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.     

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.     

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.     

Multivariate analysis of Neisseria DNA restriction endonuclease patterns

Chromosomal DNA was extracted from eleven Neisseria meningitidis and seven Neisseria gonorrhoeae isolates and cleaved with the restriction enzyme HindIII. The DNA fragments were separated according to their size, using a 4% polyacrylamide gel. The band patterns obtained were digitized and statistically analysed by the SIMCA method. To develop the models for N. meningitidis (class 1) and N. gonorrhoeae (class 2), all eleven meningococci and seven gonococci, were used. All strains were classified correctly and showed an extremely good class separation.     

Improved antiserum agar method for the serogroup differentiation of Neisseria meningitidis Y and W135

Rabbit antisera prepared to meningococcal serogroups Y and W135 strains were compared with horse antisera using the antiserum agar method (ASA) for the serogroup identification of Neisseria meningitidis. Thirty-seven group Y strains formed immunoprecipitates with the Y rabbit serum only, whereas the same Y strains formed immunoprecipitates with both the Y and W135 horse antisera. Forty-seven W135 strains formed specific immunoprecipitates with both the rabbit and horse W135 antisera by ASA. None of the 166 meningococcal isolates, representative of other meningococcal serogroups, formed immunoprecipitates with the groups Y and W135 rabbit or horse antisera. Use of specific Y and W135 rabbit antisera in ASA provides an improved technique for the serogroup differentiation of groups Y and W135 meningococci.     

Rapid molecular identification of Neisseria meningitidis isolates using the polymerase chain reaction followed by single-stranded conformation polymorphism analysis

Typing of Neisseria meningitidis strains is currently performed with conventional and molecular methods. Our objectives were: first, to develop a polymerase chain reaction (PCR) followed by single-stranded conformation polymorphism (SSCP) analysis of the PorA gene (VR1 region) to distinguish N. meningitidis subtypes and second, to evaluate the method for the identification and characterization of N. meningitidis in patient specimens. SSCP analysis of the VR1 region of the PorA1/2 gene from 126 N. meningitidis strains and 29 clinical samples identified seven SSCP types (SP-1 to SP-7); four strains were not typeable by the method. Classification according to the SSCP methods and serosubtype agreed for 122 of the 126 typeable strains (96.8%). For the 24-culture positive clinical samples, serosubtype and SSCP agreed in all cases. Five samples, which were culture-negative but obtained from children during an apparent outbreak of meningococcal disease in a primary school, presented identical SSCP classification for each sample (SP-2). PCR-SSCP is a rapid and cost-effective method for typing N. meningitidis strains that could provide important early information in the surveillance of suspected meningococcal outbreaks, particularly when culture-negative specimens constitutes the main source of material to analyze.     

Antigenic cross-reactivity between outer membrane proteins of Neisseria meningitidis and commensal Neisseria species

Two mouse sera against outer membrane proteins from a pathogenic Neisseria meningitidis strain and a commensal N. lactamica strain and two human sera from patients recovering from meningococcal meningitis were used to identify antigens common to pathogenic and commensal Neisseria species. Two major antigens of 55 kDa and 32 kDa, present in all N. meningitidis and N. lactamica strains tested, were demonstrable with all the sera used; the 55-kDa protein was iron-regulated. Demonstration of other common antigens was dependent on the serum used: a 65-kDa antigen was visualised with the human and the mouse anti-N. lactamica sera; a 37-kDa antigen identified as the meningococcal ferric binding protein (FbpA) was only detected with the mouse sera, and two antigens of 83 kDa and 15 kDa were only shown with the mouse anti-N. meningitidis serum. The results demonstrate the existence of several outer membrane antigens common to N. lactamica and N. meningitidis strains, in agreement with the hypothesis that natural immunity against meningitis is partially acquired through colonisation by commensal species, and open new perspectives for the design of vaccine formulations and the development of strategies for vaccination against meningitis.     

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.     

Identification of a 13-kDa protein associated with the polyhydroxyalkanoic acid granules from Acinetobacter spp.

