无乳链球菌GD201008-001二元调控系统RscSR的鉴定及其对细菌应激适应性和毒力特性的影响  

Identification of two-component system RscSR in Streptococcus agalactiae GD201008-001 and its influence on the bacterial stress response and virulence

作　　者：聂蒙 马可[1] 曹青[1] 黄昊 姬姝婷 刘永杰[1] NIE Meng;MA Ke;CAO Qing;HUANG Hao;JI Shuting;LIU Yongjie(College of Veterinary Medicine,Nanjing Agricultural University,Nanjing210095,China)

机构地区：[1]南京农业大学动物医学院,江苏南京210095

出　　处：《水产学报》2021年第9期1545-1554,共10页Journal of Fisheries of China

基　　金：江苏省自然科学基金(BK20171376);江苏高校优势学科建设工程项目(PAPD)。

摘　　要：为鉴定鱼源无乳链球菌GD201008-001二元调控系统(two-component system,TCS)RscSR并探究其功能,实验利用生物信息学方法预测到可能的RscSR,对其组成成分RscS和RscR的三维结构和保守结构域进行分析;构建基因缺失株ΔrscSR与互补株CΔrscSR,测定细菌的生长曲线,检测其耐酸应激、耐氧化应激、抗巨噬细胞吞噬和胞内存活能力,同时测定其对巨噬细胞的毒性和对小鼠的毒力。结果显示,RscSR具有典型的TCS结构特点,其中RscS具有组氨酸激酶结构域,RscR具有反应调节子结构;其编码基因缺失后,菌株耐酸、耐氧化、抗巨噬细胞吞噬和胞内存活能力及对巨噬细胞毒性均显著降低,而将该基因回补后各项能力均有所恢复;动物实验结果显示,野生株感染小鼠19 h内全部死亡,而ΔrscSR缺失株感染小鼠全部死亡时间为48 h。研究表明,RscSR在无乳链球菌应激适应性以及毒力方面发挥重要作用。Streptococcus agalactiae infectious disease outbreaks have occurred continuously in Oreochromis spp.farms in China since 2009. In order to further explore the regulation mechanism of S. agalactiae virulence, the function of a putative two-component system(TCS) RscSR was investigated in a highly virulent strain GD201008-001, which was isolated from moribund cultured tilapia with meningoencephalitis in Guangdong Province of China in 2010. The 3-D structures and conserved domains of histidine kinase RscS and response regulator RscR were predicted using the I-TASSER server(https://zhanglab.ccmb.med.umich.edu/I-TASSER) and SMART(https://smart. embl.de). The rscSR deletion mutant strain ΔrscSR was constructed using homologous recombination. Bacterial tolerances to acid or oxidative stress were investigated by pre-exposing to pH 5.0 or 20 mmol/L H2O2. RAW264.7 macrophages were used as cell models to evaluate the bacterial anti-phagocytosis ability, intracellular survival ability and cytotoxicity. The bacterial pathogenicity was then tested in adult mice. The bioinformatics prediction showed that RscS and RscR were a typical histidine kinase and a response regulator, respectively.The stimuli sensed by RscS might be membrane-associated or occur directly at membrane interface;RscR was predicted to bind DNA to directly affect the transcription of the target gene. Compared with the wild-type and complemented strains, the resistance to acid(pH 5.0) of ΔrscSR was significantly decreased(2.83 times and 1.65 times),and furthermore, its resistance to H2O2 was also significantly decreased(1.93 times and 1.77 times). The ΔrscSR mutant displayed significantly decreased resistance to being ingested by macrophages(2.81 times and 1.56 times)compared to that of the wild-type and complemented strains. Moreover, at 3 h after being taken up by the macrophages, the intracellular survival rate of the ΔrscSR mutant was only approximately 44%;in contrast, it was 60%both for the wild type and complemented strains. After 6 h of phagocytosis,

关 键 词：无乳链球菌 二元调控系统 耐酸应激 耐氧化应激 抗吞噬能力 毒力 

分 类 号：S941.4[农业科学—水产养殖]