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.     

Natural transformation and phase variation modulation in Neisseria meningitidis

Neisseria meningitidis has evolved the ability to control the expression-state of numerous genes by phase variation. It has been proposed that the process aids this human pathogen in coping with the diversity of microenvironments and host immune systems. Therefore, increased frequencies of phase variation may augment the organism's adaptability and virulence. In this study, we found that DNA derived from various neisserial co-colonizers of the human nasopharynx increased N. meningitidis switching frequencies, indicating that heterologous neisserial DNA modulates phase variation in a transformation-dependent manner. In order to determine whether the effect of heterologous DNA was specific to the Hb receptor, HmbR, we constructed a Universal Rates of Switching cassette (UROS). With this cassette, we demonstrated that heterologous DNA positively affects phase variation throughout the meningococcal genome, as UROS phase variation frequencies were also increased in the presence of neisserial DNA. Overexpressing components of the neisserial mismatch repair system partially alleviated DNA-induced changes in phase variation frequencies, thus implicating mismatch repair titration as a cause of these transformation-dependent increases in switching. The DNA-dependent effect on phase variation was transient and may serve as a mechanism for meningococcal genetic variability that avoids the fitness costs encountered by global mutators.     

Purification of rough-type lipopolysaccharides of Neisseria meningitidis from cells and outer membrane vesicles in spent media.

A procedure for the purification of Neisseria meningitidis lipopolysaccharide (LPS) from outer membrane vesicles (OMV) in spent growth media was developed. Five different LPS strains of group A N. meningitidis were grown in tryptic soy broth with vigorous aeration for 36-48 h, and centrifuged to collect both cells and supernatants. The amount of LPS in the OMV in the supernatants was higher or at least equal to that in the cells. The OMV in each supernatant were concentrated, pelleted by ultracentrifugation, and treated with 2% sodium deoxycholate to dissociate LPS from OMV. The LPS was then separated from capsular polysaccharides, proteins and phospholipids by gel filtration on Sephacryl S-300 column in 1% sodium deoxycholate, and precipitated from the column fractions in 70% ethanol. In addition, LPS was also extracted from cells with hot phenol-water, ultracentrifuged once after treatment with ribonuclease, and purified on Sephacryl S-300. When compared with an improved phenol-water extraction method, the LPS obtained from either OMV or cells by the above methods gave a 40-180% increase in yield. The LPS also had much higher activities in limulus amebocyte lysate assay, rabbit pyrogenic test, and enzyme-linked immunosorbent assay. The LPS purified from cells and from OMV were indistinguishable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis.     

Heterogeneity of Rhizobium lipopolysaccharides.

The lipopolysaccharides ( LPSs ) from strains of Rhizobium leguminosarum, Rhizobium trifolii, and Rhizobium phaseoli were isolated and partially characterized by mild acid hydrolysis and by polyacrylamide gel electrophoresis. Mild acid hydrolysis results in a precipitate which can be removed by centrifugation or extraction with chloroform. The supernatant contains polysaccharides which, in general, are separated into two fractions ( LPS1 and LPS2 ) by Sephadex G-50 gel filtration chromatography. The higher-molecular-weight LPS1 fractions among the various Rhizobium strains are highly variable in composition and reflect the variability reported in the intact LPSs (R. W. Carlson and R. Lee, Plant Physiol. 71:223-228, 1983; Carlson et al., Plant Physiol. 62:912-917, 1978; Zevenhuizen et al., Arch. Microbiol. 125:1-8, 1980). The LPS1 fraction of R. leguminosarum 128C53 has a higher molecular weight than all other LPS1 fractions examined. All LPS2 fractions examined are oligosaccharides with a molecular weight of ca. 600. The major sugar component of all LPS2 oligosaccharides is uronic acid. The LPS2 compositions are similar for strains of R. leguminosarum and R. trifolii, but the LPS2 from R. phaseoli was different in that it contained glucose, a sugar not found in the other LPS2 fractions or found only in trace amounts. Polyacrylamide gel electrophoretic analysis shows that each LPS contains two banding regions, a higher-molecular-weight heterogeneous region often containing many bands and a lower-molecular-weight band. The lower-molecular-weight bands of all LPSs have the same electrophoretic mobility, which is greater than that of lysozyme. The banding pattern of the heterogeneous regions varies among the different Rhizobium strains. In the case of R. leguminosarum 128C53 LPS, the heterogeneous region of a higher molecular weight than is this region from all other Rhizobium strains examined and consists of many bands separated from one another by a small and apparently constant molecular weight interval. When the heterogeneous region of R. Leguminosarum 128C53 LPS was cut from the gel and analyzed, its composition was found to be that of the intact LPS, whereas the lower-molecular-weight band contains only sugars found in the LPS2 oligosaccharide. In the case of R. leguminosarum 128C63 and R. trifolii 0403 LPSs, the heterogeneous regions are similar and consist of several band s separated by a large-molecular-weight interval with a the major band of these heterogeneous regions having the lowest molecular weight with an electrophoretic mobility near that of beta-lactoglobulin. The heterogeneous region from R. phaseoli 127K14 consists of several bands with electrophoretic mobilities near that of beta-lactoglobulin, whereas this region from R. trifolii 162S7 shows a continuous staining region, indicating a great deal of heterogeneity. The results described in this paper are discussed with regard to the reported properties of Escherichia coli and Salmonella LPSs.     

