Pilus-facilitated adherence of Neisseria meningitidis to human epithelial and endothelial cells: modulation of adherence phenotype occurs concurrently with changes in primary amino acid sequence and the glycosylation status of pilin

Adherence of capsulate Neisseria meningitidis to endothelial and epithelial cells is facilitated in variants that express pili. Whereas piliated variants of N. meningitidis strain C311 adhered to endothelial cells in large numbers (<150 bacteria/cell), derivatives containing specific mutations that disrupt pilE encoding the pilin subunit were both non-piliated and failed to adhere to endothelial cells (<1 bacterium/ cell). In addition, meningococcal pili recognized human endothelial and epithelial cells but not cells originating from other animals. Variants of strain C311 were obtained that expressed pilins of reduced apparent M_r and exhibited a marked increase in adherence to epithelial cells. Structural analysis of pilins from two hyper-adherent variants and the parent strain were carried out by DNA sequencing of their pilE genes. Deduced molecular weights of pilins were considerably tower compared with their apparent M_r values on SDS-PAGE. Hyper-adherent pilins shared unique changes in sequence including substitution of Asn-113 for Asp-113 and changes from Asn-Asp-Thr-Asp to Thr-Asp-Ala-Lys at residues 127-130 in mature pilin. Asn residues 113 and 127 of‘parental’pilin both form part of the typical eukaryotic N-glycosylation motif Asn-X-Ser/Thr and could potentially be glycosylated post-translationally. The presence of carbohydrate on pilin was demonstrated and when pilins were deglycosylated, their migration on SDS-PAGE increased, supporting the notion that variable glycosylation accounts for discrepancies in apparent and deduced molecular weights. Functionally distinct pilins produced by two fully piliated variants of a second strain (MC58) differed only in that the putative glycosylation motif Asn-60-Asn-61-Thr-62 in an adherent variant was replaced with Asp-60-Asn-61-Ser-62 in a non-adherent variant. Fully adherent backswitchers obtained from the non-adherent variant always regained Asn-60 but retained Ser-62. We propose, therefore, that functional variations in N. meningitidis pili may be modulated in large part by primary amino acid sequence changes that ablate or create N-linked glycosylation sites on the pilin subunit.     

High adhesiveness of encapsulated Neisseria meningitidis to epithelial cells is associated with the formation of bundles of pili

Pili are indispensable in adhesion of encapsulated Neisseria meningitidis (MC) to eukaryotic cells. Intrastrain variability with respect to the degree of adhesion is the result of pilin antigenic variation. We have localized the region responsible for this variability to the 20-amino-acid hypervariable domain of pilin. The replacement of an aspartic acid, located in the hypervariable region of a low-adhesive variant by a lysine restored high adhesiveness. To assess whether hyperadhesiveness confered by some pilin variants was related to the generation of a new pilus-associated ligand, high- and low-adhesive variants were purified. In a first step, low- and high-adhesive pilins were fused to maltose binding protein (MBP). These hybrid proteins bound epithelial cells with the same affinity. Truncated MBP pilin fusions identified a cell-binding domain within the 77 residues of the N-terminal end of mature pilin. This region of the protein is common to low- and high-adhesive derivatives used in this work, thus eliminating the possibility that high adhesiveness confered by some pilin variants was because of the generation of a new pilus-associated ligand. Electron-microscopic examination showed that low-adhesive derivatives expressed long and distinct pili and adhered as single cells. In contrast, pili of derivatives expressing high-adhesive pilins, either wild type or mutagenized from the low-adhesive variant, formed large bundles which bound bacteria and caused them to grow as colonies on infected mono-layers. These data demonstrate that aggregative pili promote high adhesiveness of encapsulated MC.     

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.     

