Nitric oxide metabolism in Neisseria meningitidis

Neisseria meningitidis, the causative agent of meningococcal disease in humans, is likely to be exposed to nitrosative stress during natural colonization and disease. The genome of N. meningitidis includes the genes aniA and norB, predicted to encode nitrite reductase and nitric oxide (NO) reductase, respectively. These gene products should allow the bacterium to denitrify nitrite to nitrous oxide. We show that N. meningitidis can support growth microaerobically by the denitrification of nitrite via NO and that norB is required for anaerobic growth with nitrite. NorB and, to a lesser extent, the cycP gene product cytochrome c' are able to counteract toxicity due to exogenously added NO. Expression of these genes by N. meningitidis during colonization and disease may confer protection against exogenous or endogenous nitrosative stress.     

Analysis of Neisseria lactamica antigens putatively implicated in acquisition of natural immunity to Neisseria meningitidis

Sera from healthy human volunteers, patients convalescent from meningococcal meningitis, and mice immunized with outer membrane proteins from Neisseria meningitidis and Neisseria lactamica strains were used to analyze and identify antigens cross-reactive to both neisserial species. All classes of meningococcal proteins except class 1 (PorA) and class 5 cross-reacted with N. lactamica proteins and two other proteins of 65 and 55 kDa (an iron-regulated protein). Results obtained with the mouse sera demonstrate that cross-reactive antibodies can be elicited by either N. meningitidis or N. lactamica. These results support the suggestion that N. lactamica contributes to the development of natural immunity against N. meningitidis during the first years of life. The use of vaccines containing proteins other than PorA could interfere in colonization of mucosal surfaces by N. lactamica, hampering the natural mechanisms of immunity acquisition in humans. Only convalescent sera reacted with the 55 and 65 kDa proteins, which suggests that they might be relevant for pathogenicity.     

The influence of different concentrations of melatonin on the cell surface hydrophobic characteristics of Neisseria meningitidis

The cell surface hydrophobicity of micro-organisms is a characteristic that has been associated with the colonization of mammalian epithelia and with their capacity to induce diseases. Melatonin is a hormone produced by the pineal gland that affects the immune response mechanism. This study investigated, as an expression of the virulence of Neisseria meningitidis, how its hydrophobic characteristics were affected by exposure to increasing concentrations of melatonin. An increase in the cell surface hydrophobicity of N. meningitidis was found at concentrations of 1 mmol l(-1), while lower concentrations of melatonin did not significantly affect this particular cell surface characteristic of the micro-organism. It may be concluded that melatonin clearly influences the cell surface hydrophobicity of N. meningitidis, a circumstance that should be taken into account in future studies to determine whether this hormone plays a role in the variable pathogenicity of the bacteria in different hosts.     

Inter-strain homology of pilin gene sequences in Neisseria meningitidis isolates that express markedly different antigenic pilus types

Neisseria meningitidis isolates examined in this study elaborated one of two pilus types that were antigenically markedly different. Each pilus type reacted either with SM1, a monoclonal antibody that recognizes an epitope common to all gonococcal pili, or with a polyclonal antiserum raised against meningococcal pili that did not react with SM1, but not both. Total genomic DNA from all N. meningitidis isolates analysed, irrespective of pilus type, contained at least one region with extensive homology to a gonococcal pilE probe. Different N. meningitidis strains possessed one of several configurations of genomic pilE-homologous segments. Chromosomal rearrangement of pilE-homologous sequences was associated with P+ to P- pilus phase transition in the strains examined. The arrangement of pilE-homologous segments in total genomic DNA from N. meningitidis isolated from the blood and cerebro-spinal fluid of the same patient was apparently identical.     

