从SPD_0064蛋白对荚膜多糖操纵子转录调控研究肺炎链球菌侵袭致病的分子机制

Capsule production is indispensable for pneumococcal virulence, which was significantly increased following invasive infections but with currently unknown regulatory mechanism. In our previous studies, DNA affinity purification assays (DNA pulldown) were performed to detect the candidate regulatory proteins that are bound to the promoter region of the cps operon in S. pneumoniae. Six proteins were identified as having significant protein scores by peptide mass finger printing. SPD_0064 is one of the proteins annotated as a transcription regulator in the genome sequence of S. pneumoniae D39. D39ΔSPD_0064 mutant was increased in the amount of capsular polysaccharide. Therefore, the present project is designed to analyze S. pneumoniae SPD_0064, a transcriptional regulator from the GntR family in the regulation of the cps operon and to understand whether SPD_0064 governs pneumococcal virulence by sensing the glucose in the surroundings. Electrophoretic mobility shift assay (EMSA) will be used to confirm the direct binding between SPD_0064 and the cps promoter, and DNase I footprinting assays are used to locate the specific binding site of SPD_0064 on the cps promoter. To characterize the regulatory mechanism of SPD_0064 protein on the transcription of the cps operon, qPCR and ELISA are used to compare the differences in cps transcription and CPS production among wild-type strain, ΔSPD_0064 mutant and complemented strain. In addition, bacteria will be grown in C+Y media with different concentrations of glucose, and qPCR and ELISA are used to compare the differences in cps transcription and CPS production among strains in order to clarify whether SPD_0064 is involved in the regulation of capsule production exposed to the glucose. Finally, flow cytometric assays and Western blotting will be carried out to illustrate the molecular mechanism of SPD_0064-mediated capsule production in pneumococcal virulence with both cellular and animal experiments. The experiments outlined in this proposal will provide fundamental information for understanding the role of SPD_0064-mediated capsule production in invasive pneumococcal diseases and will lay the groundwork for regulation of capsule biosynthesis as a theoretical basis for capsule industrial production and anti-virulence therapies against pneumococcal infection.

荚膜多糖是肺炎链球菌关键毒力因子，呼吸道定植菌侵袭感染后荚膜多糖含量明显增加，相关分子调控机制尚不明确。我们前期筛选到6个蛋白能与荚膜多糖操纵子启动子区结合，候选蛋白SPD_0064隶属GntR转录因子家族，缺陷后荚膜增加。本项目拟利用EMSA技术验证其与启动子结合并利用Dnase I足迹实验明确结合位点；利用qPCR和ELISA等技术对比分析野生株、缺陷株、回补株cps转录和荚膜含量辨明其调节荚膜多糖合成的分子机制；将细菌曝露于不同葡萄糖环境，通过分析SPD_0064含量变化、cps转录等明确葡萄糖是否通过SPD_0064调节荚膜多糖合成；最后，通过细胞和动物实验探讨荚膜多糖转录调控在肺炎链球菌侵袭致病中的作用。通过上述研究，试图阐明SPD_0064对荚膜多糖转录调控分子机制及其在肺炎球菌侵袭致病中的毒力机制，为从抗致病力治疗角度治疗肺炎链球菌感染和荚膜多糖工业化制备提供新的理论基础。

荚膜多糖是肺炎链球菌关键毒力因子，呼吸道定植的肺炎链球菌侵袭感染后荚膜多糖含量会明显增加，相关分子调控机制尚不明确。本项目的主要研究内容包括：1、肺炎链球菌SPD_0064蛋白对荚膜多糖操纵子的转录调控效应及其分子机制；2、葡萄糖调节荚膜多糖合成的分子机制；3、荚膜多糖合成与肺炎链球菌的侵袭致病分子机制。本项目的主要发现有：1、我们筛选获得了6个能与荚膜多糖操纵子启动子序列结合；2、SPD_0064属于GntR转录因子家族，能负性调节荚膜多糖合成相关基因转录，SPD_0064缺失株荚膜多糖含量明显增加；3、Dnase I足迹实验明确了SPD_0064与启动子序列结合的具体位点；4、通过进一步研究，我们发现了CpsR调节CPS操纵子转录的分子机理，通过EMSA研究，我们发现这一过程是通过葡萄糖作用来实现的，这种调节效应是首次在肺炎链球菌中得以明确的；5、SPD_0064介导的荚膜多糖合成在细菌侵袭致病过程中起重要作用。本项目的发现可以进一步深入了解葡萄糖碳源对肺炎链球菌致病的作用，为针对SPD_0064分子开展药物提供了理论依据；为生物制备肺炎链球菌荚膜多糖提供了理论支撑，具有较好的应用前景。目前，已发表研究论文1篇，相关研究成果已被他引10余次，并获得国家发明专利授权1次，培养硕士研究生2名，在读硕士生2名。综上所述，申请书中提出的研究目标已经顺利完成，我们预计进一步支持该领域的工作将继续增强我们对肺炎链球菌CpsR的分子和生理作用的认识。