肺炎链球菌cps操纵子的启动子序列点突变可导致细菌荚膜缺失

Identification of a point mutation in the promoter region of cps operon responsible for capsular polysaccharide deficiency in Streptococcus pneumniae SPY1

摘要:

摘要:【目的】明确肺炎链球菌荚膜多糖合成操纵子的启动子序列出现的点突变313713 T→C是否可导致细菌荚膜缺失。【方法】Western blot检测肺炎链球菌突变株SPY1 (NC_008533.1 313713 T→C)荚膜多糖含量;实时定量荧光PCR分析荚膜多糖合成基因cps2A、cps2B、cps2C以及cps2D的表达量;构建重组质粒pEVP3-cps promoterD39和pEVP3-cps promoterSPY1，分别转化D39和SPY1菌株，通过β-半乳糖苷酶活性检测来验证转入的启动子序列对细菌荚膜合成的影响，并通过电镜观察荚膜结构和荚膜肿胀试验进一步验证。【结果】肺炎链球菌SPY1 的荚膜多糖含量较野生型显著下降，其相关基因cps2A、cps2B、cps2C以及cps2D的表达量较野生型D39均显著降低;与D39-pEVP3-cps promoterD39对比，D39-pEVP3-cps promoterSPY1的β-半乳糖苷酶活性下降了76%，与SPY1-pEVP3-cps promoterD39相比，SPY1-pEVP3-cps promoterSPY1的β-半乳糖苷酶活性下降了约79%;电镜结果显示，重组SPY1-pEVP3-cps promoterD39荚膜可恢复至野生型水平，并且重组D39-pEVP3-cps promoterSPY1 (NC_008533.1 313713 T→C)荚膜肿胀试验呈阴性。【结论】荚膜多糖合成操纵子的启动子序列点突变313713 T→C可导致荚膜多糖合成基因表达显著下调，从而引起菌株SPY1的荚膜显著减少甚至缺失。

Abstract:

Abstract: ［Objective］ To determine if the point mutation of nt313713 T → C in the promoter region of capsular polysaccharide biosynthesis (cps) operon is responsible for the deficiency of capsular polysaccharide in S．pneumoniae SPY1 strain.［Methods］Western blot was used to compare the amounts of capsular polysaccharide between the wild-type strain and SPY1 strain． Ｒeal-time quantitative PCR was used to determine transcription levels of the first four genes of cps operon，cps2A，cps2B，cps2C and cps2D． The lacZ gene was used as a reporter gene to report the strength of the promoters on cps transcription．The cps promoter was amplified by PCＲ from the wild-type strain or SPY1 strain．The amplified fragments were cloned into shuttle vector pEVP3，transformed into S．pneumoniae D39 or SPY1 strain．The transcription activities of the promoters on capsular polysaccharide biosynthesis were determined by using β-galactosidase as the reporter．Transmission electron microscopy and the Neufeld test were used to reveal the changes in capsule．［Results］Compared to that in the wild-type strain，mＲNA levels of the cps genes were significantly decreased in SPY1 strain．The amount of CPS was also decreased in SPY1 strain． β-galactosidase activities in SPY1-pEVP3-cps promoterSPY1 and D39-pEVP3-cps promoterSPY1 were decreased by about 79% and 76%，respectively，compared to that of the control． Transmission electron microscopy showed that the amount of the capsular polysaccharide of SPY1-pEVP3-cps promoterD39 strain was restored to the wild-type level. In addition，capsular polysaccharide was absent in the D39-pEVP3-cps promoterSPY1 (NC_008533.1 313713 T→C) strain as determined by Neufeld test.［Conclusion］The point mutation of nt313713 T→C in the cps promoter region results in a significantly reduced transcription of the cps genes，which is responsible for the significant reduction or even absence of the biosynthesis of capsular polysaccharide in SPY1 strain.