Spd1672 Of Streptococcus Pneumoniae Modulates The Expression Of Capsular Polysaccharides

Background Streptococcus pneumoniae (S.pn) is an encapsulated opportunistic pathogen. Capsule production is indispensable for pneumococcal virulence and is strongly anti-phagocytic in non-immune hosts. Non-encapsulated S.pn is largely avirulent. And in prierty study, our group screened some in vivo induced genes in S. pn by in vivo expression technology (IVET), including a gene spd1672 which was blasted at NCBI may express lipid A core-O antigen enzyme and contains . It suggests that spd1672 gene may be another key gene for the capsular polysaccharide synthesis. In this paper, spd1672 gene would be identified in function of the capsular polysaccharide synthesis.Method Construct spd1672-deletion mutant by LFH-PCR technology. The capsule on the cell wall of mutant strain was observed by transmission electron microscopy; And the deficient strain(5×106 CFU) were used to infect BALB/c mice to determine the virulence. Non-encapsule R6 was incubated with the CPS extracted from D39Δspd1672, then the absorbance value measured using ELISA method. Additionaly, Cell wall and cytoplasm from D39 and D39Δspd1672 were prepared and then analyzed by immunoblotting. At the same time, ELISA method was adopted to further detect the amount of CPS from cell wall surface and medium supernatants of wild and mutant bacteria. At last, We examined the transcription of capsule synthesis genes in D39Δspd1672 and D39 using FQ-PCR.Result spd1672-deletion mutant was constructed successfully. The mutant strain had low survival capacity and virulence in BALB/c mice. It is observed only a large number of of phagocytic cells and lymphocyte in the peritoneal fluid smears of mice injected with deficient bacteria. The capsule structure of spd1672 deficient strain was eilimited by transmission electron microscopy. At 2 hours, the CPS on the cell wall of R6 which was incubated with the CPS was closed to D39. Additionally, ELISA and Immunoblotting analysis revealed that CPS in protoplasm and the medium supernatants of D39Δspd1672 reduction and high molecular weight capsular polysaccharide reduction, and CPS was not detected on the cell wall of D39Δspd1672. spd1672 gene can influence the gene expression of cps2D, cps2E, cps2H, cps2K , RfbC except cps2A.Conclution spd1672 gene plays a key role in regulating capsular polysaccharide synthesis mechanism. Meanwhile, protein SPD1672 (encoded by spd1672) is a ligase which was similar to lipid A core ligase and an important virulence factor. However, the polymerase function will be vertified in future.