Epithelial cell responses induced upon adherence of pathogenic Neisseria

Neisseria meningitidis and Neisseria gonorrhoeae colonize human mucosal surfaces and cause sepsis/meningitis and gonorrhoea respectively. The first step in the infection process is pilus-mediated adhesion of the bacteria to epithelial cells, followed by host cell invasion. Adhesion of pathogenic Neisseria elicits multiple responses in host cells, including cellular signalling events, cytokine production and modulation of the eukaryotic cell surface. We used microarrays to assess the respective involvement of 375 human cytokine and adhesion related genes during adhesion of piliated and non-piliated N. gonorrhoeae, and piliated encapsulated N. meningitidis to the epithelial cell line ME-180. We identified 29 differentially regulated genes not previously reported to respond to neisserial infections, many of which encode membrane proteins. Selected genes were further analysed by semiquantitative RT-PCR, and protein expression was examined by flow cytometry. We found that N. gonorrhoeae elicited a different inflammatory response than N. meningitidis and we also demonstrated that early adhesion events are responsible for the induction of specific genes. Our data create a new platform for elucidating the interaction between pathogenic Neisseria and target cells.     

Neisseria meningitidis induces the expression of the TNF-alpha gene in endothelial cells

Pilus-mediated adhesion plays a prominent role in the pathogenesis of Neisseria meningitidis by allowing the initial localized adhesion to epithelial and endothelial cells. Non-piliated bacteria are not adherent. Moreover, cytokine production during infection is a key feature of meningococcal pathogenesis. Tumour necrosis factor alpha (TNF-alpha) is known to be produced early during meningococcal infections and experimental endotoxemia. Monocytic cells are thought to be responsible for this systemic production of TNF-alpha which is involved in many aspects of meningococcal pathogenesis such as coagulopathy and activation of endothelial cells. In this report, both adherent and non-adherent N. meningitidis were shown to induce the expression of TNF-alpha gene in monocytic cells, however, only adherent N. meningitidis was able to induce the expression of TNF-alpha gene in endothelial cells. This latter induction required the presence of monocytes. These data suggest that endothelial cells may be activated selectively and efficiently by adherent N. meningitidis and can locally produce TNF-alpha upon bacterial adhesion.     

Sequence analysis and relationships between meningococcal class 3 serotype proteins and other porins from pathogenic and non-pathogenic Neisseria species

The presence of highly conserved regions within previously determined porin gene sequences from Neisseria meningitidis and Neisseria gonorrhoeae permitted the construction of oligonucleotide primers for PCR amplification of other neisserial porin genes. Although two separate porin genes (porA and porB) are present in N. meningitidis only a single fragment, corresponding to porB, could be amplified from this species. The amplified porB genes from four different meningococcal serotypes, which express the class 3 outer membrane protein, were sequenced. Amplified fragments corresponding to porin genes from N. lactamica and N. sicca were also sequenced. In common with the known neisserial porins, models of the organisation of the predicted proteins indicated trans-membrane structures with eight surface exposed loops. In the meningococcal class 3 proteins the main regions of sequence variation, which must be responsible for serotype specificity, were located on loops 5 and 7. A phylogenetic analysis of the family of porins from the Neisseria confirmed the close relationship of the meningococcal class 3 protein with the gonococcal PIA protein, while the gonococcal PIB protein was shown to be closely related to the N. lactamica porin. The close relationship seen between porins of the pathogenic and non-pathogenic Neisseriae identified no obvious virulence-associated regions in the proteins, but did suggest that the current nomenclature for neisserial porin genes may need reviewing.     

