A clonal analysis of Neisseria meningitidis serogroup A

A typing scheme has recently been developed for Neisseria meningitidis serogroup A based on the clonal population structure of these bacteria. An international strain collection consisting of 423 group A strains isolated from 23 epidemics or outbreaks since 1963, as well as from older epidemics and numerous non-epidemic situations was used in the analysis. Strains were first segregated into electrophoretic types, depending on the combined score for the electrophoretic mobilities of 7 cytoplasmic isoenzymes resolved by starch gel electrophoresis and of 2 outer membrane proteins resolved by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The bacteria were subsequently assigned to one of 21 clones after numerical analysis of their electrophoretic types.The epidemiological value of the typing scheme was assessed by examining case and carrier strains isolated during (1982–83) and subsequent to (1984–85) an epidemic in the Gambia, West Africa. The case isolates, all of which were serogroup A, were of a single clonal type. All serogroup A carrier isolates were also of this clone, while carrier strains of other serogroups showed greater clonal diversity. These results indicate that case strains during an epidemic show little clonal diversity and thus that the typing scheme is of value in distinguishing the etiology of epidemics.A retrospective epidemiological analysis of the strains in the international collection showed that most serogroup A epidemics were associated with a single or predominant clone, although some epidemics were of mixed etiology. The survey included 256 isolates from 15 African epidemics since 1963, a period which covers 3 major epidemic waves (1960–63; 1967–73 and 1981–83), thus permitting a detailed epidemiological analysis of serogroup A epidemics in this continent.Epidemiological records indicate that seven clones have been responsible for sets of epidemics throughout the world since 1915 and that at least two of these sets can be considered to represent mutually exclusive pandemics, linking numerous epidemics between 1967–75 and 1973–83, respectively.     

Mapping phosphoproteins in Neisseria meningitidis serogroup A

To investigate the phosphorylation capability of serogroup A Neisseria meningitidis (MenA) and to implement our knowledge in meningococcal biology and in bacterial post-translational modifications, cell extracts were separated by 2-DE and 51 novel phosphoproteins were revealed by the use of the highly specific Ser/Thr/Tyr-phosphorylated proteins staining by Pro-Q Diamond and identified by MALDI-ToF/MS. Our results indicate that phosphorylation in MenA is comparable to that of other bacterial species. A first functional characterization of the identified modified proteins was also given, in order to understand their role in meningococcal physiopathology.     

Molecular epidemiology of Neisseria meningitidis serogroup B in Brazil

Background

Neisseria meningitidis serogroup B has been predominant in Brazil, but no broadly effective vaccine is available to prevent endemic meningococcal disease. To understand genetic diversity among serogroup B strains in Brazil, we selected a nationally representative sample of clinical disease isolates from 2004, and a temporally representative sample for the state of São Paulo (1988–2006) for study (n = 372).

Methods

We performed multi-locus sequence typing (MLST) and sequence analysis of five outer membrane protein (OMP) genes, including novel vaccine targets fHbp and nadA.

Results

In 2004, strain B:4:P1.15,19 clonal complex ST-32/ET-5 (cc32) predominated throughout Brazil; regional variation in MLST sequence type (ST), fetA, and porB was significant but diversity was limited for nadA and fHbp. Between 1988 and 1996, the São Paulo isolates shifted from clonal complex ST-41/44/Lineage 3 (cc41/44) to cc32. OMP variation was associated with but not predicted by cc or ST. Overall, fHbp variant 1/subfamily B was present in 80% of isolates and showed little diversity. The majority of nadA were similar to reference allele 1.

Conclusions

A predominant serogroup B lineage has circulated in Brazil for over a decade with significant regional and temporal diversity in ST, fetA, and porB, but not in nadA and fHbp.     

Neisseria meningitidis serogroup B: laboratory correlates of protection

Meningococcal disease in the Western countries is frequently caused by Neisseria meningitidis serogroup B. Major efforts have been made to develop a safe and efficacious vaccine against this serogroup which is suitable for use in infants and young children. To assess the quality of the immune response after vaccination with candidate vaccines, laboratory correlates of protection are needed. For serogroups A and C, serum bactericidal activity (SBA) is a well established predictor for protection, but for serogroup B other mechanisms besides SBA may also be involved in conferring protection from disease. Several laboratory methods for identification and evaluation of the immunogenicity of possible vaccine antigens are described in this review.     

