The lipopolysaccharide (R type) as a common antigen of Neisseria gonorrhoeae. I. Immunizing properties.

The ability of R-type lipopolysaccharide (LPS), isolated from Neisseria gonorrhoeae colony type 4, to protect against infection with N. gonorrhoea colony type 1 (T1 isolates) in the mouse and chicken embryo was investigated. C57 black mice were immunized intraperitoneally with 50 microgram of LPS, and challenged intracerebrally with 10-20 LD50's of N. gonorrhoeae colony type 1. Immunized mice were significantly protected (P less than 0.01 to less than 0.05) against challenge with different T1 isolates of N. gonorrhoeae when compared with non-immunized mice. Mice, injected with succinylated or alkali-treated LPS were not protected against gonococcal challenges. In a second animal model, leghorn hens were immunized intravenously with three injections of 500 microgram of LPS followed by a booster of 2.5 mg 2 weeks later. Embryonated eggs obtained from immunized hens were protected against challenge with 5 x 10(3) - 1 x 10(4) LD50's of three different T1 isolates. When hens were injected with the chemically modified LPS, the embryos were not resistant to gonococcal challenge. The results of this study demonstrate the ability of R-type gonococcal LPS to provide protection against different T1 isolates of N. gonorrhoeae.     

Enzyme-linked immunosorbent assays for the detection of Neisseria gonorrhoeae specific antibodies.

An indirect enzyme-linked immunosorbent assay (ELISA) using rigid polystyrene microtiter plates was adapted to detect specific gonococcal antibodies against outer membrane-complex antigens extracted from Neisseria gonorrhoeae. The concentration of antigen to obtain maximum coating of the well was 10 micrograms protein per millilitre. The optimal binding of the primary antibody and enzyme-conjugated antimmunoglobulin was achieved after 1 h at 37 degrees C. Under these conditions using gonococcal antisera, no cross-reactivity was observed with outer membrane antigens extracted from Neisseria meningitidis serogroups B, C, X, Y, and W135. Neisseria meningitidis serogroup A demonstrated low levels of cross-reactivity. All the non-pathogenic Neisseria spp. tested were negative (absorbance value at 400 nm/30 min less than 0.15). The reaction of immune serum against outer membrane complex absorbed to the microwells was completely inhibited with soluble-specific antigen but not with purified N. gonorrhoeae lipopolysaccharide. Quantitative inhibition permitted the measurement of low levels of antigen (0.5 microgram/ml). The detection of N. gonorrhoeae antibody with ELISA is specific and highly sensitive.     

The production and characterization of monoclonal antibodies against the protein III of Neisseria gonorrhoeae

Monoclonal antibodies were produced against gonococcal protein III. Antibodies of two different specificities were obtained. One reacted with all Neisseria species tested (N. gonorrhoeae, N. meningitidis and five non-pathogenic species), whereas the other was specific for Neisseria gonorrhoeae and may provide the basis for improved diagnostic reagents.     

Mitogenic activity of Neisseria gonorrhoeae surface antigens in mouse splenic lymphocyte culture.

The mitogenic effects of Neisseria gonorrhoeae endotoxin, fractionated envelope componenents, and intact cells were examined on unsensitized mouse splenic lymphocytes in vitro. The stimulatory effect of these substances was measured by increased [3H]thymidine incorporation in spleen cell cultures. Intact cells, purified lipopolysaccharide (LPS), and cell envelope preparations were highly stimulatory and the stimulation index was dose dependent. Fractionated components of the envelope demonstrated variable stimulation when tested at identical LPS concentrations, reflecting the mitogenic activity of the protein moieties. The stimulatory dose responses for purified N. gonorrhoeae and Escherichia coli LPS were compared and mitogenicity was higher with gonococcal LPS at all concentrations tested. Alkaline detoxification or succinylation of N. gonorrhoeae LPS results in loss of ability to induce blast transformation. The mitogenicity of cell-surface components of N. gonorrhoeae is discussed in terms of LPS and protein content.     

