부산대학교 도서관

Abstract The outer membrane insertase of Gram-negative bacteria, BAM, is a key target for urgently needed novel antibiotics. Functional reconstitutions of BAM have so far been limited to synthetic membranes and with low throughput capacity for inhibitor screening. Here, we describe a BAM functional assay in native membrane environment capable of high-throughput screening. This is achieved by employing outer membrane vesicles (OMVs) to present BAM directly in native membranes. Refolding of the model substrate OmpT by BAM was possible from the chaperones SurA and Skp, with the required SurA concentration three times higher than Skp. In the OMVs, the antibiotic darobactin had a tenfold higher potency than in synthetic membranes, highlighting the need for native conditions in antibiotics development. The assay is successfully miniaturized for 1536-well plates and upscaled using large scale fermentation, resulting in high-throughput capacities to screen large commercial compound libraries. Our OMV-based assay thus lays the basis for discovery, hit validation and lead expansion of antibiotics targeting BAM.