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In-Depth Characterization of the Clostridioides difficile Phosphoproteome to Identify Ser/Thr Kinase Substrates
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 transd...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Society for Biochemistry and Molecular Biology
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9674922/ https://www.ncbi.nlm.nih.gov/pubmed/36252736 http://dx.doi.org/10.1016/j.mcpro.2022.100428 |
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author | Garcia-Garcia, Transito Douché, Thibaut Giai Gianetto, Quentin Poncet, Sandrine El Omrani, Nesrine Smits, Wiep Klaas Cuenot, Elodie Matondo, Mariette Martin-Verstraete, Isabelle |
author_facet | Garcia-Garcia, Transito Douché, Thibaut Giai Gianetto, Quentin Poncet, Sandrine El Omrani, Nesrine Smits, Wiep Klaas Cuenot, Elodie Matondo, Mariette Martin-Verstraete, Isabelle |
author_sort | Garcia-Garcia, Transito |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-9674922 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | American Society for Biochemistry and Molecular Biology |
record_format | MEDLINE/PubMed |
spelling | pubmed-96749222022-11-21 In-Depth Characterization of the Clostridioides difficile Phosphoproteome to Identify Ser/Thr Kinase Substrates Garcia-Garcia, Transito Douché, Thibaut Giai Gianetto, Quentin Poncet, Sandrine El Omrani, Nesrine Smits, Wiep Klaas Cuenot, Elodie Matondo, Mariette Martin-Verstraete, Isabelle Mol Cell Proteomics Research 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. American Society for Biochemistry and Molecular Biology 2022-10-14 /pmc/articles/PMC9674922/ /pubmed/36252736 http://dx.doi.org/10.1016/j.mcpro.2022.100428 Text en © 2022 The Authors https://creativecommons.org/licenses/by/4.0/This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Research Garcia-Garcia, Transito Douché, Thibaut Giai Gianetto, Quentin Poncet, Sandrine El Omrani, Nesrine Smits, Wiep Klaas Cuenot, Elodie Matondo, Mariette Martin-Verstraete, Isabelle In-Depth Characterization of the Clostridioides difficile Phosphoproteome to Identify Ser/Thr Kinase Substrates |
title | In-Depth Characterization of the Clostridioides difficile Phosphoproteome to Identify Ser/Thr Kinase Substrates |
title_full | In-Depth Characterization of the Clostridioides difficile Phosphoproteome to Identify Ser/Thr Kinase Substrates |
title_fullStr | In-Depth Characterization of the Clostridioides difficile Phosphoproteome to Identify Ser/Thr Kinase Substrates |
title_full_unstemmed | In-Depth Characterization of the Clostridioides difficile Phosphoproteome to Identify Ser/Thr Kinase Substrates |
title_short | In-Depth Characterization of the Clostridioides difficile Phosphoproteome to Identify Ser/Thr Kinase Substrates |
title_sort | in-depth characterization of the clostridioides difficile phosphoproteome to identify ser/thr kinase substrates |
topic | Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9674922/ https://www.ncbi.nlm.nih.gov/pubmed/36252736 http://dx.doi.org/10.1016/j.mcpro.2022.100428 |
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