The R-type lipopolysaccharides of Neisseria meningitidis

The lipopolysaccharides of all the different serogroups of Neisseria meningitidis are of the "R" type despite the morphologically smooth appearance and the demonstrated virulence of the organisms from which they were derived. This was confirmed when each of the lipopolysaccharides was found to be devoid of detectable O-antigen side chains, giving only a low "molecular" weight core oligosaccharide when subjected to mild acid hydrolysis. The cores were modified by dephosphorylation and subjected to sugar and methylation analysis by gas-liquid chromatography. Although all the different cores contained identical components (glucose, galactose, glucosamine, heptose, and 2-keto-3-deoxyoctonate) they could be separated into three distinct categories according to their galactose:glucose ratios. These categories are typified by the cores obtained from groups A, C, and 29-e which have galactose:glucose ratios of 1:2, 2:2, and 2:1, respectively. The modified cores were methylated and analyzed by gas chromatography--mass spectrometry and on the basis of differences in the derived methylated sugars the cores could again be divided into the same three categories as above. This structural diversity also results in some serological specificity as demonstrated by the complete serogroup specificity of the group A lipopolysaccharide.     

Alteration of epithelial cell transferrin-iron homeostasis by Neisseria meningitidis and Neisseria gonorrhoeae

Iron is an essential element for nearly all organisms. In mammals, iron is transported to body tissues by the serum glycoprotein transferrin. Transferrin-iron is internalized by binding to specific receptors followed by endocytosis. In vitro , Neisseria meningitidis and Neisseria gonorrhoeae can use iron from a variety of iron-containing compounds, including human transferrin. In vivo , transferrin is an important source of iron for N. gonorrhoeae : a mutant that is unable to bind and use transferrin-iron is unable to colonize the urethra of men or initiate disease at this site. As pathogenic Neisseria and its human host derive much of their iron from transferrin, we reasoned that a competition may exist between microbe and host epithelial cells for transferrin-iron at certain stages of infection. We therefore tested the hypothesis that N. meningitidis and N. gonorrhoeae may actively interfere with host transferrin-iron metabolism. We report that Neisseria-infected human epithelial cells have reduced levels of transferrin receptor messenger RNA and cycling transferrin receptors. The ability of infected cells to internalize transferrin receptor is also reduced. Finally, the relative distribution of surface and cycling transferrin receptors is altered in an infected cell. We conclude that Neisseria infection alters epithelial cell transferrin-iron homeostasis at multiple levels.     

The influence of recombination on the population structure and evolution of the human pathogen Neisseria meningitidis.

The extent to which recombination disrupts the bifurcating treelike phylogeny and clonal structure imposed by binary fission on bacterial populations remains contentious. Here, we address this question with a study of nucleotide sequence data from 107 isolates of the human pathogen Neisseria meningitidis. Gene fragments from 12 house-keeping loci distributed around the meningococcal chromosome were analyzed, showing that (1) identical alleles are disseminated among genetically diverse isolates, with no evidence for linkage disequilibrium; (2) different loci give distinct and incongruent phylogenetic trees; and (3) allele sequences are incompatible with a bifurcating treelike phylogeny at all loci. These observations are consistent with the hypothesis that meningococcal populations comprise organisms assembled from a common gene pool, with alleles and allele fragments spreading independently, together with the occasional importation of genetic material from other species. Further, they support the view that recombination is an important genetic mechanism in the generation new meningococcal clones and alleles. Consequently, for anything other than the short-term evolution of this species, a bifurcating treelike phylogeny is not an appropriate model.     

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.     

