Study On Biological Functions Of Capsular Polysaccharide Branches Of Streptococcus Pneumoniae Serotype 23F

Streptococcus pneumoniae is an important pathogenic bacterium for human.S.pneumoniae is surrounded by capsular polysaccharide(CPS),which is a critical virulent factor of bacteria and an antigen for host antibodies but also a basis for serotyping.Many CPSs contain branched structures that are important epitopes.The variation of CPS branches is a main way for pneumococci to produce new serotypes.However,the relationship between CPSs branches and bacterial virulence is very less known.Therefore,it is imminent to study the genetic basis and biological function of the CPS branches.Serotype 23 F,which was isolated from the patient with bacteremia and meningitis,is one member of PPV23 and PCV7 vaccines.CPS23 F contains brached rhamnose and glycerol-2-phosphate.In this study,we knocked out the genes wchA,wchX and wchW.Wch A is the first sugar transferase in CPS biosynthesis,thus,the mutant △wchA lost capsule.The capsular polysaccrides from the mutant △wchX and △wchW were isolated and purified by DEAE anion-exchange column and S300 gel-filtration.Their chemical structures were determined by NMR spectrum.The results showed that the CPS of △wchX lost glycerol-2-phosphate branch and the CPS of △wchW lost α1-2 linked rhamnose branch.Therefore,we disclose that Wch X is a transferase for glycerol-2-phosphate while Wch W is a transferase for rhamnose branch.Furthermore,by dot-blotting assay,we found that the glycerol-2-phosphate branch can be recognized by factor antiserum23 d,indicating that this glycerolphosphate acts as a dominant epitope.To find the effects of the branched strutures to serotype 23 F,we observed and compared the capsule size and permeability as well as biofilm formation among the wild type and the mutants by fluorescence confocal microscope.We also detected the bacterial adhesion to epithelial cells and macrophages by flow cytometry.Finally,the virulence of these strains was tested through animal experiments,including biofilm in mice nasopharynx and the infection in lungs and lethality.The results showed that the losing of glycerophosphate branch greatly reduced the size of capsule,and losing rhamnose branch did not affect the size of capsule but slightly increased capsule permeability.△wchX showed the significant increasing of biofilm in vitro and in vivo but also the significant increasing of bacterial adherence to host cells as well as the infection and inflammation to lung after nasal drip to mice.However,comparing to wild type,△wchX caused only 20% mice death thus intensively decreased the lethanol rates after intraperitoneal injection.On the contrary,△wchW slightly increased the biofilm and had no effect to bacterial adherence to host cells but caused 80% lethanol rate to mice.These data clearly demonstrate that the glycerol-2-phosphate branch intensively affects bacterial infection and invasion,but the rhamnose brach only have slight effects to bacterial virulence.This study lays the foundation for further investigation of the mechanism of branched structure in the virulence of S.pneumoniae,and provides a guide for predicting potential epidemic strains and designing polysaccharide-vaccines.