单核细胞增多性李斯特菌代谢物控制蛋白CcpA介导的胞内pH稳态调控机制研究

Listeria monocytogenes (L. monocytogenes) is a Gram-positive, foodborne, zoonosis and intracellular bacterium, which can survive for long periods of time in acidic conditions. The arginine deiminase (ADI) and agmatine deiminase (AgDI) pathways play critical roles in respond to acid tolerance and adaptations. The promoter regions of the ADI/AgDI controlled clusters harbor targeted CcpA (catabolite control protein) box, σB (sigma B) box and AhrC (arginine repressor) box sequences. We showed previously that σB and AhrC were primarily responsible for survival of L. monocytogenes at low pH stress. Therefore, CcpA, the other key regulator of ADI/AgDI system will be characterized in this study. The mutants corresponding to ccpA, ccpA/ahrC and ccpA/σB will be constructed. Phenotypes and intracellular pH of these mutants will be analyzed. Promoter activity will be evaluated by gfp3 and gus reporter system in the presence/absence of CcpA. The electrophoretic mobility shift assay (EMSA) will be employed to determine interactions between CcpA and promoter DNA. Protein to protein interaction, fluorescence labeling and infection biological approaches will be carried out to identify potential mechanisms of the CcpA factor on maintaining intracellular pH homeostasis and its interplay with SigB and AhrC under acidic stresses in gastrointestinal tract and macrophages after infection and engulfment. This will provide critical insights into the central role of ADI/AgDI in different physiological conditions. This is valuable to identify potential interacting partners to uncover novel regulatory and signaling networks controlling acid stress and adaptations of foodborne pathogen.

单核细胞增多性李斯特菌（Lm）是重要的人兽共患病原菌，维持胞内pH（pHi）稳态能力极强。我们前期发现，该菌精氨酸脱亚胺酶/鲱精胺脱亚胺酶（ADI/AgDI）抗酸基因簇的启动子区同时存在CcpA（代谢物控制蛋白）、σB（应激调控因子）、AhrC（精氨酸合成抑制因子）的结合位点，并且σB和AhrC均参与pHi稳态调控。为了解析CcpA在维持pHi稳态中的作用及其与σB和AhrC之间的内在联系，本项目将利用基因缺失或置换手段，研究Lm ccpA、ccpA/ahrC和ccpA/σB突变株在各种应激条件下的表型和pHi动态变化；利用gfp3和gus报告系统确定CcpA控制靶基因启动子的活性；利用胶迁移试验证实ccpA与靶基因启动子区域的结合能力；利用转录组学、蛋白互作、荧光标记、感染生物学等手段，阐明CcpA调控Lm维持pHi稳态的机制。本研究对于揭示食源性胞内病原菌的感染机制具有重要意义。

单增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。我们在前期研究中发现应激调控因子SigB和AhrC参与抗酸应激调控。CcpA属于LacI/GaIR家族的转录调控蛋白，主要通过结合靶基因启动子区保守的cre box（WTGNAANCGNWNNCW）进而发挥调控作用。前期生物信息学分析显示SigB和AhrC的启动子区可能存在cre box序列，因而我们猜测SigB、AhrC以及CcpA三者之间可能存在调控关系。为此，本项目将在此基础上继续探索CcpA在李斯特菌抗酸应激调控中的生物学作用和分子机制。体外酸应激存活试验结果显示，有机酸以及人工胃液（pH 2.5）应激下CcpA缺失导致细菌的抗酸存活能力显著降低，表明CcpA确实参与李斯特菌抗酸应激过程。利用转录组测序及荧光定量分析证实CcpA主要是通过参与调控酸应激相关基因augB、ahrC和sigB的转录进而介导抗酸应激过程。通过体外凝胶迁移试验进一步证实，CcpA能够与augB、ahrC和sigB基因的启动子区能够发生结合互作，因此初步阐明CcpA主要是通过与SigB和AhrC之间的调控互作而参与细菌酸应激耐受过程。利用细菌感染细胞试验结果表明CcpA基因的缺失不影响在巨噬细胞J774和RAW264.7中的存活和增殖能力，但反而增强了细菌在上皮细胞Caco-2上的黏附侵袭和增殖水平，表明CcpA可能还参与了李斯特菌相关毒力因子的转录调控，但具体分子机制还尚待进一步完善。本研究表明CcpA参与调控抗酸应激相关功能和调控基因的转录及表达。研究对于揭示以SigB、AhrC以及CcpA系统为中心的抗酸应激调控网络和食源性病原菌的感染机制和信号传导具有重要意义。