Identification of Efflux Substrates Using a Riboswitch-Based Reporter in Pseudomonas aeruginosa.
Identification of Efflux Substrates Using a Riboswitch-Based Reporter in Pseudomonas aeruginosa.
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作者：
铜绿假单胞菌对许多类抗生素具有内在耐药性，反映了其外膜的限制性性质及其众多外排系统的作用。然而，在这种细菌中化合物的吸收，保留和流出的动力学仍然不完全清楚。在这里，我们利用Z-核苷酸传感核糖开关的传感器能力来创建一个实验系统，该系统能够识别渗透细菌细胞的化合物的物理化学和结构特性，避免外排，并干扰叶酸循环或从头嘌呤合成。在第一步中，在流出缺陷型铜绿假单胞菌中筛选了富含抗叶酸药物的结构多样性化合物的ZTP（5-氨基咪唑-4-甲酰胺核苷5′-三磷酸）核糖开关报告活性，使我们能够鉴定进入细胞并破坏叶酸途径的化合物。这些最初的命中，然后重新筛选使用同基因外排精通细菌，使我们能够分离外排底物外排避免。我们证实了这一分类，通过测量细胞内水平的选择化合物的流出缺陷和精通应变使用高分辨率液相色谱-质谱（LC-MS）。这种简单而强大的方法，优化了高通量筛选，能够发现许多可渗透的化合物，避免外排，并为进一步完善这种多药耐药革兰氏阴性病原体中控制外排的理化和结构规则铺平了道路。重要性铜绿假单胞菌感染的治疗变得越来越具有挑战性。针对这种多重耐药细菌的新型抗生素的开发是一个优先事项，但由于其高度限制性的外膜和多个外排泵的作用，许多候选药物从未在细菌细胞中达到有效浓度。在这里，我们在铜绿假单胞菌中开发了一个强大而简单的报告系统，以筛选化学文库并识别进入细胞并留在细胞内或进入细胞并通过外排系统输出的化合物。这种方法使得能够开发铜绿假单胞菌中化合物摄取和保留的规则，这将导致更合理的新型抗生素设计。
Pseudomonas aeruginosa is intrinsically resistant to many classes of antibiotics, reflecting the restrictive nature of its outer membrane and the action of its numerous efflux systems. However, the dynamics of compound uptake, retention, and efflux in this bacterium remain incompletely understood. Here, we exploited the sensor capabilities of a Z-nucleotide-sensing riboswitch to create an experimental system able to identify physicochemical and structural properties of compounds that permeate the bacterial cell, avoid efflux, and perturb the folate cycle or de novo purine synthesis. In the first step, a collection of structurally diverse compounds enriched in antifolate drugs was screened for ZTP (5-aminoimidazole-4-carboxamide riboside 5′-triphosphate) riboswitch reporter activity in efflux-deficient P. aeruginosa, allowing us to identify compounds that entered the cell and disrupted the folate pathway. These initial hits were then rescreened using isogenic efflux-proficient bacteria, allowing us to separate efflux substrates from efflux avoiders. We confirmed this categorization by measuring intracellular levels of select compounds in the efflux-deficient and -proficient strain using high-resolution liquid chromatography-mass spectrometry (LC-MS). This simple yet powerful method, optimized for high-throughput screening, enables the discovery of numerous permeable compounds that avoid efflux and paves the way for further refinement of the physicochemical and structural rules governing efflux in this multidrug-resistant Gram-negative pathogen. IMPORTANCE Treatment of Pseudomonas aeruginosa infections has become increasingly challenging. The development of novel antibiotics against this multidrug-resistant bacterium is a priority, but many drug candidates never achieve effective concentrations in the bacterial cell due to its highly restrictive outer membrane and the action of multiple efflux pumps. Here, we develop a robust and simple reporter system in P. aeruginosa to screen chemical libraries and identify compounds that either enter the cell and remain inside or enter the cell and are exported by efflux systems. This approach enables the development of rules of compound uptake and retention in P. aeruginosa that will lead to more rational design of novel antibiotics.
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