Dataset Information

GpsB coordinates StkP signalling as a PASTA kinase adaptor in Streptococcus pneumoniae cell division

ABSTRACT: StkP, the Ser/Thr protein kinase of the major human pathogen Streptococcus pneumoniae, monitors cell wall signals and regulates growth and division in response. In vivo, StkP interacts with GpsB, a cell division protein required for septal ring formation and closure that affects StkP-dependent phosphorylation in vivo. Recent phosphoproteomic analyses revealed phosphorylation of GpsB at multiple Ser and Thr residues. Here, we confirmed that StkP directly phosphorylates GpsB in vitro and in vivo, with T79 and T83 being the major phosphorylation sites. StkP has intrinsic kinase activity, but GpsB directly stimulates the autophosphorylation of StkP and phosphorylation of StkP substrates. In vitro, GpsB phosphoablative substitutions had a reduced potential to stimulate StkP activity, whereas phosphomimetic substitutions were functional in terms of StkP activation. In vivo, substitutions of GpsB phosphoacceptor residues, either phosphoablative or mimetic, negatively affected GpsB function, resulting in reduced StkP-dependent phosphorylation and impaired cell division. Bacterial two-hybrid assay and co-immunoprecipitation of GpsB from cells with differentially active StkP indicated that increased phosphorylation of GpsB resulted in a more efficient interaction of GpsB with StkP. Our data suggest that GpsB acts as an adaptor that directly promotes StkP activity by mediating interactions within the StkP signaling hub, ensuring StkP recruitment into the complex and substrate specificity. We present a model that phosphorylation and dephosphorylation of GpsB dynamically modulate StkP activity during exponential growth and under cell wall stress of S. pneumoniae, ensuring proper functioning of the StkP signaling pathway.

INSTRUMENT(S): Q Exactive

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

SUBMITTER: Rudolf Kupcik

LAB HEAD: Marie Vajrychova

PROVIDER: PXD051731 | Pride | 2024-09-25

REPOSITORIES: pride

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Dataset's files

Source:

			Action	DRS
	RK076_GpsB_Reanalysis.msf	Msf
	RK076_Sp451_GpsB_1_1ug_01.raw	Raw
	RK076_Sp451_GpsB_1_1ug_02.raw	Raw
	RK076_Sp451_GpsB_2_1ug_01.raw	Raw
	RK076_Sp451_GpsB_2_1ug_02.raw	Raw

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GpsB Coordinates StkP Signaling as a PASTA Kinase Adaptor in Streptococcus pneumoniae Cell Division.

Stauberová Václava V Kubeša Bohumil B Joseph Merrin M Benedet Mattia M Furlan Berenice B Buriánková Karolína K Ulrych Aleš A Kupčík Rudolf R Vomastek Tomáš T Massidda Orietta O Tsui Ho-Ching T HT Winkler Malcolm E ME Branny Pavel P Doubravová Linda L

Journal of molecular biology 20240919 22

StkP, the Ser/Thr protein kinase of the major human pathogen Streptococcus pneumoniae, monitors cell wall signals and regulates growth and division in response. In vivo, StkP interacts with GpsB, a cell division protein required for septal ring formation and closure, that affects StkP-dependent phosphorylation. Here, we report that although StkP has basal intrinsic kinase activity, GpsB promotes efficient autophosphorylation of StkP and phosphorylation of StkP substrates. Phosphoproteomic analyz ...[more]

PMID: 39303764

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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.