Selective impairment of B cell function by Neisseria meningitidis

Spleen cells from CBA/J mice infected with Neisseria meningitidis displayed depressed in vitro plaque-forming cell (PFC) responses to T-dependent (sheep red blood cell; SRBC) and T-independent (TNP-LPS, TNP-Ficoll) antigens. The inhibition was observed over a wide range of antigen concentrations. The decreased responsiveness of splenocytes from infected mice was due to a selective impairment of B-cell function since helper-T-cell activity was intact in infected mice as shown by the ability of T-enriched lymphocytes to cooperate with normal B-enriched lymphocytes in the generation of an anti-SRBC response, accessory macrophage function was preserved since adherent spleen cells from bacteria-injected mice were shown to produce normal or increased levels of IL-1 and were able to cooperate with normal non-adherent spleen cells in the generation of PFC against SRBC. Addition of peritoneal cells from normal animals or extraneous IL-1 both failed to restore normal PFC responses in cultures of splenocytes from infected mice. Finally, B-enriched lymphocytes from infected mice produced poor anti-SRBC responses when cultured with either Con A supernatant or T-enriched lymphocytes from normal or infected mice. Cell-mixing experiments failed to detect the presence of suppressor cells in cultures of unfractionated spleen cells or B-enriched lymphocytes from infected mice. Therefore, the immunological unresponsiveness associated with a Neisseria meningitidis infection was attributed to a meningococcus-induced defect(s) in B-cell function. In vivo polyclonal B-cell activation leading to clonal exhaustion did not play a major role in the depression of humoral responses since meningococcal infection induced little or no polyclonal Ig secretion.     

The number of Neisseria meningitidis type IV pili determines host cell interaction

As mediators of adhesion, autoaggregation and bacteria‐induced plasma membrane reorganization, type IV pili are at the heart of Neisseria meningitidis infection. Previous studies have proposed that two minor pilins, PilV and PilX, are displayed along the pilus structure and play a direct role in mediating these effects. In contrast with this hypothesis, combining imaging and biochemical approaches we found that PilV and PilX are located in the bacterial periplasm rather than along pilus fibers. Furthermore, preventing exit of these proteins from the periplasm by fusing them to the mCherry protein did not alter their function. Deletion of the pilV and pilX genes led to a decrease in the number, but not length, of pili displayed on the bacterial surface indicating a role in the initiation of pilus biogenesis. By finely regulating the expression of a central component of the piliation machinery, we show that the modest reductions in the number of pili are sufficient to recapitulate the phenotypes of the pilV and pilX mutants. We further show that specific type IV pili‐dependent functions require different ranges of pili numbers.     

Modulation of cell surface sialic acid expression in Neisseria meningitidis via a transposable genetic element.

Cell surface-located sialic acids of the capsule and the lipooligosaccharide (LOS) are both pivotal virulence factors in Neisseria meningitidis, promoting survival and dissemination of this pathogen which can cause both sepsis and meningitis. With the aid of a unique set of isogenic meningococcal mutants defective in the expression of cell surface-located sialic acids, we have demonstrated that encapsulation hinders the primary event in the development of the disease, but the spontaneous switching of encapsulated wild-type bacteria to a capsule-negative phenotype promotes meningococcal adherence and invasion into mucosal epithelial cells. Genetic analysis of the capsule-negative, invasive bacteria revealed a unique mechanism for modulation of capsule expression based on the reversible inactivation of an essential sialic acid biosynthesis gene, siaA, by insertion/excision of a naturally occurring insertion sequence element, IS1301. Inactivation of siaA regulates both capsule expression and endogenous LOS sialylation. This is the first example of an insertion sequence element-based genetic switch mechanism in the pathogenic bacterium and is an important step in the understanding of bacterial virulence.     

Comparative study of the influence of melatonin and vitamin E on the surface characteristics of Escherichia coli

AIMS: Melatonin is a hormone produced by the pineal gland and that affects the response of various cell membranes to an oxidative stimulus. METHODS AND RESULTS: The present study evaluates the hydrophobic characteristics of Escherichia coli in response to melatonin (100 nmol l(-1), 200 micromol l(-1)) and to vitamin E (5 mg dl(-1)). A reduction was found in the surface hydrophobicity of E. coli at concentrations of 200 micromol l(-1) melatonin in a Müeller-Hinton (MH) broth. These effects were modified when a protein synthesis inhibitor (chloramphenicol) was added at sub-lethal concentrations to the broth. Vitamin E produced a greater diminution in surface hydrophobicity than melatonin. The adherence of E. coli to nitrocellulose filters increased in the presence of melatonin + chloramphenicol, and vitamin E. The effects observed were independent of the concentration of iron in the broth. CONCLUSION: Oxidative stress plays an important role in modifying the surface characteristics of E. coli, which could affect the micro-organism's capacity to adhere to epithelia. SIGNIFICANCE AND IMPACT OF THE STUDY: We think that the oxide reduction potential of the host may be a determinant factor in the bacterial colonization of animal tissue.     

