The R-type lipopolysaccharides of Neisseria meningitidis

The lipopolysaccharides of all the different serogroups of Neisseria meningitidis are of the "R" type despite the morphologically smooth appearance and the demonstrated virulence of the organisms from which they were derived. This was confirmed when each of the lipopolysaccharides was found to be devoid of detectable O-antigen side chains, giving only a low "molecular" weight core oligosaccharide when subjected to mild acid hydrolysis. The cores were modified by dephosphorylation and subjected to sugar and methylation analysis by gas-liquid chromatography. Although all the different cores contained identical components (glucose, galactose, glucosamine, heptose, and 2-keto-3-deoxyoctonate) they could be separated into three distinct categories according to their galactose:glucose ratios. These categories are typified by the cores obtained from groups A, C, and 29-e which have galactose:glucose ratios of 1:2, 2:2, and 2:1, respectively. The modified cores were methylated and analyzed by gas chromatography--mass spectrometry and on the basis of differences in the derived methylated sugars the cores could again be divided into the same three categories as above. This structural diversity also results in some serological specificity as demonstrated by the complete serogroup specificity of the group A lipopolysaccharide.     

Functional genomics of Neisseria meningitidis pathogenesis

The pathogenic bacterium Neisseria meningitidis is an important cause of septicemia and meningitis, especially in childhood. The establishment and maintenance of bacteremic infection is a pre-requisite for all the pathological sequelae of meningococcal infection. To further understand the genetic basis of this essential step in pathogenesis, we analyzed a library of 2,850 insertional mutants of N. meningitidis for their capacity to cause systemic infection in an infant rat model. The library was constructed by in vitro modification of Neisseria genomic DNA with the purified components of Tn10 transposition. We identified 73 genes in the N. meningitidis genome that are essential for bacteremic disease. Eight insertions were in genes encoding known pathogenicity factors. Involvement of the remaining 65 genes in meningocoocal pathogenesis has not been demonstrated previously, and the identification of these genes provides insights into the pathogenic mechanisms that underlie meningococcal infection. Our results provide a genome-wide analysis of the attributes of N. meningitidis required for disseminated infection, and may lead to new interventions to prevent and treat meningococcal infection.     

Serological studies of ungroupable Neisseria meningitidis

Verification that Slaterus' Neisseria meningitidis serotypes X, Y, and Z are groups distinct from each other and from groups A, B, C, and D was made by use of the tube agglutination test on absorbed and unabsorbed antisera. A significant number of meningococcal strains in this country, which could not be classified serologically with standard antisera prepared to Branham's neotype A, B, C, and D strains, were grouped specifically with antisera prepared to the Slaterus types. The strains grouped as X, Y, and Z were from various geographical areas of the United States and were isolated from both carriers and cases. Over a 2-year period, the cultures tested ranged in predominance in descending order as follows: group B, C, Y, X, Z, A, and D. It is recommended that Slaterus' types should be considered as standard groups and follow in alphabetical order with the standard A, B, C, and D groups; i.e., X would be designated as group E, Y as group F, and Z as group G. It was observed that false-grouping cross-reactions could be greatly reduced by reconstituting the lyophilized grouping antisera in 50% glycerol-water. Of 99 cultures which could not be specifically grouped with antisera reconstituted in distilled water, 19 were specifically grouped with antisera reconstituted in 50% glycerol-water.     