Characterization of the mitogenic activity elicited by Neisseria gonorrhoeae ribosomal fractions.

Ribosomal preparations from Neisseria gonorrhoeae types 1 and 4 were examined for their in vitro stimulation of mouse splenocytes to determine the ribosomal moiety or contaminant responsible for the immunoproliferative activity. In immunodiffusion tests with homologous rabbit antiserum, crude 70S ribosomes formed four precipitin bands while the purified 30S and 50S subunits showed one major line. The same antiserum reacted with lysed N. gonorrhoeae and Neisseria meningitidis A cells but no precipitation occurred with Escherichia coli cells purified N. gonorrhoeae lipopolysaccharide (LPS). No membrane or LPS contaminant was detected in the purified 30S and 50S preparations. All the ribosomal preparations from virulent and non-virulent N. gonorrhoeae consistently stimulated the murine splenocytes. The mitogenic activity of the 30S and 50S ribosomal preparation was destroyed by treatment with trypsin but only slightly decreased by ribonuclease. It is suggested that the lymphoproliferative response elicited by gonococcal ribosomes is triggered by the protein moiety of the 30S or 50S subunits.     

Transformation of leucine and rifampin traits in Neisseria gonorrhoeae with deoxyribonucleic acid from homologous and heterologous origins.

A leucine-requiring, rifampin-sensitive strain of Neisseria gonorrhoeae was transformed to a leucine-nonrequiring, rifampin-resistant phenotype with deoxyribonucleic acid (DNA) obtained from both N. meningitidis and N. gonorrhoeae. The transforming efficiency of the meningococcal DNA was about 10- to 100-fold less than that of the homologous gonococcal DNA. A chemically defined medium that would support growth of most gonococcal isolates was used as a complete medium. A minimal medium was used for selection of Leu+ transformants. N-methyl-N'-nitro-N-nitrosoguanidine was used as a mutagen for isolating leucine prototrophs from leucine-requiring isolates of N. gonorrohoeae.     

Conjugal transfer of beta-lactamase-producing plasmids of Neisseria gonorrhoeae to Neisseria meningitidis

Twenty clinical isolates of beta-lactamase-producing Neisseria gonorrhoeae from Japanese sources were studied to define their ability to serve as donors for their plasmids in conjugation with Neisseria meningitidis. These twenty strains of N. gonorrhoeae harbored the 4.5-megadalton (Mdal) beta-lactamase-producing plasmids and the 24.5-Mdal conjugative plasmids. We found that only three of twenty N. gonorrhoeae strains showed a detectable conjugation frequency (greater than 10(-5)) with N. meningitidis as the recipient although all strains were capable of mobilizing beta-lactamase-producing plasmids to N. gonorrhoeae and to Escherichia coli. The 4.5-Mdal beta-lactamase-producing plasmid was maintained in N. meningitidis, but the large 24.5-Mdal conjugative plasmid has not been found in N. meningitidis transconjugants.     

Immunologic classification of Neisseria gonorrhoeae with micro-immunofluorescence.

A reproducible immunologic classification of Neisseria gonorrhoeae strains has been achieved by the micro-immunofluorescence (Micro-IF)3 method by using formalinized whole organisms as test antigens and mouse antisera prepared by i.v. immunization with the whole organisms as antibody. Immunologic differences among Neisseria species were also distinct in this test system. Immunologic differences among gonococcal strains were not influenced by gonococcal colony type. Classification of gonococci was facilitated by use of antisera absorbed with an antigenically unique gonococcus strain. Of 180 gonococcal strains, 175 could be classified into three immunotypes: A, B, and C. Each type was further divided into subtypes designated A1, A2, A3, B1, B2, B3, C1, and C2. Minor antigenic differences still exist within each subtype. The two gonococcal isolates from each of 17 pairs of sexual contacts fell into the same subtype. Seventy-one of 73 isolates which required arginine, hypoxanthine, and uracil for growth (Arg-Hyx-Ura-) and seven of 107 other auxotypes belonged to subtypes A2 and A3. Marked geographical differences in distribution of gonococcal immunotypes were observed among those available for testing. Subtypes A2 and A3 were predominant in Seattle whereas types B and C were predominant in Southeast Asia. The only Arg-Hyx-Ura- isolates not belonging to subtypes A2 or A3 were the only two that were serum sensitive. This Micro-IF immunotyping appears potentially useful for future immunologic, epidemiologic, and genetic studies of N. gonorrhoeae.     