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.     

Neisseria meningitidis producing the Opc adhesin binds epithelial cell proteoglycan receptors

Neisseria meningitidis possesses a repertoire of surface adhesins that promote bacterial adherence to and entry into mammalian cells. Here, we have identified heparan sulphate proteoglycans as epithelial cell receptors for the meningococcal Opc invasin. Binding studies with radiolabelled heparin and heparin affinity chromatography demonstrated that Opc is a heparin binding protein. Subsequent binding experiments with purified ³⁵SO₄-labelled epithelial cell proteoglycan receptors and infection assays with epithelial cells that had been treated with heparitinase to remove glycosaminoglycans confirmed that Opc-expressing meningococci exploit host cell-surface proteoglycans to gain access to the epithelial cell interior. Unexpectedly, Opa28-producing meningococci lacking Opc also bound proteoglycans. These bacteria also bound CEA receptors in contrast to the Opc-expressing phenotype, suggesting that Opa28 may possess domains with specificity for different receptors. Opa/Opc-negative meningococci did not bind either proteoglycan or CEA receptors. Using a set of genetically defined mutants with different lipopolysaccharide (LPS) and capsular phenotype, we were able to demonstrate that surface sialic acids interfere with the Opc–proteoglycan receptor interaction. This effect may provide the molecular basis for the reported modulatory effect of capsule and LPS on meningococcal adherence to and entry into various cell types.     

Population genetics of a transformable bacterium: The influence of horizontal genetic exchange on the biology of Neisseria meningitidis

Abstract Information on the biochemistry and genetics of bacterial species, usually obtained by the study of single isolates, is enhanced by studies of populations of bacteria. Recent advances in molecular technology, particularly polymerase chain reaction-based nucleotide sequence analysis, provide powerful for the study of population genetics. Data obtained by such techniques indicate that, while some bacterial species have a clonal population structure, others are non-clonal or panmictic. Clonal populations are a consequence of asexual reproduction by binary fission; panmictic population structures results from 'horizontal' exchange of genetic material between clones. A consequence of horizontal genetic exchange is mosaic gene structures, recognisable by comparisons of nucleotide sequences. In transformable bacteria, for example the human pathogen Neisseria meningitidis , several different genes, including the gene encoding the class 1 outer membrane protein, a major surface antigen, are mosaics. This genetic process has implications both for vaccine design and in the interpretation of epidemiological data.     

Unique intermolecular bactericidal epitope involving the homosialopolysaccharide capsule on the cell surface of group B Neisseria meningitidis and Escherichia coli K1

The N-propionylated group B meningococcal polysaccharide mimics a unique bactericidal epitope on the surface of group B meningococci and Escherichia coli K1. This was confirmed when both the above organisms were able to absorb the bactericidal antibodies from a mouse-anti-N-propionylated group B meningococcal polysaccharide-tetanus toxoid conjugate serum. By using affinity columns it was possible to divide the conjugate antiserum into three distinct populations of both group B polysaccharide cross-reactive and non-cross-reactive antibodies, one of which contained most of the bactericidal activity. The cross-reactive (IgG1) antibodies were absorbed by an affinity column in which the group B polysaccharide was linked to the solid support by a long spacer arm, thereby isolating a population of non-cross-reactive (IgG1) antibodies. Surprisingly the above column also retained another population of non-cross-reactive (IgG2a) and (IgG2b) antibodies which contained most of the bactericidal activity. These latter antibodies were not absorbed by a similar group B polysaccharide-affinity column in which a short spacer arm was employed. Thus the above experiments not only effected a separation of highly bactericidal antibodies but also provided evidence that the long spacer arm is functional in the binding of the bactericidal antibodies to the affinity column. This indicates that the bactericidal epitope is mimicked by the group B polysaccharide in the presence of the long spacer arm, which supports the hypothesis that the epitope is polysaccharide-associated and is probably intermolecular in nature.     

