Previous studies have shown that human serum, guinea pig and human red cells, and human white cells contain low and high M, substances that induce gonococcal strains to become serum resistant (1-4) and change lipooligosaccharide (LOS)' pattern (5, 6) . In more recent studies, the same investigators have shown that the low Mr substance in-blood is cytidine monophospho-N-acetylneuraminic acid (CMP-NANA) or a related compound (7,8) . These studies suggest that in vivo, sufficient concentrations of CMP-NANA might induce serum resistance by sialylation of LOS. Because gonococci are not able to synthesize CMP-NANA and it is not present in the usual media, previous in vitro studies of gonococcal LOS may have dealt with different LOS structures than those that occur in vivo .Each gonococcal strain makes multiple types of LOS (9)(10)(11), and the physical (Mr) and antigenic heterogeneity of a strain's LOS reflects physicochemical differences in their glycan moieties (10, 12). mAbs 3F11 and 06B4 identify epitopes on meningococcal and gonococcal LOS that are immunochemically similar to Galo1-4G1cNAc-containing molecules present in human erythrocytes and on other human cells (13) . These epitopes are conserved on gonococcal LOS (11,14) and are variably expressed This work was supported by U. S .

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Expression of lipooligosaccharide (LOS) antigenic determinants during human gonococcal infection was studied in secretions from seven men with gonococcal urethritis. Five monoclonal antibodies with distinct gonococcal LOS specificities and an H.8 lipoprotein monoclonal antibody were used in combination with immunogold electron microscopic analysis. The LOS epitope defined by antibody 6B7 was present on all seven strains in secretions and after in vitro growth. Gonococci from six of seven patients, when grown in vitro, expressed the 6B4 LOS epitope. The 6B4 epitope is a Gal beta 1-4-GlcNAc residue, which is immunochemically similar to the precursor of the human erythrocyte i antigen. This epitope was found unmodified on gonococcal LOS in urethral secretions from two patients. The unmodified epitope could not be demonstrated on organisms in five secretions. Neuraminidase digestion exposed the 6B4 epitope on organisms in these secretions and increased the 6B4 epitope density in the two secretions, which contained the unmodified epitope. These studies indicate that in vivo modification by sialylation of gonococcal LOS Gal beta 1-4-GlcNAc residue occurs during human infection.

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Gonococcal lipooligosaccharides (LOSs) are a series of antigenically complex heteropolymers. To investigate whether all members of clonally selected populations of Neisseria gonorrhoeae express antigenically similar LOS, we studied gonococcal strains 4505 and 220 with monoclonal antibodies 6B4 and 3F11 which have specificity for different oligosaccharide epitopes on the same or comigrating LOS unit(s) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Fluorescent-antibody and immunoelectron microscopy studies indicated that all members of the clonally selected populations were not homogenous for the epitopes these antibodies recognized. Fluorescence-activated cell sorting studies of 3F11-coated strain 220 indicated that the density of epitope expression was a function of time of growth. The population could be separated into two broad groups corresponding to organisms staining strongly or weakly for the 3F11 epitope, and the epitope density decreased during the late-log and stationary phases of growth. Sequentially staining organisms on Formvar grids with 6B4 and 3F11, followed by staining with either 5- or 15-nm colloidal gold spheres conjugated to goat anti-mouse immunoglobulin M demonstrated the following populations of cells among organisms derived from a single clone: organisms which stained for both 6B4 and 3F11 epitopes and organisms which stained for either 6B4 epitopes alone or 3F11 epitopes alone. Immunofluorescence microscopy studies with rhodamine and fluorescein goat anti-mouse immunoglobulin M conjugates sequentially staining organisms on Formvar grids with 3F11 and 6B4 also demonstrated these three populations. Analysis of LOS preparations made over the last 5 years indicated no change in serotype antigen concentration or in sodium dodecyl sulfate-polyacrylamide gel electrophoresis migration pattern. These studies indicate that while clonally selected strains of Neisseria gonorrhoeae undergo phenotypic variation at the epitope level, the impact of this variation on the total LOS of the population has little overall effect on its antigenic or physicochemical properties.