Lipopolysaccharide-Dependent Membrane Permeation and Lipid Clustering Caused by Cyclic Lipopeptide Colistin

[Image: see text] Polyanionic lipopolysaccharides (LPS) play an important role in regulating the permeability of the outer membrane (OM) of Gram-negative bacteria. Impairment of the LPS-enriched OM is essential in initiating the bactericidal activity of polymyxins. We are interested in how colistin (polymyxin E) affects the membrane permeability of LPS/phospholipid bilayers. Our vesicle leakage experiment showed that colistin binding enhanced bilayer permeability; the maximum increase in the bilayer permeability was positively correlated with the LPS fraction. Addition of magnesium ions abolished the effect of LPS in enhancing bilayer permeabilization. To describe the vesicle leakage behavior from a structural perspective, we performed liquid atomic force microscopy (AFM) measurements on planar lipid bilayers. We found that colistin caused the formation of nano- and macroclusters that protruded from the bilayer by ∼2 nm. Moreover, cluster development was promoted by increasing the fraction of LPS or colistin concentration but inhibited by magnesium ions. To explain our experimental data, we proposed a lipid clustering model where colistin binds to LPS to form large-scale complexes segregated from zwitterionic phospholipids. The discontinuity (and thickness mismatch) at the edge of LPS–colistin clusters will create a passage that allows solutes to permeate through. The proposed model is consistent with all data obtained from our leakage and AFM experiments. Our results of LPS-dependent membrane restructuring provided useful insights into the mechanism that could be used by polymyxins in impairing the permeability barrier of the OM of Gram-negative bacteria.