Identification of a glutathione peroxidase homolog in Neisseria meningitidis.

Glutathione peroxidase is an antioxidant enzyme found in a diverse array of eukaryotic species. We have determined the DNA sequence of a glutathione peroxidase homolog in the pathogenic bacterium Neisseria meningitidis. The sequence displays features of a functional gene, but lacks a selenocysteine-encoding in-frame TGA codon characteristic of most mammalian glutathione peroxidase genes. The derived amino acid sequence encoded by the N. meningitidis homolog predicts a 19.9 kDa protein that displays a high level of amino acid identity with other gluathione peroxidase sequences, particularly within four conserved regions of the enzyme.

Characterization of a silent pilin gene locus from Neisseria meningitidis strain FAM18.

We cloned and characterized a silent pilin locus (pilS) in the chromosome of Neisseria meningitidis strain FAM18. This locus represents the sole region of the FAM18 chromosome with strong homology to a gonococcal pilin gene. The FAM18 pilS locus encodes two tandem, in-frame, truncated pilin genes and shares many features with the previously described pilS locus of N. meningitidis strain C114. However, DNA sequence comparison shows that different information resides in the hypervariable region of one of the gene copies between the two strains. The conservation of reading frames within silent copies and the sequence diversity in hypervariable regions are reminiscent of gonococcal pilS loci and suggest that pilS loci may be of functional importance in the meningococcus.

Distribution of specific DNA sequences among pathogenic and commensal Neisseria species.

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.

A comparative analysis of pilin genes from pathogenic and nonpathogenic Neisseria species.

Pathogenic Neisseria species elaborate type IV pili, which are considered important for virulence. In this study, we examined pilin-encoding expression loci (pilE) in nonpathogenic Neisseria species. PCR based screening detected homology to a conserved N-terminal region of pilE in 12 of 15 Neisseria species, including all human commensal isolates. The three species failing to display homology were isolated from nonhuman sources. We have also characterized complete pilE loci from the human commensal species N. lactamica and N. cinerea. As anticipated, the predicted protein sequences from these species display features typical of all type IV pilins. In addition, these commensal pilins possess two highly conserved regions, SV2 and CYS2, which are shared among all neisserial pilins. However, a comparative analysis of pilE loci from pathogenic and nonpathogenic Neisseria species reveals two distinct structural groups, one composed of the pilin genes from N. lactamica, N. cinerea, and the class II pilin-producing subset of N. meningitidis isolates, the other of gonococcal and meningococcal class I pilin-encoding genes. Since both class I and class II pilin-producing meningococci can act as pathogens, structural relationships among neisserial pilin genes do not obviously reflect either species membership or ability to cause human disease.

Characterization of the opa (class 5) gene family of Neisseria meningitidis.

Class 5 outer membrane proteins of Neisseria meningitidis show both phase- and antigenic variation of expression. The proteins are encoded by a family of opa genes that share a conserved framework interspersed with three variable regions, designated the semivariable (SV) region and hypervariable regions 1 (HV1) and 2 (HV2). In this study, we determined the number and DNA sequence of all of the opa genes of meningococcal strain FAM18, to assess the structural and antigenic variability in the family of proteins made by one strain. Pulsed field electrophoresis and Southern blotting showed that there are four opa genes in the FAM18 chromosome, and that they are not tightly clustered. DNA sequence analysis of the four cloned genes showed a modest degree of diversity in the SV region and more extensive differences in the HV1 and HV2 regions. There were four versions of HV1 and three versions of HV2 among the four genes. Each of the FAM18 opa loci contained a gene with a unique combination of SV, HV1, and HV2 sequences. We used lambda gt11 cloning and synthetic peptides to demonstrate that HV2 sequences completely encode the epitopes for two monoclonal antibodies specific for different class 5 proteins of FAM18.

Reversible phase variation of expression of Neisseria meningitidis class 5 outer membrane proteins and their relationship to gonococcal proteins II.

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.

Characterization of a class II pilin expression locus from Neisseria meningitidis: evidence for increased diversity among pilin genes in pathogenic Neisseria species.

Strains of Neisseria meningitidis elaborate one of two classes of pili. Meningococcal class I pili have many features in common with pili produced by N. gonorrhoeae, including the ability to bind monoclonal antibody SM1 and a common gene and protein structure consisting of conserved, semivariable, and hypervariable regions. Class II pili are SM1 nonreactive and display smaller subunit molecular weights than do gonococcal or meningococcal class I pili. In this study, we have determined the N-terminal amino acid sequence for class II pilin and isolated the expression locus encoding class II pilin from N. meningitidis FAM18. Meningococcal class II pilin displays features typical of type IV pili and shares extensive amino acid identity with the N-terminal conserved regions of other neisserial pilin proteins. However, the deduced class II pilin sequence displays several unique features compared with previously reported meningococcal class I and gonococcal pilin sequences. Class II pilin lacks several conserved peptide regions found within the semivariable and hypervariable regions of other neisserial pilins and displays a large deletion in a hypervariable region of the protein believed to be exposed on the pilus face in gonococcal pili. DNA sequence comparisons within all three regions of the coding sequence also suggest that the meningococcal class II pilin gene is the most dissimilar of the three types of neisserial pilE loci. Additionally, the class II locus fails to display flanking-sequence homology to class I and gonococcal genes and lacks a downstream Sma/Cla repeat sequence, a feature present in all other neisserial pilin genes examined to date. These data indicate meningococcal class II pili represent a structurally distinct class of pili and suggest that relationships among pilin genes in pathogenic Neisseria do not necessarily follow species boundaries.

The H8 antigen of pathogenic Neisseriae.

We cloned and sequenced the H8 gene from N. meningitidis FAM18. The predicted amino acid sequence included a consensus lipoprotein signal sequence processing site, consistent with lipid modification that could account for the unusual electrophoretic and solubilization properties of H8. The amino acid sequence was rich in alanine and proline, especially in an imperfectly periodic region near the amino terminus, which encompassed the epitope recognized by available monoclonal antibodies. In a panel of neisserial strains, the presence of DNA homologous to the H8 gene correlated with the expression of an H8 protein. We cloned a gene from N. meningitidis JB515 that was distinct from the H8 gene but encoded a protein also recognized by an anti-H8 monoclonal antibody. Mice were not protected from meningococcemia by passive immunization with such an antibody.