Singh Aarshi N, Wu Meishan, Ye Tiffany T, Brown Angela C, Wittenberg Nathan J
Department of Chemistry, Lehigh University, Bethlehem, PA, USA.
Department of Chemical and Biomolecular Engineering, Lehigh University, Bethlehem, PA, USA.
bioRxiv. 2024 Aug 20:2023.12.11.570829. doi: 10.1101/2023.12.11.570829.
Antibiotic resistance is a major challenge in modern medicine. The unique double membrane structure of gram-negative bacteria limits the efficacy of many existing antibiotics and adds complexity to antibiotic development by limiting transport of antibiotics to the bacterial cytosol. New methods to mimic this barrier would enable high-throughput studies for antibiotic development. In this study, we introduce an innovative approach to modify outer membrane vesicles (OMVs) from to generate planar supported lipid bilayer membranes. Our method first involves the incorporation of synthetic lipids into OMVs using a rapid freeze-thaw technique to form outer membrane hybrid vesicles (OM-Hybrids). Subsequently, these OM-Hybrids can spontaneously rupture when in contact with SiO surfaces to form a planar outer membrane supported bilayer (OM-SB). We assessed the formation of OM-Hybrids using dynamic light scattering and a fluorescence quenching assay. To analyze the formation of OM-SBs from OM-Hybrids we used quartz crystal microbalance with dissipation monitoring (QCM-D) and fluorescence recovery after photobleaching (FRAP). Additionally, we conducted assays to detect surface-associated DNA and proteins on OM-SBs. The interaction of an antimicrobial peptide, polymyxin B, with the OM-SBs was also assessed. These findings emphasize the capability of our platform to produce planar surfaces of bacterial outer membranes, which in turn, could function as a valuable tool for streamlining the development of antibiotics.
抗生素耐药性是现代医学面临的一项重大挑战。革兰氏阴性菌独特的双膜结构限制了许多现有抗生素的疗效，并且通过限制抗生素向细菌胞质溶胶的转运增加了抗生素开发的复杂性。模拟这种屏障的新方法将能够用于抗生素开发的高通量研究。在本研究中，我们引入了一种创新方法，对来自[具体来源未给出]的外膜囊泡（OMV）进行修饰，以生成平面支撑脂质双分子层膜。我们的方法首先涉及使用快速冻融技术将合成脂质掺入OMV中，以形成外膜杂交囊泡（OM - 杂交体）。随后，这些OM - 杂交体在与SiO表面接触时会自发破裂，形成平面外膜支撑双分子层（OM - SB）。我们使用动态光散射和荧光猝灭测定法评估了OM - 杂交体的形成。为了分析从OM - 杂交体形成OM - SB，我们使用了具有耗散监测的石英晶体微天平（QCM - D）和光漂白后荧光恢复（FRAP）。此外，我们进行了检测OM - SB上表面相关DNA和蛋白质的测定。还评估了抗菌肽多粘菌素B与OM - SB的相互作用。这些发现强调了我们的平台产生细菌外膜平面表面的能力，这反过来可以作为简化抗生素开发的有价值工具。
