猪链球菌AI-2信号分子受体蛋白介导的QS信号通路研究

AI-2 is an interspecies autoinducer and has been shown to regulate different bacterial physiology as well as affect virulence factor production when extracellular AI-2 combined with its receptor proteins. Our previous studies have already resolved the crystal structure of LuxS of streptococcus suis (SS) and the function of AI-2 was verified which mediates interspecies communication and facilitates regulation of bacterial virulence. However, it is still unclear how AI-2 uptake in the bacterial cell. In this study, we used the transposon mutagenesis technology to construct the SS library in which the AI-2 receptor proteins were screened. The functions of AI-2 receptor were verified through construction of the complemented strain of AI-2 receptor gene and double-gene deletion strain of luxS and AI-2 receptor gene. With DNA microarray and comparative proteomic, the target genes of SS AI-2 signaling pathway were screened. The purpose of this project is to identify the AI-2 receptor genes and target genes of SS AI-2 signaling pathway and elucidate the mechanism of the AI-2 uptake and regulation, which will help us to deeply understand LuxS/AI-2 quorum sensing system and provide the new theory basis for controlling SS infection. The discovery of AI-2 receptors is critical for the development of anti-QS agents that act on AI-2 signaling.

AI-2是细菌重要的种间信号分子，胞外的AI-2可与细菌受体蛋白结合后，转运到胞内，通过调控其下游信号通路来调控细菌毒力、生长等生理功能。项目组前期解析了猪链球菌（SS）LuxS蛋白晶体结构，发现其产生的AI-2分子参与对SS毒力等调控作用，但对其如何转运至胞内及信号转导的调控机制尚不明确。本研究以AI-2如何转运至胞内为切入点，拟通过构建SS转座子随机突变库，筛选鉴定猪链球菌AI-2的受体基因，构建受体基因互补株、luxS和受体基因双基因缺失株对其功能验证。并利用基因芯片和蛋白组学筛选AI-2及其受体基因介导的密度感应系统QS信号通路中下游靶基因，初步阐明LuxS/AI-2介导的QS信号通路。本研究旨在获知猪链球菌AI-2受体蛋白和由其介导的QS通路中靶蛋白，解析SS对AI-2的摄取及其介导的QS的分子调控机制，为寻找新型抗菌药物提供新靶点，也为控制SS感染提供新的思路。

AI-2是细菌重要的种间信号分子，胞外的AI-2可与细菌受体蛋白结合后，转运到胞内，通过调控其下游信号通路来调控细菌毒力、生长等生理功能。项目组前期解析了猪链球菌（SS）LuxS蛋白晶体结构，发现其产生的AI-2分子参与对SS毒力等调控作用，但对其如何转运至胞内及信号转导的调控机制尚不明确。通过 RNA-seq 技术分析了外源添加AI-2、缺失luxS基因后对猪链球菌转录组的调控作用，找到了一系列可能的AI-2受体蛋白。并通过构建的猪链球菌mini Tn4001转座突变库，筛选到46个AI-2信号分子变化的突变菌株。综合以上组学数据，找到了共同的4个可能的AI-2受体蛋白，并构建了其中pdh和otc的基因缺失株，验证了其可能的生物学功能。通过相关组学研究筛选鉴定AI-2受体基因介导的密度感应信号通路中下游靶基因，初步阐明猪链球菌LuxS/AI-2型密度感应系统分子调控机制。