A Molecularly Complete Planar Bacterial Outer Membrane Platform

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.

Supported Lipid Bilayers of Escherichia coli Extracted Lipids and Their Calcium Dependence

“…The result is consistent with previous studies, which showed at least ∼2 mM of Ca 2+ is necessary for the formation of PLE-SLB on SiO 2 /Si, mica, TiO 2 , indium tin oxide and gold (Dodd et al, 2008;Merz et al, 2008;Lopes et al, 2010;Unsay et al, 2013;Lind et al, 2015;Hsia et al, 2016;Konarzewska et al, 2017;Márquez and Vélez, 2017). In the previous studies of the bacterial mimic lipid bilayer, however, averaged response from the sample was mainly obtained using QCM and fluorescence microscopy (Dodd et al, 2008;Merz et al, 2008;Lopes et al, 2010;Unsay et al, 2013;Lind et al, 2015;Phan and Shin, 2015;Hsia et al, 2016;Konarzewska et al, 2017;Márquez and Vélez, 2017). The results in this study showed the heterogeneity of SLB especially at the threshold [Ca 2+ ] around 2.5 mM.…”

“…As a bacterial model cell system, reconstituted Escherichia coli (E. coli) cell membrane has been studied (Dodd et al, 2008;Merz et al, 2008;Lopes et al, 2010;Unsay et al, 2013;Lind et al, 2015;Phan and Shin, 2015;Hsia et al, 2016;Konarzewska et al, 2017;Márquez and Vélez, 2017). Understanding the bacterial membrane system helps in revealing the mechanism of molecular recognition and material transportation during bacterial infection for medical technology.…”

“…There are several previous studies on the formation of SLBs containing E. coli lipids such as polar lipid extract (PLE) or total lipid extract (TLE) (Dodd et al, 2008;Merz et al, 2008;Lopes et al, 2010;Unsay et al, 2013;Lind et al, 2015;Hsia et al, 2016;Konarzewska et al, 2017;Márquez and Vélez, 2017). Solid substrates, which are generally used for SLB formation, e.g., glass, oxidized layer on a Si wafer, and mica, are negatively charged.…”

Compatibility of GROMOS-Derived Atomic Parameters for Lipopolysaccharide Membranes with the SPC/E Water Model and Alternative Long-Range Electrostatic Treatments Using Single Nonbonded Cutoff and Atom-Based Charge Schemes

“…However, recent progresses in the development of supported asymmetric bilayers made OM models amenable to characterization via structural techniques. [18][19][20] The first MD simulation of a bacterial outer membrane was performed for the rough LPS of Pseudomonas aeruginosa using the GLYCAM93 21 and AMBER95 22 atomic parameters to describe carbohydrate and lipid moieties, respectively. 23 The simulated bilayer consisted of 16 LPS units in the outer leaflet, 40 DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine) units in the inner leaflet and a total of 104 Ca 2+ to neutralize the system.…”

Biomimetic Electronic Devices for Measuring Bacterial Membrane Disruption

“…Typically, polymyxins bind to lipids in the bacterial membrane via electrostatic interactions. Hydrophobic interactions between their N ‐terminal fatty acid tail and the membrane fatty acid lipid tails lead to insertion, membrane destabilization, and cell lysis . Electrostatic interactions are responsible for the initial binding of the antibacterial compound to the anionic surface of the bacteria.…”