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.     

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.     

Deoxyribonucleic Acid Homologies Among Species of the Genus Neisseria

Eleven aerobic species of Neisseria, a Mima sp., and a Herellea sp. were tested for deoxyribonucleic acid (DNA) homology in direct hybridization experiments. DNA labeled with either (14)C or (32)P was prepared from five species of Neisseria. Unlabeled DNA from the various microorganisms was immobilized on membrane filters, which, after pretreatment, were incubated with labeled DNA (4,000 counts per min per filter) for 14 hr at 67 C. The measure of relatedness was expressed as the relative percentage of direct binding compared to that obtained with homologous DNA. All serological types of N. meningitidis, including the newly proposed types, were homologous to the standard strain of N. meningitidis with one possible exception, type Z. The genus Neisseria is heterogeneous in nature, forming at least three distinct groups: first, N. meningitidis and N. gonorrhoeae; second, N. perflava, N. subflava, N. sicca, N. flavescens, and N. flava; third, N. catarrhalis and N. caviae. Mima and Herellea species show no significant homology with the Neisseria.     

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.     

Characterization of a stress protein from group B Neisseria meningitidis.

Increased levels of a 65-kDa stress protein (Msp65) were observed in group B Neisseria meningitidis grown under stationary-growth conditions. Electron microscopy showed two apposing rings of seven subunits, a structure typical of Escherichia coli GroEL. Msp65 was not found in either the periplasmic space or the outer membrane. Several important differences between the GroEL analogs of N. meningitidis and Neisseria gonorrhoeae are discussed.     

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.     

HmbR outer membrane receptors of pathogenic Neisseria spp.: iron-regulated, hemoglobin-binding proteins with a high level of primary structure conservation.

We have recently cloned and characterized the hemoglobin receptor gene from Neisseria meningitidis serogroup C. N. meningitidis cells expressing HmbR protein were able to bind biotinylated hemoglobin, and the binding was specifically inhibited by unlabeled hemoglobin and not heme. The HmbR-mediated hemoglobin binding activity of N. meningitidis cells was shown to be iron regulated. The presence of hemoglobin but not heme in the growth medium stimulated HmbR-mediated hemoglobin binding activity. The efficiency of utilization of different hemoglobins by the HmbR-expressing N. meningitidis cells was shown to be species specific; human hemoglobin was the best source of iron, followed by horse, rat, turkey, dog, mouse, and sheep hemoglobins, The phenotypic characterization of HmbR mutants of some clinical strains of N. meningitidis suggested the existence of two unrelated hemoglobin receptors. The HmbR-unrelated hemoglobin receptor was shown to be identical to Hpu, the hemoglobin-haptoglobin receptor of N. meningitidis. The Hpu-dependent hemoglobin utilization system was not able to distinguish between different sources of hemoglobin; all animal hemoglobins were utilized equally well. HmbR-like genes are also present in N. meningitidis serogroups A and B, Neisseria gonorrhoeae MS11 and FA19, Neisseria perflava, and Neisseria polysaccharea. The hemoglobin receptor genes from N. meningitidis serogroups A and B and N. gonorrhoeae MS11 were cloned, and their nucleotide sequences were determined. The nucleotide sequence identity ranged between 86.5% (for N. meningitidis serogroup B hmbR and MS11 hmbR) and 93.4% (for N. meningitidis serogroup B hmbR and N. meningitidis serogroup C hmbR). The deduced amino acid sequences of these neisserial hemoglobin receptors were also highly related, with overall 84.7% conserved amino acid residues. A stop codon was found in the hmbR gene of N. gonorrhoeae MS11. This strain was still able to use hemoglobin and hemoglobin-haptoglobin complexes as iron sources, indicating that some gonococci may express only the HmbR-independent hemoglobin utilization system.     

Genome analysis and strain comparison of correia repeats and correia repeat-enclosed elements in pathogenic Neisseria

