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
Stévenin, V.; Gianetto, Q.G.; Duchateau, M.; Matondo, M.; Enninga, J.; Chang, Y.Y. 2021
Macropinocytosis refers to the nonselective uptake of extracellular molecules into many different types of eukaryotic cells within large fluid-filled vesicles named macropinosomes. Macropinosomes... Show moreMacropinocytosis refers to the nonselective uptake of extracellular molecules into many different types of eukaryotic cells within large fluid-filled vesicles named macropinosomes. Macropinosomes are relevant for a wide variety of cellular processes, such as antigen sampling in immune cells, homeostasis in the kidney, cell migration or pathogen uptake. Understanding the molecular composition of the different macropinosomes formed during these processes has helped to differentiate their regulations from other endocytic events. Here, we present a magnetic purification protocol that segregates scarce macropinosomes from other endocytic vesicles at a high purity and in a low-cost and unbiased manner. Our protocol takes advantage of moderate-sized magnetic beads of 100 nm in diameter coupled to mass-spectrometry-based proteomic analysis. Passing the cell lysate through a table-top magnet allows the quick retention of the bead-containing macropinosomes. Unlike other cell-fractionation-based methodologies, our protocol minimizes sample loss and production cost without prerequisite knowledge of the macropinosomes and with minimal laboratory experience. We describe a detailed procedure for the isolation of infection-associated macropinosomes during bacterial invasion and the optimization steps to readily adapt it to various studies. The protocol can be performed in 3 d to provide highly purified and enriched macropinosomes for qualitative proteomic composition analysis. Show less
