猪链球菌小RNA rss04促进脑膜炎形成的分子机制

Streptococcus suis (SS) is considered to be a major Gram-positive pathogen worldwide in the swine industry and is also an important zoonotic agent for human. Meningitis is one of the most common features of the infection, but little is known about the mechanisms of SS meningitis. Recent studies have revealed that small RNAs (sRNAs) has emerged as key regulators of virulence in several bacteria. However, at present, there is no report about the mechanism how sRNAs regulate SS virulence. In previous study, we showed that SS sRNA rss04 was up-regulated in pig cerebrospinal fluid and rss04 deletion mutant attenuates SS virulence in a zebrafish infection model. Furthermore, rss04 contributes to SS meningitis in mouse and pig infction models. In this project, to find out the way by which rss04 contributes to meningitis, we will explore the functions of rss04 during SS interaction with mouse brain microvascular endothelial cell, microglia and neutrophil, and evaluate the effects of rss04 on SS capsule production and replication in pig cerebrospinal fluid. MS2 tagging purification, in combination with transcriptome and proteome analysis and bioinformatics prediction, will be performed to identify the rss04 targets involved in meningitis process and RNA binding proteins. To reveal the regulation mechanisms of rss04 on targets, the effects of rss04 on targets mRNA stability and translation will be evaluated, and the binding regions between rss04 and targets and roles of RNA binding proteins in regulation will be determined. These findings not only contribute to understanding the SS meningitis mechanism, but also increase the knowledges of the RNA binding proteins involved in sRNAs regulation and the mechanism of sRNAs regulation in Gram-positive bacteria.

猪链球菌（SS）是一种革兰氏阳性致病菌，可引致猪及人脑膜炎等多种疾病，目前对其脑膜炎形成机制还缺乏了解。近年来研究发现细菌小RNA是一种新型毒力调控因子，但尚未有其调控SS毒力机制的报道。申请者前期发现SS小RNA rss04在猪脑脊液中上调表达，促进猪和小鼠脑膜炎形成，该基因缺失后SS毒力降低。本项目通过探讨rss04在SS与小鼠脑微血管内皮细胞和免疫活性细胞互作中发挥的生物学功能，并分析其对SS荚膜形成能力和在猪脑脊液中增殖能力的影响，揭示rss04促进脑膜炎形成的途径；采用MS2识别序列纯化技术，结合转录组、蛋白组及生物信息学等，鉴定rss04靶位与RNA结合蛋白；分析rss04对靶位稳定性及翻译水平的影响，确定与靶位结合区域和RNA结合蛋白的功能，阐明rss04调控靶位机制。该研究有助于理解SS引致脑膜炎机制，也为革兰氏阳性菌RNA结合蛋白功能及小RNA调控靶位机制提供新的参考。

猪链球菌是一种革兰氏阳性致病菌，可引致猪及人脑膜炎等多种疾病，目前对其脑膜炎形成机制还缺乏了解。近年来研究发现细菌小RNA是一种新型毒力调控因子，但尚未有其调控猪链球菌毒力机制的报道。前期发现猪链球菌小RNA rss04在猪脑脊液中上调表达，促进猪和小鼠脑膜炎形成，该基因缺失后猪链球菌毒力降低。通过小鼠体内攻毒、比较蛋白组学分析、免疫转印、电镜观察、斑点杂交、小鼠脑微血管内皮细胞黏附与侵袭等一系列体内外实验，发现猪链球菌小RNA rss04不仅可通过抑制转录调控因子CcpA蛋白水平调控荚膜产量，从而促进猪链球菌黏附和侵袭小鼠脑微血管内皮细胞及在小鼠脑中激发更强的炎症反应，rss04还可通过正调控LuxS蛋白水平，从而影响生物被膜形成，促发脑膜炎。采用MS2识别序列纯化技术，成功建立筛选猪链球菌RNA结合蛋白的方法，结合生物信息学预测，发现9个候选RNA蛋白，为革兰氏阳性菌RNA结合蛋白功能的研究提供参考。另外，建立的小鼠蛛网膜下腔注射方法，不仅明确生物被膜在猪链球菌导致脑膜炎过程中发挥重要的作用，进一步证实猪链球菌小RNA rss04调控生物被膜从而影响脑膜炎进程。而且，与其他感染途径相比，该方法具有明显的优势：它不仅可以用于评价菌体成分在脑部对宿主免疫应答产生的影响以及模拟猪链球菌入侵中枢系统；还可以用来研究其他细菌性病原引致脑膜炎，评价抗细菌性脑膜炎药物的治疗效果。rss04是细菌小RNA研究中第一个明确与脑膜炎进程相关的sRNA，研究结果有助于理解猪链球菌引致脑膜炎机制，将猪链球菌致病机制的视野提升到新的高度。项目执行过程中共发表8篇标注的论文，其中SCI论文6篇，中文核心期刊论文2篇。