Dataset LC MS Manool disarms Streptococcus mutans by targeting ABC trasporters protein

The emergency of antibiotic resistance is serious. We must identify molecules that escape the self-defense mechanisms of microorganisms. In this study, we optimized a protocol for identifying Streptococcus mutans proteins impaired by manool, a metabolite from Salvia officinalis with antimicrobial activity. The research showed that S. mutans can evade antibiotics and grow. This study shows the potential of manool as a new antimicrobial agent and the importance of understanding the molecular mechanisms of plant-derived compounds in the fight against AMR. Samples were obtained using the Drug Affinity Responsive Target Stability (DART-S) assay. A new protocol was performed for bacterial cells that allows the DARTS assay to be applied to a prokaryotic model using a minimal medium. The DARTS assay was initially performed by incubating manool with a protein extract (pe-DARTS) from S. mutans, followed by limited proteolysis, SDS-PAGE analysis of digestion products, and proteomic identification of the separated proteins. The bc-DARTS assay involved the incubation of manool with Streptococcus mutans bacterial cells, followed by proteolysis, SDS-PAGE analysis, and LC-MS. The S. mutans overnight inoculum was centrifuged at 3000 rpm for 15 minutes, and the cells were resuspended in BHI medium to a density of 0.05 OD/ml. The cells were incubated at 37°C and cultured to the established optical densities (0.5 OD/ml and 1 OD/ml). Then cells were centrifuged at 3000 rpm for 15 minutes. After discarding the supernatant, the pellet was resuspended in M9 minimal medium 1X supplemented with 0.4% glucose for both control and treated samples, at a density of 0,5 OD/ml and 1 OD/mL. The treated samples were incubated with manool at 5 µM and 10 µM for 90 minutes.抗生素耐药性（antibiotic resistance）问题形势严峻，亟需筛选可逃逸微生物自卫机制的活性分子。本研究优化了一套用于鉴定受香紫苏醇（manool）影响的变形链球菌（Streptococcus mutans）蛋白的实验方案，香紫苏醇是药用鼠尾草（Salvia officinalis）的一种具有抗菌活性的代谢产物。研究证实变形链球菌可逃避抗生素作用并增殖，本研究揭示了香紫苏醇作为新型抗菌剂的潜力，以及阐明植物源化合物对抗微生物耐药性（Antimicrobial Resistance, AMR）分子机制的重要性。实验采用药物亲和响应靶点稳定性（Drug Affinity Responsive Target Stability, DART-S）分析技术获取样本。针对细菌细胞开发的全新实验方案，使得DARTS测定可借助基础培养基应用于原核生物模型。初始DARTS实验流程为：将香紫苏醇与变形链球菌的蛋白提取物共孵育（即pe-DARTS），随后进行有限蛋白水解、对消化产物进行SDS-聚丙烯酰胺凝胶电泳（Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis, SDS-PAGE）分析，并对分离得到的蛋白进行蛋白质组学鉴定。bc-DARTS实验流程则为：将香紫苏醇与变形链球菌菌体共孵育，随后进行蛋白水解、SDS-聚丙烯酰胺凝胶电泳分析以及液相色谱-质谱联用（Liquid Chromatography-Mass Spectrometry, LC-MS）检测。变形链球菌的过夜接种物以3000 rpm离心15分钟，菌体重悬于脑心浸液（Brain Heart Infusion, BHI）培养基中，调整至0.05 OD/ml的密度。将菌体置于37℃培养，至指定光密度值（0.5 OD/ml及1 OD/ml）。随后再次以3000 rpm离心15分钟，弃去上清液，将菌体沉淀重悬于添加了0.4%葡萄糖的1×M9基础培养基中，对照组与处理组样本均调整至0.5 OD/ml及1 OD/mL的密度。处理组样本分别与5 μM、10 μM的香紫苏醇共孵育90分钟。