Transcriptome analysis of the eukaryotic-type ser/thr kinase PknB in Staphylococcus aureus
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ABSTRACT:
ORGANISM(S): Staphylococcus aureus subsp. aureus N315 Staphylococcus aureus subsp. aureus NCTC 8325
PROVIDER: GSE15346 | GEO | 2010/03/11
SECONDARY ACCESSION(S): PRJNA116575
REPOSITORIES: GEO
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Project description:The function of the Staphylococcus aureus eukaryotic-like serine/threonine protein kinase PknB was investigated by transcriptome analysis using DNA-microarray technology and biochemical assays. The transcriptional profile reveals a strong regulatory impact of PknB on the expression of genes encoding proteins which are involved in purine and pyrimidine biosynthesis, cell wall metabolism, autolysis, and glutamine synthesis. To investigate the functional role of PknB in S. aureus DNA microarray experiments were performed. In this experiment, the impact of the deletion of PknB was investigated. Each experiment was performed 5 times including a dye swap resulting in five chips per competitive comparison to increase reproducibility. All hybridizations were done with equal amounts of cDNA probes.
2010-05-05 | E-GEOD-15346 | biostudies-arrayexpress
Project description:The eukaryotic-like Ser/Thr kinase Stk1 is crucial for virulence, cell wall biosynthesis, and drug susceptibility in methicillin-resistant Staphylococcus aureus (S. aureus) (MRSA). Importantly, MRSA lacking Stk1 become sensitive to β-lactam antibiotics, implying that Stk1 could be an alternative target for combination therapy. However, the autophosphorylation mechanism of Stk1 remains elusive. Using a phosphoproteomic study, we identified six in vivo phosphorylated activation loop residues (Ser159, Thr161, Ser162, Thr164, Thr166, and Thr172) of Stk1, which are also phosphorylated in vitro. We further showed that cis autophosphorylation of Thr172 in the GT/S motif is essential for self-activation and kinase activity of Stk1 kinase domain (Stk1-KD), whereas the trans autophosphorylation of other activation loop serines/threonines are required for the optimal kinase activity of Stk1-KD. Moreover, substitution of the activation loop serines/threonines impaired in vivo autophosphorylation activity of kinase variants, while T172A and T172D variants were unable to autophosphorylate in the cellular content, underlining the essential role of Thr172 for Stk1 activity in vivo. This study provides insights into molecular basis for regulation of Stk1 activity from S. aureus.
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Project description:The Staphylococcus aureus autoinducer-2 (AI-2) producer protein LuxS is phosphorylated by the Ser/Thr kinase Stk1 at a unique position, Thr14. The enzymatic activity of the phosphorylated isoform of LuxS was abrogated compared to that of nonphosphorylated LuxS, thus providing the first evidence of an AI-2-producing enzyme regulated by phosphorylation and demonstrating that S. aureus possesses an original and specific system for controlling AI-2 synthesis.
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Project description:Staphylococcus aureus represents an opportunistic pathogen which utilizes elaborate quorum sensing mechanisms to precisely control the expression and secretion of virulence factors. Previous studies indicated a role of the ClpXP proteolytic system in controlling pathogenesis. While detailed transcriptome data for ClpP and ClpX is available, corresponding studies on the proteome, secretome and metabolome level are largely lacking. In order to decipher the functional roles of ClpP and ClpX more globally we utilized S. aureus deletion mutants of the corresponding genes for in-depth proteomic LC-MS/MS analysis. These studies were complemented by a ClpP active site mutant strain to monitor changes solely depending on the presence and not the activity of the protein. A comparison of these strains with wild type revealed e.g. downregulation of virulence, purine/pyrimidine biosynthesis, iron uptake and stress response. Correspondingly, a reduction of a subset of purine and pyrimidine metabolite levels independently confirmed these results. Interestingly, comparison between the ClpP knockout and ClpP active site mutant strains revealed characteristic differences in UMP biosynthesis and the staphyloferrin B pathway, suggesting that the presence of the protein, although inactive, is important for maintaining these processes. These results are not only of fundamental importance to understand the cellular role of ClpXP but also have implications for the development of novel virulence inhibitor classes.
2021-03-12 | PXD018932 | Pride
Project description:We report the mRNA expression profile of Streptococcus pyogenes Type M1 strain M1T15448-derived mutant lacking Serine threonine phosphatase.
2016-10-29 | GSE74541 | GEO
Project description:We report the mRNA expression profile of Streptococcus pyogenes Type M1 strain M1T15448-derived mutant lacking eukaryote-type Serine threonine kinase.
2016-11-02 | GSE74590 | GEO
Project description:Bacteria are able to adapt to dramatically different microenvironments, but in many organisms, the signaling pathways, transcriptional programs, and downstream physiological changes involved in adaptation are not well-understood. Here, we discovered that osmotic stress stimulates a signaling network in Mycobacterium tuberculosis regulated by the eukaryotic-like receptor Ser/Thr protein kinase PknD. Expression of the PknD substrate Rv0516c was highly induced by osmotic stress. Furthermore, Rv0516c disruption modified peptidoglycan thickness, enhanced antibiotic resistance, and activated genes in the regulon of the alternative ?-factor SigF. Phosphorylation of Rv0516c regulated the abundance of EspA, a virulence-associated substrate of the type VII ESX-1 secretion system. These findings identify an osmosensory pathway orchestrated by PknD, Rv0516c, and SigF that enables adaptation to osmotic stress through cell wall remodeling and virulence factor production. Given the widespread occurrence of eukaryotic-like Ser/Thr protein kinases in bacteria, these proteins may play a broad role in bacterial osmosensing.
