CpsB Is a Modulator of Capsule-associated Tyrosine Kinase Activity in Streptococcus pneumoniae

Abstract

Tyrosine phosphorylation is associated with polysaccharide synthesis in a number of Gram-positive and Gram-negative bacteria. In Streptococcus pneumoniae, CpsB, CpsC, and CpsD affect tyrosine phosphorylation and are critical for the production of a mature capsule in vitro. To characterize the interactions between these proteins and the phosphorylation event they modulate, cps2B, cps2C, and cps2D from the capsule type 2 S. pneumoniae D39 were cloned and expressed both individually and in combination in Escherichia coli. Cps2D purified from E. coli was not phosphorylated unless it was co-expressed with its cognate transmembrane domain, Cps2C. Purified phosphorylated Cps2D had tyrosine kinase activity and could phosphorylate both dephosphorylated Cps2D and an exogenous substrate (poly-GluTyr) in the absence of ATP. Cps2B exhibited phosphatase activity against both purified phosphorylated Cps2D and p-nitrophenyl phosphate. An additional role for Cps2B as an inhibitor of Cps2D phosphorylation was demonstrated in both co-expression experiments in E. coli and in vitro experiments where it blocked the transphosphorylation of Cps2D even in the presence of the phosphatase inhibitor sodium orthovanadate. cps2C and cps2D deletion mutants in S. pneumoniae produced no detectable mature capsule during laboratory culture. Both were avirulent in systemic mouse infections and were unable to colonize the nasopharynx, suggesting that the failure to produce capsule was not dependent on the environment. Based on these results, we propose a model for capsule regulation where CpsB, CpsC, CpsD, and ATP form a stable complex that enhances capsule synthesis.

Autophosphorylation of the Escherichia coli Protein Kinase Wzc Regulates Tyrosine Phosphorylation of Ugd, a UDP-glucose Dehydrogenase

“…1). In addition, the soluble PTKs of Gram-positive bacteria have no counterpart to Tyr 569 and need to interact with a membrane protein to autophosphorylate (11,12). Phosphorylation of B. subtilis UDP-glucose dehydrogenase YwqF is more extensive when YwqD is allowed to interact with YwqC, the membrane protein modulator (26).…”

“…Indeed, although intermolecular autophosphorylation of Wzc at the C-terminal tyrosine cluster (five tyrosines from position 708 to position 715) is stimulated by intramolecular autophosphorylation at Tyr 569 (24), autophosphorylation of the S. pneumoniae kinase CpsD only occurs on the tyrosine cluster when the membrane protein modulator CpsC is also present (Fig. 1) (11,12). Several studies have been performed to assess the biological role of phosphorylation of the tyrosine cluster located at the C-terminal end of PTKs.…”

“…a transmembrane protein with limited similarity to the N-terminal domain and a soluble protein with significant similarity to the C-terminal domain of PTKs from Gram-negative bacteria. The soluble protein autophosphorylates at a tyrosine cluster located in its C terminus and also contains the Walker motifs A and B (11,25,26). The different domain organizations in PTKs from Gram-negative and Gram-positive bacteria have suggested that these enzymes might be regulated differently.…”

Tyrosine phosphorylation enhances activity of pneumococcal autolysin LytA

“…For the BY-kinase CpsD to be active it requires the polysaccharide co-polymerase protein, or kinase adaptor membrane protein, CpsC (Bender & Yother, 2001). In Staphylococcus aureus, fusion of the C-terminal cytoplasmic region of this homologous protein to the BY-kinase results in an active protein (Olivares-Illana et al, 2008;Soulat et al, 2006) We hypothesized this would also be the case in S. pneumoniae.…”

Streptococcus agalactiae Capsule Polymer Length and Attachment Is Determined by the Proteins CpsABCD

“…Interestingly, these functions appear different from those suggested for CpsA in GBS. Concerning CpsBCD, orthologous proteins in S. pneumoniae (46 -64% aa identity) were described to constitute a phosphoregulatory system, where CpsD is an autokinase and CpsB is the cognate phosphatase (18,19). By analogy, it can be argued that CPS biosynthesis in GBS is similar to S. pneumoniae overall.…”