RSH与CodY协同调控猪链球菌2型荚膜合成的分子机制研究

Streptococcus suis serotype 2 (SS2) is considered to be an important zoonotic pathogen. Capsule is one of the most important virulence factors in SS. Capsule synthesis could be regulated by environment, however, the mechanism is unknown. Previous studies on phenotypes have found that two important environmental regulator RSH and CodY co-regulate capsule synthesis in SS2. Because GTP may participate in expressional regulation of RSH and CodY, in order to study the molecular mechanism of their co-regulation on capsule synthesis, we intend to (1) identify the capsule synthesis related genes co-regulated by RSH and CodY; (2) confirm that whether GTP is mediated in the co-regulation of RSH and CodY on these genes; (3) study the roles of RSH on GTP level control and the roles of GTP on CodY regulation, reveal the tandem function of GTP on RSH and CodY co-regulation. This study will illuminate the molecular mechanism of co-regulation on capsule synthesis by RSH and CodY, and has important theoretical and practical significance in the identification of mechanism, development of new vaccines and discovery of new drug targets of infectious disease caused by SS.

猪链球菌2型（SS2）是一种重要的人兽共患病原菌，荚膜是其重要的毒力因子之一，荚膜合成受体内外环境等因素的调控，但相关调控因子调控荚膜合成的机制尚不十分清楚。申请人前期首次在表型上发现SS2中环境适应相关调控因子RSH和CodY协同调控了荚膜，本项目拟在此基础上研究该表型产生的机制。鉴于GTP可能同时参与RSH和CodY的调控作用，项目组拟首先筛选与鉴定RSH与CodY协同调控的荚膜合成基因，继而通过添加GTP合成抑制剂评估GTP是否参与RSH与CodY对以上基因的协同调控，进一步利用GTP水平检测、酶活检测和荧光蛋白报告系统等体内外试验研究RSH对GTP水平的调控和GTP在CodY调控荚膜合成基因中的作用，解析GTP如何串联RSH与CodY，阐明二者协同调控荚膜合成的分子机制，为猪链球菌毒力因子的表达调控机制研究积累科学数据，为新型疫苗和抗菌药物的设计与创制提供理论依据。

(p)ppGpp合成酶和CodY是细菌中两个重要的全局调节因子。在单核李斯特菌等病原体中，GTP连接这两个调节系统，在relA缺失菌株中继续缺失codY基因可以恢复弱毒relA缺失株的致病性。本研究在鉴定了猪链球菌的(p)ppGpp合成酶(RelA和RelQ)和CodY的基础上。为了进一步了解这两种重要的全局调控因子在猪链球菌中的相互关系，我们构建了ΔrelAΔrelQΔcodY三基因缺失突变体，并对其生长、形态和致病性进行了评价。结果表明，ΔrelAΔrelQΔcodY突变体生长缓慢，但链长部分恢复到野生株的状态，荚膜变厚和变粗糙。与ΔrelAΔrelQ、ΔcodY和WT相比，ΔrelAΔrelQΔcodY对小鼠体外全血杀伤的粘附、侵袭、抗性、致死性和定殖能力明显降低，与单核李斯特菌缺失株的特征不一致。表达调控分析表明，CodY可以不利依赖于GTP特异性结合relA启动子，并对relA的表达有正向调控作用，在relA缺失菌株中引入codY缺失进一步降低了毒力因子的表达，提示(p)ppGpp合成酶与CodY之间的关系与单核李斯特菌截然不同，而是通过CodY不依赖GTP正调控relA，介导二者的相互调控作用。本研究有助于我们进一步了解细菌中(p)ppGpp介导的严谨反应与CodY调控之间的关系。