luxS基因在冷冻致亚致死损伤的金黄色葡萄球菌修复启动中的作用及其调控机制

The most commonly detected Staphylococcus aureus causes serious food safety issues for frozen food. According to our previous studies, the sublethally injured cells can resuscitate themselves under suitable conditions by repairing the membrane and ribosomal. Meanwhile, the expression of LuxS protein increased significantly during the initiating stage of resuscitation. In the present research project, we will firstly construct the ⊿luxS deletion strain of S. aureus, and compare the differences in the recovery patterns under suitable conditions with wild strain. Then, the differentially transcriptional and proteomic characterization during resuscitation will be analyzed by employing transcriptome sequencing and liquid chromatography-tandem mass spectrometry methods, respectively, which is aim to explore the repair start-related key genes under the regulation of luxS gene. After the verification in different recovery condtions and real food environments, the results will clarify the function of luxS gene for the resuscitation of frozen sublethally injured S. aureus, by comprehensively considering the morphologically, physiologically and biochemically changing regulation of cell membrane and ribosomal. Thereafter, the regulation network of luxS gene will be established. The research results will not only be helpful for perfecting the mechanism of resuscitation initiation of frozen sublethally injured S. aureus, but also offer a theoretical basis for the establishment of effective safety detection and control technologies of pathogens in frozen food.

金黄色葡萄球菌在冷冻食品中屡有检出，严重影响人们的食用安全。本课题组前期研究结果发现，冷冻致亚致死损伤的金黄色葡萄球菌在适宜条件下，能够通过对细胞膜和核糖体的修复而回复为正常状态，而LuxS蛋白的表达量在修复启动阶段显著增大。本项目将首先构建金黄色葡萄球菌的luxS基因缺失突变株，研究突变株与出发菌株的冷冻致亚致死损伤细胞在适宜条件下修复启动的差异，并采用转录组测序和液相色谱-质谱/质谱技术，分别研究其转录组和蛋白质组差异表达谱，得出与luxS基因调控有关的修复启动关键基因。经过在不同修复环境及真实食品样品中验证后，与细胞膜和核糖体的形态及生理生化变化规律相结合，明确luxS基因在冷冻致亚致死损伤的金黄色葡萄球菌修复启动中的作用，并构建其分子调控网络。本项目研究结果不仅能够完善冷冻致亚致死损伤的金黄色葡萄球菌修复启动机制，而且能够为冷冻食品产业建立致病菌高效安全检测及控制技术提供理论依据。

在冷冻条件下，金黄色葡萄球菌能够以亚致死损伤状态长期存活，但可在适宜的温度下修复并生长，为食品安全带来严重隐患。为了阐明冷冻致亚致死细胞的修复机制，本项目首先采用扫描电镜（SEM）和透射电镜（TEM）技术研究了冷冻致亚致死细胞在修复过程中超微形态结构的变化，采用非标记高效液相色谱-质谱（HPLC-MS）技术，分析了修复过程中的差异蛋白质组，与冷冻致亚致死细胞相比，在37°C下修复1 h的金黄色葡萄球菌细胞膜完整性逐渐修复，表达上调的蛋白45个，下调的蛋白73个，细胞膜合成、氧化胁迫耐受、物质及能量代谢以及毒力因子相关蛋白的表达均发生显著变化，LuxS蛋白在冷冻致亚致死细胞修复过程中的表达显著增加。随后，本项目将luxS基因的缺失突变株ΔluxS和野生型金黄色葡萄球菌分别在-18°C条件下冻藏50 d后，测定其冷冻致亚致死细胞在不同浓度NaCl、pH、有机酸以及果汁中的修复规律，冷冻之后金黄色葡萄球菌对酸性溶液的耐受性发生明显改变，而且此抗性的改变与luxS基因的调控密切相关。经透射电镜分析，40 mg/mL抗坏血酸处理20 min可对亚致死细胞的细胞膜造成破坏，导致菌体胞质内容物流出，从而造成细胞失活。采用转录组测序（RNA-seq）技术，比较分析了金黄色葡萄球菌野生型和ΔluxS突变株的冷冻致亚致死细胞，在抗坏血酸胁迫下的转录组表达谱差异，结果发现431个基因差异表达显著，参与了细胞代谢，氨基酸代谢等生物调控通路，结果进一步表明luxS基因在金黄色葡萄球菌冷冻致亚致死细胞修复启动中具有重要的调控作用。最后，本项目也采用预测微生物学模型拟合分析策略，建立了单辛酸甘油酯对即食湿面中金黄色葡萄球菌的抑制模型、含肉桂醛猪肉糜中金黄色葡萄球菌、沙门氏菌和单核细胞增生李斯特菌热失活模型，对于食品中致病菌的高效抑制和杀灭、食品品质保持以及节能加工均具有重要意义。本项目已经发表或接收论文7篇，其中SCI收录论文4篇，JCR2区论文2篇，EI收录论文2篇；培养硕士研究生4名，经费执行合理。