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The site and mechanisms by which meningococci gain access to the CNS are unclear. In this study we determined whether production of nitric oxide (NO) is part of the host (endothelial cell) response to meningococcal cell lysate, and the consequences for endothelial cell viability. Expression of NO synthase type II (NOS-2) mRNA, protein and enzyme activity were investigated in mouse cerebrovascular endothelial cells exposed to sonicated Neisseria meningitidis. The production of nitrite peaked after 48 h of incubation, and this reflected transcriptional activation of the NOS-2 gene and increased expression of the NOS-2 protein. This endothelial response was independent of meningococcal lipopolysaccharide production. Endothelial cell death occurred as a result of NO production, and addition of a NOS inhibitor prevented cell death, but the cells did not exhibit features of apoptosis. However, inhibition of poly (ADP-ribose) polymerase (PARP) decreased the rate of cell death by more than 40%. These data indicate that N. meningitidis increases expression of NOS-2 in endothelial cells and causes cell death. Such an effect could contribute to meningococcal entry into the CNS in situ. [ABSTRACT FROM AUTHOR]

Abstract Background Glyceraldehyde 3-phosphate dehydrogenases (GAPDHs) are cytoplasmic glycolytic enzymes, which although lacking identifiable secretion signals, have also been found localized to the surface of several bacteria (and some eukaryotic organisms); where in some cases they have been shown to contribute to the colonization and invasion of host tissues. Neisseria meningitidis is an obligate human nasopharyngeal commensal which can cause life-threatening infections including septicaemia and meningitis. N. meningitidis has two genes, gapA-1 and gapA-2, encoding GAPDH enzymes. GapA-1 has previously been shown to be up-regulated on bacterial contact with host epithelial cells and is accessible to antibodies on the surface of capsule-permeabilized meningococcal cells. The aims of this study were: 1) to determine whether GapA-1 was expressed across different strains of N. meningitidis; 2) to determine whether GapA-1 surface accessibility to antibodies was dependant on the presence of capsule; 3) to determine whether GapA-1 can influence the interaction of meningococci and host cells, particularly in the key stages of adhesion and invasion. Results In this study, expression of GapA-1 was shown to be well conserved across diverse isolates of Neisseria species. Flow cytometry confirmed that GapA-1 could be detected on the cell surface, but only in a siaD-knockout (capsule-deficient) background, suggesting that GapA-1 is inaccessible to antibody in in vitro-grown encapsulated meningococci. The role of GapA-1 in meningococcal pathogenesis was addressed by mutational analysis and functional complementation. Loss of GapA-1 did not affect the growth of the bacterium in vitro. However, a GapA-1 deficient mutant showed a significant reduction in adhesion to human epithelial and endothelial cells compared to the wild-type and complemented mutant. A similar reduction in adhesion levels was also apparent between a siaD-deficient meningococcal strain and an isogenic siaD gapA-1 double mutant. Conclusions Our data demonstrates that meningococcal GapA-1 is a constitutively-expressed, highly-conserved surface-exposed protein which is antibody-accessible only in the absence of capsule. Mutation of GapA-1 does not affect the in vitro growth rate of N. meningitidis, but significantly affects the ability of the organism to adhere to human epithelial and endothelial cells in a capsule-independent process suggesting a role in the pathogenesis of meningococcal infection.