Whole genome sequences of Neisseria meningitidis strains Z2491 and MC58 and Neisseria gonorrhoeae FA1090 were analyzed for Correia repeats (CR) and CR-enclosed elements (CREE). A total of 533, 516, and 256 copies of CR and 270, 261, and 102 copies of CREE were found in these three genomes, respectively. The lengths of CREE range from 28 to 348 bp, and the lengths of multicopy CREE appear mainly in the ranges of 154 to 156 bp and 105 to 107 bp. The distribution of CREE lengths is similar between the two N. meningitidis genomes, with a greater number of 154- to 156-bp CREE (163 and 152 copies in N. meningitidis strain Z2491 and N. meningitidis strain MC58, respectively) than 105- to 107-bp CREE (72 and 77 copies). In the N. gonorrhoeae strain FA1090 genome there are relatively more 105- to 107-bp CREE (51 copies) than 154- to 156-bp CREE (36 copies). The genomic distribution of 107-bp CREE also shows similarity between the two N. meningitidis strains (15 copies share the same loci) and differences between N. meningitidis strains and N. gonorrhoeae FA1090 (only one copy is located in the same locus). Detailed sequence analysis showed that both the terminal inverted repeats and the core regions of CREE are composed of distinct basic sequence blocks. Direct TA dinucleotide repeats exist at the termini of all CREE. A survey of DNA sequence upstream of the sialyltransferase gene, lst, in several Neisseria isolates showed that 5 N. meningitidis strains contain a 107-bp CREE in this region but 25 N. gonorrhoeae strains show an exact absence of a 105-bp sequence block (i.e., the 107-bp CREE without a 5' TA dinucleotide) in the same region. Whole-genome sequence analysis confirmed that this 105-bp indel exists in many homologous 107-bp CREE loci. Thus, we postulate that all CREE are made of target TA with indels of various lengths. Analysis of 107-bp CREE revealed that they exist predominantly in intergenic regions and are often near virulence, metabolic, and transporter genes. The abundance of CREE in Neisseria genomes suggests that they may have played a role in genome organization, function, and evolution. Their differential distribution in different pathogenic Neisseria strains may contribute to the distinct behaviors of each Neisseria species.     

A Sco homologue plays a role in defence against oxidative stress in pathogenic Neisseria

Sco proteins are found in mitochondria and in a variety of oxidase positive bacteria. Although Sco is required for the formation of the Cu(A) centre in a cytochrome oxidase of the aa(3) type, it was observed that oxidases with a Cu(A) centre are not present in many bacteria that contain a Sco homologue. Two bacteria of this type are the pathogens Neisseria meningitidis and Neisseria gonorrhoeae. The sco genes of N. gonorrhoeae strain 1291 and N. meningitidis strain MC58 were cloned, inactivated by inserting a kanamycin resistance cassette and used to make knockout mutants by allelic exchange. Both N. gonorrhoeae and N. meningitidis sco mutants were highly sensitive to oxidative killing by paraquat, indicating that Sco is involved in protection against oxidative stress in these bacteria.     

Ecological separation and genetic isolation of Neisseria gonorrhoeae and Neisseria meningitidis

BACKGROUND: Classifying bacteria into species is problematic. Most microbiologists consider species to be groups of isolates that share some arbitrary degree of relatedness of biochemical or molecular (such as DNA sequence) features and that, ideally, are clearly delineated from all other groups of isolates. The main problem in applying to bacteria a biological concept of species based on the ability or inability of their genes to recombine, is that recombination appears to be rare in bacteria in nature, as indicated by the strong linkage disequilibrium between alleles found in most bacterial populations. However, there are some naturally transformable bacteria in which assortative recombination appears to be so frequent that alleles are in, or close to, linkage equilibrium. For these recombining populations a biological concept of species might be applicable. RESULTS: Populations of Neisseria gonorrhoeae and Neisseria meningitidis from Spain were analysed by multilocus enzyme electrophoresis. The data indicate that assortative recombination occurs frequently within populations, but not between populations. Similarly, the sequences of two house-keeping genes show no evidence of intragenic recombination between N. gonorrhoeae and N. meningitidis. CONCLUSIONS: N. gonorrhoeae and N. meningitidis represent extremely closely related 'sexual' populations that appear to be genetically isolated in nature, and thus conform to the biological concept of species. The extreme uniformity of N. gonorrhoeae house-keeping genes suggests that this species may have arisen recently as a clone of N. meningitidis that could colonize the genital tract. Ecological isolation - of populations that can colonize the genital tract from those that can colonize the nasopharynx - may have been an important component in speciation, leading to a lower frequency of recombination between species than within species.     

