Department of Chemical Engineering and Biotechnology, University of Cambridge, Cambridge CB3 0AS, U.K.
School of Biomedical Engineering, Cornell University, Ithaca, New York 14853, United States.
Langmuir. 2022 Jul 26;38(29):8773-8782. doi: 10.1021/acs.langmuir.2c00628. Epub 2022 Jun 24.
The rise of antibiotic resistance is a growing worldwide human health issue, with major socioeconomic implications. An understanding of the interactions occurring at the bacterial membrane is crucial for the generation of new antibiotics. Supported lipid bilayers (SLBs) made from reconstituted lipid vesicles have been used to mimic these membranes, but their utility has been restricted by the simplistic nature of these systems. A breakthrough in the field has come with the use of outer membrane vesicles derived from Gram-negative bacteria to form SLBs, thus providing a more physiologically relevant system. These complex bilayer systems hold promise but have not yet been fully characterized in terms of their composition, ratio of natural to synthetic components, and membrane protein content. Here, we use correlative atomic force microscopy (AFM) with structured illumination microscopy (SIM) for the accurate mapping of complex lipid bilayers that consist of a synthetic fraction and a fraction of lipids derived from outer membrane vesicles (OMVs). We exploit the high resolution and molecular specificity that SIM can offer to identify areas of interest in these bilayers and the enhanced resolution that AFM provides to create detailed topography maps of the bilayers. We are thus able to understand the way in which the two different lipid fractions (natural and synthetic) mix within the bilayers, and we can quantify the amount of bacterial membrane incorporated into the bilayer. We prove the system's tunability by generating bilayers made using OMVs engineered to contain a green fluorescent protein (GFP) binding nanobody fused with the porin OmpA. We are able to directly visualize protein-protein interactions between GFP and the nanobody complex. Our work sets the foundation for accurately understanding the composition and properties of OMV-derived SLBs to generate a high-resolution platform for investigating bacterial membrane interactions for the development of next-generation antibiotics.
抗生素耐药性的兴起是一个日益严重的全球性人类健康问题，具有重大的社会经济影响。了解细菌膜上发生的相互作用对于开发新的抗生素至关重要。由重建脂质囊泡制成的支持脂质双层 (SLB) 已被用于模拟这些膜，但由于这些系统的简单性质，其用途受到限制。革兰氏阴性菌来源的外膜囊泡 (OMV) 用于形成 SLB，这一突破为该领域提供了一个更具生理相关性的系统，从而打破了这一限制。这些复杂的双层系统具有很大的应用前景，但在组成、天然与合成成分的比例以及膜蛋白含量方面尚未得到充分的表征。在这里，我们使用相关原子力显微镜 (AFM) 和结构照明显微镜 (SIM) 对由合成部分和源自 OMV 的脂质部分组成的复杂脂质双层进行精确映射。我们利用 SIM 提供的高分辨率和分子特异性来识别这些双层中的感兴趣区域，并利用 AFM 提供的增强分辨率来创建双层的详细形貌图。我们能够了解两种不同脂质成分（天然和合成）在双层内混合的方式，并能够定量测量纳入双层的细菌膜的量。我们通过生成使用工程化的 OMV 制成的双层来证明该系统的可调节性，这些 OMV 包含与孔蛋白 OmpA 融合的绿色荧光蛋白 (GFP) 结合纳米抗体。我们能够直接可视化 GFP 和纳米抗体复合物之间的蛋白质-蛋白质相互作用。我们的工作为准确了解源自 OMV 的 SLB 的组成和特性奠定了基础，为开发下一代抗生素的细菌膜相互作用的高分辨率平台奠定了基础。
