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Many mucosal pathogens, including Neisseria gonorrhoeae, produce proteases that cleave immunoglobulin A (IgA), the predominant immunoglobulin class produced at mucosal surfaces. While considerable circumstantial evidence suggests that IgA1 protease contributes to gonococcal virulence, there is no direct evidence that N. gonorrhoeae requires IgA1 protease activity to infect a human host. We constructed a N. gonorrhoeae iga mutant without introducing new antibiotic resistance markers into the final mutant strain and used human experimental infection to test the ability of the mutant to colonize the male urethra and to cause gonococcal urethritis. Four of the five male volunteers inoculated with the Iga- mutant became infected. In every respect-clinical signs and symptoms, incubation period between inoculation and infection, and the proportion of volunteers infected-the outcome of human experimental infection with FA1090iga was indistinguishable from that previously reported for a variant of parent strain FA1090 matching the mutant in expression of Opa proteins, lipooligosaccharide, and pilin. These results indicate that N. gonorrhoeae does not require IgA1 protease production to cause experimental urethritis in males.

Neisseria meningitidis class 5 proteins are major outer membrane proteins that share many properties with the proteins II (P.II) of Neisseria gonorrhoeae. We generated two bactericidal monoclonal antibodies, each of which bound specifically to one of the two identified class 5 proteins produced by N. meningitidis FAM18. The monoclonal antibodies also bound to class 5 proteins of a limited number of other meningococcal strains. Using the bactericidal activity of the monoclonal antibodies, we demonstrated that expression of both class 5 proteins was subject to reversible phase variation in vitro. The N-terminal amino acid sequence of a purified class 5 protein revealed striking similarity to the N-terminal amino acid sequence of gonococcal P.II proteins. Using a cloned class 5 gene, we identified three potential class 5 gene loci in N. meningitidis FAM18. These class 5 sequences also had homology with gonococcal P.II gene sequences and contained the CTCTT repeat sequence believed to be important in the regulation of gonococcal P.II expression.

Neisseria gonorrhoeae DNA that encodes the pathogenic Neisseria H.8 common antigen was cloned in the lambda phage sep6. The recombinant phage, designated s6H.8, was detected immunologically with a monoclonal antibody that binds to the H.8 antigen. The gonococcal and s6H.8 forms of the antigen yielded identical partial proteolysis epitope maps. Neisseria species that did not manifest the H.8 antigen showed little or no DNA homology with s6H.8. This clone should facilitate investigation into the clinical utility of this antigen and determination of its possible function in gonococcal pathogenesis.

The protein II (P.II) outer membrane proteins of Neisseria gonorrhoeae, which have been implicated in gonococcal pathogenesis, have been previously shown to undergo a type of phase variation in which expression of any of several different forms of the proteins may be switched on or off. We identified six electrophoretically distinct forms of P.II proteins (designated P.IIa through P.IIf) within strain FA1090, and we isolated colonial variants of FA1090 that expressed only one of the six different P.II protein forms. Two monoclonal antibodies that bound specifically and differentially to P.II proteins were produced. One antibody bound to proteins P.IIb and P.IId and was bactericidal for all colonial variants expressing P.IIb. The second antibody bound to P.IIa and was bactericidal for colonial variants expressing P.IIa. P.II protein profiles of survivors of antibody killing indicated that multiple P.II protein species may be expressed on a single bacterium and that P.II protein switching in the gonococcus is nonrandom.

We surveyed a number of inbred mouse strains for susceptibility to meningococcemia. Mice of all strains became bacteremic after intraperitoneal injection of a serogroup C, serotype 2a human disease isolate, but the strains differed in levels of bacteremia, indicating influences of the host genome on susceptibility. There was no significant correlation between level of bacteremia and differences at major histocompatibility or immunoglobulin loci; the Salmonella susceptibility locus, Ity; the complement C5 locus, Hc; the antibody response locus, xid; or the transferrin locus, Trf. However, the Lps locus, which influences a range of host cellular responses to endotoxin and affects susceptibility to Salmonella typhimurium, did influence susceptibility to meningococcemia. There were significant differences in levels of bacteremia between C3H/HeJ (Lpsd) mice and each of the other strains (all Lpsn). We confirmed the association of the Lpsd genotype with susceptibility by using coisogenic strains from two widely separated mouse lineages: C3H and B10. Lpsd mice experienced a 1,000-fold proliferation of bacteria and were bacteremic for days before clearing the infection. In contrast, Lpsn mice cleared the bacteremia in less than 1 day. There was no difference in meningococcal growth in vitro in serum from C3H/HeJ and coisogenic C3H/HeN (Lpsn) mice, suggesting that the Lps-related difference in susceptibility may involve a cellular response.

