Data_Sheet_1_A New Perspective on the Antimicrobial Mechanism of Berberine Hydrochloride Against Staphylococcus aureus Revealed by Untargeted Metabolomic Studies.pdf

Berberine hydrochloride (BBR) is a natural product widely used in clinical medicine and animal production. It has a variety of antimicrobial effects, but its complex antimicrobial mechanism has not been clarified. This study aimed to discover the metabolic markers and gain a new perspective on the antibacterial mechanism of BBR. The effects of different inhibitory concentrations of BBR on the survival and growth of standard strain Staphylococcus aureus ATCC 25923 were analyzed by the bacteriostatic activity test. Differences in intracellular metabolites of S. aureus following 19 μg/ml BBR exposure for 1 h were investigated by combining non-targeted metabolomics techniques of gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS). The results showed that the minimum inhibitory concentration of BBR against S. aureus was 51 μg/ml. A total of 368 and 3,454 putative metabolites were identified by GC-MS and LC-MS analyses, respectively. Principal component analysis showed the separation of intracellular metabolite profiles between BBR-exposed samples and non-exposed controls. Pathway activity profiling analysis indicated a global inhibition of metabolisms by BBR exposure, while enhancement was also found in nucleic acid metabolism, amino sugar, and nucleotide sugar metabolism. Several metabolic markers were screened out mainly based on their variable importance of projection values. Two pyridine dicarboxylic acids were significantly downregulated, suggesting the reduction of stress resistance. The oxidized phospholipid (PHOOA-PE) was accumulated, while lipid antioxidant gamma-tocopherol was decreased, and farnesyl PP, the synthetic precursor of another antioxidant (staphyloxanthin), was decreased below the detection threshold. This evidence indicates that BBR reduced the antioxidant capacity of S. aureus. Accumulation of the precursors (UDP-GlcNAc, CDP-ribitol, and CDP-glycerol) and downregulation of the key metabolite D-Ala-D-Ala suggest the inhibition of cell wall synthesis, especially the peptidoglycan synthesis. Metabolites involved in the shikimate pathway (such as 3-dehydroshikimate) and downstream aromatic amino acid synthesis were disturbed. This study provides the first metabolomics information on the antibacterial mechanism of BBR against S. aureus. The key metabolic markers screened in this study suggest that the shikimate pathway, staphyloxanthin synthesis, and peptidoglycan biosynthesis are new directions for further study of BBR antibacterial mechanism in the future.

盐酸小檗碱（Berberine hydrochloride, BBR）是一种广泛应用于临床医学与动物生产领域的天然产物，具备多种抗菌活性，但其复杂的抗菌机制尚未阐明。本研究旨在挖掘代谢标志物，并为阐明盐酸小檗碱的抗菌机制提供全新视角。本研究通过抑菌活性实验，分析了不同抑制浓度的盐酸小檗碱对标准菌株金黄色葡萄球菌（Staphylococcus aureus）ATCC 25923存活与生长的影响；结合气相色谱-质谱联用（GC-MS）与液相色谱-质谱联用（LC-MS）两种非靶向代谢组学技术，探究了经19 μg/ml盐酸小檗碱处理1小时后金黄色葡萄球菌胞内代谢物的差异。结果显示，盐酸小檗碱对金黄色葡萄球菌的最低抑菌浓度为51 μg/ml。经GC-MS与LC-MS分析，分别鉴定得到368种与3454种推定代谢物。主成分分析结果显示，盐酸小檗碱处理组与未处理对照组的胞内代谢物谱存在显著分离。通路活性谱分析表明，盐酸小檗碱处理会全局性抑制菌体代谢，但同时也会增强核酸代谢、氨基糖与核苷酸糖代谢。研究人员主要基于变量重要性投影值筛选得到若干代谢标志物：两种吡啶二羧酸的表达量显著下调，提示菌体的应激抗性有所降低；氧化磷脂PHOOA-PE出现积累，而脂质抗氧化剂γ-生育酚的含量下降；另一抗氧化剂葡萄黄素（staphyloxanthin）的合成前体法尼基焦磷酸的含量降至检测阈值以下。上述证据表明，盐酸小檗碱会降低金黄色葡萄球菌的抗氧化能力。前体物质尿苷二磷酸-N-乙酰葡糖胺（UDP-GlcNAc）、胞苷二磷酸核糖醇（CDP-ribitol）与胞苷二磷酸甘油（CDP-glycerol）的积累，以及关键代谢物D-丙氨酰-D-丙氨酸（D-Ala-D-Ala）的下调，表明盐酸小檗碱抑制了菌体细胞壁合成，尤其是肽聚糖合成过程。参与莽草酸途径（如3-脱氢莽草酸）及下游芳香族氨基酸合成的代谢物均出现紊乱。本研究首次提供了盐酸小檗碱抗金黄色葡萄球菌的代谢组学相关数据。本研究筛选得到的关键代谢标志物表明，莽草酸途径、葡萄黄素合成与肽聚糖生物合成为未来解析盐酸小檗碱抗菌机制的新研究方向。