The REP2 repeats of the genome of Neisseria meningitidis are associated with genes coordinately regulated during bacterial cell interaction

Interaction with host cells is essential in meningococcal pathogenesis especially at the blood-brain barrier. This step is likely to involve a common regulatory pathway allowing coordinate regulation of genes necessary for the interaction with endothelial cells. The analysis of the genomic sequence of Neisseria meningitidis Z2491 revealed the presence of many repeats. One of these, designated REP2, contains a -24/-12 type promoter and a ribosome binding site 5 to 13 bp before an ATG. In addition most of these REP2 sequences are located immediately upstream of an ORF. Among these REP2-associated genes are pilC1 and crgA, described as being involved in steps essential for the interaction of N. meningitidis with host cells. Furthermore, the REP2 sequences located upstream of pilC1 and crgA correspond to the previously identified promoters known to be induced during the initial localized adhesion of N. meningitidis with human cells. This characteristic led us to hypothesize that at least some of the REP2-associated genes were upregulated under the same circumstances as pilC1 and crgA. Quantitative PCR in real time demonstrated that the expression of 14 out of 16 REP2-associated genes were upregulated during the initial localized adhesion of N. meningitidis. Taken together, these data suggest that these repeats control a set of genes necessary for the efficient interaction of this pathogen with host cells. Subsequent mutational analysis was performed to address the role of these genes during meningococcus-cell interaction.     

The diagnostic specificity of the antigen-binding lymphocyte test in Neisseria-induced infections

Neisseria-induce different infections, but many representatives of this genus are saprophytes. In this connection it is important to evaluate the species, and for N. meningitidis also group specificity of the immunological diagnostics of meningococcal infection and gonorrhea. In this work the specificity of the antigen-binding lymphocyte test was studied in experiments with the immunization of rabbits and the examination of patients. In the tests of indirect rosette-formation and its inhibition the role of antigenic relationships between different pathogenic and nonpathogenic Neisseria was excluded and the species and group specificity of our Neisseria immunoreagents, used in the diagnostics of meningococcal infection and gonorrhea, was proved.     

Mutagenesis of Neisseria meningitidis by in vitro transposition of Himar1 mariner

Now that the meningococcal genome sequence has been completed, the lack of a suitable method for saturation mutagenesis remains a major obstacle to the unraveling of the pathogenic propensity of Neisseria meningitidis. Here, we demonstrate that in vitro Himar1 mariner transposition on chromosomal or PCR-amplified meningococcal DNA, which is subsequently reintroduced into N. meningitidis by natural transformation, is an extremely efficient mutagenesis method. Southern blot analysis, sequencing the Himar1 insertion point in numerous transposition mutants, and a limited screening of the mutant libraries for clones impaired in maltose catabolism confirmed that Himar1 transposed randomly in N. meningitidis. Taken together, these data demonstrate that Himar1 in vitro transposition can lead to the exhaustive mutagenesis of N. meningitidis, allowing for the first time a genomic-scale mutational analysis of this important human pathogen.     

Identification and characterization of specific sequences encoding pathogenicity associated proteins in the genome of commensal Neisseria species

Abstract The distribution of distinct sequences in pathogenic and commensal Neisseria species was investigated systematically by dot blot analysis. Probes representing the genes of Rmp, pilin and IgA1 protease were found to hybridize exclusively to the chromosomal DNA of the pathogenic species, Neisseria gonorrhoeae and/or Neisseria meningitidis . In contrast, specific sequences for the genes of the porin protein Por and the opacity protein (Opa) were also detected in a panel of commensal Neisseria species such as N. lactamica, N. subflava, N, flava, N. mucosa and N. sicca . Using opa -specific oligonucleotides as probes in chromosomal blots, the genomes of the commensal Neisseria species show a totally reduced repertoire of cross-hybridizing loci compared to the complex opa gene family of N. gonorrhoeae . DNA sequence analysis of one opa -related gene derived from N. flava and N. sicca , respectively, revealed a large degree of homology with previously described gonococcal and meningococcal genes e.g., a typical repetitive sequence in the leader peptide and the distribution of the hypervariable and conserved regions. This observation, together with the finding, that the gene is constitutively transcribed, leads to the assumption that some of the commensal Neisseria species may have the potential for the expression of a protein harboring similar functions as the Opa proteins in pathogenic Neisseriae .     

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.     

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.     

