Fluorescent Detection of Lipopolysaccharide Interactions with Model Membranes
The critical importance of the lipid A moiety of LPS in resistance and pathogenesis in gram negative infections has led to the assumption that LPS interaction with target cells is due to hydrophobic interaction with plasma membranes. However, work from several laboratories, including our own, is consistent with the presence of a cell membrane structure with characterstics of a “receptor”. We have proposed a two-step model for LPS-membrane interaction which resolves the two views, and have developed a model system to control the first step (binding to membrane protein) and study the second step (intercalation into lipid bilayer). We examined the interaction of LPS with small unilamellar phosphatidylcholine vesicles labeled in the hydrophobic portion of the bilayer with the fluorescent probe diphenylhexatrine (DPH) and detected changes in the physical properties of the bilayer by measuring DPH fluorescence anisotropy (Δr). We have found that purified, phenol-extracted S. typhimurium LPS interacts with the bilayer as measured by an increase in Δr and conclude that the LPS aggregate coalesced with the lipid bilayer. The greatest change in Δr was achieved with lipid A, Ra-Re glycolipids and diphosphoryl lipid A. Monophosphoryl lipid A and lipid X were less effective. Preparations of wild-type LPS fractionated according to the length of the O-antigen side chain and unfractionated LPS had least effect on Δr. Thus other factors such as serum components or membrane proteins may be necessary to enhance the interaction of LPS with target cells.
KeywordsLipid Bilayer Acyl Chain Malachite Green Membrane Lipid Bilayer Fluorescence Anisotropy
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