Carriage of Neisseria meningitidis and Neisseria lactamica in northern Greece

In response to an increase in the number of cases of invasive meningococcal disease (IMD) in northern regions of Greece, a survey was carried out to determine if there was an increase in carriage of Neisseria meningitidis, particularly in areas where there have been increases in immigrant populations from neighbouring countries. The second objective was to determine if there was an increase in the serogroup C:2a:P1.5,2 a phenotype associated with recent outbreaks or changes in antibiotic sensitivities. As carriage of Neisseria lactamica is associated with development of natural immunity to IMD, the third objective was to determine the carriage rate of N. lactamica in this population. Among 3167 individuals tested, meningococci were isolated from 334 (10.5%). Compared with our previous studies, the proportion of meningococcal carriers was significantly increased among children in secondary education (11.3%) (chi2=9.67, P<0.005) and military recruits (37.4%) (chi2=21.11, P<0.000). Only 5/334 (1.5%) isolates expressed the phenotype associated with the increase in IMD in Greece. N. lactamica was isolated from 146/3167 (4.6%) participants. It was isolated from 71/987 (7.2%) children attending primary or nursery schools; however, the highest proportion of carriers (11.3%) was found in the boarding school for young Albanian men. In the 21-59-year age range, the majority of N. lactamica isolates (22/25, 88%) were from women, probably due to closer or more prolonged contact with children in the primary school age range. Smoking was significantly associated with isolation of meningococci from men but not from women. Penicillin-insensitive strains (25/334, 7.5%) were identified in all four regions examined; the majority (14/25, 56%) were obtained from military personnel. We conclude that there was a higher proportion of carriers in the population of northern Greece; however, the increase in carriage rate was not associated with the influx of immigrants from neighbouring countries, and there was not a higher incidence of the C:2a:P1.5,2 strain responsible for increased disease activity in Greece in either the immigrant or local populations.     

Characterization of DsbD in Neisseria meningitidis

Proper periplasmic disulfide bond formation is important for folding and stability of many secreted and membrane proteins, and is catalysed by three DsbA oxidoreductases in Neisseria meningitidis. DsbD provides reducing power to DsbC that shuffles incorrect disulfide bond in misfolded proteins as well as to the periplasmic enzymes that reduce apo-cytochrome c (CcsX) or repair oxidative protein damages (MrsAB). The expression of dsbD, but not other dsb genes, is positively regulated by the MisR/S two-component system. Quantitative real-time PCR analyses showed significantly reduced dsbD expression in all misR/S mutants, which was rescued by genetic complementation. The direct and specific interaction of MisR with the upstream region of the dsbD promoter was demonstrated by electrophoretic mobility shift assay, and the MisR binding sequences were mapped. Further, the expression of dsbD was found to be induced by dithiothrietol (DTT), through the MisR/S regulatory system. Surprisingly, we revealed that inactivation of dsbD can only be achieved in a strain carrying an ectopically located dsbD, in the dsbA1A2 double mutant or in the dsbA1A2A3 triple mutant, thus DsbD is indispensable for DsbA-catalysed oxidative protein folding in N. meningitidis. The defects of the meningococcal dsbA1A2 mutant in transformation and resistance to oxidative stress were more severe in the absence of dsbD.     

Epidemiology and pathogenesis of Neisseria meningitidis

Neisseria meningitidis, an exclusive pathogen of humans, remains the leading worldwide cause of meningitis and fatal sepsis, usually in otherwise healthy individuals. In recent years, significant advances have improved our understanding of the epidemiology and genetic basis of meningococcal disease and led to progress in the development of the next generation of meningococcal vaccines. This review summarizes current knowledge of the human susceptibility to and the epidemiology and molecular pathogenesis of meningococcal disease.     

Origin and Sequence of Chromosome Replication in Neisseria meningitidis: Influence of a Genetic Factor Determining Competence

Chromosome replication in Neisseria meningitidis was examined by enumerating mutants induced by nitrosoguanidine during synchronous replication after release from prolonged chloramphenicol inhibition. Clear maxima of mutagenesis were observed at certain times. These times were different for seven individual markers. At a definite time after the first maximum, there was a second one. The intervals between successive maxima were identical for all markers in each experiment. This permitted the construction of replication maps which were in general agreement with mapping based on marker frequency analysis by use of a transformation system. Comparison of replication in genetically competent and genetically incompetent variants substantiated the previous assumption that the change from genetic competence to genetic incompetence in this strain results in a change in the replication origin as well as in the direction of replication.     

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.     

Postgenomics of Neisseria meningitidis for vaccines development

Meningococcal disease is a global problem. Multivalent (A, C, Y, W135) conjugate vaccines have been developed and licensed; however, an effective vaccine against serogroup B has not yet become available. Outer membrane vesicle (OMV) vaccines have been used to disrupt serogroup B epidemics and outbreaks. Postgenomic technologies have been useful in aiding the discovery of new protein vaccine candidates. Moreover, proteomic technologies enable large-scale identification of membrane and surface-associated proteins, and provide suitable methods to characterize and standardize the antigen composition of OMV-based vaccines.     

