Depth-Resolved Temperature-Dependent Penetration of Polymyxin B in Phospholipids/Lipopolysaccharide Asymmetric Bilayers

The lipid matrix of the outer membrane (OM) of Gram-negative bacteria consists of a highly asymmetric lipid bilayer containing phospholipids on the inner leaflet and lipopolysaccharides (LPS) in the outer layer. The latter ensures that harmful molecules do not permeate the bacterial cell, but polymyxin B (PmB), a last-resort antibiotic, is capable of interfering with the stability of the LPS layer and overcoming the OM barrier. We have previously shown that the efficacy of PmB in disrupting isotopically asymmetric OM models (²H-phospholipids and ¹H-LPS) is regulated by the gel-to-fluid phase transition of the LPS layer. Here, we employ fully deuterated OM models (²H-phospholipids and ²H-LPS) to track the temperature-dependent penetration of PmB within the model membrane by using neutron reflectometry. We use a model-independent approach to quantify PmB penetration as a function of both concentration and temperature as well as a model-dependent analysis to localize PmB in the asymmetric bilayer. By leveraging the ability of neutrons to differentiate hydrogen from deuterium in structural biology we find that PmB hijacks LPS molecules and accumulates predominantly in the hydrophobic region of lipid A.