We have investigated the response of primary human meningothelial cells to Neisseria meningitidis. Through a transcriptome analysis, we provide a comprehensive examination of the response of meningothelial cells to bacterial infection. A wide range of chemokines are elicited which act to attract and activate the main players of innate and adaptive immunity. We showed that meningothelial cells expressed a high level of Toll-like receptor 4 (TLR4), and, using a gene silencing strategy, we demonstrated the contribution of this pathogen recognition receptor in meningothelial cell activation. Secretion of interleukin-6 (IL-6), CXCL10, and CCL5 was almost exclusively TLR4 dependent and relied on MyD88 and TRIF adaptor cooperation. In contrast, IL-8 induction was independent of the presence of TLR4, MyD88, and TRIF. Transcription factors NF-κB p65, p38 mitogen-activated protein kinase (MAPK), Jun N-terminal protein kinase (JNK1), IRF3, and IRF7 were activated after contact with bacteria. Interestingly, the protein kinase IRAK4 was found to play a minor role in the meningothelial cell response to Neisseria infection. Our work highlights the role of meningothelial cells in the development of an immune response and inflammation in the central nervous system (CNS) in response to meningococcal infection. It also sheds light on the complexity of intracellular signaling after TLR triggering.

Background: Herd immunity is important in the effectiveness of conjugate polysaccharide vaccines against encapsulated bacteria. A large multicenter study investigated the effect of meningococcal serogroup C conjugate vaccine introduction on the meningococcal population., Methods: Carried meningococci in individuals aged 15-19 years attending education establishments were investigated before and for 2 years after vaccine introduction. Isolates were characterized by multilocus sequence typing, serogroup, and capsular region genotype and changes in phenotypes and genotypes assessed., Results: A total of 8462 meningococci were isolated from 47 765 participants (17.7%). Serogroup prevalence was similar over the 3 years, except for decreases of 80% for serogroup C and 40% for serogroup 29E. Clonal complexes were associated with particular serogroups and their relative proportions fluctuated, with 12 statistically significant changes (6 up, 6 down). The reduction of ST-11 complex serogroup C meningococci was probably due to vaccine introduction. Reasons for a decrease in serogroup 29E ST-254 meningococci (from 1.8% to 0.7%) and an increase in serogroup B ST-213 complex meningococci (from 6.7% to 10.6%) were less clear., Conclusions: Natural fluctuations in carried meningococcal genotypes and phenotypes a can be affected by the use of conjugate vaccines, and not all of these changes are anticipatable in advance of vaccine introduction.

Gram-negative bacteria possess an outer membrane layer which constrains uptake and secretion of solutes and polypeptides. To overcome this barrier, bacteria have developed several systems for protein secretion. The type V secretion pathway encompasses the autotransporter proteins, the two-partner secretion system, and the recently described type Vc or AT-2 family of proteins. Since its discovery in the late 1980s, this family of secreted proteins has expanded continuously, due largely to the advent of the genomic age, to become the largest group of secreted proteins in gram-negative bacteria. Several of these proteins play essential roles in the pathogenesis of bacterial infections and have been characterized in detail, demonstrating a diverse array of function including the ability to condense host cell actin and to modulate apoptosis. However, most of the autotransporter proteins remain to be characterized. In light of new discoveries and controversies in this research field, this review considers the autotransporter secretion process in the context of the more general field of bacterial protein translocation and exoprotein function.

Objective: To determine whether mastic gum suppresses or eradicates Helicobacter pylori infection in humans., Patients and Methods: Nine patients with H. pylori infection, and without gastroduodenal ulceration, were recruited from day-case endoscopy lists and treated with mastic 1 g four times daily for 14 days. [13C]Urea breath tests (UBTs) were carried out immediately before, on day 15 and 5 weeks after treatment with mastic., Results: Mastic had no effect on H. pylori status in any of the eight completed patients; all remained H. pylori positive by UBT with no change in delta scores [pre-treatment mean +/- s.e.m. 19.1 +/- 3.7, day 15 (post-treatment) 18.7 +/- 3.8, P = 0.8, paired t-test]., Conclusion: Despite reported anti-H. pylori action in vitro, this preliminary study shows that mastic has no effect on H. pylori in humans.

