Proteome analysis of Neisseria meningitidis serogroup A

Neisseria meningitidis is an encapsulated Gram-negative bacterium responsible for significant morbidity and mortality worldwide. Meningococci are opportunistic pathogens, carried in the nasopharynx of approximately 10% of asymptomatic adults. Occasionally they enter the bloodstream to cause septicaemia and meningitis. Meningococci are classified into serogroups on the basis of polysaccharide capsule diversity, and serogroup A strains have caused major epidemics mainly in the developing world. Here we describe a two-dimensional gel electrophoresis protein map of the serogroup A strain Z4970, a clinical isolate classified as ancestral to several pandemic waves. To our knowledge this is the first systematically annotated proteomic map for N. meningitidis. Total protein samples from bacteria grown on GC-agar were electrophoretically separated and protein species were identified by matrix-assisted laser desorption/ionization time of flight spectrometry. We identified the products of 273 genes, covering several functional classes, including 94 proteins so far considered as hypothetical. We also describe several protein species encoded by genes reported by DNA microarray studies as being regulated in physiological conditions which are relevant to natural meningococcal pathogenicity. Since menA differs from other serogroups by having a fairly stable clonal population structure (i.e. with a low degree of variability), we envisaged comparative mapping as a useful tool for microevolution studies, in conjunction with established genotyping methods. As a proof of principle, we performed a comparative analysis on the B subunit of the meningococcal transferrin receptor, a vaccine candidate encoded by the tbpB gene, and a known marker of population diversity in meningococci. The results show that TbpB spot pattern variation observed in the maps of nine clinical isolates from diverse epidemic spreads, fits previous analyses based on allelic variations of the tbpB gene.     

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.     

The opcA and ΨopcB regions in Neisseria: genes, pseudogenes, deletions, insertion elements and DNA islands

Previous data have indicated that the opc gene encoding an immunogenic invasin is specific to Neisseria meningitidis (Nm) and is lacking in Neisseria gonorrhoeae (Ng). The data presented here show that Nm and Ng both contain two paralogous opc-like genes, opcA, corresponding to the former opc gene, and (psi)opcB, a pseudogene. The predicted OpcA and OpcB proteins possess transmembrane regions with conserved non-polar faces but differ extensively in four of the five surface-exposed loops. Gonococcal OpcA was expressed weakly under in vitro conditions, and it is unknown whether these bacteria can express this protein at high levels. Analysis of the sequences flanking opcA and (psi)opcB revealed a framework of conserved housekeeping genes interspersed with DNA islands. These regions also contained several pseudogenes, deletions and IS elements, attesting to considerable genome plasticity. Both opcA and (psi)opcB are located on DNA islands that have probably been imported from unrelated bacteria. A third island encodes the dcmD/dcrD R/M genes in Ng versus a small open reading frame in most strains of Nm. Rare strains of Nm were identified in which the R/M island has been imported. DNA islands in Nm and Ng seem to have been acquired by recombination via conserved flanking housekeeping genes rather than by insertion of mobile genetic elements.     

Analysis of the clonal relationships between strains of Neisseria meningitidis by pulsed field gel electrophoresis.

Fingerprint patterns were generated from strains of Neisseria meningitidis by digestion of chromosomal DNA samples with 'rare-site' restriction endonucleases and resolution of the resultant fragments by pulsed field gel electrophoresis (PFGE). The potential of this technique for the rapid establishment of the clonal relationships between different isolates of the meningococcus was investigated. The fingerprint patterns from various serogroup A strains, previously assigned to clonal subgroups on the basis of their electrophoretic types (ETs), were compared. Fingerprints generated with the endonucleases SfiI, SpeI and NheI each gave distinctive patterns for the clonal subgroups I-IV of serogroup A. Further, the endonucleases SpeI and, particularly, NheI were capable of resolving differences between various subgroup III strains isolated at different times and geographical locations. Strains isolated during the 'new wave' pandemic, which was associated with the Haj, from Europe, America, and Africa, had a characteristic fingerprint pattern and appeared to be distinct from 'old wave' pandemic strains. The PFGE technique is a relatively rapid and sensitive method for establishing clonal relationships among epidemic strains of N. meningitidis.     

Epidemiological evaluation of Neisseria meningitidis serogroup B by pulsed-field gel electrophoresis

Abstract Genomic DNA from 25 strains of serogroup B Neisseria meningitidis was subjected to pulsed-field gel electrophoresis (PFGE) after digestion with Spe I. N. meningitidis genomic DNA displayed considerable diversity. The diversity we observed among these strains was stable and included isolates from an outbreak that were phenotypically identical. This confirms the value of macrorestriction profiling and PFGE in providing epidemiologically stable strain markers for typing meningococci.     

Site-specific insertion of IS1301 and distribution in Neisseria meningitidis strains.