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Project description:Different transcriptome, metabolome, and phosphoproteome studies have already indicated a regulatory role of Ser/Thr phosphorylation in the central metabolism of S. aureus. However, it remains still unknown how this exactly tunes its metabolism. For that reason, Extreme Pathway Analysis was used to understand metabolic flux regulation by Ser/Thr phosphorylation. YANAsquare software and YANAvergence routine were chosen to calculate pathway activities of WT, ΔpknB, Δstp, and double mutant S. aureus NewmanHG strains. Gene expression data of S. aureus NewmanHG were collected taking the wild type and mutations of kinase (pknB), phosphatase (stp) or both (pknB/stp) phenotypes. The resulting flux changes were modelled with YANAvergence taking the different gene expression data and an established genome scale model of S. aureus metabolic modes as basis. The fluxes of the different strains were next calculated, taking the gene expression data into account. A concerted metabolic change of fluxes was observed. In particular the pathways for peptidoglycan, nucleotide, and aromatic amino acid synthesis as well as catabolism involving aspartate transaminase (GOT) changed significantly compared to the wild type. Single mutations of pknB and stp were compared to illustrate their different contribution to the phenotype, e.g. pyrimidine synthesis is dramatically impaired by pknB deletion but much less by loss of stp. In the double knock out strain, there is higher activity of peptidoglycan, purine, and aromatic amino acids synthesis from glucose, but lower activity of pyrimidine synthesis from glucose compared to WT. The results suggest a probable regulatory role of Ser/Thr phosphorylation in amino acid catabolism and the glycolysis/gluconeogenesis switch to provide the cell with sufficient cell wall components, nucleotides, and aromatic amino acids. Yvck is suggested to serve as regulatory protein linking Ser/Thr phosphorylation to the previous results of S. aureus metabolism. Moreover, Yvck seems in particular to be involved in the switch between glycolysis and gluconeogenesis. However, further studies based on experimental approaches are needed to demonstrate that Ser/Thr phosphorylation and Yvck are necessary for a correct cell growth under gluconeogenic conditions. The metabolic model allows not only a comprehensive representation of these changes for all involved pathways in the four strains, it further points out the function of the previously unannotated ycvk operon.. The relevance of these specific metabolic pathway adaptations regarding the pathogenicity of S.aureus and their contributions to the infection process will be analyzed and discussed.
2019-11-11 | GSE122362 | GEO
Project description:Antibiotic-resistant enterococci are major causes of hospital-acquired infections. The emergence of Enterococcus faecalis as a significant nosocomial pathogen is a consequence of its inherent resistance to certain antibiotics and of its ability to survive and proliferate in the intestinal tract. Genetic determinants of E. faecalis conferring these properties are largely unknown. Here we show that PrkC, a one-component signaling protein containing a eukaryotic-type Ser/Thr kinase domain, modulates inherent antimicrobial resistance and intestinal persistence of E. faecalis. An E. faecalis mutant lacking PrkC grows at a wild-type rate in the absence of antimicrobial stress but exhibits enhanced sensitivity to cell-envelope-active compounds, including antibiotics that target cell-wall biogenesis and bile detergents. Consistent with its bile sensitivity, the mutant was also impaired at persistence in the intestine of mice. Thus, PrkC regulates key physiological processes in E. faecalis associated with its success as a nosocomial pathogen. The predicted domain architecture of PrkC comprises a cytoplasmic kinase domain separated by a transmembrane segment from extracellular domains thought to bind uncross-linked peptidoglycan, suggesting that PrkC is a transmembrane receptor that monitors the integrity of the E. faecalis cell wall and mediates adaptive responses to maintain cell-wall integrity. Given its role in modulating traits of E. faecalis important for its ability to cause nosocomial infections, we suggest that the one-component signaling protein PrkC represents an attractive target for the development of novel therapies to prevent infections by antibiotic-resistant enterococci.
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Project description:Bacteria can respond to adverse environmental conditions by drastically reducing or even ceasing metabolic activity. They must then determine that conditions have improved before exiting dormancy, and one indication of such a change is the growth of other bacteria in the local environment. Growing bacteria release muropeptide fragments of the cell wall into the extracellular milieu, and we report here that these muropeptides are potent germinants of dormant Bacillus subtilis spores. The ability of a muropeptide to act as a germinant is determined by the identity of a single amino acid. A well-conserved, eukaryotic-like Ser/Thr membrane kinase containing an extracellular domain capable of binding peptidoglycan is necessary for this response, and a small molecule that stimulates related eukaryotic kinases is sufficient to induce germination. Another small molecule, staurosporine, that inhibits related eukaryotic kinases blocks muropeptide-dependent germination. Thus, in contrast to traditional antimicrobials that inhibit metabolically active cells, staurosporine acts by blocking germination of dormant spores.
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