Production and Visualization of Bacterial Spheroplasts and Protoplasts to Characterize Antimicrobial Peptide Localization
Production and Visualization of Bacterial Spheroplasts and Protoplasts to Characterize Antimicrobial Peptide Localization
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DOI：
10.3791/57904
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发表时间：
2018-08-01
影响因子：
1.2
通讯作者：
Darling, Louise E. O.
中科院分区：
文献类型：
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作者：
Figueroa, Dania M.;Wade, Heidi M.;Darling, Louise E. O.
使用共聚焦显微镜作为一种方法，以评估细菌内的肽定位模式通常抑制传统的光学显微镜的分辨率限制。由于给定显微镜的分辨率不易提高，我们提出了将小杆状革兰氏阴性大肠杆菌（E。coli）和革兰氏阳性巨大芽孢杆菌（B. megaterium）转化成更大的、容易成像的球形形式，称为原生质体或原生质体。这种转化允许观察者快速和清楚地确定肽是否将其自身嵌入细菌膜（即，膜定位）或穿过膜进入细胞（即，易位）。通过这种方法，我们还提出了一个系统的方法来表征肽膜定位或易位。虽然这种方法可以用于各种膜活性肽和细菌菌株，我们证明了这种协议的效用，通过观察Buforin II P11A（BF 2 P11A），抗菌肽（AMP），与E。coli原生质球和B.巨大菌原生质体。
The use of confocal microscopy as a method to assess peptide localization patterns within bacteria is commonly inhibited by the resolution limits of conventional light microscopes. As the resolution for a given microscope cannot be easily enhanced, we present protocols to transform the small rod-shaped gram-negative Escherichia coli (E. coli) and gram-positive Bacillus megaterium (B. megaterium) into larger, easily imaged spherical forms called spheroplasts or protoplasts. This transformation allows observers to rapidly and clearly determine whether peptides lodge themselves into the bacterial membrane (i.e., membrane localizing) or cross the membrane to enter the cell (i.e., translocating). With this approach, we also present a systematic method to characterize peptides as membrane localizing or translocating. While this method can be used for a variety of membrane-active peptides and bacterial strains, we demonstrate the utility of this protocol by observing the interaction of Buforin II P11A (BF2 P11A), an antimicrobial peptide (AMP), with E. coli spheroplasts and B. megaterium protoplasts.