicity. C. difficile encodes two STKs (PrkC and CD2148) and one phosphatase. We optimized a titanium dioxide phosphopeptide enrichment approach to determine the phosphoproteome of C. difficile. We identified and quantified 2500 proteins representing 63% of the theoretical proteome. To identify STK and serine/threonine phosphatase targets, we then performed comparative large-scale phosphoproteomics of the WT strain and isogenic ΔprkC, CD2148, Δstp, and prkC CD2148 mutants. We detected 635 proteins containing phosphorylated peptides. We showed that PrkC is phosphorylated on multiple sites in vivo and autophosphorylates in vitro. We were unable to detect a phosphorylation for CD2148 in vivo, whereas this kinase was phosphorylated in vitro only in the presence of PrkC. Forty-one phosphoproteins were identified as phosphorylated under the control of CD2148, whereas 114 proteins were phosphorylated under the control of PrkC including 27 phosphoproteins more phosphorylated in the ∆stp mutant. We also observed enrichment for phosphothreonine among the phosphopeptides more phosphorylated in the Δstp mutant. Both kinases targeted pathways required for metabolism, translation, and stress response, whereas cell division and peptidoglycan metabolism were more specifically controlled by PrkC-dependent phosphorylation in agreement with the phenotypes of the ΔprkC mutant. Using a combination of approaches, we confirmed that FtsK was phosphorylated in vivo under the control of PrkC and that Spo0A was a substrate of PrkC in vitro. This study provides a detailed mapping of kinase-substrate relationships in C. difficile, paving the way for the identification of new biomarkers and therapeutic targets.
艰难梭菌是导致成人抗生素后腹泻的主要原因。在感染过程中，细菌必须通过使用生存策略快速适应宿主环境。蛋白磷酸化是一种广泛用于信号转导和细胞调节的可逆翻译后修饰。Hanks 型丝氨酸/苏氨酸激酶（STKs）和丝氨酸/苏氨酸磷酸酶已成为细菌细胞信号转导和致病性的重要参与者。艰难梭菌编码两种 STKs（PrkC 和 CD2148）和一种磷酸酶。我们优化了二氧化钛磷酸肽富集方法来确定艰难梭菌的磷酸蛋白质组。我们鉴定和定量了 2500 种蛋白质，代表理论蛋白质组的 63%。为了鉴定 STK 和丝氨酸/苏氨酸磷酸酶的靶标，我们随后对 WT 菌株和同源缺失突变体ΔprkC、CD2148、Δstp 和 prkC CD2148 进行了比较大规模的磷酸蛋白质组学分析。我们检测到含有磷酸化肽的 635 种蛋白质。我们表明 PrkC 在体内多个位点磷酸化并在体外自身磷酸化。我们无法在体内检测到 CD2148 的磷酸化，而这种激酶仅在存在 PrkC 的情况下在体外被磷酸化。41 种磷酸蛋白被鉴定为受 CD2148 控制的磷酸化，而 114 种蛋白质受 PrkC 控制，包括在Δstp 突变体中更磷酸化的 27 种磷酸蛋白。我们还观察到在Δstp 突变体中更磷酸化的磷酸肽中富含磷酸苏氨酸。两种激酶都靶向代谢、翻译和应激反应所需的途径，而细胞分裂和肽聚糖代谢则更受 PrkC 依赖性磷酸化的特异性控制，这与ΔprkC 突变体的表型一致。使用多种方法相结合，我们证实 FtsK 在体内受 PrkC 控制磷酸化，并且 Spo0A 是 PrkC 的体外底物。这项研究提供了艰难梭菌中激酶-底物关系的详细图谱，为鉴定新的生物标志物和治疗靶标铺平了道路。
