Double-Shell Giant Vesicles Mimicking Gram-Negative Cell Wall Behavior during Dehydration

Abstract

A biomimetic system modeling the behavior of Gram-negative bacteria under hyperosmotic stress was developed. To this end, we introduced a two-step electroswelling procedure for encapsulation of giant unilamellar vesicles by an additional membrane. Both membranes of the resulting double-shell vesicles (DSVs) were fluid. Additionally, the outer membrane was rigidified by a monolayer of streptavidin forming a two-dimensional crystal. For strong attachment of this protein layer, the outer membrane contained biotinylated lipids. This reinforced protein-lipid compound membrane served to model the assembly of the murein wall and outer membrane of Gram-negative bacteria. We characterized DSVs by confocal laser scanning microscopy. Furthermore, DSVs were exposed to hyperosmotic media (osmotic difference 0-1100 mosm/L), and the resulting shapes were analyzed. DSVs coated with streptavidin were much less deformed or destroyed by osmotic stress than bare DSVs or DSVs coated with noncrystalline avidin. Osmotically stressed DSVs coated with streptavidin displayed weak wrinkling of the outer membrane and formed small daughter vesicles of the inner membrane. Both features and the toughness against hyperosmotic stress are well described characteristics of Gram-negative bacteria.