Genes associated with meningococcal capsule complex are also found in Neisseria gonorrhoeae.

A homolog of the meningococcal cps locus region E has been identified in Neisseria gonorrhoeae immediately upstream of the gonococcal region D locus. Region E has no detectable function in capsule biosynthesis in Neisseria meningitidis or in lipopolysaccharide biosynthesis in either organism. The open reading frame is homologous to proteins of unknown function in Escherichia coli and Haemophilus influenzae. Further analysis of the N. meningitidis cps cluster has identified a second copy of region D encoding three additional open reading frames, including homologs of DNA methyltransferases. The organization of the region D and E genes in N. gonorrhoeae and N. meningitidis in relation to the cps genes provides some insight into the evolutionary origin of encapsulation in N. meningitidis.     

A study by high-resolution two-dimensional polyacrylamide gel electrophoresis of relationships between Neisseria gonorrhoeae and other bacteria

High-resolution two-dimensional polyacrylamide gel electrophoresis was used to analyse the soluble proteins from seven strains of Neisseria gonorrhoeae, six strains of Neisseria meningitidis and one or two strains of twelve other species. Approximately 200 individual polypeptides could be visualized as Coomassie Blue stained spots on an electrophoretogram of N. gonorrhoeae and similar numbers were found for the other bacteria. Each species of bacterium had a distinctly different pattern of spots which could be recognized. Quantitative comparisons of 48 selected spots derived from one strain of N. gonorrhoeae with those of five other strains of gonococcus, three strains of N. meningitidis and one of Branhamella catarrhalis, showed relationships in agreement with their current taxonomic classification but with a higher level of discrimination than that of previously used methods. It was also possible to distinguish the individual gonococcal strains. It is suggested that the method could be useful for bacterial classification and identification.     

ComE, a competence protein from Neisseria gonorrhoeae with DNA-binding activity

Neisseria gonorrhoeae is naturally able to take up exogenous DNA and undergo genetic transformation. This ability correlates with the presence of functional type IV pili, and uptake of DNA is dependent on the presence of a specific 10-bp sequence. Among the known competence factors in N. gonorrhoeae, none has been shown to interact with the incoming DNA. Here we describe ComE, a DNA-binding protein involved in neisserial competence. The gene comE was identified through similarity searches in the gonococcal genome sequence, using as the query ComEA, the DNA receptor in competent Bacillus subtilis. The gene comE is present in four identical copies in the genomes of both N. gonorrhoeae and Neisseria meningitidis, located downstream of each of the rRNA operons. Single-copy deletion of comE in N. gonorrhoeae did not have a measurable effect on competence, whereas serial deletions led to gradual decrease in transformation frequencies, reaching a 4 x 10(4)-fold reduction when all copies were deleted. Transformation deficiency correlated with impaired ability to take up exogenous DNA; however, the mutants presented normal piliation and twitching motility phenotype. The product of comE has 99 amino acids, with a predicted signal peptide; by immunodetection, a 8-kDa protein corresponding to processed ComE was observed in different strains of N. gonorrhoeae and N. meningitidis. Recombinant His-tagged ComE showed DNA binding activity, without any detectable sequence specificity. Thus, we identified a novel gonococcal DNA-binding competence factor which is necessary for DNA uptake and does not affect pilus biogenesis or function.     

