The overall goals of this project are to determine the structure and permeability of membranes composed of lipopolysaccharides (endotoxins) isolated from various Gram-negative bacteria. The outer monolayer of the outer membrane of these bacteria contains a unique lipid called lipopolysaccaride (LPS), which consists of specific polysaccarides linked to lipid A, which contains 4 to 7 saturated fatty acid chains. LPS is essential for the growth and survival of the bacterium and is also responsible for a variety of pathological reactions in humans. Moreover, LPS forms a tight permeability barrier which is thought to be critical for the resistance of these bacteria to various antibiotics. Mutant bacteria contain modified LPSs with different numbers of saccharides and polar constitutents. It has been found that specific mutant bacteria have quite different susceptibilities to hydrophobic antibiotics.
The specific aims of this proposal are to determine and correlate the structure and permeability properties of bilayers composed of LPSs from mutant bacteria which exhibit a range of antibiotic susceptibilities. The research involves the combined use of X-ray diffraction, differential scanning calorimetry, and antibiotitic permeability measurements of LPS and LPS:bacterial phospholipid bilayers in the presence and absence of divalent cations. The experiments are designed to test systematically several hypotheses concerning the role of the LPS polysaccharide chains and in-plane intermolecular interactions to the susceptibility of bacteria to antibiotics. Such fundamental information should be useful in understanding the physical basis of the barrier function of the bacterial outer membrane and the mechanisms involved in antibiotic entry into the cell.
