Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi, Japan.
Sci Rep. 2017 Apr 25;7(1):1129. doi: 10.1038/s41598-017-01267-5.
Because of the scanty pipeline of antibiotics newly obtained from nature, chemical modification of established drugs is one of the major streams of current antibacterial research. Intuitive and easy-to-use assays are critical for identifying drug candidates with novel modes of action. In this study, we demonstrated that metabolic fluorescent staining of growing cell walls is a powerful tool for mode-of-action analyses of antibiotics using Streptococcus pyogenes. A set of major cell-wall-inhibiting antibiotics (bacitracin, D-cycloserine, flavomycin, oxacillin, ramoplanin, and vancomycin) was employed to validate the potential of the assay. The mechanistic differences of these antibiotics were successfully observed. For instance, D-cycloserine treatment induced fluorescently stained, excessive peripheral cell wall growth. This may indicate that the switch from the peripheral growth stage to the succeeding septal growth was disturbed by the treatment. We then applied this assay to analyze a series of vancomycin derivatives. The assay was sufficiently sensitive to detect the effects of single-site chemical modification of vancomycin on its modes of action. This metabolic fluorescent labeling method is easy to perform, especially because it does not require radiolabeled substrates. Thus, it is suitable for the preliminary evaluation of antibacterial mechanisms during antibacterial research.
由于从自然界新获得的抗生素数量有限，因此化学修饰已有的药物是当前抗菌研究的主要方向之一。直观易用的检测方法对于确定具有新型作用模式的药物候选物至关重要。在这项研究中，我们证明了使用酿脓链球菌对生长中的细胞壁进行代谢荧光染色是分析抗生素作用模式的有力工具。我们使用了一组主要的细胞壁抑制抗生素（杆菌肽、D-环丝氨酸、氟霉素、苯唑西林、瑞他帕林和万古霉素）来验证该检测方法的潜力。成功观察到这些抗生素的机制差异。例如，D-环丝氨酸处理会诱导荧光染色的、过度的外周细胞壁生长。这可能表明，处理干扰了从外周生长阶段到随后的隔膜生长的转变。然后，我们将该检测方法应用于一系列万古霉素衍生物的分析。该检测方法非常灵敏，可以检测到万古霉素的单点化学修饰对其作用模式的影响。这种代谢荧光标记方法易于操作，特别是因为它不需要放射性标记的底物。因此，它适用于抗菌研究中抗菌机制的初步评估。
