Con A修饰型抗菌肽脂质体对单增李斯特菌生物被膜的靶向作用及机制研究

Listeria monocytogenes is a food-borne pathogen which is widely distributed in the environment. L. monocytogenes is capable of attaching to the surfaces of food and equipments, and forming biofilm in food processing, storage and transportation process. The cells in the biofilm are more resistant and more difficult to completely remove, which endangers food security. Antimicrobial peptides are expected to become potential alternatives because they can overcome the limitations of traditional biofilm control methods. Based on our previous result that the anchovy antibacterial peptide liposome exhibited anti- L. monocytogenes biofilm activity, the liposome is proposed to be surface-modified by concanavalin A (Con A) according to the specific affinity interaction between Con A and the carbohydrates in the extracellular polymers of the biofilm, in order to achieve active targeting delivery and improve the peptide concentration on the surface of the biofilm. The objectives of the research are as follows: to find the effect of preparation condition on the physicochemical properties and activities of the antimicrobial peptide liposome; to establish the surface monolayer coverage model to analyze the targeted interaction between the liposome and the biofilm; to investigate the influence of the liposome on the surface adhesion and the expression of quorum sensing-related genes of the Listeria cells; to reveal the L. monocytogenes biofilm control mechanism of the Con A modified antimicrobial peptides liposome. The results will lay the foundation for the realization of effective control of food-borne pathogens biofilm.

单增李斯特菌是一种自然界较为常见的食源性致病菌，在食品加工、储运过程中可黏附于食品、设备表面形成生物被膜。被膜菌具有更强的抗逆性，难以彻底清除，危及食品安全。抗菌肽能克服传统生物被膜控制方法的局限性，有望成为潜在的替代手段。本研究在证实鳀鱼抗菌肽脂质体具有抗单增李斯特菌生物被膜活性的基础上，基于刀豆球蛋白A(Concanavalin A, Con A)与被膜胞外多聚物中糖单元的特异性亲和作用，利用Con A对该抗菌肽脂质体进行表面修饰，实现脂质体的主动靶向传递，提高其在生物被膜表面的作用浓度。考察不同制备条件对Con A修饰型抗菌肽脂质体理化性质和抗被膜活性的影响；建立表面单层覆盖模型，分析抗菌肽脂质体与被膜的靶向性相互作用；通过研究抗菌肽脂质体对单增李斯特菌细胞表面黏附性及群体感应相关基因的影响，明确该脂质体对单增李斯特菌生物被膜的调控机制，为实现食源性致病菌生物被膜的有效控制奠定基础。

单增李斯特菌是一种自然界较为常见的食源性致病菌，在食品加工、储运过程中可形成生物被膜，危及食品安全。项目基于表面修饰的抗菌肽脂质体对单增李斯特菌的刺激响应性和靶向亲和性，利用刀豆球蛋白A (Concanavalin A, ConA)、壳聚糖对不同食品来源抗菌肽脂质体进行表面修饰，采用粒径分析、荧光探针、微结构表征等手段研究了脂质体的理化性质，探讨了表面修饰对其稳定性的调控机制；分析了抗菌肽脂质体对单增李斯特菌及其生物被膜的靶向性吸附及抑制活性；研究了抗菌肽脂质体对该菌细胞表面黏附性及群体感应相关基因表达的影响。结果表明：ConA修饰型抗菌肽AAP（GLSRLFTALK）脂质体可引起细菌细胞结构的明显坍塌、细胞膜破损、胞内物质外溢，从而抑制生物被膜的形成。ConA修饰的抗菌肽脂质体与生物被膜中的胞外多糖存在特异性结合，其至生物被膜的吸附是自发性放热过程。一定浓度的壳聚糖可降低脂质体双层膜的流动性和通透性，从而增强其稳定性。壳聚糖修饰的抗菌肽Apep10（GLARCLAGTL）脂质体与单增李斯特菌之间存在静电引力和疏水作用。ConA与壳聚糖修饰均可实现抗菌肽脂质体的靶向递送。单增李斯特菌分泌的溶血素LLO可实现抗菌肽从脂质体中的触发性释放。表面修饰型抗菌肽脂质体处理使得单增李斯特菌生物被膜形成相关基因——转录激活因子prfA表达量下调。上述结果可为实现食源性致病菌生物被膜的有效控制奠定基础。