The development of new antibiotics is particularly problematic in Gram-negative bacteria due to the presence of the outer membrane (OM), which serves as a permeability barrier. Recently, the β-barrel assembly machine (BAM), located in the OM and responsible for β-barrel type OM protein (OMP) assembly, has been validated as a novel target for antibiotics. Here, we identified potential BAM complex inhibitors using a screening approach that reports on cell envelope σ^E and Rcs stress in Escherichia coli. Screening a library consisting of 316 953 compounds yielded five compounds that induced σ^E and Rcs stress responses, while not inducing the intracellular heat-shock response. Two of the five compounds (compounds 2 and 14) showed the characteristics of known BAM complex inhibitors: synergy with OMP biogenesis mutants, decrease in the abundance of various OMPs, and loss of OM integrity. Importantly, compound 2 also inhibited BAM-dependent OMP folding in an in vitro refolding assay using purified BAM complex reconstituted in proteoliposomes.

中文翻译：

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在筛选方法中结合细胞包膜应激报告基因检测来鉴定 BAM 复合物抑制剂

由于革兰氏阴性菌具有作为渗透性屏障的外膜（OM），新抗生素的开发尤其成问题。最近，位于 OM 中负责 β 桶型 OM 蛋白（OMP）组装的 β 桶组装机（BAM）已被验证为抗生素的新靶点。在这里，我们使用报告大肠杆菌细胞包膜 σ ^E和 Rcs 应激的筛选方法鉴定了潜在的 BAM 复合物抑制剂。筛选由 316 953 种化合物组成的库，产生了 5 种可诱导 σ ^E和 Rcs 应激反应，但不诱导细胞内热休克反应的化合物。五种化合物中的两种（化合物 2 和 14）显示出已知 BAM 复合物抑制剂的特征：与 OMP 生物发生突变体协同作用、各种 OMP 丰度降低以及 OM 完整性丧失。重要的是，在使用蛋白脂质体中重构的纯化 BAM 复合物进行的体外重折叠测定中，化合物 2 还抑制了 BAM 依赖性 OMP 折叠。