In a search for immunogenic virulence factors in Neisseria meningitidis, we have identified a gene encoding a predicted 160 kDa protein with homology to the autotransporter family of proteins. Members of this family are secreted or surface exposed and are often associated with virulence in Gram-negative bacterial pathogens. We named the gene adhesion and penetration protein (app), because of its extensive homology to the hap gene of Haemophilus influenzae. We reconstructed the gene with reference to genomic sequence data and cloned and expressed the protein in Escherichia coli. Rabbit antiserum raised against recombinant App reacted with proteins in all meningococcal isolates examined, which represented clonal groups responsible for the majority of meningococcal invasive disease. Antibodies to the protein were detected in the sera of patients convalescing from meningococcal infection. Purified App had strong stimulating activity for T cells isolated from a number of healthy donors and from one convalescent patient. We confirmed that App is surface localized, cleaved and secreted by N. meningitidis. Importantly, the rabbit anti-App serum killed the organism in the presence of complement. Thus, App is conserved among meningococci, immunogenic in humans and potentially involved in virulence. It therefore merits further investigation as a component of a future multivalent vaccine.

Fluorescent amplified-fragment length polymorphism (FAFLP), a genotyping technique with phylogenetic significance, was applied to 123 isolates of Neisseria meningitidis. Nine of these were from an outbreak in a British university; 9 were from a recent outbreak in Pontypridd, Glamorgan; 15 were from sporadic cases of meningococcal disease; 26 were from the National Collection of Type Cultures; 58 were carrier isolates from Ironville, Derbyshire; 1 was a disease isolate from Ironville; and five were representatives of invasive clones of N. meningitidis. FAFLP analysis results were compared with previously published multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) results. FAFLP was able to identify hypervirulent, hyperendemic lineages (invasive clones) of N. meningitidis as well as did MLST. PFGE did not discriminate between two strains from the outbreak that were classified as similar but distinct by FAFLP. The results suggest that high resolution of N. meningitidis for outbreak and other epidemiological analyses is more cost efficient by FAFLP than by sequencing procedures.

A meningococcal genomic expression library was screened for potent CD4+ T-cell antigens, using patients' peripheral blood lymphocytes (PBLs). One of the most promising positive clones was fully characterized. The recombinant meningococcal DNA contained a single, incomplete, open reading frame (ORF), which was fully reconstructed with reference to available genomic sequence data. The gene was designated autA (auto-transporter A) as its peptide sequence shares molecular characteristics of the auto-transporter family of proteins. Only a single copy of this gene was detected in the meningococcal, and none in the gonococcal, genomic sequence databases. The complete autA gene, when cloned into an expression vector, expressed a protein of approximately 68 kDa. Purified rAutA recalled strong secondary T-cell responses in PBLs of patients and some healthy donors, and induced strong primary T-cell responses in healthy donors. The human B-cell immunogenicity and cross-reactivity of AutA, purified under native conditions, was confirmed in dot immunoblot experiments. Immunoblots with rabbit polyclonal antibodies to rAutA demonstrated the conserved nature, antigenicity and cross-reactivity of AutA amongst meningococci of different serogroups and strains representing different hypervirulent lineages. AutA showed homology with another meningococcal and gonococcal ORF (designated AutB). AutB was cloned and expressed and used to raise an autB-specific antiserum. Immunoblot experiments indicated that AutB is not expressed in meningococci and does not cross-react with AutA. Thus, AutA, being a potent CD4+ T-cell and B-cell-stimulating antigen, which is highly conserved, deserves further investigation as a potential vaccine candidate.

