Application of bacterial cytological profiling to crude natural product extracts reveals the antibacterial arsenal of Bacillus subtilis.
Application of bacterial cytological profiling to crude natural product extracts reveals the antibacterial arsenal of Bacillus subtilis.
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DOI：
10.1038/ja.2015.116
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发表时间：
2016-05
期刊：
影响因子：
--
通讯作者：
Pogliano K
中科院分区：
文献类型：
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作者：
Nonejuie P;Trial RM;Newton GL;Lamsa A;Ranmali Perera V;Aguilar J;Liu WT;Dorrestein PC;Pogliano J;Pogliano K
尽管大多数临床使用的抗生素均源自天然产物，但由于这些提取物的复杂性以及将生物活性与特定分子相关联的工具有限，从天然产物提取物中鉴定新的抗菌分子很困难。在这里，我们证明细菌细胞学分析 (BCP) 提供了一种快速方法，用于确定平板和复杂天然产物提取物的作用机制以及活性引导纯化。我们制备了枯草芽孢杆菌 3610 的提取物，可杀死大肠杆菌 lptD 突变体，并使用 BCP 观察未分级提取物中的两种生物活性：翻译抑制和细胞质膜透化。我们使用 BCP 指导纯化负责每种活性的分子，鉴定翻译抑制剂 bacillaene 和 bacillaene B（糖基化 bacillaene），并证明两个分子有助于细胞通透，即细菌素枯草溶菌素和环肽孢子形成杀伤因子。我们的结果表明 bacillaene 介导翻译停滞，并表明 bacillaene B 的最低抑制浓度比未修饰的 bacillaene 高 10 倍。最后，我们证明 BCP 可用于在琼脂平板上筛选菌株，无需提取物制备，大大节省时间并提高通量。因此，即使存在多种活性，BCP 也可以通过鉴定具有有趣生物活性的菌株、粗提物和级分来简化新型天然产物的分离，从而使研究人员能够将劳动密集型步骤集中在具有所需活性的那些上。
Although most clinically used antibiotics are derived from natural products, identifying new antibacterial molecules from natural product extracts is difficult due to the complexity of these extracts and the limited tools to correlate biological activity with specific molecules. Here, we show that bacterial cytological profiling (BCP) provides a rapid method for mechanism of action determination on plates and in complex natural product extracts and for activity-guided purification. We prepared an extract from Bacillus subtilis 3610 that killed the Escherichia coli lptD mutant and used BCP to observe two types of bioactivities in the unfractionated extract: inhibition of translation and permeablization of the cytoplasmic membrane. We used BCP to guide purification of the molecules responsible for each activity, identifying the translation inhibitors bacillaene and bacillaene B (glycosylated bacillaene) and demonstrating that two molecules contribute to cell permeabilitization, the bacteriocin subtilosin and the cyclic peptide sporulation killing factor. Our results suggest that bacillaene mediates translational arrest, and show that bacillaene B has a minimum inhibitory concentration 10 × higher than unmodified bacillaene. Finally, we show that BCP can be used to screen strains on an agar plate without the need for extract preparation, greatly saving time and improving throughput. Thus, BCP simplifies the isolation of novel natural products, by identifying strains, crude extracts and fractions with interesting bioactivities even when multiple activities are present, allowing investigators to focus labor-intensive steps on those with desired activities.