Cultivation of Neisseria meningitidis serogroup A on semisynthetic liquid nutrient media]

In cultivation of meningococcus of serological group A in fluid semisynthetic medium of simple composition prepared on the basis of purified acid casein hydrolysate with profound splitting there were obtained microbial cultures with a density of 4-5 x 10(9) microbial cells per 1 ml after 20-24 hours of cultivation with shaking. Alkalinity of the medium increased (to pH 8.0-8.2 during the stationary phase) with increase of the microbial cell concentration. A study of the accumulation of group-specific thermostable polysaccharide antigen in dynamics of meningococcus cultivation on semisynthetic medium tested showed the preparations obtained by alcoholic precipitation to be colourless and to contain much antigen (by inhibition of indirect hemagglutination), particularly at the phasees of negative growth acceleration and at the stationary phase. The suggested fluid semisynthetic medium of simple composition could be used for production of diagnostic and prophylactic meningococcus preparations belonging to the serological group A.     

Whole-genome sequence of the transformable Neisseria meningitidis serogroup A strain WUE2594

Serogroup A meningococci are a leading cause of bacterial meningitis in children and young adults worldwide. However, the genetic basis of serogroup A strains' virulence and their epidemiological properties remain poorly understood. Therefore, we sequenced the complete genome of the transformable Neisseria meningitidis serogroup A strain WUE2594.     

Structure of the capsular antigen of Neisseria meningitidis serogroup H

The capsular polysaccharide of Neisseria meningitidis serogroup H is composed of the following repeating unit, Gro = glycerol; (formula; see text) Partial O-acetylation of the D-Galp moieties is found for C-2 (21%) and C-3 (57%). The structural elucidation of the biopolymer is based on sugar analysis, methylation analysis, partial acid hydrolysis, using gas-liquid chromatography/mass spectrometry studies, and NMR spectroscopy with 1H, 13C and 31P.     

The Neisseria meningitidis serogroup A capsular polysaccharide O-3 and O-4 acetyltransferase

Neisseria meningitidis serogroup A capsular polysaccharide (CPS) is composed of a homopolymer of O-acetylated, alpha1-->6-linked ManNAc 1-phosphate that is distinct from the capsule structures of the other meningococcal disease-causing serogroups, B, C, Y, and W-135. The serogroup A capsule biosynthetic genetic cassette consists of four open reading frames, mynA-D (sacA-D), that are specific to serogroup A, but the functions of these genes have not been well characterized. mynC was found to encode an inner membrane-associated acetyltransferase that is responsible for the O-acetylation of the CPS of serogroup A. The wild-type CPS as revealed by 1H NMR had 60-70% O-acetylated ManNAc residues that contained acetyl groups at O-3, with some species acetylated at O-4 and at both O-3 and O-4. A non-polar mynC mutant generated by introducing an aphA-3 kanamycin resistance cassette produced CPS with no O-acetylation. A serogroup A capsule-specific monoclonal antibody was shown to recognize the wild-type O-acetylated CPS, but not the CPS of the mynC mutant, which lacked O-acetylation. MynC was C-terminally His-tagged and overexpressed in Escherichia coli to obtain the predicted approximately 26-kDa protein. The acetyltransferase activity of purified MynC was demonstrated in vitro using [14C]acetyl-CoA. MynC O-acetylated the O-acetylated CPS of the mynC mutant and further acetylated the wild-type CPS of serogroup A meningococci, but not the CPS of serogroup B or C meningococci. Genetic complementation of the mynC mutant confirmed the function of MynC as the serogroup A CPS O-3 and O-4 acetyltransferase. MynC represents a new subclass of O-acetyltransferases that utilize acetyl-CoA to decorate the D-mannosamine capsule of N. meningitidis serogroup A.     

Polysaccharide-protein complexes isolated from different strains of Neisseria meningitidis serogroup B

Fractionation of the biomass of 3 serogroup B N. meningitidis strains, obtained from solid serum-free and liquid synthetic media, by increasing concentrations of cetavlone revealed that the formation of natural polysaccharide-protein complexes with the ratio of their components approaching 1:1 was possible under the conditions ensuring the intensive synthesis of capsular polysaccharide. Two strains, 125 and 1642, grown on a solid amino peptide-containing medium regularly produced two polysaccharide-protein complexes with the protein/polysaccharide ratio approaching 1:1. One of these complexes passed easily into the supernatant fluid and could be precipitated with 0.1% cetavlone. The second complex was more firmly bound to the outer membrane of the cell and could be precipitated with 1% cetavlone. In most experiments an additional fraction with high protein content in relation to sialic acid was isolated from the biomass.     

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.     

Genome sequence of Neisseria meningitidis serogroup B strain H44/76

Neisseria meningitidis is an obligate human pathogen. While it is a frequent commensal of the upper respiratory tract, in some individuals the bacterium spreads to the bloodstream, causing meningitis and/or sepsis, which are serious conditions with high morbidity and mortality. Here we report the availability of the genome sequence of the widely used serogroup B laboratory strain H44/76.     