Abstract Transferrin-binding proteins from Neisseria meningitidis vary among different isolates. We have identified and studied a hypervariable region adjacent to the carboxyl-end of the transferrin-binding domain of the Tbp2 molecule. The tbp2 genes from six strains of N. meningitidis were cloned and sequenced in this particular region. Sequence analysis of these regions along with five other sequences available from pathogenic Neisseria showed a common organisation of seven highly variable nucleotide stretches interspersed with six conserved nucleotide stretches. The variable regions correlated with the location of immunoreactive epitopes in polyclonal antisera raised to transferrin-binding proteins identified by peptide pin technology. Sequence analysis suggested a mosaic-like organisation of the tbp2 genes. Taken together, these data suggest that the antigenic variation in this part of the protein may result from a strong host immune pressure.     

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.     

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.     

Detection of the lst gene in different serogroups and LOS immunotypes of Neisseria meningitidis

The sialylation of the lipooligosaccharide (LOS) of Neisseria meningitidis is mediated by the LOS sialyltransferase enzyme encoded by the lst gene. PCR using four sets of primers that targeted to different regions of the lst gene was used to survey the distribution of lst in different serogroups and LOS immunotypes of N. meningitidis as well as other Neisseria species. While the lst gene was found in N. meningitidis strains regardless of their capsular serogroup and LOS structures, the gene is also found in N. gonorrhoeae, N. lactamica, N. polysaccharea, and N. subflava biovar subflava. The presence of the lst gene in these organisms and the sialylation of their LOS antigens were discussed.     

Characteristics of Neisseria meningitidis strains isolated from patients with symptoms of meningococcal meningitis in Poland in 1995-2000

Phenotype and genotype identification of 179 Neisseria meningitidis strains isolated from cerebrospinal fluid or blood of patients with meningococcal infection, hospitalized in Poland, was performed. This is the first analysis of that type conducted in Poland. We analyzed strains collected in 1995-2000 from laboratories located all over the country. Phenotype Neisseria meningitidis B:22:P1.14 was the predominant among analyzed invasive strains in Poland. Type 22 is characteristic to most of the strains isolated in our country. No strain from analyzed group belonged to known epidemic clusters. One penicillin resistant strain (MIC = 2 mg/l) and about 27% strains with decreased susceptibility to penicillin (0.1 = < MIC < 1.0 mg/l) were present among 166 N. meningitidis tested. All strains were susceptible to ciprofloxacin and rifampicin.     

Evaluation of the antiserum agar method for the serogroup identification of Neisseria meningitidis.

The antiserum agar method (ASA), which is based on the formation of immunoprecipitates around bacterial growth on agar containing meningococcal hyperimmune horse serum, was evaluated for serogroup identification of Neisseria meningitidis. Four hundred meningococcal stains were serogrouped by ASA employing horse antisera to serogroups A, B, C, Y, W135, Z, and 29E and compared to serogroup identification by bacterial slide agglutination (BA) employing rabbit antisera. Overall, there was 95% agreement between the two methods. The ASA proved to be more accurate than BA since 15 strains which cross-reacted with Y and W135 rabbit antisera by BA were specifically serogrouped as either Y or W135 by ASA. In addition, 5 out of 75 strains which were ungroupable by BA were serogrouped as either B or 29E by ASA. Repeat serogroup identification of 100 meningococcal strains by ASA provided identical results thus showing the reproducibility of the method. The ASA is advantageous to BA since it is more reliable, utilizes standard antisera which do not have to be absorbed to remove cross-reactions, does not require the preparation of standardized bacterial antigen, and is simple to perform.     

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.     

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.     

Iron and outer membrane proteins in the susceptibility of Neisseria meningitidis to human serum

The proportion of carrier-isolated Neisseria meningitidis strains sensitive to human serum (37.2%) was found to be significantly higher than that of case-isolated ones (4.1%), although the difference is too low to consider serum-resistance responsible for invasion in this microorganism. Serum-susceptibility was not related to the existence of specific outer membrane proteins, as is the case of N. gonorrhoeae. Iron restriction induced iron-regulated outer membrane proteins in each strain (but not the same proteins in all strains) but without any detectable effect on serum-susceptibility. Iron excess was also unable to induce changes in the susceptibility of N. meningitidis to human serum.     

