Genomic Rearrangements in Neisseria meningitidis Strains of the ET-5 Complex

A physical map of the chromosome of Neisseria meningitidis strain 44/76, which belongs to the epidemic clone ET-5, was constructed. DNA fragments obtained after SfiI and NheI digestion were resolved by pulsed field gel electrophoresis (PFGE). The overall arrangement of 26 genetic markers localized on the 2.3-Mb chromosome was conserved in comparison with that in meningococcal strains B1940 and Z2491. Simplified physical maps of 29 additional strains belonging to the ET-5 complex isolated from various parts of the world were compared with that of strain 44/76. Ten distinct patterns of hybridization were identified. While two of the seven probes hybridized to fragments of the same size in all strains, the remaining probes hybridized to different fragments, in some cases to fragments not adjacent on the chromosome of 44/76. These results indicated the occurrence of genetic rearrangements in the genome of the ET-5 meningococcal clone in the course of its epidemic spread. Received: 17 November 1999 / Accepted: 28 December 1999     

Hemagglutination by Neisseria meningitidis

The direct agglutination of erythrocytes by Neisseria meningitidis was studied as a marker for adherence. Hemagglutination (HA) was studied by slide test (5-min incubation) and by dilutions in microtitre plates (20-h incubation). Meningococci that were freshly isolated from subjects agglutinated only human cells by slide test but human, dog, rabbit, guinea pig, and rat cells were agglutinated in the microtitre system. Newly isolated strains were piliated and HA positive but pili were lost after 10 passages on agar, and bacteria became HA negative. HA could be maintained by "affinity culturing," which selected markedly adhesive bacteria: erythrocytes with adherent meningococci were isolated and cultured on agar. This procedure was repeated daily. HA titres were unaffected by mannose but were reduced by sonic disruption, trypsinization, ultraviolet irradiation, heating (65 degrees C), and formaldehyde. Encapsulated (serogroupable) bacteria had low HA titres compared with nongroupable strains, and purified capsular polysaccharides A and C inhibited HA. Meningococcal HA is probably mediated by pili and modified by other factors such as encapsulation. Colonial variation was not a reliable indicator of piliation, and HA is best used for this purpose.     

NarE: a novel ADP-ribosyltransferase from Neisseria meningitidis

Mono ADP-ribosyltransferases (ADPRTs) are a class of functionally conserved enzymes present in prokaryotic and eukaryotic organisms. In bacteria, these enzymes often act as potent toxins and play an important role in pathogenesis. Here we report a profile-based computational approach that, assisted by secondary structure predictions, has allowed the identification of a previously undiscovered ADP-ribosyltransferase in Neisseria meningitidis (NarE). NarE shows structural homologies with E. coli heat-labile enterotoxin (LT) and cholera toxin (CT) and possesses ADP-ribosylating and NAD-glycohydrolase activities. As in the case of LT and CT, NarE catalyses the transfer of the ADP-ribose moiety to arginine residues. Despite the absence of a signal peptide, the protein is efficiently exported into the periplasm of Neisseria. The narE gene is present in 25 out of 43 strains analysed, is always present in ET-5 and Lineage 3 but absent in ET-37 and Cluster A4 hypervirulent lineages. When present, the gene is 100% conserved in sequence and is inserted upstream of and co-transcribed with the lipoamide dehydrogenase E3 gene. Possible roles in the pathogenesis of N. meningitidis are discussed.     

Candidate Neisseria meningitidis NspA vaccine

The highly conserved NspA protein has been found in the outer membrane of every Neisseria meningitidis strain tested so far. Two monoclonal antibodies (MAbs) directed against this protein were used to demonstrate that biologically important epitopes of the NspA protein are exposed at the surface of serologically distinct meningococcal strains. Analysis of sera collected from mice that survived a deadly meningococcal challenge following immunization with recombinant NspA protein (rNspA) revealed the presence of cross-reactive antibodies which efficiently attached to and killed the four serogroup B strains tested. These data are additional proof that the NspA protein is exposed at the surface of intact meningococcal cells, which is an important characteristic for a vaccine candidate.     

Sulphur acquisition by Neisseria meningitidis

Group B Neisseria meningitidis (SD1C) was grown on defined medium supplemented with each of a variety of sulphur compounds as the sole source of sulphur. The organism grew on sulphate, sulphite, bisulphite, thiosulphate, dithionite, hydrosulphide, thiocyanate, L-cysteine, L-cystine, reduced glutathione, methionine, mercaptosuccinate, and lanthionine, but not on dithionate unless previously sulphur starved. Good growth was seen on concentrations of sulphate or thiosulphate as low as 10 microM. When pregrown on and subsequently starved for sulphate, the meningococcus showed enhanced transport capacity for this ion. Optimal conditions for assessing sulphur transport by active sulphur-limited cells were determined. The maximal sulphate uptake velocity was 9.3 nmol sulphate X mg protein-1 X min-1, and the apparent Km was 1.4 microM, far below human nasopharyngeal or serum sulphate levels.     

