超声场诱导大肠杆菌磷脂酶基因pldA高表达促进纳米百里香油乳液杀菌的机理

大肠杆菌对食品安全造成重大威胁。申请人前期研究发现，超声与纳米百里香油乳液(TEON)协同可显著提升大肠杆菌致死率，同时检测到磷脂酶(pldA)的表达水平明显上调，但机理尚不清楚。为了解释这一现象，本项目提出了超声诱导pldA高表达引起细胞膜脂质代谢紊乱促进TEON渗入杀菌的假说。项目拟采用激光共聚焦显微镜、RT-qPCR等手段，分析在超声作用下pldA的表达变化规律及其与致死率的相关性；构建大肠杆菌pldA缺失株(∆pldA)和过表达株(pldA*)，检测其对TEON的敏感性，并运用低温冷冻电镜等表征细胞膜微观结构、渗透性等膜特性，比较其与野生菌株的异同；同时采用脂质组学等技术检测细胞膜脂肪酸、磷脂以及脂多糖的组成变化，分析超声下pldA调控的脂质代谢通路和关键基因；通过上述研究明确pldA在协同杀菌中的作用，解析协同杀菌的机制，为超声物理场与TEON协同微生物控制理论提供理论依据。

Escherichia coli (E. coli) poses a major threat to food safety. The applicant’s previous research on the subject found that combination of ultrasound and thyme essential oil nanoemulsions (TEON) increased its lethality towards E. coli significantly, while the expression of phospholipase(pldA)gene and protein levels of the E. coli are upregulated under the ultrasound treatment, yet the related mechanisms have not been fully explored. To explain this synergistic phenomenon, it is hypothesized that ultrasound-induced pldA high expression in disturbed cell membrane lipid metabolism and allow TEON to infiltrate the cell membrane, leading to the death of microorganisms. In this proposal, laser confocal microscopy, RT-qPCR and other/similar technologies will be applied to analyze the change of pldA expression level under ultrasound and its correlation with lethality rate. E. coli pldA-deleted strain(∆pldA) and over-expressed strain(pldA*) will be constructed, the sensitivity of all strains to TEON will be detected, cryo-electron microscopy and other/similar techniques also will be employed to characterize microstructure, permeability and fluidity of cell membrane, compared to those of wild type strain. Lipidomics and other/similar techniques will be implemented to detect changes of fatty acids, phospholipids and lipopolysaccharides composition in cell membranes, which will permit to analyze the corresponding lipid metabolic pathway and identify the key lipid metabolism-related genes. Finally elucidating the function of pldA and molecular mechanism in the synergistic effects of ultrasonic field and TEON providing a theoretical basis for the ultrasonic physical field and TEON cooperative microbial control concept.