Combining Cell Envelope Stress Reporter Assays In A Screening Approach To Identify Bam Complex Inhibitors

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 beta-barrel assembly machine (BAM), located in the OM and responsible for beta-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 sigma(E) and Rcs stress in Escherichia coli. Screening a library consisting of 316 953 compounds yielded five compounds that induced sigma(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.