卷曲乳杆菌S层蛋白裂解腐生葡萄球菌细胞壁的机制研究

Surface layers (S-layers) are monomolecular crystalline arrays composed of protein or glycoprotein subunits present as the outermost component of cell wall in bacteria. The studies of Lactobacillus S-layer protein have been mainly focused on its adhesion and immune regulatory function. Our previous research showed that S-layer protein (SlpB) of Lactobacillus crispatus exhibited a novel function of lysing cell wall. SlpB could significantly inhibit the growth of common spoilage bacteria in meat such as Staphylococcus saprophyticus and Pseudomonas, showing a broad antimicrobial spectrum and stable antibacterial activity. Hence, it could be widely applied in the field of meat preservation. However, the hydrolysis mechanism of SlpB on the bacterial cell wall remains unknown. The current project aims to study the antibacterial activity and investigate the antibacterial mechanism of SlpB using Staphylococcus saprophyticus as the indicator bacteria. To determine the binding site, the binding constants of SlpB with cell wall components will be obtained using single-molecule force spectroscopy combined with blocking test. The peptidoglycan cleavage site will also be determined by analyzing the hydrolysis products of SlpB using HPLC-MS. Furthermore, a set of N- and C-terminally truncated recombinant SlpB proteins will be constructed to investigate the cell wall binding domain and catalytic domain. Subsequently, co-expression of active domains will be employed to verify the enzymatic activity. Based on the study, the project will illustrate the molecular mechanism of cell wall lysis of SlpB on S. saprophyticus, and provide a theoretical basis for the application of novel and natural antibacterial agents.

S层是细菌细胞外由蛋白或糖蛋白亚单位组成的单分子层晶格状结构，目前有关乳杆菌S层蛋白的研究主要集中于黏附和免疫调节功能。申请者前期研究发现，卷曲乳杆菌的S层蛋白（SlpB）具有裂解细胞壁的新功能，可显著抑制腐生葡萄球菌、假单胞菌等肉品中常见腐败菌的生长，其抑菌谱宽，稳定性强，在肉品保鲜领域具有广阔的应用前景。但SlpB如何与细胞相互作用进而裂解细胞壁未见报道。本项目拟以腐生葡萄球菌为指示菌，聚焦SlpB的抗菌活性，采用原子力单分子力谱结合阻断法分析SlpB与细胞壁组分的结合常数，确定其结合位点；采用HPLC-MS分析SlpB水解指示菌肽聚糖的产物，推断活性靶点；进而通过分段截取SlpB的不同片段，确定其细胞壁结合结构域和催化结构域。在此基础上，融合表达SlpB活性结构域，进行功能验证，阐明其裂解腐生葡萄球菌细胞壁的分子机制，为新型天然抗菌剂的应用研究提供理论依据。

本项目首先分析SlpB对腐生葡萄球菌细胞壁的损伤作用，随后利用蛋白互作仪分析SlpB与细胞壁组分的结合常数，确定其结合位点；采用HPLC-MS分析SlpB水解指示菌肽聚糖的产物，推断活性靶点；进而通过分段截取SlpB的不同片段，确定其细胞壁结合结构域和催化结构域。在此基础上，研究SlpB对肉品的保鲜效果。结果表明SlpB能裂解腐生葡萄球菌的细胞壁，协助膜损伤细菌素nisin进入胞内，共同起到杀死细菌的效果。蛋白互作仪分析显示SlpB与肽聚糖有强的结合能力，且结合常数与浓度呈正比说明SlpB的结合位点为肽聚糖。HPLC-MS分析显示SlpB水解肽聚糖产物为四肽的肽链，所以SlpB可能为酰胺酶或内肽酶活性。SlpB C端323-501 AA具有完整的细胞壁结合能力，说明该段为SlpB细胞壁结合结构域。SlpB N端1-359 AA具有肽聚糖水解活性，说明该段为SlpB的催化结构域。将SlpB与nisin应用于冷鲜鸡肉品保鲜，SlpB能显著增强nisin的保鲜效果，在nisin的基础上，将冷鲜鸡货架期延长3天，本项目通过阐明SlpB裂解腐生葡萄球菌细胞壁的分子机制，为新型天然抗菌剂的应用研究提供理论依据。