Analyses of gonococcal H8 antigen. Surface location, inter- and intrastrain electrophoretic heterogeneity, and unusual two-dimensional electrophoretic characteristics.

Abstract

The H8 protein is a surface-exposed antigen that is found, among members of the Neisseria genus, primarily on pathogenic species. In this study, the surface exposure of H8 was reassessed by four techniques. Results of slide agglutination, indirect fluorescent antibody binding, absorption of sera with whole gonococci, and immune electron microscopy all confirmed the presence of H8 in the outer membrane. The degree to which protein A-gold-labeled monoclonal antibodies bound to H8 was marked, and suggested that this antigen was present in abundant amounts in the outer membrane. Also in this study, the electrophoretic heterogeneity of this common surface antigen was examined. Because H8 stains poorly, electrophoretic mobility was assessed using polyclonal antibodies and a monoclonal antibody that recognizes a common H8 epitope. H8 was analyzed with respect to protein I, lipopolysaccharide (LPS), and pilus and opacity phenotypic variation; results confirmed that heterogeneity of Mr was the rule among strains (21 were examined), however, the variability in Mr was independent of protein I or LPS Mr. In one strain (FA1090), the heterogeneity of H8 was examined among 10 piliation/opacity variants; the H8 (and LPS) Mr was identical in all variants; similar data were generated in strains JS3 and JS1. The electrophoretic mobility of H8 was altered in serum-resistant and neutrophil enzyme-resistant gonococci compared to the sensitive gonococci. Some of the unusual electrophoretic migration characteristics of the antigen were also examined. H8 formed a unique mushroom-shaped band in one-dimensional gels; in a two-dimensional electrophoresis system, the antigen migrated aberrantly, very similarly to LPS. Also seen in the two-dimensional electrophoresis profile were multimers of the H8 antigen; in strain JS3 (Mr 23,500), these migrated at 43,600, 86,000, and greater than 150,000. In other strains, the Mr of the multimers differed depending upon the Mr of the monomer. The two-dimensional migration characteristics (as measured by antigenicity) were completely destroyed by proteinase K digestion. Activity of H8 polyclonal antibodies to the antigens in two-dimensional gels was completely removed by adsorption of formalin-fixed whole cells, but was not affected by adsorption with LPS. These electrophoretic characteristics may reflect the close association of some nonprotein constituent, perhaps lipid or carbohydrate or both.

The Lip Lipoprotein from Neisseria gonorrhoeae Stimulates Cytokine Release and NF-κB Activation in Epithelial Cells in a Toll-like Receptor 2-dependent Manner

“…As Fig. 1A demonstrates, immunoblotting with mAb 2C3 revealed the presence of a protein with an apparent molecular mass of ϳ18 kDa in each step of the purification, which is consistent with the H.8/Lip previously reported in other strains of Neisseria (23,28,43). This band was absent from the F62 H.8 deletion mutant (data not shown).…”

Interaction of Neisseria gonorrhoeae with classical complement components, C1-inhibitor, and a monoclonal antibody directed against the Neisserial H.8 antigen.

“…H.8 is interesting for several reasons: (a) it has a common, antigenically invariant epitope that is immunoaccessible on viable bacteria in vitro and in vivo (16,(19)(20)(21); (b) the antigen (15, 16) and the gene (22) appear to be restricted to the pathogenic (and opportunistic) members of the genus; (c) the H.8 antigen gene contains a repetitive 15-bp sequence constituting > 90% of the structural gene (23), six copies of which are found in another virulence-associated gene, the azurin gene (24, 25); and (d) H.8 appears to be a lipoprotein, the first described in Neisseria species, and may be important in membrane architecture and integrity. Finally, the H.8 antigen is immunogenic in humans: H.8-specific antibody responses have been detected in patients with local and disseminated gonococcal and meningococcal disease (26,27).…”

“…MAb 10 directed against Neisserial surface antigen H.8 was prepared as described by Hitchcock et al (16). Tissue culture supernatant was concentrated 20-fold using a 100-ml cell and PM100 membrane (Amicon Corp., Danvers Chemicals, Piscataway, NJ) (36).…”

Conserved lipoproteins of pathogenic Neisseria species bearing the H.8 epitope: lipid-modified azurin and H.8 outer membrane protein

“…The conserved antigens that have been identified have been the subject of considerable study in an effort to determine whether they play a direct role in neisserial pathogenesis and whether they might be targets of a protective immune response. The existence of a protein epitope that is conserved among pathogenic Neisseria species was revealed by the binding of a monoclonal antibody (MAb) designated H.8 (8), as well as other MAbs with similar specificities (14,31). These MAbs bind to all gonococci and meningococci that have been tested and to N. lactamica and N. cinerea strains, but not to strains of other commensal Neisseria species (1,8,14,31).…”

“…The existence of a protein epitope that is conserved among pathogenic Neisseria species was revealed by the binding of a monoclonal antibody (MAb) designated H.8 (8), as well as other MAbs with similar specificities (14,31). These MAbs bind to all gonococci and meningococci that have been tested and to N. lactamica and N. cinerea strains, but not to strains of other commensal Neisseria species (1,8,14,31). There has been much recent progress in identifying and characterizing the proteins that are recognized by H.8-specific MAbs, particularly through the use of recombinant deoxyribonucleic acid (DNA) approaches.…”