The long term objective of this program is to relate the physical and chemical structure of Neisseria gonorrhoeae lipooligosaccharides (LOS) to antigenic structures that are immunologically recognized by humans and to use this information to study the epidemiology and immunopathogenesis of gonococcal disease. These structurally and immunologically complex molecules differ in several important aspects from those of enteric bacilli. Epitope expression within their oligosaccharides (OS) is governed by their tertiary and quaternary structure, or conformation, that is influenced by the chemical milieu. Because of this, primary and secondary structure, or linkages between glycose constituents, are an insufficient basis for assigning and characterizing epitopes. During the next five years we will concentrate on defining the conformational basis of epitope expression within gonococcal LOS. By using strains selected for differences in LOS composition and complement interactions, we will be able to correlate conformational structures, as we determine them, with biologic phenomena. We will use mass spectrometry and nuclear magnetic resonance to determine the physical, chemical and conformational requirements for epitope expression in gonococcal LOS. These techniques will include GC/MS analysis of permethylated OS, proton-proton and proton-13C 2-D NMR, and fast atom bombardment mass spectrometry. Individual LOS components and their OS will be separated using aqueous and organic solvent HPLC technology. The effect of interactions of divalent cations with LOS OS on epitope expression will be explored by titration of divalent cations with the respective material and determination of stereochemical organization by circular dichroism, and of mass-shift patterns of OS derived from cation-depleted and cation-replete native LOS by FAB/MS. The requirement for a hydrophobic milieu for optimal epitope expression will be explored by degradative and substitutive techniques. The goal of these experiments will be to determine if there is a minimal length, degree of hydroxylation, degree of unsaturation, specific linkage or degree of substitution that restores epitope expression to delipidated OS. We will use a battery of monoclonal antibodies and a series of isogenic mutants that express different proteins II to determine whether hydrophobic interactions between LOS and OS and surface expressed peptide sequences of proteins II influence epitope expression. Finally, we will determine whether multiple epitopes are expressed on a single LOS and whether multiple LOS components are expressed on single bacteria.