Physiology of sialic acid capsular polysaccharide synthesis in serogroup B Neisseria meningitidis

The pathway for biosynthesis of sialic acid capsular polysaccharide was examined in Neisseria meningitidis serogroup B strain M986 and in strain PRM102, an isogenic mutant defective in polysaccharide production. Strain PRM102 was found to possess only 25% of the level of sialyltransferase activity that was found in strain M986, but it had wild-type levels of both the N-acetylneuraminic acid (NANA) condensing enzyme and the CMP-NANA synthetase. A new meningococcal enzyme, a CMP-NANA hydrolase, was found in both meningococcal strains. This enzyme generated CMP and NANA from CMP-NANA, had a Km of 0.88 microM, had a Vmax of 10.75 nmol of NANA produced per h per mg of protein, and was completely inhibited by 45.3 microM CMP. The sialyltransferase, which also had CMP-NANA as substrate, was insensitive to CMP addition. Subcellular fractionation and purification of cytoplasmic and outer membranes on sucrose density gradients revealed that both the sialyltransferase and the CMP-NANA hydrolase were cytoplasmic membrane associated. The NANA condensing enzyme and the CMP-NANA synthetase were found to be cytosolic. A working hypothesis for the regulation of sialic acid polysaccharide synthesis was developed. The CMP-NANA hydrolase with its high affinity for CMP-NANA regulates polysaccharide formation by the sialyltransferase, whereas CMP, a product of both the sialyltransferase and the CMP-NANA hydrolase, modulates the activity of the hydrolase on the cytoplasmic membrane.     

Purification of capsular polysaccharide from Neisseria meningitidis serogroup C by liquid chromatography

Neisseria meningitidis serogroup C capsular polysaccharide (MenCPS) is an important antigen against meningococcal infection. This paper describes a new purification methodology employing liquid chromatography that resulted in a polysaccharide showing the characteristics recommended by the World Health Organization for vaccine purposes. In this method, steps of the traditional procedure that yield low recovery and use toxic materials were modified. The present process consists in the following steps: (1) continuous flow centrifugation of the culture for removal of the cells; (2) supernatant concentration by tangential filtration (100 kDa cutoff); (3) addition of 0.5% DOC, heating to 55 degrees C during 30 min and tangential filtration (100 kDa cutoff); (4) anion exchange chromatography (Source 15Q) and (5) size exclusion chromatography (Sepharose CL-4B). The polysaccharide C fraction obtained in that way was dialyzed and freeze-dried. The structural identity of the polysaccharide was demonstrated by (1)H-NMR spectrometry.     

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.     

The molecular characterization of serogroup C Neisseria meningitidis strains circulating in Beijing

The aim of this study was to characterize the molecular features of serogroup C Neisseria meningitidis strains circulating in Beijing, China. Twenty out of 23 strains belonged to ST 4821. The causative serosubtype for meningococcal meningitis was P1.12-1,16-8. All of the strains expressed class 3 PorB protein. Among the five pulsed-field gel electrophoresis patterns observed, pattern III predominated.     

Biofilm formation on human airway epithelia by encapsulated Neisseria meningitidis serogroup B

Neisseria meningitidis is the etiologic agent of meningococcal meningitis. We compared 48-h biofilm formation by N. meningitidis serogroup B strains NMB, MC58, C311 and isogenic mutants defective in capsule formation on SV-40 transformed human bronchial epithelial (HBE) cells in a flow cell. We demonstrated that strains NMB and NMB siaA-D were defective in biofilm formation over glass, and there was a partial rescue of biofilm growth for strain NMB on collagen-coated coverslips at 48 h. We demonstrated all three serogroup B strains form biofilms of statistically equivalent average height on HBE cells as their isogenic capsular mutants. Strain NMB also formed a biofilm of statistically equivalent biomass as the NMB siaA-D mutant on HBE cells at 6 and 48 h. These biofilms are significantly larger than biofilms formed over glass or collagen. Verification that strain NMB expressed capsule in biofilms on HBE cells was demonstrated by staining with 2.2.B, a monoclonal antibody with specificity for the serogroup B capsule. ELISA analysis demonstrated that strains MC58 and C311 also produced capsules during biofilm growth. These findings suggest that encapsulated meningococci can form biofilms on epithelial cells suggesting that biofilm formation may play a role in nasopharyngeal colonization.     

Nitric oxide participates in the immune response against Neisseria meningitidis serogroup B

The present report explores the role of nitric oxide into the immune response against Neisseria meningitidis serogroup B. Here we show that NO mediates the alphaTNF increase induced by N. meningitidis derived lipopolysaccharides (LPS), at the same time that participates in the bactericidal activity of resting or gammaIFN activated macrophages and plays a role in the specific DTH and IgG response induced by a commercial anti-meningococcal vaccine. Our findings suggest a positive role for NO at the final effector mechanisms and in the early events driving the immunity against N. meningitidis, suggesting also an insight into its role in endotoxic shock.