Mismatch repair and the regulation of phase variation in Neisseria meningitidis

Neisseria meningitidis controls the expression of several genes involved in host adaptation by a process known as phase variation. The phase variation frequency of haemoglobin (Hb) receptors among clinical isolates of serogroups A, B and C differed drastically, ranging from approximately 10(-6) to 10(-2) cfu-1. Frequencies of phase variation are a genetic trait of a particular strain, as two unlinked Hb receptors, hpuAB and hmbR, phase varied with similar frequencies within a given isolate. Based on these frequencies, six Neisserial clinical isolates could be grouped into three distinct classes; slow, medium and fast. An increase in phase variation frequency was accompanied by high rates of spontaneous mutation to rifampicin and nalidixic acid resistance in one medium and one fast strain. The remaining three medium strains displayed elevated levels of phase variation without increases in overall mutability, as they possessed low rates of spontaneous mutation to drug resistance. The mismatch repair system of N. meningitidis was found to play an important role in determining the overall mutability of the clinical isolates. Inactivation of mismatch repair in any strain, regardless of its original phenotype, increased mutability to a level seen in the fast strain. Insertional inactivation of mutS and mutL in the slow strain led to 500- and 250-fold increases in hmbR switching frequency respectively. Concurrently, the frequency of spontaneous point mutations of mutS and mutL mutants from the slow strain was increased 20- to 30-fold to the level seen in the high strain. The status of Dam methylation did not correlate with either the phase variation frequency of Hb receptors or the general mutability of Neisserial strains. Analysis of an expanded set of isolates identified defects in mismatch repair as the genetic basis for strains displaying both the fast Hb switching and high mutation rate phenotypes. In conclusion, elevated frequencies of phase variation were accompanied by increased overall mutability in some N. meningitidis isolates including strains shown to be mismatch repair defective. Other isolates have evolved mechanisms that seem to affect only the switching frequency of phase-variable genes without an accompanied increased accumulation of spontaneous mutations.     

Genetic characterization of pilin glycosylation and phase variation in Neisseria meningitidis