The insertion element IS1301 has been shown to mediate capsule phase variation in Neisseria meningitidis found in N. serogroup B by reversible insertional inactivation of the siaA gene. We have determined the target site specificity of this element by cloning and sequencing the insertion sites of 12 identical IS1301 copies found in N. meningitidis B1940. A target consensus core of 5'-AYTAG-3' was identified, with the central TA being duplicated following insertion. Additional features around the target sites, including extended palindromic symmetry, stem-loop formation, and the high incidence of AT tracts, indicate that other factors, such as DNA secondary structure, are involved in target recognition. The left inverted repeat of an IS1016-like element acts as a hot spot for insertion, with one insertion element combination located upstream of their gene. According to further sequence analysis, we were able to place IS1301 in the IS5 subgroup within the IS4 family of elements. A survey of 135 Neisseria strains indicated the presence of IS1301 in 27.9 to 33.3% of N. meningitides serogroup B, C, and W135 strains and in 86.7% of serogroup Y strains. IS1301 did not occur in serogroup A strains, in Neisseria gonorrhoeae, or in apathogenic Neisseria spp.     

Induction of the anti-meningitis immunity with synthetic peptides. III. Immunoactive synthetic fragments of NspA protein from Neisseria meningitidis

Four potentially immunoactive peptide fragments of the NspA protein from the outer membrane of the bacterium Neisseria meningitidis were synthesized in order to create a synthetic vaccine against the meningococcal infection by the serogroup B bacterium. Mice of various lines were immunized with the free peptides nonconjugated with a protein carrier. All the synthetic peptides were shown to induce the production of the antipeptide antibodies in mice. A peptide capable of inducing a decrease in the number of bacteria in blood and the protection of infected animals from death was found in the experiments on the protection of the animals infected with two strains of the Neisseria meningitidis serogroup B. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2002, vol. 28, no. 4; see also http://www.maik.ru.     

Whole-genome organization and functional properties of miniature DNA insertion sequences conserved in pathogenic Neisseriae.

The chromosome of pathogenic Neisseriae is peppered by members of an abundant family of small DNA sequences known as Correia elements. These DNA repeats, that we call nemis (for neisseria miniature insertion sequences) can be sorted into two major size classes. Both unit-length (154-158 bp) and internally rearranged (104-108 bp) elements feature long terminal inverted repeats (TIRs), and can potentially fold into robust stem-loop structures. Nemis are (or have been) mobile DNA sequences which generate a specific 2-bp target site duplication upon insertion, and strictly recall RUP, a repeated DNA element found in Streptococcus pneumoniae. The subfamilies of 26L/26R, 26L/27R, 27L/27R and 27L/26R elements, found by wide-genome computer surveys in both the Neisseria meningitidis and the Neisseria gonorrhoeae genomes, originate from the combination of TIRs which vary in length (26-27 bp) as in sequence content (L and R types). In both species, the predominant subfamily is made by the 26L/26R elements. The number of nemis is comparable in the N. meningitidis Z2491 (A serogroup) and the MC58 (B serogroup) strains, but is sharply reduced in the N. gonorrhoeae strain F1090. Consequently, several genes which are conserved in the two pathogens are flanked by nemis DNA in the meningococcus genome only. More than 2/3 of nemis are interspersed with single-copy DNA, and are found at close distance from cellular genes. Both primer extension and RNase protection data lend support to the notion that nemis are cotranscribed with cellular genes and subsequently processed, at either one or both TIRs, by a specific endoribonuclease, which plausibly corresponds to RNase III.     

Identification of two major families of transferrin receptors among Neisseria meningitidis strains based on antigenic and genomic features

Abstract The transferrin receptor or transferrin-binding proteins (Tbps) of 50 strains of Neisseria meningitidis belonging to different serogroups were examined by Western blotting using two rabbit antisera raised against Tbp purified from N. meningitidis strains B16B6 and M982. On the basis of the reactivity of Tbp2 with the antisera two patterns were observed and allowed the classification of 74% of the strains in group I (M982-like strains) and 26% in group II (B16B6-like strains). Southern blot analysis was performed on the genomic DNA of 16 meningococcal strains and showed that under stringent conditions, the tbp2 probes were specific for each group identified. Both immunological and genomic analyses have led to the identification within N. meningitidis strains of two major families distinguished on the basis of the characteristics of Tbp2 molecules, independently of serogroup, type or subtype.     

