Autophosphorylation of the Bacterial Tyrosine-Kinase CpsD Connects Capsule Synthesis with the Cell Cycle in Streptococcus pneumoniae

Abstract

Bacterial capsular polysaccharides (CPS) are produced by a multi-protein membrane complex, in which a particular type of tyrosine-autokinases named BY-kinases, regulate their polymerization and export. However, our understanding of the role of BY-kinases in these processes remains incomplete. In the human pathogen Streptococcus pneumoniae, the BY-kinase CpsD localizes at the division site and participates in the proper assembly of the capsule. In this study, we show that the cytoplasmic C-terminal end of the transmembrane protein CpsC is required for CpsD autophosphorylation and localization at mid-cell. Importantly, we demonstrate that the CpsC/CpsD complex captures the polysaccharide polymerase CpsH at the division site. Together with the finding that capsule is not produced at the division site in cpsD and cpsC mutants, these data show that CPS production occurs exclusively at mid-cell and is tightly dependent on CpsD interaction with CpsC. Next, we have analyzed the impact of CpsD phosphorylation on CPS production. We show that dephosphorylation of CpsD induces defective capsule production at the septum together with aberrant cell elongation and nucleoid defects. We observe that the cell division protein FtsZ assembles and localizes properly although cell constriction is impaired. DAPI staining together with localization of the histone-like protein HlpA further show that chromosome replication and/or segregation is defective suggesting that CpsD autophosphorylation interferes with these processes thus resulting in cell constriction defects and cell elongation. We show that CpsD shares structural homology with ParA-like ATPases and that it interacts with the chromosome partitioning protein ParB. Total internal reflection fluorescence microscopy imaging demonstrates that CpsD phosphorylation modulates the mobility of ParB. These data support a model in which phosphorylation of CpsD acts as a signaling system coordinating CPS synthesis with chromosome segregation to ensure that daughter cells are properly wrapped in CPS.

Phosphorylation systems in symbiotic nitrogen-fixing bacteria and their role in bacterial adaptation to various environmental stresses

“…Bacterial TKs are characterized as polysaccharide co-polymerases participating in the polymerization of EPS subunits or recurring O-antigen subunits in LPS molecules (Marczak et al, 2017). This group includes the following proteins: Wzc of E. coli, CpsD of Streptococcus pneumoniae, Ptk of Acinetobacter johnsonii, and AmsA of Erwinia amylovora (Soulat et al, 2007;Grangeasse, Nessler & Mijakovic, 2012;Nourikyan et al, 2015). Unfortunately, only a small number of TKs (e.g., ExoP of S. meliloti and PssP of R. leguminosarum) and TPs have been described in symbiotic bacteria (Mazur et al, 2002;Medeot et al, 2016).…”

Expression of Active <i>Staphylococcus aureus</i> Tyrosine Kinases in a Human Cell Line

“…BY-kinases have been implicated in various events, including stress responses, the cell cycle, and DNA replication, [5][6][7][8] but their best-studied function is their essential contribution to polysaccharide synthesis and transport. The majority of BY-kinases are encoded in the operons that regulate polysaccharide production.…”

Importance of protein Ser/Thr/Tyr phosphorylation for bacterial pathogenesis

“…While the CapA1B1 BY-kinase complex activates CPS synthesis via phosphorylation of the biosynthetic enzymes CapO [73], CapE, and CapM, the Ser/Thr kinase Stk1 promotes peptidoglycan synthesis and reduces CPS production by inhibiting CapA1B1 kinase and CapM glycosyltransferase activities [72]. BY-kinases are also able to coordinate capsule formation with cell division [74]. In S. pneumoniae, autophosphorylation of the BY-kinase CpsD, which forms part of a larger CPS assembly complex, is required for proper capsule production at the septum.…”

“…In S. pneumoniae, autophosphorylation of the BY-kinase CpsD, which forms part of a larger CPS assembly complex, is required for proper capsule production at the septum. Furthermore, by modulating the mobility of the chromosome partitioning protein ParB, phosphorylated CpsD acts to coordinate cell constriction with the encapsulation of daughter cells [74].…”