Abstract
The bacterial outer membrane (OM) is a barrier containing membrane proteins and liposaccharides that fulfill crucial functions for Gram-negative bacteria. With the advent of drug-resistant bacteria, it is necessary to understand the functional role of this membrane and its constituents to enable novel drug designs. Here we report a simple method to form an OM-like supported bilayer (OM-SB), which incorporates native lipids and membrane proteins of gram-negative bacteria from outer membrane vesicles (OMVs). We characterize the formation of OM-SBs using quartz crystal microbalance with dissipation (QCM-D) and fluorescence microscopy. We show that the orientation of proteins in the OM-SB matches the native bacterial membrane, preserving the characteristic asymmetry of these membranes. As a demonstration of the utility of the OM-SB platform, we quantitatively measure antibiotic interactions between OM-SBs and polymyxin B, a cationic peptide used to treat Gram-negative infections. This data enriches understanding of the antibacterial mechanism of polymyxin B, including disruption kinetics and changes in membrane mechanical properties. Combining OM-SBs with microfluidics will enable higher throughput screening of antibiotics. With a broader view, we envision that a molecularly complete membrane-scaffold could be useful for cell-free applications employing engineered membrane proteins in bacterial membranes for myriad technological purposes.
Highlights
Details on the preparation of lipid vesicles, outer membrane vesicles (OMVs) size and surface charge characterization, Polydimethylsiloxane (PDMS) well fabrication, fluorescent labeling of OMVs and liposomes, preparation of glass coverslips used as supports for supported bilayers, formation of Supported lipid bilayers (SLBs) from pure liposomes, fluorescence recovery after photobleaching (FRAP), and Proteinase K susceptibility assays for the determination of ClyA-green fluorescent protein (GFP) orientation in outer membrane (OM)-SB are provided in the Supplemental Information
To induce OMVs to fuse to a glass surface, we modified a procedure reported previously by our group for the formation of proteinaceous bilayers from mammalian cell blebs[36,43]. This involved first adsorbing OMVs onto the glass followed by addition of PEG-liposomes to the system to catalyze OMV rupture
We demonstrate how OM-like supported bilayer (OM-SB) is applied to probe the antimicrobial mechanism of polymyxin B (PMB), an amphipathic peptide composed of a cationic cyclic portion and a hydrophobic fatty acyl tail
Summary
We report a simple method to form an OM-like supported bilayer (OM-SB), which incorporates native lipids and membrane proteins of gram-negative bacteria from outer membrane vesicles (OMVs). The resulting OM-SBs were studied in detail, including bilayer property characterization, protein orientation, and an investigation of bilayer formation kinetics using several surface-sensitive techniques: total internal reflection fluorescence microscopy, fluorescence recovery after photobleaching (FRAP), and quartz crystal microbalance with dissipation (QCM-D).
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