Dataset Information

In-depth characterization of the Clostridioides difficile phosphoproteome to identify Ser/Thr kinases substrates

ABSTRACT: Clostridioides difficile is the leading cause of intestinal nosocomial post-antibiotic infections in adults. During infection, the bacterium must rapidly respond and 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. Recently, Hanks-type Serine/Threonine kinases (STKs) and phosphatases (STPs), have emerged as important players in bacterial cell signaling and pathogenicity. However, characterization of STKs targets in prokaryotes remained a difficult challenge. Here, we applied and adapted the TiO2 phosphopeptide enrichment approach to determine the phosphoproteome of C. difficile. We have identified and quantified 2497 proteins representing 63,1% of the theoretical proteome. Among these proteins, 649 contained phosphorylated peptides with a localization probability >0,75. This pathogen encodes two STKs (PrkC and CD2148) and one associated phosphatase (STP), PrkC was shown to be crucial in the regulation of cell wall homeostasis, antibiotic resistance and cell division. Comparative phosphoproteomics of wild-type, prkC, CD2148, stp and double prkC CD2148 strains were performed in order to determinate STKs targets. 104 proteins were identified as specific substrates of PrkC, 46 of CD2148 and 94 of both kinases. Using a combination of in vivo and in vitro approaches, FtsK and Spo0A were validated as a direct target of PrkC. This study provides a detailed mapping of kinase-substrate relationships which may aid in the identification of new biomarkers and therapeutic targets.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Peptoclostridium Difficile (strain 630) (clostridium Difficile)

TISSUE(S): Culture Condition:anaerobically-grown Cell, Prokaryotic Cell

SUBMITTER: Thibaut Douché

LAB HEAD: Mariette Matondo

PROVIDER: PXD029827 | Pride | 2022-12-08

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	20190311_Q2_NEO_ColQ2-53-DifKin_TiO2-2mg-CD-10-1-HF-5.raw	Raw
	20190311_Q2_NEO_ColQ2-53-DifKin_TiO2-2mg-CD-10_1-HF-4.raw	Raw
	20190311_Q2_NEO_ColQ2-53-DifKin_TiO2-2mg-CD-4-1-HF-3.raw	Raw
	20190311_Q2_NEO_ColQ2-53-DifKin_TiO2-2mg-CD-4_1-HF-1.raw	Raw
	20190719_Q1_NEO_ColQ1-77-DifKin_TiO2-2mg-10MIN-incub-1st-elution_eFASP-HF-6.raw	Raw

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Publications

In-Depth Characterization of the Clostridioides difficile Phosphoproteome to Identify Ser/Thr Kinase Substrates.

Garcia-Garcia Transito T Douché Thibaut T Giai Gianetto Quentin Q Poncet Sandrine S El Omrani Nesrine N Smits Wiep Klaas WK Cuenot Elodie E Matondo Mariette M Martin-Verstraete Isabelle I

Molecular & cellular proteomics : MCP 20221014 11

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. diffici ...[more]

PMID: 36252736

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Project description:Clostridium difficile is an anaerobic spore-forming rod-shaped gram-positive bacterium that can infect both humans and animals. Most studies on the pathogenesis of C. difficile have focused on its toxins and their effect on the host cells. Recently, we utilized microarrays to identify conserved and divergent genes associated with virulence in C. difficile isolates from humans and animals. Our data provided the first clue toward a complex mechanism underlying host adaptation and pathogenesis. Microarray technology offers an efficient high-throughput tool to study the transcriptional profiles of pathogens and infected host cells. Transcriptomes of C. difficile after exposure to environmental and antibiotic stresses and those of human epithelial colorectal Caco-2 cells upon TcdA treatment have been analyzed. To our knowledge, there are still no reports on the transcriptomic study of host-pathogen interactions for C. difficile infection (CDI). In vitro analyses of interplay between host and pathogen are essential to unravel the mechanisms of infection and to investigate the host response to infection. We therefore employed microarrays to study both bacterial and human cellular transcriptome kinetics during CDI to Caco-2 cells. Here we present a large-scale analysis of transcriptional profiles to reveal molecular determinants playing a role in C. difficile pathogenesis and the host response. We found that there were 254 and 224 differentially-expressed genes after CDI in C. difficile and Caco-2 cells, respectively. These genes are clustered according to their functional categories and their potential roles in pathogenesis and host response are discussed. Our results will not only increase our understanding on the host-pathogen interaction, but may also provide targets for drug development. Clostridium difficile: Control vs Infection (time course) mRNA with genomic DNA of tested and reference strains Caco-2 cells: Control vs Infected with Clostridium difficile Time-course experiments of Caco-2 cells infected with C. difficile for 30, 60 and 120 min