植物乳杆菌羊毛硫细菌素Plantaricin ZJ316的抗菌作用靶点和作用机制

The lantibiotics, a specific class of antibiotics that is produced by some lactic acid bacteria. The members of this family are all ribosomally produced, however they posses some intriguing chemical features that sets them apart from normal peptides. This is the presence of didehydroamino acids and lanthionine residues that are post-translationally introduced in the peptides. In particularly, the lantibiotics from lactic acid bacteria (LAB) have been widely explored and investigated as to their potential applications as preservatives in the food industry. We have recently purified and characterized a novel bacteriocin, plantaricin ZJ316 from a lactic-acid-producing strain named Lb. plantarum ZJ316. With this research program I aim to uncover the secrets behind the success of the lantibiotics: to obtain a fundamental understanding of how it work at molecular level. In particular I aim to have detailed knowledge of mode of action and preferably also structural information on the plantaricin ZJ316 bound to its target by several approaches on biochemistry, molecular biology, physiology and structural chemistry. By learning how this lantibiotic work, the knowledge that will be obtained is in that it will provide not only insight into the mode of action of the lantibiotic, but likely also new important biological target will be uncovered. Moreover, the basic knowledge of this lantibiotic work will provide possibility for applying in the food industry.

由某些乳酸菌产生的羊毛硫细菌素（lantibiotic）是一类具有独特抗菌作用的翻译后修饰肽，作为新型的食品防腐剂有很好的应用前景。该类细菌素的抗菌作用机制的确立能为细菌素的应用及微生物源天然防腐剂的开发提供生物学基础。研究组已在植物乳杆菌（Lactobacillus plantarum ZJ316）中分离到一个新的小分子量羊毛硫细菌素plantaricin ZJ316，拟通过与已知作用机制的细菌素比较，运用生物化学、分子生物学、生理学、结构化学等进行细菌素对敏感菌抗菌作用机制的研究，确定抗菌肽的抗菌作用位点和抑制细胞组成成分合成的途径，揭示细菌素的抗菌作用分子靶点和作用机制。项目的完成不仅有助于我们更全面系统地认识乳酸菌细菌素抗菌作用模式，还可为乳酸菌源lantibiotic在食品工业中的应用提供坚实的理论基础。

乳酸菌细菌素具有抗菌活性强、不易产生耐药性等优点，作为新型食品防腐剂有很好的应用前景。本实验室自行筛选、鉴定的乳酸菌细菌素plantaricin ZJ316，抑菌谱广、热稳定性强，尤其对金黄色葡萄球菌等食源性致病菌抑菌效果显著，具有很好的应用开发潜力。本项目通过研究细菌素抗菌作用机制，发现plantaricin ZJ316通过使细胞内钾离子全部外泄，细菌膜上形成孔洞导致细菌内容物外泄而死亡；脂质体II及其合成酶MraY和MurG不是该抗菌肽的作用靶点。全基因组测序发现植物乳杆菌ZJ316还具有合成双肽细菌素Plantaricin NC8的能力，首次发现该细菌素对革兰氏阴性菌肠炎沙门氏菌抑菌作用显著。本项目在大肠杆菌中异源表达纯化Plantaricin NC8，并研究其对沙门氏菌的杀菌作用机制。以肠炎沙门氏菌CGMCC1.1552为指示菌，发现Plantaricin NC8处理后指示菌跨膜电势差 (ΔΨ) 和质子浓度梯度 (ΔpH) 瞬间坍塌，胞内 ATP 水平迅速消失，胞外电导率急剧升高，质子电动势 (PMF)坍塌；扫描电镜和透射电镜观察发现Plantaricin NC8处理的沙门氏菌细胞表面和剖面形态结构均有显著变化。Plantaricin NC8 诱导指示菌细胞膜通透性增加，胞内离子泄露，致使细胞膜破损，内容物外泄，最终导致其死亡。运用荧光泄漏试验进一步探究脂质体II是否为Plantaricin NC8结合靶点，结果表明与nisin不同，添加过量人工合成的脂质体II并没有结合Plantaricin NC8，进而抑制指示菌细胞膜穿孔引起的荧光泄露，因此脂质体II不是该细菌素结合靶点。综上，本研究揭示了植物乳杆菌ZJ316产乳酸菌细菌素的清晰遗传背景和作用机制，可望为其在食品工业作为天然防腐剂的开发应用奠定基础。