双功能小RNA rss03促进猪链球菌在猪血中存活的分子机制

Streptococcus suis (SS) is a major bacterial pathogen of pigs worldwide. It is also an emerging zoonotic agent. Septicaemia and meningitis are common features of the infection. To cause infection and diseases, SS must survive in host blood, but little is known about the mechanisms of SS survival in pig blood. Dual-function small RNAs (sRNAs) have emerged as key regulators of virulence in several bacteria. They not only function as riboregulators that base pair with and regulate translation or stability of mRNA targets, but also encode polypeptides. However, at present, there is no report about SS dual-function sRNAs. In the previous study, we have showed that dual-function sRNA rss03 contributes to SS survival in pig blood. In this project, rss03 targets involved in SS survival in pig blood will be identified. To reveal regulation mechanisms of rss03 on targets as a riboregulator, the effects of rss03 on targets mRNA stability and translation will be evaluated. To illustrate the roles of rss03 polypeptide in SS survival in pig blood, the orthologues of rss03 polypeptide will be searched in other bacteria, and the conserved amino acids that are essential for SS survival in blood will be revealed. Interaction partners of rss03 polypeptide will be identified, and the effect of polypeptide on the protein stability of interaction partners will be demonstrated. These findings not only contribute to further understanding the SS pathogenesis, but also increase the knowledges of dual-function sRNAs functions and regulation mechanisms of sRNAs.

猪链球菌（SS）可引致猪及人败血症、脑膜炎等，在血液中存活对SS致病至关重要，但是在血液中存活机制还缺乏了解。细菌双功能小RNA是一种新型毒力调控因子，不仅可以RNA调节子形式调控靶位mRNA稳定性或者翻译，还可以编码多肽。目前尚未见SS双功能小RNA的报道。申请者前期研究发现，双功能小RNA rss03促进SS在猪血中存活。本项目拟鉴定rss03与SS在猪血中存活相关的直接靶位，分析rss03对靶位稳定性及翻译水平的影响，阐明rss03以RNA调节子调控靶位的机制；寻找rss03多肽在其它细菌中的同源蛋白，确定该多肽与SS在猪血中存活相关的关键氨基酸；筛选与多肽互作蛋白，明确多肽在蛋白水平对互作蛋白稳定性影响，揭示多肽促进SS在猪血中存活的途径。本研究不仅有助于进一步揭示SS致病机理，为防控该类疫病提供新视角，而且为细菌双功能小RNA生物学功能提供新参考，丰富细菌小RNA调控毒力机制。

猪链球菌是一种革兰阳性致病菌，可引致猪及人败血症、脑膜炎等多种疾病，目前对其致病机制还缺乏了解。近年来研究发现细菌小RNA是一种新型毒力调控因子。申请者前期研究发现，小RNA rss03促进猪链球菌在猪血中存活，且该基因缺失后猪链球菌毒力降低，但其靶位和调控毒力机制未知。生物信息学分析发现，rss03分布于猪链球菌多种血清型及其亲缘关系较近的副猪链球菌和反刍链球菌，且在猪链球菌2型中rss03在基因组中位置和序列较为保守，提示其在猪链球菌2型中具相似的生物学功能。结合CopraRNA软件预测、MS2亲和纯化结合RNA测序方法（MAPS）以及蛋白组学筛选rss03靶位，并通过凝胶阻滞试验，明确了rss03可通过结合多个直接靶位基因发挥调控作用，并且发现MAPS在猪链球菌sRNA靶位筛选中具有明显优势。通过凝胶阻滞、mRNA稳定性、氧化应激存活、胞内H2O2含量测定、小鼠体内感染、巨噬细胞胞内存活等一系列体内外实验，发现rss03可通过促进其直接靶位甘油通道蛋白glpF mRNA稳定性，进而有助于猪链球菌摄取甘油和外排H2O2，促进猪链球菌在宿主体内的存活。rss03还可通过正调控其间接靶位铁硫簇合成基因簇sufCDSUB的蛋白水平，增强猪链球菌在限铁条件下存活和抗氧化应激能力。此外，rss03还可以通过抑制转录调控因子glpR的蛋白水平促进猪链球菌对甘油的摄取和抑制转录调控因子deoR的蛋白水平进一步促进glpF的表达。而且，还发现了rss03候选靶位碳酸酐酶（SCA）、YbaB/EbfC家族核苷相关蛋白（YED）和2-氨基-4-羟基-6-羟甲基二氢蝶啶二磷酸激酶（AHH）具有促进猪链球菌抗氧化应激能力。本研究揭示了小RNA rss03不仅可通过增强猪链球菌抗氧化应激能力，从而促进其在血液中存活，还可通过摄取甘油和增强猪链球菌在限铁条件下的存活能力促进其毒力。上述研究不仅将猪链球菌致病机制的视野提升到新的高度，而且丰富了细菌小RNA调控毒力机制。项目执行过程中共发表8篇标注的论文，其中SCI论文3篇，中文核心期刊论文5篇。