Name: Phosphoproteome upon Salmonella infection
Keywords: Human

Dataset Information

Phosphoproteome upon Salmonella infection

ABSTRACT: HCT116 cells were grown in SILAC-DMEM (arginine and lysine-free medium), supplemented with dialyzed FCS (10%) and lysine [74µg/ml] and arginine [42µg/ml] as “light K0, R0”, “medium K4, R6” or “heavy K8, R10”. Cells were left uninfected or infected with Salmonella and lysed 30 min or 2h post-infection.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

SUBMITTER: Marcel Hahn

LAB HEAD: Ivan Dikic

PROVIDER: PXD023703 | Pride | 2021-05-06

REPOSITORIES: Pride

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Publications

SIK2 orchestrates actin-dependent host response upon Salmonella infection.

Hahn Marcel M Covarrubias-Pinto Adriana A Herhaus Lina L Satpathy Shankha S Klann Kevin K Boyle Keith B KB Münch Christian C Rajalingam Krishnaraj K Randow Felix F Choudhary Chunaram C Dikic Ivan I

Proceedings of the National Academy of Sciences of the United States of America 20210501 19

Salmonella is an intracellular pathogen of a substantial global health concern. In order to identify key players involved in Salmonella infection, we performed a global host phosphoproteome analysis subsequent to bacterial infection. Thereby, we identified the kinase SIK2 as a central component of the host defense machinery upon Salmonella infection. SIK2 depletion favors the escape of bacteria from the Salmonella-containing vacuole (SCV) and impairs Xenophagy, result ...[more]

PMID: 33947818

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Project description:Analysis of the response to arginine of the Escherichia coli K-12 transcriptome by microarray hybridization and real-time quantitative PCR provides a first coherent quantitative picture of the ArgR-mediated repression of arginine biosynthesis and uptake genes. Transcriptional repression was shown to be the major control mechanism of the biosynthetic genes, leaving only limited room for additional transcriptional or post-transcriptional regulations. The art genes coding for the specific arginine uptake system are subject to ArgR-mediated repression, ; with a strong repression of artJ, coding for the periplasmic binding protein of the system. The hisJQMP genes of the histidine transporter (part of the LAO uptake system) were discovered to be a part of the arginine regulon. Analysis of their control region with reporter gene fusions and electrophoretic mobility shift in the presence of pure ArgR repressor showed the involvement in repression of the ArgR protein and of an ARG box 120 bp upstream of hisJ. No repression of the genes of the AO uptake system was observed. Finally, comparing the time course of arginine repression of gene transcription with the evolution of the specific ; activities of the cognate enzymes showed that while full genetic repression was achieved two minutes after arginine addition, enzyme concentrations were diluted at the rate of cell ; division. This emphasizes the importance of the feedback-inhibition of the first enzymatic ; step of the pathway in controlling the metabolic flow through the biosynthesis in the period following the onset of repression. Experiment Overall Design: 3 groups of 3 replicate samples were analyzed: Experiment Overall Design: (1) Wild type on minimal medium (strain P4X, slides 1753, 1765, 1989), this created a reference condition Experiment Overall Design: (2) Wild type grown in the presence of arginine (strain P4X plus 100Âµg/ml of arginine, slides 1754, 1766, 1990), this gave a list of all the genes repressed by the system: ArgR+arginine Experiment Overall Design: (3) Mutant where no active repressor is present; grown in the presence of arginine (strain P4XB2 (argR-) plus 100Âµg/ml arginine, slides 1992, 1993, 1994). This condition told us which genes are indeed regulated by the system: ArgR+arginine. Those genes are in this case derepressed.

Project description:Most available knowledge on fungal arginine metabolism is derived from studies on Saccharomyces cerevisiae, in which arginine catabolism is initiated by releasing urea via the arginase reaction. Orthologs of the S. cerevisiae genes encoding the first three enzymes in the arginase pathway were cloned from Kluyveromyces lactis and shown to functionally complement the corresponding deletion in S. cerevisiae. Surprisingly, deletion of the single K. lactis arginase gene KlCAR1 did not completely abolish growth on arginine as nitrogen source. Growth rate of mutant strongly increased during serial transfer in shake-flask cultures. A combination of RNAseq-based transcriptome analysis and 13C-15N-based flux analysis was used to elucidate the arginase-independent pathway. Isotopic 13C15N-enrichment in ?-aminobutyrate revealed succinate as the entry point in the TCA cycle of the alternative pathway. Transcript analysis combined with enzyme activity measurements indicated increased expression in the Klcar1? mutant of a guanidinobutyrase (EC.3.5.3.7), an enzyme not previously demonstrated in fungi. Expression of the K. lactis KLLA0F27995g (renamed KlGBU1) encoding guanidinobutyrase enabled S. cerevisiae to use guanidinobutyrate as sole nitrogen source and its deletion in K. lactis almost completely abolish growth on this nitrogen source. Phylogenetic analysis suggests that this enzyme activity is widespread in fungi. The goal of the present study was to characterize arginine catabolism in K. lactis. To this end, CAR1, CAR2 and PRO3 orthologs in K. lactis were identified and functionally analysed by deletion, expression in S. cerevisiae and enzyme activity assays. Since deletion of the arginase gene in K. lactis was found not to completely abolish growth on arginine as a sole nitrogen source, the alternative pathway for arginine catabolism operating in this yeast was studied by a combination of transcriptome analysis, 13C and 15N isotope-based flux analysis and enzyme activity assays in cell extracts. To investigate arginine metabolism in the arginase-negative K. lactis strain, strains GG1632 (Klku80? KlCAR1 reference strain) and IMS0367 (Klcar1? Arg+) were grown in aerobic bioreactor batch cultures on glucose chemically defined medium with arginine as sole nitrogen source. RNA sequencing of samples taken during the exponential phase of growth on glucose-arginine media of the reference strain G1631 and the arginase less strain IMS0367 were compared resulting in the characterization of a new function.

Dataset Information

Phosphoproteome upon Salmonella infection

Publications

SIK2 orchestrates actin-dependent host response upon <i>Salmonella</i> infection.

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