PDF(Pubmed)
Abstract：
Phenotypic analysis assays such as bacterial cytological profiling (BCP) have become increasingly popular for antibiotic mode of action analysis. A plethora of dyes, protein fusions, and reporter strains are available and have been used for this purpose, enabling both rapid mode of action categorization and in-depth analysis of antibiotic mechanisms. However, non-expert researchers may struggle choosing suitable assays and interpreting results. This is a particular problem for antibiotics that have multiple or complex targets, such as the bacterial cell envelope. Here, we set out to curate a minimal set of accessible and affordable phenotypic assays that allow distinction between membrane and cell wall targets, can identify dual-action inhibitors, and can be implemented in most research environments. To this end, we employed BCP, membrane potential, fluidity, and cell wall synthesis assays. To assess specificity and ease of interpretation, we tested three well-characterized and commercially available reference antibiotics: the potassium ionophore valinomycin, the lipid II-binding glycopeptide vancomycin, and the dual-action lantibiotic nisin, which binds lipid II and forms a membrane pore. Based on our experiments, we suggest a minimal set of BCP, a membrane-potentiometric probe, and fluorescent protein fusions to MinD and MreB as basic assay set and recommend complementing these assays with Laurdan-based fluidity measurements and a PliaI reporter fusion, where indicated. We believe that our results can provide guidance for researchers who wish to use phenotypic analysis for mode of action studies but do not possess the specialized equipment or expert knowledge to employ the full breadth of possible techniques.IMPORTANCEPhenotypic analysis assays using specialized fluorescence fusions and dyes have become increasingly popular in antibiotic mode of action analysis. However, it can be difficult to implement these methods due to the need for specialized equipment and/or the complexity of bacterial cell biology and physiology, making the interpretation of results difficult for non-experts. This is especially problematic for compounds that have multiple or pleiotropic effects, such as inhibitors of the bacterial cell envelope. In order to make phenotypic analysis assays accessible to labs, whose primary expertise is not bacterial cell biology, or with limited equipment and resources, a set of simple and broadly accessible assays is needed that is easy to implement, execute, and interpret. Here, we have curated a set of assays and strains that does not need highly specialized equipment, can be performed in most labs, and is straightforward to interpret without knowing the intricacies of bacterial cell biology.
摘要：
诸如细菌细胞学谱分析(BCP)的表型分析测定对于抗生素作用模式分析已经变得越来越流行。大量的染料,蛋白质融合，和报告菌株可用，并已用于此目的，实现快速的作用模式分类和对抗生素机制的深入分析。然而,非专业研究人员可能难以选择合适的分析和解释结果。对于具有多个或复杂靶标的抗生素来说，这是一个特殊的问题，如细菌细胞包膜。这里,我们着手策划一套最小的可访问和负担得起的表型测定，允许区分膜和细胞壁的目标，可以识别双重作用抑制剂，并且可以在大多数研究环境中实施。为此,我们雇佣了BCP,膜电位，流动性,和细胞壁合成试验。为了评估特异性和易于解释，我们测试了三种特征明确且市售的参考抗生素：钾离子载体缬氨酸霉素，脂质II结合糖肽万古霉素，和双重作用的抗生素乳酸链球菌素，结合脂质II并形成膜孔。根据我们的实验,我们建议一组最小的BCP，膜电位探针，并将荧光蛋白融合到MinD和MreB作为基本测定集，并建议使用基于Laurdan的流动性测量和PliaI报告融合来补充这些测定，whereindicated.我们相信，我们的结果可以为希望将表型分析用于行动模式研究但不具备专用设备或专家知识来采用全部可能技术的研究人员提供指导。使用专门的荧光融合和染料的表型分析测定在抗生素作用模式分析中变得越来越流行。然而,由于需要专门的设备和/或细菌细胞生物学和生理学的复杂性，实施这些方法可能很困难。使非专家难以解释结果。这对于具有多重或多效性的化合物尤其成问题。如细菌细胞包膜的抑制剂。为了使实验室可以使用表型分析测定，他们的主要专长不是细菌细胞生物学，或者设备和资源有限，需要一组简单且易于实现的测定，execute,和解释。这里,我们已经策划了一套不需要高度专用设备的检测和菌株，可以在大多数实验室进行，并且在不了解细菌细胞生物学的复杂性的情况下直接解释。
