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.     

Functional genomics of Neisseria meningitidis pathogenesis

The pathogenic bacterium Neisseria meningitidis is an important cause of septicemia and meningitis, especially in childhood. The establishment and maintenance of bacteremic infection is a pre-requisite for all the pathological sequelae of meningococcal infection. To further understand the genetic basis of this essential step in pathogenesis, we analyzed a library of 2,850 insertional mutants of N. meningitidis for their capacity to cause systemic infection in an infant rat model. The library was constructed by in vitro modification of Neisseria genomic DNA with the purified components of Tn10 transposition. We identified 73 genes in the N. meningitidis genome that are essential for bacteremic disease. Eight insertions were in genes encoding known pathogenicity factors. Involvement of the remaining 65 genes in meningocoocal pathogenesis has not been demonstrated previously, and the identification of these genes provides insights into the pathogenic mechanisms that underlie meningococcal infection. Our results provide a genome-wide analysis of the attributes of N. meningitidis required for disseminated infection, and may lead to new interventions to prevent and treat meningococcal infection.     

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 Opc invasin binds to the cytoskeletal protein α-actinin

Neisseria meningitidis Opc protein is an effective invasin for human endothelial cells. We have investigated novel human endothelial receptors targeted by Opc and observed that Opc-expressing bacteria interacted with a 100 kDa protein in whole-cell lysates of human endothelial and epithelial cells. The identity of the protein was established as α-actinin by mass spectrometry. Opc expression was essential for the recognition of α-actinin whether provided in a purified form or in cell extracts. The interaction of the two proteins did not involve intermediate molecules. As there was no demonstrable expression of α-actinin on the surfaces of any of the eight cell lines studied, the likelihood of the interactions after meningococcal internalization was examined. Confocal imaging demonstrated considerable colocalization of N. meningitidis with α-actinin especially after a prolonged period of internalization. This may imply that bacteria and α-actinin initially occur in separate compartments and co-compartmentalization occurs progressively over the 8 h infection period used. In conclusion, these studies have identified a novel and an intracellular target for the N. meningitidis Opc invasin. Since α-actinin is a modulator of a variety of signalling pathways and of cytoskeletal functions, its targeting by Opc may enable bacteria to survive/translocate across endothelial barriers.     

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.     

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.     

NafA negatively controls Neisseria meningitidis piliation

Bacterial auto-aggregation is a critical step during adhesion of N. meningitidis to host cells. The precise mechanisms and functions of bacterial auto-aggregation still remain to be fully elucidated. In this work, we characterize the role of a meningococcal hypothetical protein, NMB0995/NMC0982, and show that this protein, here denoted NafA, acts as an anti-aggregation factor. NafA was confirmed to be surface exposed and was found to be induced at a late stage of bacterial adherence to epithelial cells. A NafA deficient mutant was hyperpiliated and formed bundles of pili. Further, the mutant displayed increased adherence to epithelial cells when compared to the wild-type strain. In the absence of host cells, the NafA deficient mutant was more aggregative than the wild-type strain. The in vivo role of NafA in sepsis was studied in a murine model of meningococcal disease. Challenge with the NafA deficient mutant resulted in lower bacteremia levels and mortality when compared to the wild-type strain. The present study reveals that meningococcal NafA is an anti-aggregation factor with strong impact on the disease outcome. These data also suggest that appropriate bacterial auto-aggregation is controlled by both aggregation and anti-aggregation factors during Neisseria infection in vivo.     

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.     

Interaction of Neisseria meningitidis with human brain microvascular endothelial cells: role of MAP- and tyrosine kinases in invasion and inflammatory cytokine release

Neisseria meningitidis traversal across the blood-cerebrospinal fluid barrier is an essential step in the pathogenesis of bacterial meningitis. We have previously shown that invasion of human brain microvascular endothelial cells (HBMEC) by meningococci is mediated by bacterial outer membrane protein Opc that binds fibronectin, thereby anchoring the bacterium to the integrin alpha 5 beta 1-receptor on the endothelial cell surface. However, subsequent signal transduction mechanisms essential for or regulated by N. meningitidis adhesion and invasion, or HBMEC responses to N. meningitidis are unknown. In this report we investigated the role of c-Jun N-terminal kinases 1 and 2 (JNK1 and JNK2), p38 mitogen-activated (MAP) kinase and protein tyrosine kinases in endothelial-N. meningitidis interaction. Binding of meningococci to HBMEC phosphorylated and activated JNK1 and JNK2 and p38 MAPK as well as their direct substrates c-Jun and MAP kinase activated kinase-2 (MAPKAPK-2), respectively. Non-invasive meningococcal strains lacking opc gene (opc mutants and sequence type 11 complex meningococci) still activated p38 MAPK, however, failed to activate JNK. Inhibition of JNK1 and JNK2 significantly reduced internalization of N. meningitidis by HBMEC without affecting its adherence. Blocking the endothelial integrin alpha 5 beta 1 also decreased N. meningitidis-induced JNK activation in HBMEC. These findings indicate the crucial role of JNK signalling pathway in N. meningitidis invasion in HBMEC. In contrast, p38 MAPK pathway was important for the control of interleukin-6 (IL-6) and IL-8 release by HBMEC. Genistein, a protein tyrosine kinase inhibitor, decreased both invasion of N. meningitidis into HBMEC and IL-6 and IL-8 release, indicating that protein tyrosine kinases, which link signals from integrins to intracellular signalling pathways are essential for both bacterial internalization and cytokine secretion by HBMEC.     

A novel interpretation of structural dot plots of genomes derived from the analysis of two strains of Neisseria meningitidis

Neisseria meningitidis is the agent of invasive meningococcal disease, including cerebral meningitis and septicemia. Because the diseases caused by different clonal groups (sequence types) have their own epidemiological characteristics, it is important to understand the differences among the genomes of the N. meningitidis clonal groups. To this end, a novel interpretation of a structural dot plot of genomes was devised and applied; exact nucleotide matches between the genomes of N. meningitidis serogroup A strain Z2491 and serogroup B strain MC58 were identified, leading to the specification of various structural regions. Known and putative virulence genes for each N. meningitidis strain were then classified into these regions. We found that virulence genes of MC58 tend more to the translocated regions (chromosomal segments in new sequence contexts) than do those of Z2491, notably tending towards the interface between one of the translocated regions and the collinear region. Within the col-linear region, virulence genes tend to occur within 16 kb of gaps in the exact matches. Verification of these tendencies using genes clustered in the cps locus was sufficiently supportive to suggest that these tendencies can be used to focus the search for and understanding of virulence genes and mechanisms of pathogenicity in these two organisms.     

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.