基因岛LHR对大肠杆菌耐压性的影响及机制研究

Pathogenic bacteria acquire genes protecting against environmental insults by lateral gene transfer, thus increasing resistance to food preservation technologies. Locus of heat resistance (LHR) is a transmissible genomic island, which encodes proteins of stress response including sHSP20 and ClpK, and thus very likely confers pressure resistance to Escherichia coli. Current studies regarding to high hydrostatic pressure (HHP) mainly focus on inactivation and mechanisms of microorganisms, while less studies focus on genes of pressure resistance. In this project, LHR and the fragments of LHR positive E. coli strains will be constructed to figure out the key protein modules for pressure resistance. Furthermore, the alterations of E. coli cells before and after cloning of LHR, including the cellular morphology and structure, and permeability of membrane as well, will be observed to explore their relationships with pressure resistance. Mechanisms of pressure resistance relate to protein folding and aggregation will be further analyzed through the effects of LHR on the formation and proteins expression of inclusion bodies. Consequently, this study will explain the pressure resistance of LHR and comprehensively propose its related mechanisms, thus provide theoretical basis for the safety of HHP application.

致病菌通过水平基因转移获得抗性基因以对抗环境压力，从而对食品加工技术提出挑战。LHR（locus of heat resistance）是一段可转移的基因岛，可编码sHSP20和ClpK等压力反应蛋白，因此很可能会为大肠杆菌（Escherichia coli）提供耐压性。目前对超高压（high hydrostatic pressure，HHP）加工技术的研究主要集中在杀菌效果和机制，而对耐压基因的研究较少。本项目拟首先通过含有片段和全段LHR菌株的构建，明确对E. coli耐压性起关键作用的蛋白模块。继而通过研究LHR克隆前后细胞外部形态、内部结构、以及细胞膜通透性的变化，进一步明确耐压性与细胞变化的关系。从蛋白水平上研究LHR对HHP处理的细胞包涵体的形成和蛋白表达的影响，进而提出蛋白折叠和聚集相关的耐压机制，从而全面解析LHR的耐压性和耐压机制，为HHP的安全应用提供理论基础。

致病性大肠杆菌有相当一部分菌株即使经过热或超高压处理后依然能够存活下来，当这部分大肠杆菌通过基因水平转移获得一些特殊的毒性因子以后，将会对消费者的健康造成严重的威胁。因此，对大肠杆菌的耐压基因和耐压机制进行全面解析对保证食品安全尤为重要。本项目首先以高耐压性菌株LHR阳性菌株为目标菌株，针对其全段和片段基因ORF1-7, ORF8-10, ORF11-16以及ORF1-16，构建完成四株突变体菌株，而后分析了LHR的全段或片段对大肠杆菌耐压性的影响；研究了不同食品基质中各突变菌株的耐压性差异；明确了LHR对大肠杆菌耐压性起作用的基因片段和关键蛋白，包括热休克蛋白HSP和解聚集蛋白ClpK组成的蛋白稳态模块，尤其是硫氧还蛋白TRX和钠/氢交换子KefB等组成的氧化反应蛋白模块。发现了氨基葡萄糖、植物精油百里香酚和甲壳素等天然抑菌剂可有效降低大肠杆菌的耐压性，提出了对具有高度耐压性大肠杆菌进行有效控制的技术。该项研究全面解析了与基因岛LHR相关的大肠杆菌的耐压性、耐压机制以及控制技术，分析了超高压技术的应用、机制和挑战，有助于对超高压技术的深入理解，为超高压技术的安全应用提供理论基础。