Neisseria lactamicus sp. n., a Lactose-fermenting Species Resembling Neisseria meningitidis

The biochemical and serological characteristics of lactose-utilizing strains of Neisseria were determined. These organisms were found in the nasopharynx of man and grew well on Thayer-Martin Selective Medium. They were compared with N. meningitidis to ascertain whether they were variants of this species. Differences between the lactose-using strains and the recognized species of Neisseria were considered significant enough to warrant designation of a new species, Neisseria lactamicus. This group has not been widely recognized as being separate from N. meningitidis; therefore, the normal incidence and clinical significance of these organisms has not been fully established. These organisms are oxidase-positive and positive for beta-D-galactosidase activity; they demonstrate fermentation in King Oxidation-Fermentation Medium; and they produce acid from only glucose, lactose, and maltose, of the 27 substrates incorporated in Cystine Trypticase Agar. Individual strains vary in their ability to grow on Nutrient Agar at both 25 and 37 C and in their pigmentation on Loeffler Medium. Results indicated that these organisms are serologically distinct from the N. meningitidis serogroups. Only 34 of 116 strains of N. lactamicus were smooth and could be tested by slide agglutination. None of the 34 could be grouped as N. meningitidis group A, B, C, D, X, Y, or Z. Thirty-one of these strains could, however, be specifically grouped with antisera prepared with N. lactamicus strains. Cross absorptions confirmed that N. lactamicus is serologically distinguishable from N. meningitidis.     

Nmel, a restriction endonuclease from Neisseria meningitidis

A restriction endonuclease, Nmel, present in Neisseria meningitidis was partially purified by passing through a blue 2-cross linked agarose column; no contaminating nucleases remained detectable. This enzyme cleaved phage lambda, adenovirus type 2 and phi x 174 DNA but did not cleave SV40 DNA. It had an absolute requirement for Mg2+ for its activity and was inhibited by high concentrations of NaCl or MgCl2. Nmel activity was completely abolished after 1 h of incubation at 65 degrees C. S-adenosyl-L-methionine and ATP had no effect on its activity suggesting that Nmel is a type II restriction endonuclease enzyme. It is the first report of a restriction enzyme present in N. meningitidis.     

Vaccine development against Neisseria meningitidis

Meningococcal disease is communicable by close contact or droplet aerosols. Striking features are high case fatality rates and peak incidences of invasive disease in infants, toddlers and adolescents. Vaccine development is hampered by bacterial immune evasion strategies including molecular mimicry. As for Haemophilus influenzae and Streptococcus pneumoniae, no vaccine has therefore been developed that targets all serogroups of Neisseria meningitidis. Polysaccharide vaccines available both in protein conjugated and non‐conjugated form, have been introduced against capsular serogroups A, C, W‐135 and Y, but are ineffective against serogroup B meningococci, which cause a significant burden of disease in many parts of the world. Detoxified outer membrane vesicles are used since decades to elicit protection against epidemic serogroup B disease. Genome mining and biochemical approaches have provided astounding progress recently in the identification of immunogenic, yet reasonably conserved outer membrane proteins. As subcapsular proteins nevertheless are unlikely to immunize against all serogroup B variants, thorough investigation by surrogate assays and molecular epidemiology approaches are needed prior to introduction and post‐licensure of protein vaccines. Research currently addresses the analysis of life vaccines, meningococcus B polysaccharide modifications and mimotopes, as well as the use of N. lactamica outer membrane vesicles.     

Vascular colonization by Neisseria meningitidis

Bacterial infection of human vasculature can lead to unregulated systemic activation of coagulation and innate immunity and rapidly becomes life threatening. Neisseria meningitidis is a vascular pathogen that causes fatal sepsis and meningitis. Post-mortem histological analysis of tissues from individuals infected with N. meningitidis show large bacterial aggregates in close association with the vascular wall of small vessels. The ability of this bacterium to colonize blood vessel endothelium is likely to impact its capacity to both multiply in the blood stream and reach the brain. This process will be referred to as vascular colonization. Recent work has described a number of early steps in N. meningitidis vascular colonization, from attachment to proliferation and dissemination, focusing on the bacterial-host interaction.     

Mechanistic studies of a retaining alpha-galactosyltransferase from Neisseria meningitidis

Lipopolysaccharyl alpha-galactosyltransferase from Neisseria meningitidis catalyzes the transfer of a galactosyl moiety from the activated donor UDP-Gal to glycoconjugates to yield an elongated saccharide product with net retention of anomeric configuration relative to the donor substrate. Through kinetic analyses in which the concentrations of both substrates are independently varied and through inhibition studies with dead-end analogues of both substrates and with the oligosaccharide product, we have demonstrated that this enzyme follows an ordered bi-bi kinetic mechanism. Various aspects of the chemical mechanism including the possible formation of a covalent glycosyl-enzyme intermediate were also probed using an assortment of strategies. While the results of these investigations were unable to clearly delineate the chemical mechanism of this enzyme, they provide important insights into the catalytic machinery surrounding the events involved in catalysis.     

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.     

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.     

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.     

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.     

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.