PilC of Neisseria meningitidis is involved in class II pilus formation and restores pilus assembly, natural transformation competence and adherence to epithelial cells in PilC-deficient gonococci

Type 4 pili produced by the pathogenic Neisseria species constitute primary determinants for the adherence to host tissues. In addition to the major pilin subunit (PilE), neisserial pili contain the variable PilC proteins represented by two variant gene copies in most pathogenic Neisseria isolates. Based upon structural differences in the conserved regions of PilE, two pilus classes can be distinguished in Neisseria meningitidis . For class I pili found in both Neisseria gonorrhoeae and N. meningitidis , PilC proteins have been implicated in pilus assembly, natural transformation competence and adherence to epithelial cells. In this study, we used primers specific for the pilC2 gene of N. gonorrhoeae strain MS11 to amplify, by the polymerase chain reaction, and clone a homologous pilC gene from N. meningitidis strain A1493 which produces class II pili. This gene was sequenced and the deduced amino acid sequence showed 75.4% and 73.8% identity with the gonococcal PilC1 and PilC2, respectively. These values match the identity value of 74.1% calculated for the two N. gonorrhoeae MS11 PilC proteins, indicating a horizontal relationship between the N. gonorrhoeae and N. meningitidis pilC genes. We provide evidence that PilC functions in meningococcal class II pilus assembly and adherence. Furthermore, expression of the cloned N. meningitidis pilC gene in a gonococcal pilC1,2 mutant restores pilus assembly, adherence to ME-180 epithelial cells, and transformation competence to the wild-type level. Thus, PilC proteins exhibit indistinguishable functions in the context of class I and class II pili.     

Multilocus sequencing--a new method of genotyping bacteria and first results of its use

Comparative characterization (molecular typing) of isolates within a bacterial species is one of the major problems in microbiology and epidemiology. However, it is rather difficult to correlate data obtained in various laboratories, because traditional, including molecular, methods employed in typing pathogenic microorganisms cannot be standardized. In 1998, Maiden et al. proposed multilocus sequence typing (MLST); through which alleles of several housekeeping genes are directly assessed by nucleotide sequencing, each unique allele combination determining a sequence type of a strain. The advantages of this approach are that the culturing of pathogenic microorganisms is avoided, as their gene fragments are amplified directly from biological samples, and that the sequencing data are unambiguous, easy to standardize, and electronically portable. The latter makes it possible to generate an expandable global database for each species at an Internet site, in order to use it for the purposes of genotyping pathogenic bacteria (and other infectious agents). MLST protocols have been elaborated for Neisseria meningitidis, Streptococcus pneumoniae, and Helicobacter pylori; those for Streptococcus pyogenes, Staphylococcus aureus, and Haemophilus influenzae are now being developed. Basic principles and the first results of MLST have been reviewed, including data on the distribution and microevolution of N. meningitidis clones causing epidemic meningococcal infection, the relative recombination and mutation rates in the N. meningitidis genome, the identification of antibiotic-resistant S. pneumoniae clones causing severe generalized infection, the grouping of H. pylori isolates from various geographic regions, etc.     

Post-genomics of Neisseria meningitidis: an update

Neisseria meningitidis infection still remains a major life-threatening bacterial disease worldwide. The availability of bacterial genomic sequences generated a paradigm shift in microbiological and vaccines sciences, and post-genomics (comparative genomics, functional genomics, proteomics and a combination/evolution of these techniques) played important roles in elucidating bacterial biological complexity and pathogenic traits, at the same time accelerating the development of therapeutic drugs and vaccines. This article summarizes the most recent technological and scientific advances in meningococcal biology and pathogenesis aimed at the development and characterization of vaccines against the pathogenic meningococci.     

Interactions between Neisseria meningitidis and human cells that promote colonisation and disease

Neisseria meningitidis is the leading cause of bacterial meningitis, a potentially fatal condition that particularly affects children. Multiple steps are involved during the pathogenesis of infection, including the colonisation of healthy individuals and invasion of the bacterium into the cerebrospinal fluid. The bacterium is capable of adhering to, and entering into, a range of human cell types, which facilitates its ability to cause disease. This article summarises the molecular basis of host-pathogen interactions at the cellular level during meningococcal carriage and disease.