Pili of Neisseria meningitidis are a key virulence factor, being the major adhesin of this capsulate organism and contributing to specificity for the human host. Pili are post-translationally modified by addition of either an O-linked trisaccharide, Gal (beta1-4) Gal (alpha1-3) 2,4-diacetamido-2,4,6-trideoxyhexose or an O-linked disaccharide Gal (alpha1,3) GlcNAc. The role of these structures in meningococcal pathogenesis has not been resolved. In previous studies we identified two separate genetic loci, pglA and pglBCD, involved in pilin glycosylation. Putative functions have been allocated to these genes; however, there are not enough genes to account for the complete biosynthesis of the described structures, suggesting additional genes remain to be identified. In addition, it is not known why some strains express the trisaccharide structure and some the disaccharide structure. In order to find additional genes involved in the biosynthesis of these structures, we used the recently published group A strain Z2491 and group B strain MC58 Neisseria meningitidis genomes and the unfinished Neisseria meningitidis group C strain FAM18 and Neisseria gonorrhoeae strain FA1090 genomes to identify novel genes involved in pilin glycosylation, based on homology to known oligosaccharide biosynthetic genes. We identified a new gene involved in pilin glycosylation designated pglE and examined four additional genes pglB/B2, pglF, pglG and pglH. A strain survey revealed that pglE and pglF were present in each strain examined. The pglG, pglH and pglB2 polymorphisms were not found in strain C311 musical sharp 3 but were present in a large number of clinical isolates. Insertional mutations were constructed in pglE and pglF in N. meningitidis strain C311 musical sharp 3, a strain with well-defined lipopolysaccharide (LPS) and pilin-linked glycan structures. Increased gel migration of the pilin subunit molecules of pglE and pglF mutants was observed by Western analysis, indicating truncation of the trisaccharide structure. Antisera specific for the C311 musical sharp 3 trisaccharide failed to react with pilin from these pglE and pglF mutants. GC-MS analysis of the sugar composition of the pglE mutant showed a reduction in galactose compared with C311 musical sharp 3 wild type. Analysis of amino acid sequence homologies has suggested specific roles for pglE and pglF in the biosynthesis of the trisaccharide structure. Further, we present evidence that pglE, which contains heptanucleotide repeats, is responsible for the phase variation between trisaccharide and disaccharide structures in strain C311 musical sharp 3 and other strains. We also present evidence that pglG, pglH and pglB2 are potentially phase variable.     

Patterns of sequence variation within the Neisseria meningitidis lactoferrin binding proteins

Lactoferrin binding proteins A and B (LbpA and LbpB) compose the lactoferrin receptor of the obligate human pathogen Neisseria meningitidis . This receptor is thought to be important for colonization and initiation of invasive disease because of its role in acquiring host iron and providing protection from the cationic peptide, lactoferricin. By virtue of its function, the receptor is accessible to the host immune system and displays substantial sequence variation. In this study, we analyzed a broad collection of LbpAs (62) and LbpBs (101) to determine the distribution of sequence variation within each protein and to search for patterns between sequence similarity and strain typing. The sequence variation in LbpA was predominantly observed in 3 surface loops and, surprisingly, in the N-terminal region immediately upstream of the predicted TonB box. The analysis of LbpB revealed that the variability was distributed throughout the protein, particularly in the highly variable negatively charged regions in the C-lobe, but otherwise was greater in the N-lobe than the C-lobe. There was no readily identifiable correlation between the sequence variation within LbpA, LbpB, multi-locus sequence type, or serogroup.     

Iron transport systems in Neisseria meningitidis.

Acquisition of iron and iron complexes has long been recognized as a major determinant in the pathogenesis of Neisseria meningitidis. In this review, high-affinity iron uptake systems, which allow meningococci to utilize the human host proteins transferrin, lactoferrin, hemoglobin, and haptoglobin-hemoglobin as sources of essential iron, are described. Classic features of bacterial iron transport systems, such as regulation by the iron-responsive repressor Fur and TonB-dependent transport activity, are discussed, as well as more specific features of meningococcal iron transport. Our current understanding of how N. meningitidis acquires iron from the human host and the vaccine potentials of various components of these iron transport systems are also reviewed.     

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.     

Interactions between encapsulated Neisseria meningitidis and host cells.

A major feature of Neisseria meningitidis is its ability to invade human brain meninges. To access the meninges, the bacteria must cross the blood-brain barrier (BBB), which is one of the tightest barriers in the body. Therefore, N. meningitidis must have evolved some type of sophisticated means to bypass the physical properties of this cellular barrier. As N. meningitidis is encapsulated when present in the bloodstream, this review will focus on the mechanisms that encapsulated N. meningitidis has developed to interact with host cells and will suggest ways in which these mechanisms may be helpful for crossing the BBB.     