Hybridomas secreting monoclonal antibodies against an apparent strain-specific cell surface antigen of Neisseria gonorrhoeae were produced. Spleen cells from BALB/c mice immunized with whole gonococci were fused with mouse myeloma cell line Sp2/0, and hybrid cells were selected in culture. One hybridoma that secreted antibodies reactive with the immunizing strain was cloned by limiting dilution to obtain cell lines secreting monoclonal antibodies. These antibodies reacted with purified outer membranes from the immunizing strain as well as with whole gonococci. Binding of antibodies to whole gonococci was highly strain specific, with most gonococcal strains showing less than 1% of the binding with the immunizing strain. Antibodies did not bind to the other Neisseria species tested. Binding of monoclonal antibodies to whole gonococci of the immunizing strain was not dependent on state of piliation. The extent of antibody binding did vary in different colonial variants of the immunizing strain. Antibody bound to cells from colonies that were transparent or of intermediate opacity, but did not bind to cells from deeply opaque colony variants.

This study showed that a protein II (PII) of Neisseria gonorrhoeae FA1090 appeared to act as a mediator of attachment to HeLa cells. Two colony variants of FA1090 were selected. Both gonococcal variants were nonpiliated, but one contained a PII and the other did not. A monoclonal antibody (1090-10.1), which was directed against the PII, inhibited the apparent PII-mediated attachment to HeLa cells. Antibodies produced from clone 1035-4, which had no PII specificity, did not inhibit the attachment and were used as controls. Inhibition of gonococcal attachment by the 1090-10.1 monoclonal antibodies was demonstrated by fluorescent microscopy analysis. Monoclonal antibody 1090-10.1 appeared to cause agglutination of the PII-containing organism. To block the clumping caused by the PII-specific monoclonal antibodies, Fab fragments of goat anti-mouse IgG were incubated with gonococci and the 1090-10.1 monoclonal antibodies. The results showed that the goat anti-mouse IgG Fab fragments partially blocked the agglutination caused by the PII-specific monoclonal antibody. The effect of the 1090-10.1 antibodies on attachment was also determined by monitoring the HeLa cells with attached iodinated gonococci. The monoclonal antibody appeared to inhibit the PII-mediated attachment.

Gonococci express a family of related outer membrane proteins designated protein II (P.II), which undergo both phase and antigenic variation. Six P.II proteins have been identified in strain FA1090. We developed monoclonal antibodies specific for each P.II protein. Using these antibodies as probes, we purified the six different P.II proteins of this strain. Despite the relatedness of the proteins, we could not purify all of them by a single purification scheme. Four P.II proteins were purified by chromatofocusing, and the remaining two proteins were purified by hydrophobic interaction chromatography on phenyl-Sepharose. The N-terminal amino acid sequence of the proteins showed a high degree of sequence conservation. However, there was variability at specific amino acid residues, giving each P.II protein a unique N-terminal amino acid sequence. Thus P.II proteins of one strain differ among themselves not only in antigenic determinants and primary structure, but also in other characteristics affecting their properties in different chromatographic systems.

Several traits, including pili and the outer membrane proteins P.II and H.8, have been associated with pathogenic Neisseria species. We examined several Neisseria species for DNA sequence homology to cloned pilin, P.II, and H.8 genes. Strains of Neisseria gonorrhoeae and N. meningitidis showed hybridization to all of these genes. Commensal strains showed little hybridization to any of these genes. Strains of N. lactamica and N. cinerea showed intermediate patterns of hybridization. Generally, organisms that expressed a given trait showed DNA homology to the corresponding cloned gene. However, we observed pili on some commensal strains that did not show hybridization to the cloned gonococcal pilin gene.

To determine the ultrastructural distribution and immunological accessibility of proteins II (P.IIs) on the surfaces of whole gonococci, anti-P.II gold probes were developed and used in electron microscopic studies of viable P.II-expressing variants of Neisseria gonorrhoeae FA1090. Anti-P.II probes clearly marked the surfaces of organisms and their associated outer membrane blebs. The surface-exposed portion of P.II is antigenically variable. With the use of two different sizes of gold probes, it was demonstrated that individual gonococcal cells can express more than one antigenic type of P.II simultaneously.

Previous work indicates that strains of Neisseria gonorrhoeae isolated in Seattle, Wash., and Atlanta, Ga., show an association between serotypes 1 and 2 of protein I of the outer membrane and the ability to cause disseminated infection (T.M. Buchanan and J.F. Hildebrandt, Infect. Immun. 32:985-994, 1981). By using the same serotyping system, we confirmed the association between those serotypes and both disseminated infection and serum resistance in strains from North Carolina. Some strains of the same serotype had protein I species with different apparent molecular weights.

A hybridoma derived from a mouse immunized with gonococcal outer membranes produced an antibody, designated H.8, that bound to all strains of Neisseria gonorrhoeae and N. meningitidis tested, and to N. lactamica and N. cinerea, but only rarely to other nonpathogenic Neisseria species. Studies with the gonococcal strain used in production of the antibody showed that the antibody bound to a surface-exposed, protease-sensitive, and heat-modifiable outer membrane antigen that we believe is distinct from previously described gonococcal outer membrane proteins.