Antigenic variation of pilin regulates adhesion of Neisseria meningitidis to human epithelial cells

Pili have been shown to play an essential role in the adhesion of Neisseria meningitidis to epithelial cells. However, among piliated strains, both inter- and intrastrain variability exist with respect to their degree of adhesion to epithelial cells in vitro (Virji et al., 1992). This suggests that factors other than the presence of pili per se are involved in this process. The N. meningitidis pilin subunit undergoes extensive antigenic variation. Piliated low- and high-adhesive derivatives of the same N. meningitidis strain were selected and the nucleotide sequence of the pilin gene expressed in each was determined. The highly adhesive derivatives had the same pilin sequence. The alleles encoding the pilin subunit of the low-adhesive derivatives were completely different from the one found in the high-adhesive isolates. Using polyclonal antibodies raised against one hyperadhesive variant, it was confirmed that the low-adhesive piliated derivatives expressed pilin variants antigenically different from the highly adhesive strains. The role of antigenic variation in the adhesive process of N. meningitidis was confirmed by performing allelic exchanges of the pilE locus between low-and high-adhesive isolates. Antigenic variation has been considered a means by which virulent bacteria evade the host immune system. This work provides genetic proof that a bacterial pathogen, N. meningitidis, can use antigenic variation to modulate their degree of virulence.     

Neisseria meningitidis pili induce type-IIA phospholipase A2 expression in alveolar macrophages

Induction of type-IIA secreted phospholipase A2 (sPLA2-IIA) expression by bacterial components other than lipopolysaccharide has not been previously investigated. Here, we show that exposure of alveolar macrophages (AM) to Neisseria meningitidis or its lipooligosaccharide (LOS) induced sPLA2-IIA synthesis. However, N. meningitidis mutant devoid of LOS did not abolish this effect. In addition, a pili-defective mutant exhibited significantly lower capacity to stimulate sPLA2-IIA synthesis than the wild-type strain. Moreover, pili isolated from a LOS-defective strain induced sPLA2-IIA expression and nuclear factor kappa B (NF-kappaB) activation. These data suggest that pili are potent inducers of sPLA2-IIA expression by AM, through a NF-kappaB-dependent process.     

Expression of the Opc protein correlates with invasion of epithelial and endothelial cells by Neisseria meningitidis

Whereas capsulate strains of Neisseria meningitidis are dependent on pili for adhesion to human endothelial and epithelial cells, strains which lacked assembled pili and were partially capsule-deficient adhered to and invaded human endothelial and epithelial cells if they expressed the Opc protein. Bacteria expressing low or undetectable levels of Opc protein failed to adhere to or invade eukaryotic cells. In addition, the presence of OpaAC751 protein on the surface of bacteria did not increase bacterial interactions with host cells. Association of Opc-expressing bacteria was inhibited by antibodies against Opc. Invasion was dependent on the host-cell cytoskeletal activity and was inhibited by cytochalasin D. In some cells, infected at the apical surface, bacteria emerging from basal surface were detected by electron microscopy. Opc is found in diverse meningococci and may represent a common virulence factor which facilitates adherence and invasion by these bacteria.     

The role of pili in the interactions of pathogenic Neisseria with cultured human endothelial cells

The influence of the two surface structures of Neisseria meningitidis, capsule and pili, in bacterial interactions with human endothelial cells was investigated. Increased association correlated with the presence of pili on bacteria while capsule type had no apparent effect. Strains expressing both Class I and Class II pili associated with endothelial cells in significantly larger numbers compared with the non-piliated variants of the same strains (greater than 10x). Variants of Neisseria gonorrhoeae strain P9 expressing antigenically distinct pili also associated with endothelial cells in larger numbers (greater than 30x) compared with the non-piliated variant. Electron microscopic studies confirmed these data and showed that gonococci were internalized more frequently compared with meningococci. One consequence of increased association was an increase in the cytopathic effect of bacteria on the target cells.     

