Probing the properties of lipopolysaccharide monolayers and their interaction with the antimicrobial peptide polymyxin B by atomic force microscopy.

In contrast to the majority of all known cell types, Gram-negative bacteria have a second membrane, the outer membrane, which is an asymmetric bilayer composed of a phospholipid inner leaflet and a glycolipid outer leaflet. The glycolipid layer, in most cases being composed of a lipopolysaccharide (LPS), is the first target for antimicrobial agents. To get a basic understanding of the membrane-forming properties of LPS, we reconstituted monolayers of deep rough mutant LPS from Salmonella enterica serova Minnesota (R595 LPS), its lipid A moiety, and of the synthetic tetraacyl compound 406 (resembling the biosynthetic lipid A precursor IVa) at the air-water interface of a film balance. The liquid-expanded (LE) and liquid-condensed (LC) domains in the coexisting region were investigated with epifluorescence and, after transferring the monolayer onto mica, as a Langmuir-Blodgett film, with atomic force microscopy (AFM). The fluorescence and the AFM images showed identical domain structure. The higher resolution of the AFM images, however, contained more topographic details. Different heights and adhesion forces between the LE and LC domains could be observed. Differences in the adhesion forces between the AFM tip and the sample were determined in the repulsive and the attractive dynamic scanning modes, demonstrating the importance of a careful interpretation of height images. We propose that an increase in the lateral pressure causing the LE-LC transition of the monolayers leads to a reorientation of the molecules due to a tilt angle between the alkyl chains and the diglucosamine backbone. LPS monolayers have been utilized as a simplified reconstitution model of the outer membrane to study the interaction with antimicrobial agents. We investigated the action of the polycationic peptide polymyxin B (PMB) and found dramatic influences on the domain structures.