Epidemiology,hypermutation, within-host evolution and the virulence of Neisseria meningitidis

Many so-called pathogenic bacteria such as Neisseria meningitidis, Haemophilus influenzae, Staphylococcus aureus and Streptococcus pneumoniae are far more likely to colonize and maintain populations in healthy individuals asymptomatically than to cause disease. Disease is a dead-end for these bacteria: virulence shortens the window of time during which transmission to new hosts can occur and the subpopulations of bacteria actually responsible for disease, like those in the blood or cerebral spinal fluid, are rarely transmitted to new hosts. Hence, the virulence factors underlying their occasional pathogenicity must evolve in response to selection for something other than making their hosts sick. What are those selective pressures? We address this general question of the evolution of virulence in the context of phase shifting in N. meningitidis, a mutational process that turns specific genes on and off, and, in particular, contingency loci that code for virulence determinants such as pili, lipopolysaccharides, capsular polysaccharides and outer membrane proteins. We use mathematical models of the epidemiology and the within-host infection dynamics of N. meningitidis to make the case that rapid phase shifting evolves as an adaptation for colonization of diverse hosts and that the virulence of this bacterium is an inadvertent consequence of short-sighted within-host evolution, which is exasperated by the increased mutation rates associated with phase shifting. We present evidence for and suggest experimental and retrospective tests of these hypotheses.     

Meningococcus Hijacks a β2-adrenoceptor/β-Arrestin pathway to cross brain microvasculature endothelium

Following pilus-mediated adhesion to human brain endothelial cells, meningococcus (N. meningitidis), the bacterium causing cerebrospinal meningitis, initiates signaling cascades, which eventually result in the opening of intercellular junctions, allowing meningeal colonization. The signaling receptor activated by the pathogen remained unknown. We report that N. meningitidis specifically stimulates a biased β2-adrenoceptor/β-arrestin signaling pathway in endothelial cells, which ultimately traps β-arrestin-interacting partners, such as the Src tyrosine kinase and junctional proteins, under bacterial colonies. Cytoskeletal reorganization mediated by β-arrestin-activated Src stabilizes bacterial adhesion to endothelial cells, whereas β-arrestin-dependent delocalization of junctional proteins results in anatomical gaps used by bacteria to penetrate into tissues. Activation of β-adrenoceptor endocytosis with specific agonists prevents signaling events downstream of N. meningitidis adhesion and inhibits bacterial crossing of the endothelial barrier. The identification of the mechanism used for hijacking host cell signaling machineries opens perspectives for treatment and prevention of meningococcal infection.     

LPS from Neisseria meningitidis is crucial for inducing monocyte- and microparticle-associated tissue factor activity but not for tissue factor expression

Neisseria meningitidis causes sepsis with coagulopathy. The present study evaluated the tissue factor (TF)-inducing capacity of bacterial LPS in different presentation forms, i.e. membrane-bound LPS versus purified LPS, and of non-LPS components of N. meningitidis. By using a wild-type N. meningitidis, a mutant N. meningitidis lacking LPS (LPS-deficient N. meningitidis), purified LPS from N. meningitidis and Escherichia coli, we measured TF-expression and TF-activity on human monocytes and microparticles (MPs). The effect of TF-modulators, such as phosphatidylserine (PS), tissue factor pathway inhibitor (TFPI) and recombinant IL-10 (rhIL-10) was investigated. In plasmas from meningococcal patients, fibrinopeptide A (FPA), LPS and IL-10 were quantified. Monocytes and MPs exposed to purified LPS or wild-type N. meningitidis had much higher TF-activity than monocytes and MPs exposed to LPS-deficient N. meningitidis (clot formation assay). Incubation with wild-type N. meningitidis, but also LPS-deficient N. meningitidis, resulted in TF-expression on monocytes (flow cytometry, qRT-PCR). Increased cellular TF-activity is associated with coincident surface-exposure of PS and the number of monocytes positive for both PS and TF was significantly higher for monocytes exposed to wild-type N. meningitidis (7.6%) compared with monocytes exposed to LPS-deficient N. meningitidis (1.8%). Treatment with rhIL-10 reduced monocyte- and MP-associated TF-activity, the number of monocytes positive for both TF and PS, and microvesiculation. Patients with meningococcal septicemia had significantly higher levels of LPS, FPA and IL-10 than patients with distinct meningitis. Our results indicate that LPS from N. meningitidis is crucial for inducing TF-activity, but not for monocyte- and MP-associated TF-expression. TF-activity seems to require coincident expression of TF and PS on monocytes, and LPS induces such double-positive monocytes.     

Neisseria meningitidis tonB, exbB, and exbD genes: Ton-dependent utilization of protein-bound iron in Neisseriae.

