Label-Free Quantitative Proteomics Reveals the Multitargeted Antibacterial Mechanisms of Lactobionic Acid against Methicillin-Resistant Staphylococcus aureus (MRSA) using SWATH-MS Technology

The objective of the present study was to reveal the antibacterial mechanism of lactobionic acid (LBA) against methicillin-resistant Staphylococcus aureus (MRSA) using quantitative proteomics by sequential window acquisition of all theoretical mass spectra (SWATH-MS) to analyze 100 differentially expressed proteins after LBA treatment. Furthermore, multiple experiments were conducted to validate the results of the proteomic analysis including reactive oxygen species (ROS), virulence-associated gene expression, and the relative quantification of target proteins and genes by parallel reaction monitoring and quantitative real-time PCR. Combining the ultrastructure observations, proteomic analysis, and our previous research, the mode of LBA action against MRSA was speculated as cell wall damage and loss of membrane integrity; inhibition of DNA repair and protein synthesis; inhibition of virulence factors and biofilm production; induction of oxidative stress; and inhibition of metabolic pathways. These results suggest potential applications for LBA in food safety and pharmaceuticals, considering its multitarget effects against MRSA.本研究旨在通过全理论质谱序列窗口采集（sequential window acquisition of all theoretical mass spectra, SWATH-MS）定量蛋白质组学技术，分析乳糖酸（lactobionic acid, LBA）处理耐甲氧西林金黄色葡萄球菌（methicillin-resistant Staphylococcus aureus, MRSA）后产生的100种差异表达蛋白，以揭示乳糖酸抗MRSA的作用机制。此外，本研究通过多项实验对蛋白质组学分析结果进行验证，包括活性氧（reactive oxygen species, ROS）检测、毒力相关基因表达分析，以及采用平行反应监测（parallel reaction monitoring, PRM）与实时定量聚合酶链反应（quantitative real-time PCR）对目标蛋白与基因进行相对定量。结合超微结构观察结果、蛋白质组学分析及本团队前期研究，推测乳糖酸抗MRSA的作用模式为：破坏细胞壁并导致细胞膜完整性丧失；抑制DNA修复与蛋白质合成；抑制毒力因子与生物膜生成；诱导氧化应激；以及抑制代谢通路。鉴于乳糖酸对MRSA具有多靶点作用效果，本研究结果提示其在食品安全与医药领域具备潜在应用价值。