Angew. Chem. Int. Edn. Engl. doi:10.1002/anie.201504287

Credit: JEREMY LASKY

Gram-negative bacteria have a cellular envelope consisting of an asymmetric outer lipid bilayer (OM) and an inner bilayer (IM). The OM comprises cation-crosslinked lipopolysaccharide (LPS) molecules in its outer leaflet that create a physical barrier to penetration by antimicrobial molecules. The OM and IM also both provide an additional hydrophobic barrier. Although the chemical composition of the envelope is well defined, its physical and dynamic properties have been difficult to study because few in vitro models recapitulate the intact system. Clifton et al. have now added to this toolbox by generating an asymmetric model of the complete E. coli envelope. To do this, they coated a gold surface with a self-assembled monolayer, and sequentially modified this to create a floating supported bilayer (FSB) consisting of a deuterium-labeled phospholipid, DPPC, and unlabeled LPS. The authors were then able to monitor formation of the FSB and verify its asymmetry by deuterium-sensitive neutron scattering. They found that the FSB thickness and stability agreed with measurements from simpler model membranes. In addition, the response of the model to the removal of stabilizing cations was consistent with in vivo OM behavior. The authors were also able to use the OM model to probe the structural consequences of the antimicrobial protein (AMP) lactoferrin for the first time. Lactoferrin is thought to disrupt LPS crosslinks, and the authors saw large perturbations in the asymmetry and LPS thickness when it was added to the model membrane. Similar experiments with another AMP, lysozyme, demonstrated its ability to bind electrostatically to the anionic outer leaflet, in agreement with its previously demonstrated more limited OM-disruptive effects. These results suggest that the OM model recapitulates the intact OM system and enables studies of OM interactions with other antimicrobials. As well, the model should be amenable to techniques such as AFM and FRET to monitor further biophysical and chemical properties of the complex cellular envelope.