We have recently cloned and characterized the hemoglobin (Hb) receptor gene, hmbR, from Neisseria meningitidis. To identify additional proteins that are involved in Hb utilization, the N. meningitidis Hb utilization system was reconstituted in Escherichia coli. Five cosmids from N. meningitidis DNA library enabled a heme-requiring (hemA), HmbR-expressing mutant of E. coli to use Hb as both porphyrin and iron source. Nucleotide sequence analysis of DNA fragments subcloned from the Hb-complementing cosmids identified four open reading frames, three of them homologous to Pseudomonas putida, E. coli, and Haemophilus influenzae exbB, exbD, and tonB genes. The N. meningitidis TonB protein is 28.8 to 33.6% identical to other gram-negative TonB proteins, while the N. meningitidis ExbD protein shares between 23.3 and 34.3% identical amino acids with other ExbD and TolR proteins. The N. meningitidis ExbB protein was 24.7 to 36.1% homologous with other gram-negative ExbB and TolQ proteins. Complementation studies indicated that the neisserial Ton system cannot interact with the E. coli FhuA TonB-dependent outer membrane receptor. The N. meningitidis tonB mutant was unable to use Hb, Hb-haptoglobin complexes, transferrin, and lactoferrin as iron sources. Insertion of an antibiotic cassette in the 3' end of the exbD gene produced a leaky phenotype. Efficient usage of heme by N. meningitidis tonB and exbD mutants suggests the existence of a Ton-independent heme utilization mechanism. E. coli complementation studies and the analysis of N. meningitidis hmbR and hpu mutants suggested the existence of another Hb utilization mechanism in this organism.     

Olfactory nerve--a novel invasion route of Neisseria meningitidis to reach the meninges

Neisseria meningitidis is a human-specific pathogen with capacity to cause septic shock and meningitis. It has been hypothesized that invasion of the central nervous system (CNS) is a complication of a bacteremic condition. In this study, we aimed to characterize the invasion route of N. meningitidis to the CNS. Using an intranasally challenged mouse disease model, we found that twenty percent of the mice developed lethal meningitis even though no bacteria could be detected in blood. Upon bacterial infection, epithelial lesions and redistribution of intracellular junction protein N-cadherin were observed at the nasal epithelial mucosa, especially at the olfactory epithelium, which is functionally and anatomically connected to the CNS. Bacteria were detected in the submucosa of the olfactory epithelium, along olfactory nerves in the cribriform plate, at the olfactory bulb and subsequently at the meninges and subarachnoid space. Furthermore, our data suggest that a threshold level of bacteremia is required for the development of meningococcal sepsis. Taken together, N. meningitidis is able to pass directly from nasopharynx to meninges through the olfactory nerve system. This study enhances our understanding how N. meningitidis invades the meninges. The nasal olfactory nerve system may be a novel target for disease prevention that can improve outcome and survival.     

Humanized Mouse Model to Study Bacterial Infections Targeting the Microvasculature

Neisseria meningitidis causes a severe, frequently fatal sepsis when it enters the human blood stream. Infection leads to extensive damage of the blood vessels resulting in vascular leak, the development of purpuric rashes and eventual tissue necrosis. Studying the pathogenesis of this infection was previously limited by the human specificity of the bacteria, which makes in vivo models difficult. In this protocol, we describe a humanized model for this infection in which human skin, containing dermal microvessels, is grafted onto immunocompromised mice. These vessels anastomose with the mouse circulation while maintaining their human characteristics. Once introduced into this model, N. meningitidis adhere exclusively to the human vessels, resulting in extensive vascular damage, inflammation and in some cases the development of purpuric rash. This protocol describes the grafting, infection and evaluation steps of this model in the context of N. meningitidis infection. The technique may be applied to numerous human specific pathogens that infect the blood stream.     

Cytosolic proteins contribute to surface plasminogen recruitment of Neisseria meningitidis

Plasminogen recruitment is a common strategy of pathogenic bacteria and results in a broad-spectrum surface-associated protease activity. Neisseria meningitidis has previously been shown to bind plasminogen. In this study, we show by several complementary approaches that endolase, DnaK, and peroxiredoxin, which are usually intracellular proteins, can also be located in the outer membrane and act as plasminogen receptors. Internal binding motifs, rather than C-terminal lysine residues, are responsible for plasminogen binding of the N. meningitidis receptors. Recombinant receptor proteins inhibit plasminogen association with N. meningitidis in a concentration-dependent manner. Besides binding purified plasminogen, N. meningitidis can also acquire plasminogen from human serum. Activation of N. meningitidis-associated plasminogen by urokinase results in functional activity and allows the bacteria to degrade fibrinogen. Furthermore, plasmin bound to N. meningitidis is protected against inactivation by alpha(2)-antiplasmin.     

