非编码RNA参与2型猪链球菌89K毒力岛致病作用的分子机制研究

Streptococcus suis 2 (S. suis 2, SS2) has evolved into a highly infectious entity, which is responsible for a wide range of serious diseases in both piglets and humans, including meningitis, septicemia, arthritis, etc. Two recent large-scale outbreaks of SS2 infections in China (one in Jiangsu Province, 1998, and the other in Sichuan Province, 2005) were characterized by streptococcal toxic shock syndrome (STSS), and raised significant international concern among the public health professionals. However, the molecular pathogenesis of SS2-caused disease is still poorly understood. The smallnoncoding RNA（sRNA）acting by base-pairing with target mRNAs, resulting in post-transcriptionally regulating gene expression, is the important regulator in the bacterial response to stress, virulence and metabolism.To shed light on the mystery of high virulence of the epidemic outbreak strains of SS2, our joint research group completed a comprehensive study of comparative genomics, decoding the whole genome sequences oftwo virulent SS2 strains(98HAH12 and 05ZYH33) isolated from Chinese infected patients（Plos One，2007）. We analyzed the genome sequences of four classic SS2 strains (98HAH12,05ZYH33,S735,P1/7)and identified a series of sRNA genes and validated the expression of most of them. Screening by RT-PCR analysis revealed that four out of the five sRNA candidates specific to the Chinese endemic strains were transcribed, from which two candidates (SR36 and SR37) are located in IGRs of PAI89K. We thus investigated the possible role and molecular mechanism of the PAI89K specific sRNAs in virulence control and environment adaptation. we'd like to carry out a comprehensive study on contribution of sRNAs to the biological function of PAI89K island， and to elucidatethe molecular mechanism of sRNAs in regulation to environment adaptation and pathogenicity of highly pathogenic SS2 strain,through a series of experimental analhyses by using gene knockout tcchnique, high-throughput sequencing of the transcriptome(RNA-seq)， electron microscopy studies,molecular and cellucular assay,and animal infection experiments. Our study will facilitate further understanding of SS2 pathogenesis and points to directions of development on some effective strategies to combat highly pathogenic SS2 infections in China.

2型猪链球菌（SS2）是一种危害严重的人畜共患病原菌，江苏和四川曾暴发较大规模的猪群及从业人群感染，流行株高致病的机制尚未阐明。非编码小RNA(sRNA)是一类新型调控分子，与细菌环境适应、毒力调控等密切相关。课题组前期发现国内SS2强毒株携带特有的89K毒力岛（PLOS ONE，2007），对4株SS2全基因序列综合预测，在89K岛核心功能区发现2个候选sRNA（SR36、SR37），可能参与该岛致病性等功能的调控。本研究拟荧光定量PCR分析候选sRNA及其潜在靶基因转录水平与环境压力的关联，构建候选RNA及转录显著关联靶基因的缺失株和互补株，系统比较突变株与野生株在溶血黏附等生物特性、与宿主免疫杀伤细胞相互作用、模型动物致病性等方面的差异；RNA-seq系统分析突变株与野生株转录谱的差异，探索sRNA 在SS2环境适应中的作用，初步解析sRNA参与89K岛致病作用的分子调控机理。

SS2是一种重要的人兽共患病病原菌，给养猪业造成巨大经济损失。SS2感染的临床症状有脑膜炎，心内膜炎，关节炎，急性感染时甚至猝死。然而SS2的致病机制仍然未知。细菌sRNA能参与细菌细胞膜的重排、生物膜的形成、糖代谢、铁离子稳态维持和氨基酸代谢等。在病原细菌中，sRNA还参与毒力调控。在2型猪链球中鲜有关于sRNA的报道。因此本研究旨在发现新sRNA分子，并探讨新sRNA分子与细菌致病性的关系。本研究主要取得了一下进展：.1 SS2 05ZYH33中新sRNA分子的预测和鉴定：生物信息学预测并筛选得到了40个候选sRNA分子。RT-PCR和Northern blot验证了其中20个sRNA分子的表达。BLASTN对其做了同源性检测。RNAfold预测了这20个sRNA分子的二级结构。TargetRNA2对9个新鉴定的sRNA分子进行靶标预测的结果表明，sRNA的靶标分布很广泛，包括DNA合成酶，氨基酸代谢，糖代谢以及细菌的毒力相关的基因。.2 SsRNA34特征鉴定以及敲除株和互补株的构建：SsRNA34为基因间区的sRNA，全长扩增发现SsRNA34的全为长526 nt。同源性检测发现SsRNA34在猪链球菌中分布广泛。为对sRNA34进行后续的功能研究，构建了同源重组敲除质粒pUC18-SsRNA34。电转化敲除质粒pUC18-SsRNA34到05ZYH33的感受态细胞，筛选得到敲除株∆SsRNA34。构建了回复突变质粒pVA838-SsRNA34。电转化穿梭质粒pVA838-SsRNA34到∆SsRNA34感受态细胞，构建了回复突变株为C∆SsRNA34。.3 SsRNA34对细菌表型和毒力的影响：生长曲线表明∆SsRNA34比野生株慢。革兰氏染色发现∆SsRNA34的链长明显增长。体外毒力实验发现∆SsRNA34抗小鼠巨噬细胞RAW264.7的吞噬明显减弱，小鼠存活实验和小鼠竞争性感染实验表明，∆SsRNA34毒力显著低于野生型。.4 SsRNA34靶标预测：RNA-seq分析发现SsRNA34缺失后，141个基因的表达发生了明显的变化，其中70个基因发生上调，71个基因的表达发生下调。RT-PCR证实了组蛋白乙酰转移酶HPA2和谷氨酰胺代谢相关的基因GlnP/Q的下调表达，这三个基因的表达变化倍数最多, 提示SsRNA34可能对这三个基因有直接的调控关系。