Bacterial Outer-Membrane-Mimicking Giant Unilamellar Vesicle Model for Detecting Antimicrobial Permeability

Abstract

The construction of bacterial outer membrane models with native lipids like lipopolysaccharide (LPS) is a barrier to understanding antimicrobial permeability at the membrane interface. Here, we engineer bacterial outer membrane (OM)mimicking giant unilamellar vesicles (GUVs) by constituting LPS under different pH conditions and assembled GUVs with controlled dimensions. We quantify the LPS reconstituted in GUV membranes and reveal their arrangement in the leaflets of the vesicles. Importantly, we demonstrate the applications of OM vesicles by exploring antimicrobial permeability activity across membranes. Model peptides, melittin and magainin-2, are examined where both peptides exhibit lower membrane activity in OM vesicles than vesicles devoid of LPS. Our findings reveal the mode of action of antimicrobial peptides in bacterial-membrane-mimicking models. Notably, the critical peptide concentration required to elicit activity on model membranes correlates with the cell inhibitory concentrations that revalidate our models closely mimic bacterial membranes. In conclusion, we provide an OM-mimicking model capable of quantifying antimicrobial permeability across membranes.

Engineering semi-permeable giant liposomes

“…The permeability of individual molecules is quantified in terms of the permeabilization percent of the vesicle population, where vesicles are designated as permeabilized when I in / I out > 0.98 (ESI†). 34 The GUVs (DPhPC 50 mol%, POPC 50 mol%) without DOTAP do not allow diffusion of small molecules investigated here revealed by single vesicle images and low percent of vesicle permeabilization (Fig. 2A and B).…”

Dynamic Control of Functional Coacervates in Synthetic Cells

“…The vesicles are prepared using the PVA gel-assisted method. ,, GUVs with semipermeable membranes are prepared with a defined lipid composition of 44.8 mol % 1,2-diphytanyl- sn -glycero-3-phosphocholine (DPhPC), 44.8 mol % 1-palmitoyl-2-oleoyl- sn -glycerol-3-phosphocholine (POPC), 10 mol % dimethyldioctadecylammonium bromide (DDA-B) or 1,2-dioleoyl-3-trimethylammonium-propane (chloride salt) (DOTAP), and 0.4 mol % 1,2-dioleoyl- sn -glycerol-3-phosphoethanolamine labeled with (ATTO 390-DOPE)/0.05 mol % ATTO 550-DOPE. The lipid composition is chosen such that it is the best suited for encapsulating the coacervate forming components such as the polycation PLL and enabling the permeation of small molecules without the need of reconstituting membrane pores .…”

Engineering Planar Gram-Negative Outer Membrane Mimics Using Bacterial Outer Membrane Vesicles

“…Firstly, it provides a valuable tool for assessing interactions between potential antibiotics, such as AMPs, and the bacterial membrane using a variety of analytical techniques, such as surface-based biosensors atomic force microscopy 9,10 , fluorescence microscopy 11 , and quartz crystal microbalance 12 . In recent years, the utilization of synthetic lipids to create planar bilayers has emerged as a significant biotechnological advancement, and it has also been employed in the past for testing the effect of AMPs on gram-negative bacteria, where bacterial lipids with known composition were mixed to prepare vesicles [13][14][15] . However, synthetic lipid vesicles have several disadvantages, including that their composition is not an exact mimic of the outer membrane.…”