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 titanium 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).
Highlights
Streptococcus pneumoniae is a commensal bacterium of the human nasopharynx but has emerged as a serious opportunistic human pathogen that can colonize the human upper respiratory tract and can transmigrate into the major organs blood and the nervous system, causing several severe and invasive infections including pneumonia, otitis media, bacteremia and meningitis (Mitchell, 2000; Bogaert et al, 2004; Ramirez et al, 2015)
Notable changes in the proteome of the kinase mutant were especially observed in proteins belonging to the categories “Amino acid metabolism,” “Energy metabolism,” “Regulatory functions,” “Signal transduction,” and “Transcription.” In the proteome of the phosphatase mutant, proteins of the mentioned categories were found to be remarkably changed in abundance
This study provides a complex and comprehensive protein repository of high proteome coverage of S. pneumoniae D39 including identification of serine/threonine/tyrosine phosphorylation, which will facilitate further investigations of this important human pathogen
Summary
Streptococcus pneumoniae (pneumococcus) is a commensal bacterium of the human nasopharynx but has emerged as a serious opportunistic human pathogen that can colonize the human upper respiratory tract and can transmigrate into the major organs blood and the nervous system, causing several severe and invasive infections including pneumonia, otitis media, bacteremia and meningitis (Mitchell, 2000; Bogaert et al, 2004; Ramirez et al, 2015). The first PhpP mutant in the encapsulated S. pneumoniae D39 strain was generated and characterized, thereby demonstrating that PhpP is not essential for pneumococcal growth or survival (Agarwal et al, 2012) Another successful construction of a phpP mutant in the non-encapsulated S. pneumoniae Rx1 strain by Ulrych et al (2016) led to the identification of a novel PhpP substrate, the putative RNA binding protein Jag. the pneumococcal phosphatase PhpP is known to dephosphorylate the cognate kinase StkP. A current approach toward the characterization of the pneumococcal phosphatase PhpP suggests that PhpP and StkP cooperatively regulate cell division of S. pneumoniae, the authors revealed a novel PhpP substrate, SPD_1849, a putative RNA binding protein Jag (SpoIIIJ-associated protein) (Ulrych et al, 2016). To obtain further information on the activity, targets and target sites of PhpP and StkP we combined the advantages of phosphopeptide enrichment using titanium dioxide and spectral library based data evaluation for an in-depth analysis of the phosphoproteome
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