Tyrosine phosphorylation of CpsD negatively regulates capsular polysaccharide biosynthesis in Streptococcus pneumoniae

In Streptococcus pneumoniae, the first four genes of the capsule locus (cps A to cpsD) are common to most serotypes. By analysis of various in-frame de letion and site-directed mutants, the function of their gene products in ca psular polysaccharide (CPS) biosynthesis was investigated. We found that wh ile CpsB, C and D are essential for encapsulation, CpsA is not. CpsC and Cp sD have similarity to the amino-terminal and carboxy-terminal regions, resp ectively, of the autophosphorylating protein-tyrosine kinase Wzc from Esche richia coli. Alignment of CpsD with Wzc and other related proteins identifi ed conserved Walker A and B sequence motifs and a tyrosine rich domain clos e to the carboxy-terminus. We have shown that CpsD is also an autophosphory lating protein-tyrosine kinase and that point mutations in cpsD affecting e ither the ATP-binding domain (Walker A motif) or the carboxy-terminal [YGX] (4) repeat domain eliminated tyrosine phosphorylation of CpsD. We describe, for the first time, the phenotypic impact of these two mutations on polysa ccharide production and show that they affect CPS production differently. W hereas a mutation in the Walker A motif resulted in loss of encapsulation, mutation of the tyrosines in the [YGX](4) repeat domain resulted in an appa rent increase in encapsulation and a mucoid phenotype. These data suggest t hat autophosphorylation of CpsD at tyrosine attenuates its activity and red uces the level of encapsulation. Additionally, we demonstrated that CpsC is required for CpsD tyrosine phosphorylation and that CpsB influences dephos phorylation of CpsD. These results are consistent with CpsD tyrosine phosph orylation acting to negatively regulate CPS production. This has implicatio ns for the function of CpsC/CpsD homologues in both Gram-positive and Gram- negative bacteria and provides a mechanism to explain regulation of CPS pro duction during pathogenesis.