Engineering\nPlanar Gram-Negative Outer Membrane Mimics\nUsing Bacterial Outer Membrane Vesicles

Antibiotic resistance is a major challenge in modern\nmedicine.\nThe unique double membrane structure of Gram-negative bacteria limits\nthe efficacy of many existing antibiotics and adds complexity to antibiotic\ndevelopment by limiting transport of antibiotics to the bacterial\ncytosol. New methods to mimic this barrier would enable high-throughput\nstudies for antibiotic development. In this study, we introduce an\ninnovative approach to modify outer membrane vesicles (OMVs) from Aggregatibacter actinomycetemcomitans, to generate\nplanar supported lipid bilayer membranes. Our method first involves\nthe incorporation of synthetic lipids into OMVs using a rapid freeze–thaw\ntechnique to form outer membrane hybrid vesicles (OM-Hybrids). Subsequently,\nthese OM-Hybrids can spontaneously rupture when in contact with SiO<sub>2</sub> surfaces to form a planar outer membrane supported bilayer\n(OM-SB). We assessed the formation of OM-Hybrids using dynamic light\nscattering and a fluorescence quenching assay. To analyze the formation\nof OM-SBs from OM-Hybrids we used quartz crystal microbalance with\ndissipation monitoring (QCM-D) and fluorescence recovery after photobleaching\n(FRAP). Additionally, we conducted assays to detect surface-associated\nDNA and proteins on OM-SBs. The interaction of an antimicrobial peptide,\npolymyxin B, with the OM-SBs was also assessed. These findings emphasize\nthe capability of our platform to produce planar surfaces of bacterial\nouter membranes, which in turn, could function as a valuable tool\nfor streamlining the development of antibiotics.