Nanomaterials Research Division, Research Institute of Electronics, Shizuoka Universitygrid.263536.7, Shizuoka, Japan.
Integrated Bioscience Section, Graduate School of Science and Technology, Shizuoka Universitygrid.263536.7, Shizuoka, Japan.
Microbiol Spectr. 2022 Aug 31;10(4):e0011422. doi: 10.1128/spectrum.00114-22. Epub 2022 Jul 13.
Antimicrobial peptides (AMPs) inhibit the proliferation of or kill bacterial cells. To measure these activities, several methods have been used, which provide only the average value of many cells. Here, we report the development of a method to examine the antimicrobial and bactericidal activities of AMPs at the single-cell level (i.e., single-cell analysis) and apply this strategy to examine the interaction of an AMP, magainin 2 (Mag), with Escherichia coli cells. Using this method, we monitored the proliferation of single cells on agar in a microchamber and measured the distribution of the number of cells in each microcolony using optical microscopy. For method A, we incubated cells in the presence of various concentrations of AMPs for 3 h. The fraction of microcolonies containing only a single cell, , increased with the Mag concentration and reached 1 at a specific concentration, which corresponded to the MIC. For method B, after the interaction of a cell suspension with an AMP for a specific time, an aliquot was diluted to stop the interaction, and the proliferation of single cells then was monitored after a 3-h incubation; this method permits the definition of (), the fraction of dead cells after the interaction. For the interaction of Mag with E. coli cells, () increased with the interaction time, reaching ~1 at 10 and 20 min for 25 and 13 μM Mag, respectively. Thus, these results indicate that a short interaction time between Mag and E. coli cells is sufficient to induce bacterial cell death. To elucidate the activity of antimicrobial peptides (AMPs) against bacterial cells, it is important to estimate the interaction time that is sufficient to induce cell death. We have developed a method to examine the antimicrobial and bactericidal activities of AMPs at the single-cell level (i.e., single-cell analysis). Using this method, we monitored the proliferation of single cells on agar in a microchamber and measured the distribution of the number of cells in each microcolony using optical microscopy. We found that during the interaction of magainin 2 (Mag) with E. coli cells, the fraction of dead cells, (), increased with the interaction time, rapidly reaching 1 (e.g., 10 min for 25 μM Mag). This result indicates that Mag induces cell death after a short time of interaction.
抗菌肽 (AMPs) 抑制细菌细胞的增殖或杀死细菌细胞。为了测量这些活性，已经使用了几种方法，这些方法仅提供了许多细胞的平均值。在这里，我们报告了一种在单细胞水平上检测 AMPs 的抗菌和杀菌活性的方法的开发（即单细胞分析），并将该策略应用于检测抗菌肽 magainin 2 (Mag) 与大肠杆菌细胞的相互作用。使用这种方法，我们在微室中的琼脂上监测单细胞的增殖，并使用光学显微镜测量每个微菌落中细胞数量的分布。对于方法 A，我们在存在各种浓度的 AMP 的情况下孵育细胞 3 小时。含有单个细胞的微菌落的分数，，随着 Mag 浓度的增加而增加，并在特定浓度下达到 1，该浓度对应于 MIC。对于方法 B，在细胞悬浮液与 AMP 相互作用特定时间后，取一份稀释以停止相互作用，然后在 3 小时孵育后监测单细胞的增殖；这种方法允许定义（），相互作用后死亡细胞的分数。对于 Mag 与大肠杆菌细胞的相互作用，（）随着相互作用时间的增加而增加，对于 25 和 13 μM Mag，分别在 10 和 20 分钟达到约 1。因此，这些结果表明，Mag 与大肠杆菌细胞之间的短相互作用时间足以诱导细菌细胞死亡。为了阐明抗菌肽 (AMPs) 对细菌细胞的活性，重要的是估计诱导细胞死亡所需的相互作用时间。我们已经开发了一种在单细胞水平上（即单细胞分析）检测 AMP 的抗菌和杀菌活性的方法。使用这种方法，我们在微室中的琼脂上监测单细胞的增殖，并使用光学显微镜测量每个微菌落中细胞数量的分布。我们发现，在 magainin 2 (Mag) 与大肠杆菌细胞相互作用期间，死亡细胞的分数，（）随着相互作用时间的增加而增加，迅速达到 1（例如，对于 25 μM Mag，为 10 分钟）。该结果表明，Mag 在短时间相互作用后诱导细胞死亡。
