精氨酸抑制因子ArgR与应激调控因子SigB介导的单增李斯特菌抗酸应激调控机制研究

Listeria monocytogenes (Lm) is a Gram-positive, foodborne, zoonosis and intracel-lular bacterium, which can survive and replicate in mildly and lethally acidic environ-ments or within the host. The stress regulatory factor SigB and arginine deiminase (ADI) system in Lm have been found to play critical roles in acid tolerance and adaptations to environmental stress. Lm arginine repressor (ArgR) contributes to regula-tion of arginine metabolism by binding to operator sites (ArgR box), leading to repression of arginine biosynthetic genes and activation of catabolic genes in the presence of arginine. In previous studies, we found the SigB and ArgR boxes both exist in the promoter regions of SigB and ADI system related genes. However, the regulatory mechanisms mediated by ArgR and SigB in acid tolerance of Lm remain unknown. Therefore, the roles of proteins involved in ArgR and SigB regulatory network will be characterized by protein-protein, protein-DNA interactions, and enzymatic activity analysis as well. To this end, a number of deletion mutants and genetic replacement mutants for key stress-related regulatory genes will be constructed. The phenotypes under growth conditions, virulence to epithelial cells and mice, and intracellular pH (pHi) dynamic changes of the mutant strains under various acidic environments will be further investigated. The ultimate goal of this study is to elucidate central roles of the regulatory network between ArgR and SigB in listerial acid responses and the mechanism causing foodborne infection. This will provide insights into the role of ArgR and SigB in different physiological situations, in vitro and in vivo host niche acidic environments, underscoring the central role of ADI system in foodborne disease. It will be helpful in future pursuit to identify potential interacting partners of arginine metabolic enzymes to uncover novel regulatory and signaling net-works controlling acid stress and adaptations of intracellular pathogens.

单增李斯特菌是重要的人兽共患食源性胞内寄生菌，可以在宿主消化道等体内外酸应激环境下长期存活，并能通过抗酸基因簇精氨酸脱亚胺酶系统（ADI）和应激调控因子SigB调控细菌对环境应激的适应性。ArgR为李斯特精氨酸合成抑制因子，在SigB以及ADI系统相关基因的启动子区域存在SigB box和ArgR box。SigB以及ArgR是如何互相调控并影响细菌的抗酸应激呢？分子作用机制如何？为此，我们将通过缺失株构建、蛋白互作与突变分析、酶学活性分析、质子跨膜流动监测以及ArgR和SigB蛋白对拟调控基因启动子区的识别和结合分析等技术，系统地研究单增李斯特菌ArgR与SigB之间的调控关系及其分别在酸应激环境以及消化道和巨噬细胞内对相关酸耐受通路的调控机制，并最终阐明以ArgR和SigB为中心的抗酸应激调控网络和单增李斯特菌经由消化道感染的传播机制。本研究将为食源性病原菌的防控提供理论依据。

单核细胞增多性李斯特菌是自然界广泛存在的食源性病原菌，可以突破胃肠道、血脑和胎盘屏障。在体外生存及体内感染过程中，李斯特菌会遭受到诸多不利应激因素，尤其是酸性环境应激。李斯特菌主要依赖多种机制协同参与抗酸应激，并成功建立感染。本项目主要研究了李斯特菌应激调控因子SigB与精氨酸抑制因子ArgR对酸应激相关基因的调控机制和网络。ArgR属于ArgR/AhrC转录调控因子家族，参与精氨酸代谢的调控。ArgR与其他同源蛋白的亲缘性比较近，且存在该家族蛋白两个重要的结构域（启动子结合区“SR”和精氨酸结合区“GTIAGDTT”）。ArgR与细菌的酸应激存活能力密切相关，ArgR缺失后导致细菌在致死性酸应激下的存活能力显著增强。EMSA显示，ArgR能够与arcA、sigB、argC和argG启动子区的ArgR box发生结合，结合能力存在明显的差异，即argC≥argG>sigB>arcA。ArgR的“SR”氨基酸置换（S42A和R43A）后与被调控基因启动子的结合能力明显减弱或丧失，证实这两个位点为ArgR与启动子结合的关键氨基酸。酸应激显著上调argR的转录水平，argR缺失显著提高argG的转录和蛋白表达量，表明其对精氨酸合成起负调控作用。酸应激条件下argR缺失株的arcA转录和表达水平显著提高，同样提高的还有sigB转录和表达水平，初步表明ArgR也负调控精氨酸的分解代谢，但是由于arcA的表达同样受sigB正调控，ArgR对arcA的负调控并不一定是直接作用，可能是通过sigB起作用。当添加外源性精氨酸后，argG在应激和非应激下的转录和表达同样受ArgR显著抑制，而ArgR对sigB和arcA的抑制效应却解除。本研究表明ArgR在非应激和酸应激状态下能够反馈负调控精氨酸合成代谢通路基因argC和argG，且首次发现ArgR缺失能够增强细菌的酸应激存活能力，并首次证实该变化是由于ArgR参与负调控抗酸应激功能和调控基因（arcA和sigB）的转录及表达而引起。本项目系统研究了李斯特菌SigB与精氨酸抑制因子ArgR间的互作机制及对酸应激相关基因的调控机制关系，为进一步深入探索李斯特菌经消化道感染的抗应激和传播机制奠定了重要基础，对于防控李斯特菌及其它食源性病原微生物具有重要意义。