Fibronectin mediates Opc-dependent internalization of Neisseria meningitidis in human brain microvascular endothelial cells

A central step in the pathogenesis of bacterial meningitis caused by Neisseria meningitidis (the meningococcus) is the interaction of the bacteria with cells of the blood-brain barrier. In the present study, we analysed the invasive potential of two strains representing hypervirulent meningococcal lineages of the ET-5 and ET-37 complex in human brain-derived endothelial cells (HBEMCs). In contrast to previous observations made with epithelial cells and human umbilical vein-derived endothelial cells (HUVECs), significant internalization of encapsulated meningococci by HBMECs was observed. However, this uptake was found only for the ET-5 complex isolate MC 58, and not for an ET-37 complex strain. Furthermore, the uptake of meningococci by HBMECs depended on the presence of human serum, whereas serum of bovine origin did not promote the internalization of meningococci in HBMECs. By mutagenesis experiments, we demonstrate that internalization depended on the expression of the opc gene, which is present in meningococci of the ET-5 complex, but absent in ET-37 complex meningococci. Chromatographic separation of human serum proteins revealed fibronectin as the uptake-promoting serum factor, which binds to HBMECs via alpha 5 beta 1 integrin receptors. These data provide evidence for unique molecular mechanisms of the interaction of meningococci with endothelial cells of the blood-brain barrier and contribute to our understanding of the pathogenesis of meningitis caused by meningococci of different clonal lineages.     

Nasal secretions of Neisseria lactamica carriers have an inhibitory effect on Neisseria meningitidis attachment to human oroepithelial cells

Nasal secretions of volunteers colonized by N. lactamica impaired the attachment of N. lactamica and of meningococci of groups A and B to oroepithelial cells. Bacterial adherence was found to be mediated by nonpiliated adhesins with antigen(s) which probably are shared by the strains tested. Although a strong attachment-inhibiting activity arises in their nasal secretions, volunteers remained colonized by N. lactamica. This evidence suggest that the eradication of Neisseria carriage is a multifactorial event.     

The effect of gangliosides on the adhesive interaction of Neisseria meningitidis with human cells

The influence of a number of gangliosides and sialic acid on the adhesive interaction of meningococci and human cells have been studied. Sialic acid has been found to produce no influence on adhesion, and the preliminary treatment of meningococci with gangliosides or their preparations suppresses the capacity of meningococci for attachment to epithelial cells and erythrocytes. At the same time the degree of the inhibition of adhesion depends on the kind and concentration of gangliosides. On the contrary, after the treatment of target cells with gangliosides (1.25 micrograms/ml) the adhesion indices of meningococci with respect to these cells increase 5- to 8-fold. These data are indicative of the participation of gangliosides in the adhesive interaction of meningococci and human cells.     

I-domain-containing integrins serve as pilus receptors for Neisseria gonorrhoeae adherence to human epithelial cells

Two pilus receptors are identified for the pathogenic Neisseria, CD46 and complement receptor 3. An intimate association between the asialoglycoprotein receptor and gonococcal lipooligosaccharide mediates invasion of primary, male urethral epithelial cells (UECs); however, studies to identify pilus receptors on these cells have not been performed. Based on our previous studies we reasoned that the I-domain-containing (IDC), alpha(1)- and alpha(2)-integrins might serve as pilus receptors on UECs and on urethral tissue. Confocal microscopy revealed colocalization of pilus with alpha(1) and alpha(2) integrins on UECs and tissue. We found that recombinant I-domain and antibodies directed against the alpha(1)- and alpha(2)-integrins inhibited gonococcal association with UECs and with immortal cell lines of variable origin. Gonococcus-integrin colocalization occurred at early time points post infection, but this interaction dissociated with extended infection. Similarly, Western Blot analyses revealed that gonococcal pilin coimmunoprecipitates with alpha(1)- and alpha(2)-integrins. However, studies performed in parallel and that were designed to capture CD46-pilus immune complexes indicated that a CD46-pilus interaction did not occur. Collectively, these data suggest that while CD46 might be able to bind gonococcal pilus, IDC integrins are preferentially used as the initial docking site for gonococci on UECs, on urethral tissue and on some immortal cell lines.     

