The Regulation Effect Of CcpA Protein On The Biosynthesis Of Capsular Polysaccharide In Streptococcus Pneumoniae

ObjectiveThe capsular polysaccharide(capsule) is a critical virulence factor of streptococcus pneumonia required for both asymptomatic colonization and invasive disease, which protects the bacterium by interfering with host phagocytic killing. The biosynthetic genes of Streptococcus pneumoniae capsular polysaccharide is an operon structure, namely cps locus,which synthesized by the the regulation in the promoter region of cps locus. But it is not clear that which regulated protein can influence the expression of capsule’s biosynthetic genes and the synthesis of capsular polysaccharide by the regulation in the promoter region of cps locus.Our previous study showed that there may be interaction between CcpA and the cps locus of Streptococcus pneumonia.CcpA is a transcriptional regulator associated with glucose metabolism and the expression of a number of genes. This study was performed to investigate the interaction and the regulation effect of the glycometabolic protein, CcpA, on the biosynthesis of capsular polysaccharide(CPS) in S. pneumoniae.MethodsHis-tagged CcpA protein was expressed in E. coli BL21(DE3) and purified by Ni2+ affinity chromatography. The anti-CcpA serum was obtained from immunized mice and the antibody titer was determined by ELISA. The conservation of CcpA was determined by Western blotting. In addition, binding of CcpA protein to the promoter region of cps locus was verified by EMSA. The amount of capsular polysaccharide was determined by ELISA and compared among wild type D39 strain, ccpA mutant and the complement strains.ResultsCcpA protein was conserved in different pneuococcal serotypes included in this study. EMSA assay revealed that CcpA protein was able to bind the promoter region of the cps locus in a dose-dependent manner. The absence of ccpA gene led to an increased expression of capsular polysaccharide, and complement expression of CcpA protein significantly reduced the amount of capsular polysaccharide.ConclusionCcpA is conserved in S. pneumoniae, which plays a role in regulation of the expression of the capsular polysaccharide.