Enzyme-linked immunosorbent assays for the detection of Neisseria gonorrhoeae specific antibodies.

An indirect enzyme-linked immunosorbent assay (ELISA) using rigid polystyrene microtiter plates was adapted to detect specific gonococcal antibodies against outer membrane-complex antigens extracted from Neisseria gonorrhoeae. The concentration of antigen to obtain maximum coating of the well was 10 micrograms protein per millilitre. The optimal binding of the primary antibody and enzyme-conjugated antimmunoglobulin was achieved after 1 h at 37 degrees C. Under these conditions using gonococcal antisera, no cross-reactivity was observed with outer membrane antigens extracted from Neisseria meningitidis serogroups B, C, X, Y, and W135. Neisseria meningitidis serogroup A demonstrated low levels of cross-reactivity. All the non-pathogenic Neisseria spp. tested were negative (absorbance value at 400 nm/30 min less than 0.15). The reaction of immune serum against outer membrane complex absorbed to the microwells was completely inhibited with soluble-specific antigen but not with purified N. gonorrhoeae lipopolysaccharide. Quantitative inhibition permitted the measurement of low levels of antigen (0.5 microgram/ml). The detection of N. gonorrhoeae antibody with ELISA is specific and highly sensitive.     

The drug sensitivity of meningococci and the characteristics of its determination

The results obtained in the study of antibiotic and sulfamide sensitivity of 197 Neisseria meningitidis strains of groups A, B and C, isolated from the spinal fluid and blood of patients with meningococcal infection hospitalized in the 2nd Clinico-Infectious Hospital, Moscow, in 1984-1989 and studied with the use of the disc diffusion method and the method of serial dilutions of antibiotics in solid culture media, are presented. As revealed in this study, N. meningitidis strains retained their high sensitivity to penicillin and ampicillin (MIC50 = 0.016 and 0.032 micrograms/ml respectively). Sensitivity to tetracycline decreased (MIC50 = 0.5 micrograms/ml) and to rifampicin increased (MIC50 = 0.063 micrograms/ml). 48.5% of strains were resistant to streptomycin. In recent years the proportion of N. meningitidis, resistant to sulfanilamide preparations, significantly decreased and MIC50 was equal to 2.5 micrograms/ml in comparison with 5-10 micrograms/ml in the preceding period. The results of testing sensitivity to antibiotics by both methods coincided. Still the disc diffusion method can be used in epidemiological surveillance on meningococcal infection, while for more exact differentiation of N. meningitidis strains the use of the method of serial dilutions is necessary.     

Transformational exchanges in the dihydropteroate synthase gene of Neisseria meningitidis: a novel mechanism for acquisition of sulfonamide resistance.

The nucleotide sequences of the chromosomal dihydropteroate synthase (dhps) genes in sulfonamide-susceptible and sulfonamide-resistant strains of Neisseria meningitidis of serogroups A, B and C were determined. The molecular weights and the amino acid sequences showed similarity to those of all other known dihydropteroate synthase polypeptides. Sequence comparison of the N. meningitidis dhps genes indicated horizontal transfer of DNA segments rather than point mutations as the cause for resistance in meningococci. The dhps genes in three of four sulfonamide-resistant meningococci contained identical central regions of 424 bp. Compared with the corresponding genes in susceptible strains, each central region included an insert of 6 bp. In one of the sulfonamide-resistant strains, the dhps gene was similar to the corresponding genes in the sensitive strains in its NH2-terminal and C-terminal parts. Its central region, however, was identical to the corresponding regions of two of the other resistant genes, and thus it could be seen as a hybrid dhps gene. Transformation experiments and mapping of transformed dhps genes indicated the existence of a novel mechanism for the dissemination of sulfonamide resistance in N. meningitidis. The origin of the resistance-mediating segment of the gene is unknown, but hybridization results showed the presence of homologous dhps genes in Neisseria gonorrhoeae and N. lactamica but not in N. subflava or Branhamella catarrhalis.