DNA结合蛋白XtgS调控无乳链球菌毒力基因的分子机制

DNA-binding proteins, widely existing in bacteria, are important transcription factors which could modulate the transcription of virulence related genes. Streptococcus agalactiae has been recognized as one of the major causes of pneumonia, meningitis in neonates, mastitis in cows and meningoencephalitis in fish. In our previous research, a novel DNA binding protein, named XtgS, was found in the S. agalactiae strain GD201008-001. In order to elucidate the molecular mechanism of this DNA binding protein XtgS in regulating the S. agalactiae virulence genes, a gene deletion mutant of XtgS will be constructed. The effect of XtgS in bacteria virulence will be evaluated and the differences on gene expression between wild strain and mutant will be screened by RNA sequencing. Moreover EMSA and ChIP will be performed to find the target sequences combined with XtgS. This research will for the first time articulate clearly the relationship between S.agalactiae virulence and the DNA binding protein. The findings will increase our knowledge about the pathogenesis of S. agalactiae and contribute to the prevention and control of this bacteria.

DNA结合蛋白广泛存在于细菌中，是重要的转录调节因子，调控毒力相关基因的转录。无乳链球菌是一类人、牛、鱼共患的重要病原，可引起人类新生儿败血症、脑膜炎，奶牛乳腺炎以及鱼类的脑膜脑炎。申请人前期在无乳链球菌GD201008-001基因组中发现一个新的DNA结合蛋白XtgS，该蛋白对无乳链球菌毒力基因的调控有待阐明。本课题通过构建基因缺失株△XtgS，评价缺失对无乳链球菌的毒力影响；同时应用转录组测序，分析亲本株与缺失株的基因转录的差异；并通过EMSA和ChIP试验，精确定位XtgS所结合的靶基因序列，进而阐明XtgS调控该菌毒力基因的分子机制。本项目将首次明确无乳链球菌毒力与DNA结合蛋白的关系，进一步揭示无乳链球菌的分子致病机理，为该病原的防控提供理论依据。

申请人前期在无乳链球菌GD201008-001基因组中发现一段10bk特有序列，这段序列中存在一个DNA结合蛋白XtgS，推测该蛋白在该10kb中发挥重要作用，参与无乳链球菌的毒力基因调控。本课题通过构建基因缺失株△10 kb, 缺失株△XtgS 和互补株 C△XtgS，分析比较各菌株的生物学形状和毒力变化，明确XtgS蛋白的缺失可明显增强无乳链球菌的毒力(P<0.001)；并应用转录组测序，分析过表达XtgS的菌株A909XtgS与亲本株的基因转录的差异，初步确定可能受XtgS 蛋白调控的基因；后续通过Promoter-pTCV-lacZ质粒的β-半乳糖苷酶活性比较，EMSA等试验，精确定位XtgS所结合的靶基因序列，确定XtgS蛋白直接结合在一个靶基因（A964_1958）启动子的回文序列上，阻遏该基因的转录。同时XtgS蛋白还间接阻遏无乳链球菌2个转铁系统的转录。综合以上研究进展，本项目首次阐明XtgS蛋白的调控作用和结合的靶序列，初步证实该DNA结合蛋白与无乳链球菌毒力的关系，为后续深入研究调控基因的功能，进一步揭示无乳链球菌的分子致病机制奠定基础。