Bacterial adherence to human endothelial cells

The adult respiratory distress syndrome (ARDS) is frequently caused by exposure of the lung endothelium to circulating endotoxin (lipopolysaccharide, LPS) and pulmonary infections frequently develop during the course of ARDS. The present studies demonstrate that LPS and interleukin 1 (IL-1, a mediator released by endothelial cells after exposure to LPS) enhance the adherence of Staphylococcus aureus to human umbilical vein endothelial cells. gamma-Interferon, another mediator that induces expression of some cell surface antigens on endothelial cells, had no effect on bacterial adherence. The adherence of bacteria to endothelium was increased by prior opsonization of the bacteria with fresh human serum and was reduced by prior absorption of the serum with bacteria before the use of the serum for opsonization. The capacity of LPS to increase bacterial adherence was time dependent and was maximally expressed after 6 h of exposure; it was blocked by exposure of endothelial cells to LPS in the presence of reduced temperature or dactinomycin (Actinomycin D). These observations suggest that circulating LPS not only can trigger the development of ARDS but also may predispose the lung to the development of pulmonary infections by increasing adherence of bacteria to endothelium.     

A functional two-partner secretion system contributes to adhesion of Neisseria meningitidis to epithelial cells

Neisseria meningitidis is a frequent commensal of the human nasopharynx causing severe invasive infections in rare cases. A functional two-partner secretion (TPS) system in N. meningitidis, composed of the secreted effector protein HrpA and its cognate transporter HrpB, is identified and characterized in this study. Although all meningococcal strains harbor at least one TPS system, the hrpA genes display significant C-terminal sequence variation. Meningococcal genes encoding the TPS effector proteins and their transporters are closely associated and transcribed into a single mRNA. HrpA proteins are translocated across the meningococcal outer membrane by their cognate transporters HrpB and mainly released into the environment. During this process, HrpA is proteolytically processed to a mature 180-kDa form. In contrast to other known TPS systems, immature HrpA proteins are stable in the absence of HrpB and accumulate within the bacterial cell. A small percentage of mature HrpA remains associated with the bacteria and contributes to the interaction of meningococci with epithelial cells.     

Variation in the expression of pili and outer membrane protein by Neisseria meningitidis during the course of meningococcal infection

The occurrence of antigenic shift during meningococcal infection has been investigated by comparison of paired isolates obtained from the blood, cerebrospinal fluid or nasopharynx of patients. Isolates from any individual produced identical DNA 'fingerprints' and showed stability in expression of both class 2 outer membrane protein and an antigen common to pathogenic Neisseria, confirming their origin as a single strain. One of the four strains examined produced variants which differed in the molecular mass of their class 5 outer membrane proteins. Three of the strains produced pili containing the epitope recognized by monoclonal antibody SM1 and two of these gave rise to variants which expressed pili of differing subunit molecular masses. The two variants of the remaining strain produced pilins lacking the common epitope detected by antibody SM1 but radioimmune precipitation with polyclonal anti-pilus antiserum revealed that variation in the molecular mass of the pilin expressed also occurred with this second class of pili. Antigenic variation in expression of both class 5 outer membrane proteins and pili therefore appears to be a common occurrence during meningococcal infection.     

Neisseria meningitidis infection of human endothelial cells interferes with leukocyte transmigration by preventing the formation of endothelial docking structures

Neisseria meningitidis elicits the formation of membrane protrusions on vascular endothelial cells, enabling its internalization and transcytosis. We provide evidence that this process interferes with the transendothelial migration of leukocytes. Bacteria adhering to endothelial cells actively recruit ezrin, moesin, and ezrin binding adhesion molecules. These molecules no longer accumulate at sites of leukocyte-endothelial contact, preventing the formation of the endothelial docking structures required for proper leukocyte diapedesis. Overexpression of exogenous ezrin or moesin is sufficient to rescue the formation of docking structures on and leukocyte migration through infected endothelial monolayers. Inversely, expression of the dominant-negative NH(2)-terminal domain of ezrin markedly inhibits the formation of docking structures and leukocyte diapedesis through noninfected monolayers. Ezrin and moesin thus appear as pivotal endothelial proteins required for leukocyte diapedesis that are titrated away by N. meningitidis. These results highlight a novel strategy developed by a bacterial pathogen to hamper the host inflammatory response by interfering with leukocyte-endothelial cell interaction.     