Conserved virulence of C to B capsule switched Neisseria meningitidis clinical isolates belonging to ET-37/ST-11 clonal complex

Capsule switching in Neisseria meningitidis is thought to occur by horizontal DNA exchange between meningococcal strains. Antigenic variants may be generated by allelic replacement of the siaD gene; the variants may then be selected by specific immunity against the capsular antigen. There were several vaccination campaigns against serogroup C in France in 2002, following an increase in the prevalence of invasive isolates of serogroup C of the phenotype C:2a:P1.5 and C:2a:P1.5,2 belonging to the ET-37/ST-11 clonal complex. We evaluated the emergence of capsule variants by the detection of B:2a:P1.5 and B:2a:P1.5,2 meningococcal isolates of the ET-37/ST-11 clonal complex. These isolates were significantly more frequent after the year 2002. Pulsed field gel electrophoresis profiles of the serogroup B (ET-37/ST-11) isolates differed from that of serogroup C (ET-37/ST-11) isolates by the bands that harbor the siaD genes responsible for the serogroup specificity. However, serogroup B and C, ET37/ST-11 isolates both express similar virulence as assessed from colonization and invasiveness in a mouse model. Our results indicate that capsule switching events within the same clonal complex can arise frequently with no alteration in virulence. This justifies an enhanced system of surveillance by molecular typing of such isolates, particularly after serogroup-specific vaccination.     

Characterization of serogroup C meningococci isolated from 14 provinces of China during 1966-2005 using comparative genomic hybridization

Neisseria meningitidis is a major cause of bacterial meningitis and septicemia worldwide. In China, serogroup A strains were responsible for over 95% of the cases, while serogroup B strains were mainly the cause of localized outbreaks and sporadic cases. Before 2003, serogroup C strains were only recovered from a few sporadic cases. However, a sudden increase in the number of cases due to serogroup C strains occurred during 2003-2005 in Anhui Province, China. Many cases were found in other provinces at the same time. Multilocus sequence typing (MLST) results indicated that the unique sequence type 4821 clone meningococci, a new hyper-virulent lineage, was responsible for the serogroup C meningitis outbreaks. We have completed the project of sequencing the whole genome of the Chinese N. meningitidis serogroup C representative isolate 053442. We fabricated a whole-genome microarray of N. meningitidis isolate 053442 and analyzed the genome composition differences among 81 serogroup C isolates which were isolated from 14 provinces of China during 1966-2005. The comparative genomic hybridization (CGH) result shows that the genome compositions of nearly all serogroup C isolates are similar to that of 053442. The products of many absent open reading frames (ORFs) are conserved hypothetical proteins. The results will provide a valuable resource from which one can analyze the genome composition and genetic background of serogroup C meningococci in China.     

Immunochemical studies and genetic background of two Neisseria meningitidis isolates expressing unusual capsule polysaccharide antigens with specificities of both serogroup Y and W135

We described 2 unusual Neisseria meningitidis strains isolated from epidemiologically unrelated invasive meningococcal disease cases in Ontario, Canada. Both isolates have features typical of serogroup Y N. meningitidis: are of serotype 2c, are of the multi-locus sequence types typical of the serogroup Y strains in Canada, and are genotyped as serogroup Y based on a previously described PCR-ELISA method that detects the serogroup-Y-specific siaD gene. However, both strains were poly-agglutinable in both anti-Y and anti-W135 antisera. Further studies on 1 of these 2 isolates showed the presence of glucose and galactose as well as sialic acids in its purified capsular polysaccharide, suggesting the presence of both serogroup Y and serogroup W135 polysaccharides. Rabbit antisera produced to this strain contained antibodies to both purified serogroup Y and serogroup W135 capsular polysaccharides. Absorption experiments with either serogroup Y or serogroup W135 bacteria confirmed the presence of antibodies to these 2 different polysaccharides. DNA sequencing of the cps operon from both isolates revealed a siaD gene with 99.7% homology to the published siaD sequence from a serogroup Y strain but with 3 point mutations that all resulted in amino acid changes. How these strains may affect results of routine surveillance, PCR diagnosis, and immuno-protection by vaccination are discussed.     

Neisseria meningitidis serogroup B lipooligosaccharide genotyping reveals high prevalence of L2 strains in Spain and unexpected relationship with factor H-binding protein expression

Neisseria meningitidis may be classified according to the lipooligosaccharide immunotype. We show that this classification can be achieved by PCR genotyping of the genes involved in the lipooligosaccharide inner-core biosynthesis, lpt3, lpt6, lgtG and lot3. Genotyping data correlated well (90-100%) with mass spectrometry data and was, therefore, applied to screen a random subset of recent N. meningitidis serogroup B isolates from Europe. Analysis of the proportion of the different lipooligosaccharide types highlighted the predominance of L3 strains. Surprisingly, high rates of L2 type strains were found in Spain (17%, versus 2.5% in Germany and 1.9% in the United Kingdom). Therefore, we also investigated further these Spanish L2 strains in an attempt to explain such prevalence despite the known sensitivity of L2 immunotype to complement. We explored the hypothesis that these strains express high amounts of factor H-binding protein (fHbp), but we found, on the contrary, that L2 strains express low or undetectable amounts of fHbp. Our findings suggest that, in addition to a genetic analysis, a multivalent approach may be necessary to estimate the effectiveness of a N. meningitidis serogroup B vaccine.     