Slide Agglutination Method for the Serological Identification of Neisseria gonorrhoeae with Anti-Gonococcal Antibodies Adsorbed to Protein A-Containing Staphylococci

A rapid slide agglutination test has been developed for the identification of Neisseria gonorrhoeae that are primarily detected as oxidase-positive colonies in gonococcal cultures. The technique is based on the specific nonimmune reactivity between the Fc portion of immunoglobulin (Ig)G and staphylococcal protein A. IgG molecules adsorbed to stabilized staphylococci will thereby become oriented with their antigen-reactive sites that are directed outwards. Protein A-containing staphylococci with unabsorbed anti-gonococcal antibodies gave positive co-agglutination reactions with gonococci but also with meningococci, some Moraxella, Haemophilus, and Pseudomonas strains. These crossreactions were eliminated by absorption of the anti-gonococcal antiserum with meningococcal and Moraxella organisms prior to the coating of reagent staphylococci. In the routine culture diagnosis of N. gonorrhoeae the use of specific gonococcal reagent staphylococci gave concordant results with fermentation procedures and immunofluorescent techniques.     

Antibody to an artificial disaccharide antigen cross-reactive with Neisseria gonorrhoeae lipopolysaccharide.

An artificial antigen was prepared from 4-O-beta-I-galactopyranosyl-D-glucose (lactose) and 8-ethoxycarbonyloctanol. Covalent attachment to bovine serum albumin provided an antigen that elicited antilactose antibody in rabbits and goat. These antibodies were active against Neisseria gonorrhoeae lipopolysaccharide in passive hemagglutination tests. The same antibody agglutinated cells of Streptococcus faecalis, strain N, and precipitated the lactose-containing cell wall diheteroglycan of this organism. Fractionation of rabbit and goat antibody raised against the synthetic antigen of S. faecalis vaccine provided two antibody fractions only one of which, eluted from the immunoadsorbent by galactose, was active against N. gonorrhoeae lipopolysaccharide.     

PilC of Neisseria meningitidis is involved in class II pilus formation and restores pilus assembly, natural transformation competence and adherence to epithelial cells in PilC-deficient gonococci

Type 4 pili produced by the pathogenic Neisseria species constitute primary determinants for the adherence to host tissues. In addition to the major pilin subunit (PilE), neisserial pili contain the variable PilC proteins represented by two variant gene copies in most pathogenic Neisseria isolates. Based upon structural differences in the conserved regions of PilE, two pilus classes can be distinguished in Neisseria meningitidis . For class I pili found in both Neisseria gonorrhoeae and N. meningitidis , PilC proteins have been implicated in pilus assembly, natural transformation competence and adherence to epithelial cells. In this study, we used primers specific for the pilC2 gene of N. gonorrhoeae strain MS11 to amplify, by the polymerase chain reaction, and clone a homologous pilC gene from N. meningitidis strain A1493 which produces class II pili. This gene was sequenced and the deduced amino acid sequence showed 75.4% and 73.8% identity with the gonococcal PilC1 and PilC2, respectively. These values match the identity value of 74.1% calculated for the two N. gonorrhoeae MS11 PilC proteins, indicating a horizontal relationship between the N. gonorrhoeae and N. meningitidis pilC genes. We provide evidence that PilC functions in meningococcal class II pilus assembly and adherence. Furthermore, expression of the cloned N. meningitidis pilC gene in a gonococcal pilC1,2 mutant restores pilus assembly, adherence to ME-180 epithelial cells, and transformation competence to the wild-type level. Thus, PilC proteins exhibit indistinguishable functions in the context of class I and class II pili.     

A variable genetic island specific for Neisseria gonorrhoeae is involved in providing DNA for natural transformation and is found more often in disseminated infection isolates