The regulating action of oxygen on the nutritional requirements of Neisseria meningitidis

The study has revealed regularities in changing nutritional requirements of Neisseria meningitidis with changes in the degree of the oxygen saturation of the culture medium in a fermenter under the conditions of the controlled cultivation of N. meningitidis in a synthetic culture medium in the process of batch, semicontinuous and continuous flow cultivation. As shown in this study, when oxygen supply is limited, the consumption of carbohydrates prevails, while in the presence of surplus oxygen the prevalence of the consumption of amino nitrogen is observed.     

Expression of epithelial cell iron-related genes upon infection by Neisseria meningitidis

Infection by the obligate human pathogens Neisseria meningitidis (MC) and Neisseria gonorrhoeae (GC) reduces the expression of host epithelial cell transferrin receptor 1 (TfR-1) (Bonnah et al., 2000, Cellular Microbiology 2: 207-218). In addition, the rate and pattern of TfR-1 cycling is altered, leading to diminished uptake of Tf-iron by infected host cells. As Tf-iron is important for maintaining iron homeostasis in the eukaryotic cell, these findings raised the possibility that Neisseria infection might affect further pathways of epithelial cell iron metabolism. We used a specialized cDNA microarray platform, the 'IronChip', to investigate the expression of genes involved in iron transport, storage and regulation. We show that mRNA expression of several host genes involved in iron homeostasis is altered. Surprisingly, the general mRNA expression profile of infected cells closely resembled that of uninfected cells grown in an iron-limited environment. An important exception to this profile is TfR-1, the mRNA level of which is strongly reduced. Low TfR-1 expression may be explained in part by decreased activity of the iron-regulatory proteins (IRPs) in MC-infected cells, which may result in the destabilization of TfR-1 mRNA. Intriguingly, low IRP activity contrasts with the decrease in H-ferritin protein levels in infected cells. This finding suggests that low IRP activity may be responsible in part for the decrease in TfR-1 mRNA levels. A discussion of these novel findings in relation to MC infection and virulence is provided.     

Localization of tetramethylphenylenediamine-oxidase in the outer cell wall layer of Neisseria meningitidis.

A highly active tetramethylphenylenediamine-oxidase has been found in association with the cell wall blebs, evaginations of the outer wall membrane, of Neisseria meningitidis. Isolated wall blebs consumed oxygen in the presence of ascorbate-tetramethylphenylenediamine but not in the presence of succinate, whereas cell envelope preparations are active in both substrates. The ratio of succinate dehydrogenase/tetramethylphenylenediamine-oxidase activities in preparations of envelopes was approximately 100 times that in isolated wall blebs, indicating that the outer membrane preparations were highly purified.     

Chemical and antigenic analysis of the cell walls of Neisseria meningitidis group B

Vedros, Neylan A. (Naval Medical Research Institute, Bethesda, Md.), and Paul R. Hill. Chemical and antigenic analysis of the cell walls of Neisseria meningitidis group B. J. Bacteriol. 91:1992-1997. 1966.-Cell walls have been isolated from Neisseria meningitidis group B, by rapid freeze-thawing or treatment with sodium deoxycholate. Chemical analysis of the cell walls indicated that the amino acid composition, and content of hexosamines and of lipids, were similar to those reported for Escherichia coli, except for higher concentrations of alanine, methionine, glutamic acid, and phenylalanine. The meningococcal cell walls showed groupspecificity in the complement-fixation test, were nontoxic for rabbits, and in rabbits produced antibodies which protected mice from challenge with the homologous strain.     

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.