Dataset Information

Proteomic investigation uncovers potential targets and target sites of pneumococcal serine-threonine kinase StkP and phosphatase PhpP

ABSTRACT: Like eukaryotes, different bacterial species express one or more Ser/Thr kinases and phosphatases that operate in various signaling networks by catalyzing phosphorylation and dephosphorylation of proteins that can immediately regulate biochemical pathways by altering protein function. The human pathogen Streptococcus pneumoniae encodes a single Ser/Thr kinase-phosphatase couple known as StkP-PhpP, which has shown to be crucial in the regulation of cell wall synthesis and cell division. In this study, we applied proteomics to further understand the physiological role of pneumococcal PhpP and StkP with an emphasis on phosphorylation events on Ser and Thr residues. Therefore, the proteome of the non-encapsulated D39 strain (WT), a kinase (ΔstkP) and phosphatase mutant (ΔphpP) were compared in a mass spectrometry based label-free quantification experiment. Results show that a loss of function of PhpP causes an increased abundance of proteins in the phosphate uptake system Pst. Quantitative proteomic data demonstrated an effect of StkP and PhpP on the two-component systems ComDE, LiaRS, CiaRH and VicRK. To obtain further information on the function, targets and target sites of PhpP and StkP we combined the advantages of phosphopeptide enrichment using titan dioxide and spectral library based data evaluation for sensitive detection of changes in the phosphoproteome of the wild type and the mutant strains. According to the role of StkP in cell division we identified several proteins involved in cell wall synthesis and cell division that are apparently phosphorylated by StkP. Unlike StkP, the physiological function of the co-expressed PhpP is poorly understood. For the first time we were able to provide a list of previously unknown putative targets of PhpP. Under these new putative targets of PhpP are, among others, five proteins with direct involvement in cell division (DivIVA, GpsB) and peptidoglycan biosynthesis (MltG, MreC, MacP).

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Streptococcus Pneumoniae Serotype 2 (strain D39 / Nctc 7466)

SUBMITTER: Thomas Sura

LAB HEAD: Dörte Becher

PROVIDER: PXD015268 | Pride | 2020-01-22

REPOSITORIES: Pride

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Publications

Proteomic Investigation Uncovers Potential Targets and Target Sites of Pneumococcal Serine-Threonine Kinase StkP and Phosphatase PhpP.

Hirschfeld Claudia C Gómez-Mejia Alejandro A Bartel Jürgen J Hentschker Christian C Rohde Manfred M Maaß Sandra S Hammerschmidt Sven S Becher Dörte D

Frontiers in microbiology 20200204

Like eukaryotes, different bacterial species express one or more Ser/Thr kinases and phosphatases that operate in various signaling networks by catalyzing phosphorylation and dephosphorylation of proteins that can immediately regulate biochemical pathways by altering protein function. The human pathogen <i>Streptococcus pneumoniae</i> encodes a single Ser/Thr kinase-phosphatase couple known as StkP-PhpP, which has shown to be crucial in the regulation of cell wall synthesis and cell division. In ...[more]

PMID: 32117081

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Project description:Pneumococcal bacteriocins (pneumocins) are antibacterial toxins that mediate intra- and interspecies competition within the human host. What triggers pneumocin expression is poorly understood. Using RNA-sequencing, we mapped the regulon of the pneumocin cluster (blp) of Streptococcus pneumoniae D39 and show that several antibiotics activate the blp-genes. Real-time gene expression measurements showed that while the promoter driving expression of the two-component regulatory system blpS/H was constitutive, the remaining blp-promoters (e.g. controlling pneumocin expression, immunity and the inducer peptide BlpC) was pH-dependent, induced in the late exponential phase and occurred in all cells. Intriguingly, competence for genetic transformation, mediated by the ComD/E two-component quorum system, was induced by the same stimuli. To test for regulatory interplay, we utilized synthetic BlpC and competence-stimulating peptide (CSP). Strikingly, immediately upon addition of CSP, the blp-promoters were activated in a comD/E dependent manner. After a delay, blp-expression was highly induced but strictly dependent on the presence of blpRH and blpC. This raised the question how BlpC is exported since phylogenetic analysis showed that the putative exporter for BlpC, blpAB, is not intact in strain D39 and most other strains. However, all sequenced pneumococcal strains contain intact comAB genes, encoding the transport system for CSP. In fact, we show that high expression of the blp-genes requires comAB. Together, we demonstrate that regulation of pneumocin expression is intertwined with competence, explaining why certain antibiotics induce blp-expression. Antibiotic-induced pneumocin expression might promote survival under antibiotic stress by killing and liberating DNA from neighboring (antibiotic-resistant) bacteria, which can then be used for transformation or repair. Pairwise comparison of untreated and antibiotic-treated cells (either DNA-seq or RNA-seq). It was performed with deep sequencing, using an Illumina HiSeq 2000 machine with 100 nt paired-end reads. Control and HPUra-treated samples were analysed from duplicate samples, while other conditions were from single samples.

Dataset Information

Proteomic investigation uncovers potential targets and target sites of pneumococcal serine-threonine kinase StkP and phosphatase PhpP

Publications

Proteomic Investigation Uncovers Potential Targets and Target Sites of Pneumococcal Serine-Threonine Kinase StkP and Phosphatase PhpP.

Similar Datasets

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