ClassIIb类细菌素PlnJ/K对金黄色葡萄球菌作用靶点及抑菌分子机理研究

Staphylococcus aureus infection, especially infection of methicillin-resistant S. aureus (MRSA) is serious threat to food safety and food security, thus it has been quite urgent to find novel inhibitors to MRSA. PlnJ/K, a class IIb lactic acid bacteria (LAB) bacteriocin purified and characterized in our lab, is with great thermal stability and antibacterial activity, and can effectively inhibit food-borne pathogens, such as MRSA. It could be considered as a potential candidate to treat the multiple drugs resistance pathogens and as a food preservative. Uncover the binging target of Pln J/K and its antibacterial mechanism would be critical for its application in food industry. In this project, we intend to establish a novel system to efficiently identify the bind target of PlnJ/K. We will generate PlnJ/K-resistant mutations, followed by whole genome sequencing based strategy (including whole genome sequencing, genome-wide comparative analysis and single nucleotide polymorphisms analysis) to analyze and search the resistance-related mutant genes. A strategy of knock out the resistance-related genes in wild type strain will be utilized to verify the “predicted binding target” after RT-qPCR analysis. In vitro binding assay will be applied to identify the “binding target”. Fluorescence leakage and ITC assays will be used to reveal the mode of action of the Pln J/K against Staphylococcus aureus. The findings in this proposal will not only provide insight into the mechanism of the novel LAB bacteriocin Pln J/K against Staphylococcus aureus, but also provide a view to the use of Pln J/K for food (preservatives) applications in the furure.

金黄色葡萄球菌尤其是“超级细菌”（MRSA）严重威胁食品安全，寻找有效抗菌剂迫在眉睫。PlnJ/K为class IIb类细菌素，热稳定、抗菌活性强，能高效抑制MRSA等食源性致病菌，有望成为新型食品防腐剂。明确作用靶点及抑菌分子机理将为其开发应用提供生物学基础。本研究拟建立class II细菌素作用靶点鉴定新体系来筛选PlnJ/K作用靶点：以抗PlnJ/K MRSA突变株为出发菌株，通过全基因组测序、比较基因组及单核苷酸多态性等分析寻找抗性突变基因，结合RT-qPCR、基因敲除等分子生物学技术鉴定“候选作用靶点”，运用细菌素结合试验明确作用靶点。通过等温滴定量热和荧光泄露等试验揭示PlnJ/K与靶蛋白结合方式及其抑菌作用模式，阐明PlnJ/K对MRSA的抑菌分子机理。本研究不仅有助于我们更全面系统地认识乳酸菌源细菌素抑菌机理，还可为PlnJ/K在食品工业中的应用提供坚实的理论基础。

本研究围绕食源性病原菌耐甲氧西林金黄色葡萄球菌（MRSA）引起的食物中毒及其耐药性这一全球共同关注的食品安全热点和难点问题，旨在揭示本实验室分离到的新型乳酸菌源细菌素 PlnJ/K 对 MRSA 的作用靶点及其抑菌机制，以期为该细菌素作为新型食品防腐剂的应用奠定基础。研究运用食品级乳酸菌载体系统，实现了 PlnJ/K 的高效制备，明确该异源表达的细菌素的分子特性、稳定性及抗菌活性；运用定向增加细菌素浓度的方法，筛选获得 5 株抗 PlnJ/K 的MRSA 抗性突变株；通过全基因组测序、比较基因组及单核苷酸多态性等分析找到2个抗性突变基因，结合RT-qPCR等分子生物学技术鉴定出glycosyltransferase family 4 protein为“候选作用靶点”。异源表达纯化“候选作用靶点”，进一步运用结合试验、荧光泄露试验，发现glycosyltransferase family 4 protein不是 PlnJ/K的靶点。创新性的建立 GST Pull-down 与 iTRAQ-LC-MS/MS 技术相结合的候选靶蛋白筛选体系，发现 2 个跨膜蛋白可能为 PlnJ/K 的作用靶点。进一步研究PlnJ/K 抗菌模式和分子作用机制，发现其通过促使靶细菌跨膜电势差(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，细胞膜通透性增加，胞内离子泄露，细胞膜破损，内容物外泄，最终导致其死亡。本项目建立了一套Class IIb类抗菌作用靶点研究体系，可为同类细菌素的作用靶点研究提供参考，同时该研究可为乳酸菌源细菌素 PlnJ/K 的应用打下理论基础。