Objective: To determine the rates of, and risk factors for, meningococcal carriage and acquisition among university students., Design: Repeated cross sectional study., Participants: 2,507 students in their first year at university., Main Outcome Measures: Prevalence of carriage of meningococci and risk factors for carriage and acquisition of meningococci., Results: Carriage rates for meningoccoci increased rapidly in the first week of term from 6.9% on day 1, to 11.2% on day 2, to 19.0% on day 3, and to 23.1% on day 4. The average carriage rate during the first week of term in October among students living in catered halls was 13.9%. By November this had risen to 31.0% and in December it had reached 34. 2%. Independent associations for acquisition of meningococci in the autumn term were frequency of visits to a hall bar (5-7 visits: odds ratio 2.7, 95% confidence interval 1.5 to 4.8), active smoking (1.6, 1.0 to 2.6), being male (1.6, 1.2 to 2.2), visits to night clubs (1. 3, 1.0 to 1.6), and intimate kissing (1.4, 1.0 to 1.8). Lower rates of acquisition were found in female only halls (0.5, 0.3 to 0.9). The most commonly acquired meningococcal strain was C2a P1.5 (P1.2), which has been implicated in clusters of invasive meningococcal disease at other UK universities., Conclusions: Carriage rates of meningococci among university students increase rapidly in the first week of term, with further increases during the term. The rapid rate of acquisition may explain the increased risk of invasive meningococcal disease and the timing of cases and outbreaks in university students.

Clinical microbiologists do not normally prescribe prophylactic antibiotics for travellers' diarrhoea, and when questioned at a Hospital Infection Society (HIS) meeting only 2% admitted doing so. During a HIS visit to India, 5 (28%) members took daily ciprofloxacin prophylaxis and none of them developed diarrhoea.

Neisseria meningitidis, when cultured in iron-restricted environments, synthesises new outer-membrane proteins, many of which are necessary for their survival and growth. Some of these proteins e.g. transferrin-binding proteins 1 and 2 (Tbp1 and Tbp2), are required for the acquisition of iron from transferrin and are examples of important iron-regulated meningococcal surface antigens which are not expressed after growth in common laboratory media. The antigenicity and antigenic heterogeneity of these proteins have been extensively studied, and the bactericidal activity of antibodies directed to them have been studied. In this work we analysed the ability of such antibodies to inhibit transferrin binding and to block iron uptake from human transferrin. Antisera from mice immunized with either meningococcal outer membrane vesicles, purified Tbp1/2 complexes, or purified Tbp2, were incorporated in radiolabeled-iron uptake assays. Uptake was blocked by more than 80% in the homologous strains, but blocked much less efficiently in some heterologous strains, correlating well with inhibition of transferrin binding and with an inhibitory effect on bacterial growth. Inhibition of iron uptake from citrate was unaffected which suggests that this effect is due to antibodies against the components of the transferrin binding system, specially Tbp2. Our results support the importance of these proteins and their suitability to be considered in the development of effective vaccines against serogroup B meningococci.

It has been reported that a 126 kDa protein of Listeria monocytogenes binds human transferrin. It is evident from results presented here that this is not so and that the observation of others resulted from interaction of this 126 kDa protein with streptavidin.

Neisseria meningitidis expresses several novel outer membrane proteins (OMPs) in vivo and when grown under iron limitation in vitro. One of the most prominent is a 70 kDa iron-regulated protein (FrpB). FrpB was purified by elution from SDS-polyacrylamide gels and rabbit polyclonal antiserum (R-70) was raised against it. R-70 was bactericidal against homologous, but not heterologous, strains in the presence of human complement. The bactericidal activity was retained when R-70 was adsorbed with formaldehyde-fixed iron-replete cells (i.e. not expressing FrpB), but lost when absorbed with fixed iron-restricted cells (which express FrpB). A murine monoclonal anti-FrpB antibody (mAb M70) was raised against a common epitope which showed complete cross-reaction on Western blots of OMPs from other serogroups and serotypes of N. meningitidis and some commensal Neisseriae species. However, it failed to kill the organism. Immunogold electron microscopy on ultrathin sections, using the R-70 antiserum adsorbed with fixed iron-replete cells, showed labelling on 40% of the cells, whereas the R-70 adsorbed with fixed iron-restricted cells and mAb M70 failed to label. However, none of these sera labelled whole cells, suggesting lack of surface accessibility. It appears that the highly conserved cross-reactive epitopes of FrpB only become exposed in the process of generating the antigen, whereas the surface-exposed epitopes recognized in killing assays are immunologically variable among different strains.(ABSTRACT TRUNCATED AT 250 WORDS)