Serotypes among Neisseria meningitidis serogroups B and C strains isolated in Canada

Antisera made to prototype serogroup B strains of Neisseria meningitidis were used to serotype, by agar gel double diffusion, 262 meningococcal serogroups B and C strains isolated in Canada. The strains included 93 from patients and 169 from carriers. Serotype 2 was associated with 39 of 75 (52%) of group B strains and 14 of 18 (77.8%) of group C strains isolated from patients. The group B strains were mainly (87.2%) serotype 2b, while the majority (92.2%) of group C strains was serotype 2a. Other serotypes (including a new provisional serotype) represented 25.3 and 5.5% of groups B and C strains, respectively. The new serotype accounted for 13% of the group B strains. Approximately 23% of the strains isolated from patients were nontypable. The distribution of serotype 2, nontype 2 (other serotypes), and nontypable strains isolated from carriers was 2.1, 36.6, and 61.3%, respectively, for group B meningococci and 22.2, 29.6, and 48.25, respectively, for group C meningococci. Serotype 11 was the most prominent of the strains isolated from carriers. Approximately 7% of all the strains were multiple serotypes. Serotype 2 is an important virulence marker associated with meningococcal groups B and C disease in Canada, with serotypes 2a and 2b being markedly associated with groups C and B meningococcal disease, respectively.     

Bioinformatic analysis of outer membrane proteome of Neisseria meningitidis and Neisseria lactamica.

Two-dimensional electrophoresis (isoelectric focusing/SDS-PAGE) and Western-blotting techniques were used to analyze and compare common and/or specific outer-membrane proteins and antigens from Neisseria meningitidis and Neisseria lactamica. Bioinformatic image analyses of proteome and immunoproteome maps indicated the presence of numerous proteins and several antigens shared by N. meningitidis and N. lactamica, although the inter-strain variation in the maps was of similar magnitude to the inter-species variation, and digital comparison of the maps did not reveal proteins found to be identical by MALDI-TOF fingerprinting analysis. PorA and RmpM, two relevant outer-membrane antigens, manifested as various spots at several different positions. While some of these were common to all the strains analyzed, others were exclusive to N. meningitidis and their electrophoretic mobilities were different than expected. One such spot, with a molecular mass of 19 kDa, may be the C-terminal fragment of RmpM (RmpM-Cter). The results demonstrate that computer-driven analysis based exclusively on spot positions in the proteome or immunoproteome maps is not a reliable approach to predict the identity of proteins or antigens; rather, other identification techniques are necessary to obtain accurate comparisons.     

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.     

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.     

Cellular and free lipopolysaccharides of some species of Neisseria.

Cultures of eight non-pathogenic species of Neisseria grown in simple defined media released lipopolysaccharide (free lipopolysaccharide) by a process distinct from cellular autolysis. Analyses of the pure cellular and free lipopolysaccharides obtained from six species of Neisseria revealed that they were remarkably similar and were devoid of detectable O-antigen side chains. Three distinct types of core-oligosaccharides were demonstrated. Type I core-oligosaccharide was a branched structure of alpha-D-glucopyranosyl units (7 mol) terminated by a reducing end group of 3-deoxy-D-manno-octulosonic acid. Type II core-oligosaccharide contained D-glucose, 2-deoxy-2-amino-D-glucose, L-rhamnose, L-glycero-D-manno-heptose, 3-deoxy-D-manno-octulosonic acid, phosphate, and ethanolamine in a molar ratio of 3:2:1:1:1:1:1. Type III coreoligosaccharide was composed of D-glucose, L-glycero-D-manno-heptose, 3-deoxy-D-manno-octulosonic acid, and phosphate in a molar ratio of 3:3:1:1. Lipopolysaccharides of N. caviae and N. sicca contained type I core-oligosaccharides exclusively, while those of N. flava and N. perflava contained only type II core-oligosaccharide. Cellular lipopolysaccharide from N. cinerea contained core-oligosaccharides of types I and II in a ratio of 27:73, while the analogous preparation from N. flavescens contained core-oligosaccharide types II and III in a ratio of 21:4. Free lipopolysaccharides from these two organisms contained only one type of coreoligosaccharide. Lipid A components of all the lipopolysaccharide preparations were very similar being composed of about 25% by weight of dodecanoic acid, 3-hydroxy-dodecanoic acid, and 3-hydroxy-tetradecanoic acid.