噬菌体多糖解聚酶介导的肠炎沙门氏菌生物被膜消除机制

Salmonella enterica is one of the most common foodborne bacteria. Salmonella which exist in biofilm is highly resistant to common disinfection methods. Thus, eliminating the biofilm in the realm of food science is considered as a bottleneck problem nowadays. Our team’s recent studies represent that Salmonella enterica phage polysaccharide Depolymerase (SepD) plays a key role in eliminating Salmonella enterica biofilm. Whereas, the exact function and molecular mechanism of SepD are not clear. Therefore, in this research, the role of SepD is planned to be revealed from three levels as follows: (1) At genomic and bioinformatic level, the complete genomes of phages with different capacities to eliminate biofilm will be sequenced, as well as the regulatory regions, putative active sites, the core domains and protein structure of SepD will be explored; (2) At protein level, SepD protein will be expressed and interacted with the biofilm. The molecular mechanism of SepD on eliminating biofilm will be discussed; (3) At phage level, phage candidates with high capacity of eliminating biofilm, which is applicable in the field of food, will be selected. The study will figure out the elimination mechanism of Salmonella enterica biofilm mediated by SepD and provide an important scientific basis for developing novel methods, so as to effectively prevent and control Salmonella enterica biofilm in the field of food science.

肠炎沙门氏菌是最重要的食源性病原菌之一，而生物被膜状态下的肠炎沙门氏菌对常见消毒方法具有抗性，消除其生物被膜是当下食品领域亟待解决的重要问题。申请人前期发现肠炎沙门氏菌噬菌体多糖解聚酶(SepD)在消除该菌生物被膜中发挥重要作用，然而具体功能和分子作用机理尚不清楚。本研究拟从三个层次展开：(1)基因组和生物信息层面，对具有不同生物被膜消除能力的噬菌体进行全基因组测序，分析SepD表达的调控序列、SepD酶活位点、结构域和蛋白质三级结构对噬菌体消除生物被膜能力的影响；(2)蛋白质层面，体外表达SepD并与生物被膜互作，解析该基因的功能和该酶的分子基础；(3)噬菌体层面，筛选和构建具有高效清除生物被膜能力的、可供食品安全领域应用的候选噬菌体。以上研究为食品领域高效控制肠炎沙门氏菌生物被膜提供科学依据和新思路。

肠炎沙门氏菌是最重要的食源性病原菌之一，而生物被膜状态下的肠炎沙门氏菌对常见消毒方法具有抗性，同时近年来发现食品生产链中的耐药沙门氏菌比例显著升高，亟需新型防控技术。本项目开展了如下研究：(1)筛选了100余株噬菌体，经试验发现5株噬菌体具有较好抑菌能力和较宽的裂解谱。并且对耐药沙门氏菌具有较好的裂解效果。同时测定了这些噬菌体的形态，pH稳定性、温度稳定性、一步生长曲线等生物学特性。（2）完成60株噬菌体全基因组测序，筛选不携带有害基因并能够有效表达多糖解聚酶的噬菌体。其中，发现一株新型噬菌体LPSEYT，对其进行功能注释和种属鉴定。克隆多糖解聚酶SpeD分析其结构，并证明该酶对生物被膜状态下的致病性大肠杆菌和沙门氏菌具有杀菌效果。（3）研究这些噬菌体对不同食品接触表面和食品基质（奶、肉和生菜）中生物被膜状态肠炎沙门氏菌的防控效果。发现在4℃中效果最好，可在1-6h 内完全杀灭牛奶和生菜中的肠炎沙门氏菌，而在蛋黄中的杀菌效果普遍较差。以上研究为食品安全领域提供了候选噬菌体，为高效控制可形成生物被膜的肠炎沙门氏菌提供了科学依据和新思路。