Clostridioides difficile is the leading cause of postanti-biotic diarrhea in adults. During infection, the bacterium must rapidly adapt to the host environment by using sur-vival strategies.... Show moreClostridioides difficile is the leading cause of postanti-biotic diarrhea in adults. During infection, the bacterium must rapidly adapt to the host environment by using sur-vival 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 en-codes two STKs (PrkC and CD2148) and one phosphatase. We optimized a titanium dioxide phosphopeptide enrich-ment approach to determine the phosphoproteome of C. difficile. We identified and quantified 2500 proteins rep-resenting 63% of the theoretical proteome. To identify STK and serine/threonine phosphatase targets, we then per-formed comparative large-scale phosphoproteomics of the WT strain and isogenic AprkC, CD2148, Astp, and prkC CD2148 mutants. We detected 635 proteins containing phosphorylated peptides. We showed that PrkC is phos-phorylated on multiple sites in vivo and autophosphor-ylates in vitro. We were unable to detect a phosphorylation for CD2148 in vivo, whereas this kinase was phosphory-lated in vitro only in the presence of PrkC. Forty-one phosphoproteins were identified as phosphorylated under the control of CD2148, whereas 114 proteins were phos-phorylated under the control of PrkC including 27 phos-phoproteins more phosphorylated in the Astp mutant. We also observed enrichment for phosphothreonine among the phosphopeptides more phosphorylated in the Astp 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 phosphoryla-tion in agreement with the phenotypes of the AprkC 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. Show less