Pathogenic neisseriae can use hemoglobin, transferrin, and lactoferrin independently of the tonB locus

Redundant TonB systems which function in iron transport from TonB-dependent ligands have recently been identified in several gram-negative bacteria. We demonstrate here that in addition to the previously described tonB locus, an alternative system exists for the utilization of iron from hemoglobin, transferrin, or lactoferrin in Neisseria meningitidis and Neisseria gonorrhoeae. Following incubation on media containing hemoglobin, N. meningitidis IR3436 (tonB exbB exbD deletion mutant) and N. gonorrhoeae PD3401 (tonB insertional mutant) give rise to colonies which can grow with hemoglobin. Transfer of Hb(+) variants (PD3437 or PD3402) to media containing hemoglobin, transferrin, and/or lactoferrin as sole iron sources resulted in growth comparable to that observed for the wild-type strains. Transformation of N. meningitidis IR3436 or N. gonorrhoeae PD3401 with chromosomal DNA from the Hb(+) variants yielded transformants capable of growth with hemoglobin. When we inactivated the TonB-dependent outer membrane hemoglobin receptors (HmbR or HpuB) in the Neisseria Hb(+) variants, these strains could not grow with hemoglobin; however, growth was observed with transferrin and/or lactoferrin. These results demonstrate that accumulation of iron from hemoglobin, transferrin, and lactoferrin in the pathogenic neisseriae can occur via a system that is independent of the previously described tonB locus.     

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.     

The lipopolysaccharide (R type) as a common antigen of Neisseria gonorrhoeae. II. Use of hen antiserum to gonococcal lipopolysaccharide in a rapid slide test for the identification of N. gonorrhoeae from primary isolates and secondary cultures.

An antiserum has been prepared in hens to R-type gonococcal lipopolysaccharide (LPS) and used in a simple slide-agglutination test for the identification of Neisseria gonorrhoeae. Anti-LPS serum agglutinated gonococcal cells representative of the four colony types of N. gonorrhoeae. Absorption of the antiserum with LPS removed the agglutinating activity. Secondary cultures (1120) were tested without observation of the colony type and all were agglutinated. No agglutination occurred with strains of Neisseria meningitidis, Neisseria lactamica, non-pathogenic Neisseria. Pseudomonas aeruginosa, Branhamella catarrhalis, or with species of lactobacilli and Acinetobacter. Cross-reactivity of the antiserum occurred with some streptococci. The anti-LPS serum was used to identify N. gonorrhoeae in primary isolates from the cervix, urethra, and pharynx. Of 251 gonococcal isolates tested, 249 were agglutinated by the antiserum, while all of the corresponding second cultures were agglutinated. The antiserum did not agglutinate N. meningitidis found in primary isolates from pharyngeal specimens. Anti-LPS hen serum should be useful for the rapid identification of N. gonorrhoeae in primary isolates or secondary cultures.     

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.     

Genetic diversity of the iron-binding protein (Fbp) gene of the pathogenic and commensal Neisseria

Abstract The pathogenic Neisseria and most commensal Neisseria species produce an iron-binding protein (Fbp) when grown under iron-limited conditions. In the current study, we confirmed the presence of Fbp, as well as DNA sequences homologous to the gonococcal fbp , in strains of N. gonorrhoeae, N. meningitidis, N. cinerea, N. lactamica, N. subflava, N. kochii and N. polysaccharea . The fbp genes from these strains were amplified by the polymerase chain reaction, digested with Stu I or Rsa I, and the restriction patterns examined. The patterns for the gonococcal and meningococcal fbp were virtually identical; however, variations were observed in the fbp sequences of the commensal Neisseria species. N. flavescens, N. mucosa, N. sicca, N. ovis and Branhamella catarrhalis , did not produce Fbp as detected by sodium dodecyl sulfate-polyacrylamide gel electropheris and reactivity with an Fbp specific monoclonal antibody, nor did they hybridize to an fbp -specific DNA probe.     

Epithelial cell responses induced upon adherence of pathogenic Neisseria

Neisseria meningitidis and Neisseria gonorrhoeae colonize human mucosal surfaces and cause sepsis/meningitis and gonorrhoea respectively. The first step in the infection process is pilus-mediated adhesion of the bacteria to epithelial cells, followed by host cell invasion. Adhesion of pathogenic Neisseria elicits multiple responses in host cells, including cellular signalling events, cytokine production and modulation of the eukaryotic cell surface. We used microarrays to assess the respective involvement of 375 human cytokine and adhesion related genes during adhesion of piliated and non-piliated N. gonorrhoeae, and piliated encapsulated N. meningitidis to the epithelial cell line ME-180. We identified 29 differentially regulated genes not previously reported to respond to neisserial infections, many of which encode membrane proteins. Selected genes were further analysed by semiquantitative RT-PCR, and protein expression was examined by flow cytometry. We found that N. gonorrhoeae elicited a different inflammatory response than N. meningitidis and we also demonstrated that early adhesion events are responsible for the induction of specific genes. Our data create a new platform for elucidating the interaction between pathogenic Neisseria and target cells.