In-Depth characterization of the clostridioides difficile phosphoproteome to identify Ser/Thr kinase substrates
| Autor: | Transito Garcia-Garcia, Thibaut Douché, Quentin Giai Gianetto, Sandrine Poncet, Nesrine El Omrani, Wiep Klaas Smits, Elodie Cuenot, Mariette Matondo, Isabelle Martin-Verstraete |
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| Přispěvatelé: | Pathogénèse des Bactéries Anaérobies / Pathogenesis of Bacterial Anaerobes (PBA (U-Pasteur_6)), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Plateforme de Protéomique / Proteomics platform, Université Paris Cité (UPCité)-Spectrométrie de Masse pour la Biologie – Mass Spectrometry for Biology (UTechS MSBio), Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité)-Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, Institut Pasteur [Paris] (IP)-Université Paris Cité (UPCité), MICrobiologie de l'ALImentation au Service de la Santé (MICALIS), AgroParisTech-Université Paris-Saclay-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Department of Medical Microbiology, Leiden University Medical Center (LUMC), Universiteit Leiden-Universiteit Leiden, Universiteit Leiden, Institut Universitaire de France (IUF), Ministère de l'Education nationale, de l’Enseignement supérieur et de la Recherche (M.E.N.E.S.R.), This work was funded by the Institut Pasteur, the Université Paris Cité, and grants from the ITN Marie Curie, Clospore (grant no.: H2020-MSCA-ITN-2014 642068), and the ANR DifKin (grant no.: ANR-17-CE15-0018-01)., ANR-17-CE15-0018,DifKin,Une Ser/Thr kinase impliquée dans la résistance à des composés antimicrobiens importants pour la colonization chez Clostridium difficile(2017), European Project: 642068,H2020,H2020-MSCA-ITN-2014,CLOSPORE(2015) |
| Jazyk: | angličtina |
| Rok vydání: | 2022 |
| Předmět: |
Threonine
cell division Proteome envelope homeostasis Clostridioides difficile [SDV]Life Sciences [q-bio] Protein Serine-Threonine Kinases Phosphoproteins Biochemistry serine phosphorylation Analytical Chemistry phosphatase Clostridioides Bacterial Proteins Hanks kinase Phosphoprotein Phosphatases Serine threonine phosphorylation Phosphorylation Molecular Biology |
| Zdroj: | Molecular & Cellular Proteomics, 21(11). ELSEVIER Molecular and Cellular Proteomics Molecular and Cellular Proteomics, 2022, 21, ⟨10.1016/j.mcpro.2022.100428⟩ |
| ISSN: | 1535-9476 1535-9484 |
| Popis: | International audience; 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. |
| Databáze: | OpenAIRE |
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