新型抗菌肽F1对超级大肠杆菌（MCR-1）的抗菌作用过程及其自溶酶大量表达的分子机制

新型抗菌肽及其抗菌机理已成为食源有害微生物控制研究的重要方向和热点。申请人前期研究发现L. paracasei FX-6发酵物中新型抗菌肽F1对超级细菌（耐黏菌素大肠杆菌MCR-1）有很强的抗菌作用。通过初步超微观察、生化分析及蛋白组学等研究发现，F1可能通过“接触—透膜—自溶”作用过程，使菌膜快速裂解，但作用具体过程及自溶酶（CLpP、CLpX）大量表达机制尚不清晰。本研究采用多种生化指标检测、动态超微分析，辅以计算机模拟与验证等方法研究抗菌肽的具体抗菌作用过程。以“自溶酶”大量表达为突破点，研究抗菌肽诱导超级细菌自溶酶大量表达的信号物质，发现其相关基因及表达通路；通过Red同源重组基因技术获得与自溶酶大量表达相关基因缺失菌，抗菌肽作用后检测其自溶酶活性、生化指标和超微结构变化，进一步验证抗菌肽对超级细菌的分子作用机制。该研究有望揭示抗菌肽新的抗菌过程与机制，为抗菌肽应用提供理论基础。

Novel antimicrobial peptides and their antibacterial mechanisms have attracted increasing research attention in control of foodborne pathogens. In our previous studies, we have found that the new antibacterial peptide F1 isolated from L. paracasei FX-6 fermentation had a strong antibacterial activity on super bacteria (colistin resistant Escherichia coli. MCR-1). The results of cryo-SEM, biochemical analysis and proteomic analysis indicated that F1 might rapidly rupture the bacterial membrane through "contact-penetration-autolysis" mechanism. However, details of antibacterial process and mechanisms of expression of autolysin (CLpP, CLpX) are still not clear. In this study, numerous techniques were used including biochemical indicators assays, dynamic ultrastructure visual analysis, and computer simulation prediction and verification to investigate the detailed steps of antibacterial process. Taken the expression of autolytic enzyme as the breakthrough point, the signal substances induced the expression of autolysin in super bacteria were isolated, purified and identified. And the related genes and expression pathways of signal molecules were further to elucidate. The autolysin expression-related gene expression was knocked out by homologous recombination technique. Changes in autolysin activity, biochemical indexes and microstructures in MCR-1 were detected to verify the molecular mechanism of antibacterial peptides on super bacteria before and after treatment of F1. This study is expected to reveal the new antibacterial process and molecular mechanism of antibacterial peptides, providing theoretical basis for the application of antibacterial peptides in food industry.