Imaging Bacterial Cell Death Induced by Antimicrobial Peptides in Real Time Using High Speed AFM.
Imaging Bacterial Cell Death Induced by Antimicrobial Peptides in Real Time Using High Speed AFM.
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DOI：
10.1017/s1431927610062422
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发表时间：
2010-07
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抗菌肽（AMP）是一类很有前途的抗菌剂，用于对抗对传统抗生素产生耐药性的细菌。这些肽杀死细菌的机制仍然知之甚少。肽与细菌细胞壁的相互作用被认为是杀菌作用的原因[1,2,3]。到目前为止，这种相互作用还没有被可视化，也没有很多关于这种相互作用动力学的数据。传统的原子力显微镜（AFM）是观察活细胞细胞壁变化的有效工具，但由于其图像采集速度慢，无法深入了解其动力学机制。我们使用基于小悬臂[5]的定制AFM组件首次实时成像抗菌肽的杀菌作用。利用该系统，我们以纳米级的空间分辨率和秒级的时间分辨率研究了嵌合AMP CM15在活大肠杆菌细胞上的活性。我们观察到注射AMP后细胞表面形态的快速变化（见图1），在同一图像中，单个单克隆细菌的响应时间不同（见图2）。结合AFM和荧光显微镜，我们将细胞形态的变化与细胞壁通透性和细胞死亡bb0联系起来。本研究结果表明，高速原子力显微镜可以揭示个体细胞的不同行为，而不是与整体细胞的行为相比。在抗菌肽的情况下，培养阶段的存在为抗菌肽攻击细菌的机制提供了新的指示。我们相信这项技术将为表征和研究合成抗菌肽的有效性提供一种全新的方法。
Antimicrobial peptides (AMP) are a promising class of antimicrobial agents in the battle against bacteria that have built up resistance to conventional antibiotics. The mechanism by which these peptides kill bacteria is still poorly understood. The interaction of the peptides with the bacterial cell walls has been suggested to be responsible for the bactericidal effect [1, 2, 3]. Thus far, this interaction has not been visualized nor is there much data on the kinetics of this interaction. Traditional Atomic Force Microscopy (AFM) is a useful tool for observing the changes in the cell wall of living cells [4], but due to its slow image acquisition speed it cannot provide insight into the dynamics of the mechanism. We have used custom-built AFM components based on small cantilevers [5] to image for the first time the bactericidal action of antimicrobial peptides in real time. With this system, we investigated the activity of the chimeric AMP CM15 with nanometer spatial-and seconds temporal-resolution on live E. coli cells. We observed rapid changes in surface morphology of the cells after injection of the AMP (see figure 1), with a response time that differs between individual monoclonal bacteria in the same image (see figure 2). Using combined AFM and fluorescence microscopy, we correlated the change in cell morphology to cell wall permeability and cell death [6]. The results of this study show that high-speed atomic force microscopy can reveal distinctively different behavior of individual cells than compared to the behavior of bulk cells. In the case of the antimicrobial peptides, the presence of an incubation-phase gives new indications for the mechanism by which the antimicrobial peptide attacks the bacterium. We believe that this technique will enable a whole new method of characterizing and studying the effectiveness of synthetic antimicrobial peptides.