Bacterial Multidrug Exporters
Bacterial Multidrug Exporters
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DOI：
10.1007/978-1-4939-7454-2
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发表时间：
2018
期刊：
影响因子：
--
通讯作者：
A. Yamaguchi;K. Nishino
中科院分区：
文献类型：
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作者：
A. Yamaguchi;K. Nishino
抗性诱导细胞分裂（RND）蛋白家族是与周质衔接蛋白和外膜孔蛋白结合以影响革兰氏阴性菌中来自细胞质和周质的底物转运的内膜转运蛋白。各种结构不同的化合物是RND转运蛋白的底物。沿着它们在抗生素抗性中的显著作用，这些转运蛋白对于生态位定殖、群体感应和毒力以及脂肪酸和胆汁盐的去除是必不可少的。因此，RND是抗菌剂开发的有吸引力的目标。然而，虽然用RND抑制剂增强抗生素的效用是一个吸引人的概念，但只有一小部分核心化学型被确定为外排泵抑制剂（EPI）。因此，我们的主要目标是开发和验证RND模型系统的外排分析和发现策略。在这里，我们描述了一种流式细胞仪染料积累试验，使用荧光素二乙酸酯（FDA）询问模型革兰氏阴性病原体大肠杆菌，Francisella tularensis，和类鼻疽伯克霍尔德氏菌。在存在已知外排抑制剂和RND缺失菌株的情况下，荧光染料保留增加。该测定可以以高通量的形式用于评估染料底物候选物的外排，并筛选化学文库中的新型EPI。使用微量稀释培养板在选择试剂生物安全等级-3（BSL 3）环境中测试抑制致病菌株外排的分类化合物的生长抑制和抗生素增效作用。这种组合的方法证明了实用的流式细胞术分析外排活性，并提供了一个有用的平台，在其中表征致病性革兰氏阴性菌的外排。筛选小分子库的新EPI候选人提供了发现新类别的抗菌化合物的潜力。
The resistance nodulation cell division (RND) family of proteins are inner membrane transporters that associate with periplasmic adaptor proteins and outer membrane porins to affect substrate transport from the cytosol and periplasm in Gram-negative bacteria. Various structurally diverse compounds are substrates of RND transporters. Along with their notable role in antibiotic resistance, these transporters are essential for niche colonization, quorum sensing, and virulence as well as for the removal of fatty acids and bile salts. As such, RNDs are an attractive target for antimicrobial development. However, while enhancing the utility of antibiotics with an RND inhibitor is an appealing concept, only a small core of chemotypes has been identified as efflux pump inhibitors (EPIs). Thus, our key objective is the development and validation of an efflux profiling and discovery strategy for RND model systems. Here we describe a flow cytometric dye accumulation assay that uses fluorescein diacetate (FDA) to interrogate the model Gram-negative pathogensEscherichia coli,Franscisella tularensis, andBurkholderia pseudomallei. Fluorochrome retention is increased in the presence of known efflux inhibitors and in RND deletion strains. The assay can be used in a high-throughput format to evaluate efflux of dye-substrate candidates and to screen chemical libraries for novel EPIs. Triaged compounds that inhibit efflux in pathogenic strains are tested for growth inhibition and antibiotic potentiation using microdilution culture plates in a select agent Biosafety Level-3 (BSL3) environment. This combined approach demonstrates the utility of flow cytometric analysis for efflux activity and provides a useful platform in which to characterize efflux in pathogenic Gram-negative bacteria. Screening small molecule libraries for novel EPI candidates offers the potential for the discovery of new classes of antibacterial compounds.