Neisseria meningitidis NadA is a new invasin which promotes bacterial adhesion to and penetration into human epithelial cells

Neisseria meningitidis is a human pathogen, which is a major cause of sepsis and meningitis. The bacterium colonizes the upper respiratory tract of approximately 10% of humans where it lives as a commensal. On rare occasions, it crosses the epithelium and reaches the bloodstream causing sepsis. From the bloodstream it translocates the blood-brain barrier, causing meningitis. Although all strains have the potential to cause disease, a subset of them, which belongs to hypervirulent lineages, causes disease more frequently than others. Recently, we described NadA, a novel antigen of N. meningitidis, present in three of the four known hypervirulent lineages. Here we show that NadA is a novel bacterial invasin which, when expressed on the surface of Escherichia coli, promotes adhesion to and invasion into Chang epithelial cells. Deletion of the N-terminal globular domain of recombinant NadA or pronase treatment of human cells abrogated the adhesive phenotype. A hypervirulent strain of N. meningitidis where the nad A gene was inactivated had a reduced ability to adhere to and invade into epithelial cells in vitro. NadA is likely to improve the fitness of N. meningitidis contributing to the increased virulence of strains that belong to the hypervirulent lineages.     

Interactions of Neisseria meningitidis with cells of the human meninges

The interaction of Neisseria meningitidis with the meninges that surround and protect the brain is a pivotal event in the progression of bacterial meningitis. Two models of the human meninges were established in vitro, using (i) sections of fresh human brain and (ii) cultures of viable cells grown from human meningiomas. Neisseria meningitidis showed a specific predilection for binding to the leptomeninges and meningeal blood vessels in human brain and not to the cerebral cortex. There was a close correlation between the adherence of different Neisseria species to leptomeninges and cultured cells. The major ligand that mediated adherence was the pilus, and pilin variation modulated the interactions. The presence of Opa protein increased the association of Cap+ meningococci that expressed low-adhesive pili, but did not influence the association of high-adhesive pili. In contrast, Opc did not influence the adherence of Cap+ meningococci, whereas loss of capsule was associated with a more intimate interaction between the bacteria and the meningioma cell that was not apparent with Cap+ meningococci. There was no evidence of internalization of meningococci by meningioma cells in vitro, an observation that is consistent with the barrier properties of the leptomeninges to N. meningitidis observed in vivo.     

Kingella kingae expresses type IV pili that mediate adherence to respiratory epithelial and synovial cells

Kingella kingae is a gram-negative bacterium that colonizes the respiratory tract and is a common cause of septic arthritis and osteomyelitis. Despite the increasing frequency of K. kingae disease, little is known about the mechanism by which this organism adheres to respiratory epithelium and seeds joints and bones. Previous work showed that K. kingae expresses long surface fibers that vary in surface density. In the current study, we found that these fibers are type IV pili and are necessary for efficient adherence to respiratory epithelial and synovial cells and that the number of pili expressed by the bacterium correlates with the level of adherence to synovial cells but not with the level of adherence to respiratory cells. In addition, we established that the major pilin subunit is encoded by a pilA homolog in a conserved region of the chromosome that also contains a second pilin gene and a type IV pilus accessory gene, both of which are dispensable for pilus assembly and pilus-mediated adherence. Upon examination of the K. kingae genome, we identified two genes in physically separate locations on the chromosome that encode homologs of the Neisseria PilC proteins and that have only a low level homology to each other. Examination of mutant strains revealed that both of the K. kingae PilC homologs are essential for a wild-type level of adherence to both respiratory epithelial and synovial cells. Taken together, these results demonstrate that type IV pili and the two PilC homologs play important roles in mediating K. kingae adherence.