A gene library of Neisseria meningitidis B15 P1.16 DNA was established in lambda Zap II and clones containing DNA encoding transferrin binding protein 1 (TBP-1) identified following hybridisation with a 63-bp DNA probe based on the codon assignment for the first 21 N-terminal amino acids of TBP-1. Sequencing of the cloned DNA demonstrated that all of the intergenic DNA (i.e. upstream of tbp-1 running through to the 3' end of the transferrin-binding protein 2 gene) and approx. 15% of tbp-1 had been cloned. The complete gene was generated using a polymerase chain reaction, with the primer for the 3' end being based on tbp-A of N. gonorrhoeae, and the approx. 2.9-kb DNA product cloned into pGem-3Z. The expressed protein (approx. 100 kDa) reacted with antiserum to an N-terminal peptide of TBP-1. In addition, the native product was surface-expressed by Escherichia coli and bound human transferrin.

The NH2-terminal amino sequence through the first 20 amino acids was obtained for transferrin-binding protein (TBP)1 from three strains of Neisseria meningitidis. These were identical except for a glutamine to a glycine substitution at residue 6 in one case. The sequences of the NH2-terminal 20 amino acids of TBP2 from the same three strains were also determined; one TBP2 had a M(r) of 68,000 and the other two of 78,000. Sequences were identical up to residue 13 in all three proteins. Peptides based on the NH2-terminal sequences of TBP1 and 2 were synthesized, linked to Keyhole Limpet haemocyanin and used to raise antibodies in rabbits. Anti-peptide antibodies cross-reacted on immunoblotting with the respective TBPs from all meningococcal strains tested, as well as with those from N. gonorrhoeae, suggesting that the NH2-terminals of these proteins are well conserved in the Neisseria. Neither anti-peptide serum reacted with the analogous TBP1 and 2 from Haemophilus influenzae, although common epitopes have previously been shown to exist.

The genes encoding transferrin-binding proteins (TBPs) 1 and 2 of Neisseria meningitidis and N. gonorrhoeae were used as model loci in a novel method of cloning (twin N-terminal polymerase chain reaction; TNT-PCR) involving amplification between the 5' ends of two genes. Primers were based on N-terminal amino-acid sequences. A 2.1-kb product amplified from N. meningitidis strain SD (B15 P1.16) was cloned into a plasmid vector and partially sequenced. Translated sequence immediately downstream of the primer at both ends of this product correlated to the additional known N-terminal amino acids of TBP-1 and 2. The protein encoded by the cloned sequence reacted with TBP-2-specific antiserum. The size of products generated in TNT-PCR correlated exactly with the different sized TBP-2 produced by 10 strains of the Neisseria spp. examined, indicating successful cloning of the gene for TBP-2 and showing it to be adjacent to and preceding TBP-1 on the chromosome for both N. meningitidis and N. gonorrhoeae.

Neisseria meningitidis is able to chelate iron from human transferrin (HTF), the main sequestrator of extracellular iron in vivo. Previous workers have reported that a ca. 70 kilodalton (kDa) iron regulated outer membrane protein (FeRP-70) is a highly specific receptor for HTF. We have examined the interaction between the iron regulated outer membrane proteins (OMP's) and HTF, using HTF and rabbit anti HTF, as well as gold labelled HTF (Au-HTF) to blot OMP's of various serogroups and serotypes of N. meningitidis. Also, we used monospecific rabbit anti FeRP-70 in competitive experiments to determine the role of FeRP-70 in HTF-binding. Single proteins (molecular weights range ca. 60 to ca. 90 kDa) were identified in the OMP's from each strain which reacted with HTF. HTF failed to block the reaction between FeRP-70 and the OMP's, conversely anti FeRP-70 failed to block the HTF-binding reaction. We believe that the 70 kDa iron regulated protein of N. meningitidis is not a human transferrin receptor.