Post-genomics of Neisseria meningitidis: an update

Neisseria meningitidis infection still remains a major life-threatening bacterial disease worldwide. The availability of bacterial genomic sequences generated a paradigm shift in microbiological and vaccines sciences, and post-genomics (comparative genomics, functional genomics, proteomics and a combination/evolution of these techniques) played important roles in elucidating bacterial biological complexity and pathogenic traits, at the same time accelerating the development of therapeutic drugs and vaccines. This article summarizes the most recent technological and scientific advances in meningococcal biology and pathogenesis aimed at the development and characterization of vaccines against the pathogenic meningococci.     

Hemagglutination Inhibition for Serogrouping of Neisseria meningitidis

A hemagglutination inhibition (HI) test has been studied as an alternative to bacterial agglutination (BA) for serogrouping strains of Neisseria meningitidis isolated from clinical specimens. The HI test consists of polysaccharide antigens adsorbed to sheep red blood cells which were then agglutinated by group-specific antisera. Supernatant fluids from suspensions of meningococci were used to inhibit the agglutination. Results of the two tests agreed for 381 (80%) carrier strains. Of the remaining 95 strains, 82 (86%) were identified by HI although they were nongroupable by BA. Thus, the HI test has been shown to be more highly specific and sensitive and to be more economical of reagents and time than the BA test.     

Isolation of Bacteriophages Active Against Neisseria meningitidis

Five distinct bacteriophages have been isolated from strains of Neisseria meningitidis. Filtrates with titers of 10(-4) to 10(-6) were produced with a modified Swanstrom and Adams semisolid agar procedure, employing Eugonbroth with added agar and an incubation temperature of 30 C. Of 49 strains of N. meningitidis (groups B and C), 25 were lysed by one or more of the phages, but there was no lysis of other Neisseria and Mima polymorpha strains.     

Postgenomics of Neisseria meningitidis for vaccines development

Meningococcal disease is a global problem. Multivalent (A, C, Y, W135) conjugate vaccines have been developed and licensed; however, an effective vaccine against serogroup B has not yet become available. Outer membrane vesicle (OMV) vaccines have been used to disrupt serogroup B epidemics and outbreaks. Postgenomic technologies have been useful in aiding the discovery of new protein vaccine candidates. Moreover, proteomic technologies enable large-scale identification of membrane and surface-associated proteins, and provide suitable methods to characterize and standardize the antigen composition of OMV-based vaccines.     

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.     

Loss of endotoxin liberation in Neisseria meningitidis

Four strains of Neisseria meningitidis were studied during serial passage. Upon subcultivation, two of them lost the ability to liberate endotoxin. Ultrastructurally, the two parent endotoxin liberating strains exhibited quantitatively more free cell wall membranes and blebs in the medium than their non-liberating variants. Similarly, the endotoxin-releasing original strains exhibited higher sulfonamide resistance than their variants, and had markedly more sticky cells, which showed pronounced adherence to the surfaces of plastic and heated blood agar.     

Response of Neisseria meningitidis to iron limitation

In the human body, the concentration of free iron is limiting for bacterial growth, since iron is bound to transport and storage proteins such as transferrin and lactoferrin. When grown under iron starvation, Neisseria meningitidis produces receptors for these proteins in the outer membrane. These receptors are presently being characterized at the molecular level. Here, we summarize our current knowledge of these receptors, with special emphasis on the LbpA and FrpB proteins, which are studied in our laboratories. Furthermore, the genetic and antigenic variability of these proteins and their vaccine potential are discussed.     

Bacteriocin production by strains of Neisseria meningitidis

Kingsbury, David T. (Naval Medical Research Institute, Bethesda, Md.). Bacteriocin production by strains of Neisseria meningitidis. J. Bacteriol. 91:1696-1699. 1966.-Strains of Neisseria meningitidis produce substances inhibitory to other strains of meningococcus. These substances are nontransmissible and show a high degree of strain specificity. The properties of one of these substances resemble those of the class of bacterial inhibitors called bacteriocins. Synthesis of this "meningocin" can be increased as much as 200-fold by induction with mitomycin C. It shows a high degree of heat stability and is sensitive to proteolytic enzymes. Six bacteriocins from strains of N. meningitidis have been used to type meningococci. By use of this procedure, strains that were identical serologically were placed into distinct bacteriocin groups.