Characterization of a GpsB-associated regulator of PBP1a reveals the organization of the cell wall remodeling complex of Streptococcus pneumoniae
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Exploris 480
ORGANISM(S): Streptococcus Pneumoniae (ncbitaxon:1313)
SUBMITTER: 
Christophe GRANGEASSE
PROVIDER: MSV000098614 | MassIVE | Wed Jul 23 06:02:00 BST 2025
SECONDARY ACCESSION(S): PXD066468
REPOSITORIES: MassIVE
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Project description:Streptococcus pneumoniae is a human commensal bacterium that causes serious diseases. Peptidoglycan (PG) is the critical component of bacteria that maintains cell shape, size, chaining, and turgor resistance. Because of its surface exposure and eubacterial-specific mechanism of biosynthesis, PG biosynthesis remains an outstanding target for discovery of new antibiotics to resistant “superbugs,” like S. pneumoniae. Pneumococcal PG biosynthesis is carried out by a balance of septal and peripheral (side-wall-like) PG synthesis mediated by bPBP2x and bPBP2b, respectively, that emanates from midcell regions. We selected for additional suppressor mutations, besides mltG, that eliminate the requirement for essential bPBP2b in peripheral PG synthesis. We found that mutations that inactivate a putative RNA binding protein, designated KhpA, suppressed the requirement for some, but not all of the essential proteins required for peripheral PG synthesis. KhpA was used as bait in co-IP experiments to demonstrate interactions with a second RNA binding protein, designated KhpB, in cells. Mutations in khpA or khpB phenocopy each other and result in slower growth rates, smaller cell size with normal aspect ratios, and induction of cell wall stress responses. RIP-Seq analyses confirmed KhpAB as RNA binding protein in cells. Another suppressor of Δpbp2b implicated induction of a cell division operon as a way to bypass the requirement for bPBP2b. We show that increased expression of this cell division operon occurs in ΔkhpAB mutants and that this induction is necessary and sufficient to account for suppression of Δpbp2b. Comparisons of relative transcript amounts and protein levels indicate that induction of this cell division operon in ΔkhpAB mutants occurs primarily at the post-transcriptional level. Together, our results show that KhpAB, which are virulence factors in S. pneumoniae and are conserved in other Gram-positive pathogens, acts as a new class of RNA binding protein, with properties analogous to Hfq in Gram-negative bacteria.
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Project description:Membrane eukaryotic-like Ser/Thr protein-kinases play a pivotal role in different aspects of bacterial physiology. In contrast to the diversity of their extracellular domains, their cytoplasmic catalytic domains are highly conserved. However, the function of a long juxtamembrane domain (JMD), which connects the catalytic domain to the transmembrane helix, remains elusive. In this study, we investigated the function of the JMD of the Ser/Thr protein-kinase StkP in cell division of Streptococcus pneumoniae. We observed that the deletion of the JMD did not affect StkP kinase activity. However, it impaired the ability of StkP to phosphorylate its endogenous substrates, which resulted in significant cell morphogenesis defects. Furthermore, multiple threonine residues were identified as being phosphorylated in the JMD. To investigate the functional significance of these phosphorylation sites, we conducted an integrative analysis, combining structural biology, proteomics and bacterial cell imaging. Our results revealed that the phosphorylation of the JMD did not affect the phosphorylation of StkP substrates. However, we observed that it influenced the timing of StkP localization at the division septum and the dynamics of cell constriction. We further demonstrated that phosphorylation of the JMD facilitated the recruitment of several cell division proteins, suggesting that it is required for the assembly of the division machinery at the division septum. In conclusion, this study demonstrates that the function of the JMD of StkP is modulated by phosphorylation and critical for the cell division of S. pneumoniae. These observations may serve as a model for understanding the regulatory function of other bacterial Ser/Thr protein-kinases.
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Project description: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.
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