单核细胞增生李斯特菌RsbS通过调控SigB介导抗应激机制研究

Listeria monocytogenes (L. monocytogenes) is a foodborne and zoonosis bacterium, which can survive in various food and environments due to its strong stress-resistance. Human and animal listeriosis manifested as gastroenteritis, meningitis and encephalitis. The general stress-responsive alternative sigma factor sigma B (σB), which is the principal stress regulator of L. monocytogenes, plays a pivotal role in its survival and infection. Rsbs are new-found uptream regulators of σB, which control σB activation and inactivation. In our previous study, we demonstrated that RsbX is a negative regulator of σB during the recovery period after the primary stress or in the stationary phase. In this study, we will focus on the function of the regulator RsbS in stress resistance and its regulatory mechenism on σB. The rsbS deletion and complement mutants will be constructed. Phenotypes of stress resistance of these mutants will be analyzed. Promoter activity of σB will be evaluated by gfp reporter system. Binding ability of RsbS and σB promoter region will be tested by EMSA. The transcriptional and translational analysis will be carried out to determine σB expression in the presence/absence of RsbS. The Site-directed mutagenesis，phosphorylation and dephosphorylation tests and Protein to protein interactions will be performed to indenify mechanisms of RsbS on σB regulation. Our study is valuable to clarify the regulatory networks controlling stress resistance of L. monocytogenes, which is helpful to the establishment of new techniques for foodborne pathogen control.

单核细胞增生李斯特菌是重要的人兽共患食源性病原菌，依靠极强的抗应激能力存在于食品、环境中，引起人和动物的胃肠炎、脑膜炎、流产等，危害极大。Sigma B（σB）为李斯特菌最重要的应激调控因子，对其存活和感染至关重要。Rsbs调控操纵子是近年来发现的一串σB上游调控子，调控σB的激活和失活。我们已证实调控操纵子RsbX在应激后恢复期和生长平台期负调控σB。在此基础上，本项目将重点研究调控操纵子RsbS的抗应激功能及其通过调控σB介导的抗应激机制。本研究利用基因缺失和回补技术，研究RsbS对细菌抗应激表型的影响；综合运用报告系统、凝胶迁移、转录和蛋白水平检测研究应激应答和失活过程中RsbS对σB启动子和表达水平的影响；利用定点突变、磷酸化/去磷酸化检测和蛋白互作等方法研究RsbS参与调控σB的机制。本研究对于阐明李斯特菌抗应激调控网络意义重大，为建立食源性病原菌的防控新技术打下基础。

单核细胞增生李斯特菌是重要的人兽共患食源性病原菌，能抵抗自然环境、食品加工环境和宿主胃肠道中各种应激，最终建立感染。Sigma B（σB）作为单增李斯特菌最重要的应激调控因子，亦受到其调控操纵子Rsbs的精密调控。我们在前期研究中发现，调控操纵子RsbX在应激后恢复期和生长平台期负调控σB。在此基础上，本项目重点研究了调控操纵子RsbS在抗应激过程中的作用及其通过调控σB介导的抗应激机制。本研究利用基因缺失和回补手段，研究了RsbS对细菌抗应激表型的影响；利用gfp报告系统研究了RsbS缺失对σB启动子活性的影响；利用转录水平和蛋白水平的分析研究应激应答中RsbS对σB表达水平的影响；利用定点突变、磷酸化/去磷酸化检测和蛋白互作等方法研究RsbS参与调控σB的机制。结果表明，RsbS通过负调控σB参与单增李斯特菌的抗应激过程；RsbS与RsbR存在体外互作，且RsbR-T205可能是关键的结合位点；RsbX可对RsbS发生体外去磷酸化作用。本研究对于阐明李斯特菌抗应激调控网络以及食源性病原菌的防控具有重要意义。