Inventory of the proteins in Neisseria meningitidis serogroup B strain MC58

A protein inventory of Neisseria meningitidis strain MC58, a meningococcal strain belonging to the serogroup B, was performed by proteomics. A differential extraction procedure was employed and 238 protein species were identified by 2D mini-maps and MALDI-ToF analyses. In this catalog, we detected protein products from 33 genes, which were not yet annotated in previous N. meningitidis proteomic studies. This approach is suitable for high-throughput studies on differential expression of N. meningitidis genomes.     

Generation of capsule-deficient Neisseria meningitidis strains by homologous recombination

Capsule-deficient mutants of Neisseria meningitidis serogroup B strain B1940 were constructed by allelic replacement using the plasmids pMF120 and pMF121, which contain the flanking regions of the gene locus for the biosynthesis pathway of the group B meningococcal capsular polysaccharide. Southern blot analysis of chromosomal DNA of the capsule-deficient meningococcal strains confirmed the generation of large deletions in the chromosomal cps gene complex. The same strategy proved useful in constructing meningococcal strains with capsular types A, C, W135, Y and Z.     

Toxicity of an algal mucopolysaccharide for Escherichia coli and Neisseria meningitidis strains

Several bacterial strains of clinical significance have been tested to assess the toxic effect of a lectin-like algal mucopolysaccharide from Fucus vesiculosus on their growth. The toxic effect of the mucopolysaccharide has been found to be exerted only on Escherichia coli and Neisseria meningitidis strains. The degree of toxicity, measured by the effect on the growth of the bacteria, is variable depending on the strains of E. coli tested, whereas with N. meningitidis the results obtained indicate homogeneity between the strains, without significative variations among different serotypes even in the same serogroup.     

Transferrin-binding protein B of Neisseria meningitidis: sequence-based identification of the transferrin-Binding site confirmed by site-directed mutagenesis

A sequence-based prediction method was employed to identify three ligand-binding domains in transferrin-binding protein B (TbpB) of Neisseria meningitidis strain B16B6. Site-directed mutagenesis of residues located in these domains has led to the identification of two domains, amino acids 53 to 57 and 240 to 245, which are involved in binding to human transferrin (htf). These two domains are conserved in an alignment of different TbpB sequences from N. meningitidis and Neisseria gonorrhoeae, indicating a general functional role of the domains. Western blot analysis and BIAcore and isothermal titration calorimetry experiments demonstrated that site-directed mutations in both binding domains led to a decrease or abolition of htf binding. Analysis of mutated proteins by circular dichroism did not provide any evidence for structural alterations due to the amino acid replacements. The TbpB mutant R243N was devoid of any htf-binding activity, and antibodies elicited by the mutant showed strong bactericidal activity against the homologous strain, as well as against several heterologous tbpB isotype I strains.     

Genetic isolation of meningococci of the electrophoretic type 37 complex

Neisseria meningitidis (the meningococcus) is a naturally competent bacterial species in which intra- and interspecific horizontal gene transfer is a major source of genetic diversity. In strains of the electrophoretic type 37 (ET-37) complex and of the A4 cluster, we identified genomic DNA coding for a novel restriction-modification system and for the tail of a previously unidentified prophage. Furthermore, a novel 7.2-kb DNA segment restricted to clones of the ET-37 complex and the A4 cluster was isolated and shown to occur both as a plasmid (pJS-B) and as a chromosomal integration. Neither the genomic loci nor pJS-B was present in ET-5 complex, lineage 3, or serogroup A meningococci. The differential distribution of the DNA segments described herein, as well as of opcA, porB, nmeAI, nmeBI, and nmeDI described previously, supports the concept of genetic isolation of hypervirulent lineages responsible for most cases of serogroup C disease worldwide.     

Characterization of Neisseria meningitidis strains isolated from carriers

Neisseria meningitidis carriers strains were isolated from 17-19 teenagers (n = 14) and recruits (n = 267). The longitudinal study comprises three meningococcal carriage trials performed on healthy young men during two--six months of their service in Polish military units. Altogether 54, 124 and 89 meningococcal strains were obtained during spring 1998 and autumn 1998, 1999 trials. Sixty two percent of meningococcal carrier strains were non-groupable, however among the remaining strains, serogroup B was predominant (29.5%). During spring 1998 and autumn 1999 trials the predominant phenotypes were N. meningitidis NG:21:P1.7, but during the autumn 1998 NG:21:P1.7 or NG:NT:P1.5. Ribotyping of type 21 and/or subtype P1.7 strains (n = 27) showed presence of 2 main ribotypes. Pulsed Field Gel Electrophoresis of consecutive isolates recovered from the same carrier showed great similarity of the patterns.