LPxTG酶突变在B族链球菌CC17侵袭性中的功能及机制研究

CC17 is the most popular invasive GBS, The exactly molecular mechanism is not clear. By sequence analysis of multiple invasive GBS strains, and found that several SNPs in genes of the LPxTG enzyme named SAP and ScpB were significantly different from the colonized strains. Bioinformatics analysis indicated that the mutant amino acids occurred in the core domains of SAP and ScpB, Evolutionary analysis showed that the mutant SNP strains aggregated into clade1，all stains are CC17 in clade1, and the mutated gene sequence is totally consistent with the popular invasive strain COH1. It is hypothesized that the LPxTG enzyme mutation affects the invasive function of CC17 and the SNPs are related to the evolution. In this study, six mutant strains were constructed by using COH1 and A909 as recipient strains, and the invasive differences of mutant strains in in vitro functional experiments and in vivo experiments were compared, and the specific functions of LPxTG enzyme mutations in CC17 invasion were clarified. Revealing the mechanism of joint mutation of SAP and ScpB leading to the invasion of CC17; continue to complete the local saved GBS strains genome sequencing, establish the published GBS-WGS database, and clarify the specific characteristics of LPxTG enzyme gene SNPs in GBS epidemic and evolution, To provide evidence for the study of CC17 pathogenesis.

CC17是最主要流行的侵袭性GBS群，其致病的确切分子机制尚不明确。课题组前期完成多个GBS侵袭株测序，发现LPxTG酶SAP和ScpB基因的SNP与定植株差异显著，生物信息学分析提示突变氨基酸发生在SAP和ScpB的核心结构域，进化分析显示突变SNP株聚集为一个Clade1，均为CC17，突变基因序列与高侵袭流行株COH1比对高度一致，提出LPxTG酶突变影响CC17侵袭性功能并与其遗传进化有关假说。本研究拟以COH1和定植株A909为受体菌构建6种突变菌株，比较突变菌株在体外功能实验及小鼠体内实验中的侵袭性差异，明确LPxTG酶突变在CC17侵袭性中的具体功能，揭示SAP和ScpB联合突变导致CC17侵袭性增强的机制；继续完成本地已保存GBS全基因组测序，建立已公布GBS-WGS数据库，阐明LPxTG酶基因SNP在GBS流行与进化中的具体特征，为研究CC17致病机制提供有力证据。

CC17是最主要流行的侵袭性GBS群，其致病的确切分子机制尚不明确。课题组前期完成多 个GBS侵袭株测序，发现LPxTG酶SAP和ScpB基因的SNP与定植株差异显著，生物信息学分析提示 突变氨基酸发生在SAP和ScpB的核心结构域，进化分析显示突变SNP株聚集为一个Clade1，均为 CC17，突变基因序列与高侵袭流行株COH1比对高度一致，提出LPxTG酶突变影响CC17侵袭性功能并与其遗传进化有关假说。课题期间完成阐明不同LPxTG酶突变（SAP）的标准菌株（COH1和A909）体外功能，部分结果有显著差异。完成对T7Iq:pET28a:sap(COH1); T7Iq:pET28a:sap(A909)两个菌株的较大培养量（1L）的SAP蛋白提取和完成后续纯化（采用FPLC平台），进行纯化蛋白的体外功能试验DNS，显示纯化蛋白具有相应功能，并显示出功能有差异，P＝0.0003。建立SAP蛋白提取及纯化平台，并进一步从蛋白层面证实不同SNP模型的SAP体外功能具有差异在菌株层面和纯化蛋白层面的体外实验验证LPXTG相关的毒力因子SAP功能研究。构建完成本地GBS-WGS检测平台和生物信息分析平台，选取本地侵袭性GBS菌株共111株，2021年孕产妇定植GBS菌株120株及早期定植菌株22株，共计253株进行WGS, 目前已完成133株GBS数据本地化存储与分析，其中侵袭性菌株全部完成生物信息分析。完成构建既定GBS菌株基因组的同线性分析模型及特异性SNP位点的定位，完成构建既定GBS菌株基因组的进化树结合其它重要信息的集成可视图形化分析模型。并完成建立网页版可视化生物信息分析，明确LPxTG酶突变SNP在GBS中流行分布规律和遗传特征，为研究CC17致病机制提供有力证据。