Neisseria gonorrhoeae (the gonococcus) is the causative agent of the sexually transmitted disease gonorrhoea. Most gonococcal infections remain localized to the genital tract but, in a small proportion of untreated cases, the bacterium becomes systemic to produce the serious complication of disseminated gonococcal infection (DGI). We have identified a large region of chromosomal DNA in N. gonorrhoeae that is not found in a subset of gonococcal isolates (a genetic island), in the closely related pathogen, Neisseria meningitidis or in commensal Neisseria that do not usually cause disease. Certain versions of the island carry a serum resistance locus and a gene for the production of a cytotoxin; these versions of the island are found preferentially in DGI isolates. All versions of the genetic island encode homologues of F factor conjugation proteins, suggesting that, like some other pathogenicity islands, this region encodes a conjugation-like secretion system. Consistent with this hypothesis, a wild-type strain released large amounts of DNA into the medium during exponential growth without cell lysis, whereas an isogenic strain mutated in a peptidoglycan hydrolase gene (atlA) was drastically reduced in its ability to donate DNA for transformation during growth. This genetic island constitutes the first major discriminating factor between the gonococcus and the other Neisseria and carries genes for providing DNA for genetic transformation.     

Intrageneric transformation of neisseria gonorrhoeae and neisseria perflava to streptomycin resistance and nutritional independence.

Auxotrophic mutants of Neisseria gonorrhoeae and Neisseria perflava were transformed to prototrophy using homologous and heterologous deoxyribonucleic acid (DNA). Within either species the efficiencies of transformation for nutritional markers were found to be very similar to the values obtained for transformation to streptomycin resistance. The number of transformants in the interspecific N. perflava (donor) - - leads to N. gonorrhoeae (recipient) cross was 100-fold lower than the number obtained in the intraspecific N. gonorrhoeae - - leads to N. gonorrhoeae cross for streptomycin resistance, as well as for several nutritional markers. In the reciprocal experiment the difference in the number of transformants in the interspecific N. gonorrhoeae - - leads to N. perflava cross and the number obtained in the intraspecific N. perflava - - leads to N. perflava cross varied from 600 to 1,000-fold for the streptomycin resistance marker. Of greater interest was the finding that N. perflava auxotrophs, although transformable to prototrophy with wild-type N. perflava DNA, were not transformed to nutritional independence by gnoncoccal DNA. These same mutants were transformable to streptomycin resistance using the heterologous gonococcal DNA. When the DNAs of N. meningitidis, N. flava, and N. lactamicus were used to transform N. gonorrhoeae to prototrophy or streptomycin resistance, the transformation frequencies obtained fell along a gradient that in general reflected taxonomic relationships. On the other hand, with N. perflava as the recipient for these same DNAs, only N. flava DNA could transform auxotrophs to prototrophy, although transformation to streptomycin resistance occurred in all cases. DNA from N. perflava - - leads to N. gonorrheae streptomycin-resistant or Ade+ intergenotic transformants transformed N. gonorrhoeae cells at a 100-fold-higher efficiency than did DNA from N. perflava. Our findings suggest that (i) N. gonorrhoeae and N. perflava are more closely related than hitherto suspected and (ii) N. perflava is more selective with respect to heterologous DNA than is N. gonorrhoeae.     

Rapid identification of pathogenic neisserias using the Identicult-Neisseria test

A rapid enzymatic method using chromogenic substrates for the rapid identification of pathogenic neisseria (Identicult-Neisseria, Scott Laboratories Inc., CA, USA) was tested in parallel with the rapid carbohydrate utilization test (RCUT) and the Phadebact Monoclonal GC Test against 198 consecutive clinical isolates of oxidase-positive Gram-negative diplococci (118 Neisseria gonorrhoeae , 76 N. meningitidis and four N. lactamica ). On initial testing the Identicult-Neisseria gave a 95% overall concordance (97.5% N. gonorrhoeae , 90.8% N. meningitidis ). with the RCUT and Phadebact tests; the corresponding figures after repeat testing were 98% overall concordance (98.3% N. gonorrhoeae , 97.4% N. meningitidis ). Two of the three strains of N. gonorrhoeae mis-identified as N. meningitidis on primary testing were also mis-identified on repeat testing. Seven strains of N. meningitidis were mis-identified on initial testing (six as Moraxella catarrhalis and one as N. lactamica ) and two on repeat testing (both as Mor. catarrhalis ). We conclude that the Identicult-Neisseria is not sufficiently reliable for the culture confirmation of gonococci and meningococci.