Cultivation of Neisseria meningitidis serogroups A, B and C in a synthetic nutrient broth

The processes of the cultivation of N. meningitidis, serogroups A, B and C, in a liquid synthetic culture medium have been studied. Strictly group-specific biomass has been obtained. The maximum productivity at all stages of the batch cultivation of N. meningitidis strains 125 and 133 in this medium does not differ from that at similar stages of cultivation in modified Cohen-Wheller semisynthetic medium. In the serotype antigen preparations obtained from N. meningitidis strain 125 grown in the above-mentioned liquid synthetic culture medium basic polypeptides with a molecular weight of 33000, 36000 and 41000 D have been detected. Their presence in N. meningitidis cells is linked with the growth phase of the population.     

Reliability of the detection of meningococcal gamma-glutamyl transpeptidase as an identification marker for Neisseria meningitidis

Detection of gamma-glutamyl transpeptidase (GGT; ggt ) activity is one of the useful methods for a specific identification of Neisseria meningitidis. However, we previously happened to isolate a ggt -deficient N. meningitidis strain (NIID113) from a healthy carrier. In this study, in order to re-examine the reliability of the marker, we again investigated the GGT activity of 245 N. meningitidis human isolates and identified two other GGT-defective N. meningitidis isolates besides NIID113. The isolation frequency (1.2%) of ggt mutants among human isolates strongly confirmed the 98.8% reliability of GGT activity as the identification marker for N. meningitidis.     

Level of IgG antibodies to bacteria of 12 species in the blood sera of rabbits, immunized with preparations obtained from Neisseria meningitidis, grown under the stress conditions

The blood sera of rabbits, immunized with preparations obtained from N. meningitidis of serogroups A, B or C, cultivated under the stress conditions, were studied. These sera were found to contain IgG antibodies not only to N. meningitidis antigens, but also to the bacterial antigens of 12 species. The sera of rabbits, immunized with meningococcal preparation of serogroup A, were found to have the elevated levels of IgG antibodies, in comparison with the control, to the antigens of 3 other bacterial species; the blood sera of rabbits, immunized with meningococcal preparation of serogroup B, were found the elevated levels of IgG antibodies to the antigens of 11 other bacterial species; and the blood sera of rabbits, immunized with meningococcal preparation of serogroup C, to the antigens of 9 other bacterial species. The study of serogroup B meningococci, used as an example, revealed the influence of the growth phase of the culture on the content of cross-reacting antigens. Their greatest amount was determined at the stationary phase when the stressor effect on the culture reached its maximum and their least amount, at the exponential phase when the stressor effect on the culture was minimal. It was, therefore, found to be expedient to obtain immunodiagnostic and test systems from N. meningitidis cultures, grown to middle of the exponential phase of growth.     

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.     

Evaluation of blood culture bottles seeded with X-V factors for the detection of Neisseria meningitidis and Haemophilus influenzae

The purpose of this in vitro study was to determine the ability of seeded and not-seeded commercial pediatric blood culture bottles to support the growth of the most frequently responsible microorganisms for bacterial meningitides (Neisseria meningitidis, and Haemophilus influenzae). Tests have been carried out with an automated colorimetric pediatric blood culture system, BacTAlert, Organon Teknika. Bottles were inoculated with X-V factors and serial dilutions of the each bacterium in six times (10(1)-10(6) colony forming unit [CFU]/ml). The bottles, which were supplemented with X-V factors, proved to be effective and time to detection (TTD) was shorter than the un-seeded bottles (p0.05). Time difference between seeded and not-seeded bottles was getting greater at high dilutions of both bacteria. We consider that in presence of a few bacteria, the seeding of bottles with X-V factors is very critical obtaining N. meningitidis, and H. influenzae as the causative agents of meningitidis. The recovery rate of the microorganisms, which were isolated from cerebrospinal fluid by using the X-V factor-seeded blood culture bottles, is therefore higher than with the conventional culture methods.     

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.     

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.