Natalie J. Griffiths

Cellular and molecular biology of Neisseria meningitidis colonization and invasive disease.

The human species is the only natural host of Neisseria meningitidis, an important cause of bacterial meningitis globally, and, despite its association with devastating diseases, N. meningitidis is a commensal organism found frequently in the respiratory tract of healthy individuals. To date, antibiotic resistance is relatively uncommon in N. meningitidis isolates but, due to the rapid onset of disease in susceptible hosts, the mortality rate remains approx. 10%. Additionally, patients who survive meningococcal disease often endure numerous debilitating sequelae. N. meningitidis strains are classified primarily into serogroups based on the type of polysaccharide capsule expressed. In total, 13 serogroups have been described; however, the majority of disease is caused by strains belonging to one of only five serogroups. Although vaccines have been developed against some of these, a universal meningococcal vaccine remains a challenge due to successful immune evasion strategies of the organism, including mimicry of host structures as well as frequent antigenic variation. N. meningitidis express a range of virulence factors including capsular polysaccharide, lipopolysaccharide and a number of surface-expressed adhesive proteins. Variation of these surface structures is necessary for meningococci to evade killing by host defence mechanisms. Nonetheless, adhesion to host cells and tissues needs to be maintained to enable colonization and ensure bacterial survival in the niche. The aims of the present review are to provide a brief outline of meningococcal carriage, disease and burden to society. With this background, we discuss several bacterial strategies that may enable its survival in the human respiratory tract during colonization and in the blood during infection. We also examine several known meningococcal adhesion mechanisms and conclude with a section on the potential processes that may operate in vivo as meningococci progress from the respiratory niche through the blood to reach the central nervous system.

Meningococcal interactions with the host.

Neisseria meningitidis interacts with host tissues through hierarchical, concerted and co-ordinated actions of a number of adhesins; many of which undergo antigenic and phase variation, a strategy that helps immune evasion. Three major structures, pili, Opa and Opc predominantly influence bacterial adhesion to host cells. Pili and Opa proteins also determine host and tissue specificity while Opa and Opc facilitate efficient cellular invasion. Recent studies have also implied a role of certain adhesin-receptor pairs in determining increased host susceptibility to infection. This chapter examines our current knowledge of meningococcal adhesion and invasion mechanisms particularly related to human epithelial and endothelial cells which are of primary importance in the disease process.

Neisseria meningitidis Opc Invasin Binds to the Sulphated Tyrosines of Activated Vitronectin to Attach to and Invade Human Brain Endothelial Cells

The host vasculature is believed to constitute the principal route of dissemination of Neisseria meningitidis (Nm) throughout the body, resulting in septicaemia and meningitis in susceptible humans. In vitro, the Nm outer membrane protein Opc can enhance cellular entry and exit, utilising serum factors to anchor to endothelial integrins; but the mechanisms of binding to serum factors are poorly characterised. This study demonstrates that Nm Opc expressed in acapsulate as well as capsulate bacteria can increase human brain endothelial cell line (HBMEC) adhesion and entry by first binding to serum vitronectin and, to a lesser extent, fibronectin. This study also demonstrates that Opc binds preferentially to the activated form of human vitronectin, but not to native vitronectin unless the latter is treated to relax its closed conformation. The direct binding of vitronectin occurs at its Connecting Region (CR) requiring sulphated tyrosines Y56 and Y59. Accordingly, Opc/vitronectin interaction could be inhibited with a conformation-dependent monoclonal antibody 8E6 that targets the sulphotyrosines, and with synthetic sulphated (but not phosphorylated or unmodified) peptides spanning the vitronectin residues 43–68. Most importantly, the 26-mer sulphated peptide bearing the cell-binding domain 45RGD47 was sufficient for efficient meningococcal invasion of HBMECs. To our knowledge, this is the first study describing the binding of a bacterial adhesin to sulphated tyrosines of the host receptor. Our data also show that a single region of Opc is likely to interact with the sulphated regions of both vitronectin and of heparin. As such, in the absence of heparin, Opc-expressing Nm interact directly at the CR but when precoated with heparin, they bind via heparin to the heparin-binding domain of the activated vitronectin, although with a lower affinity than at the CR. Such redundancy suggests the importance of Opc/vitronectin interaction in meningococcal pathogenesis and may enable the bacterium to harness the benefits of the physiological processes in which the host effector molecule participates.

IFN‐γ amplifies NFκB‐dependent Neisseria meningitidis invasion of epithelial cells via specific upregulation of CEA‐related cell adhesion molecule 1

Temporal relationship between viral and bacterial infections has been observed, and may arise via the action of virus‐induced inflammatory cytokines. These, by upregulating epithelial receptors targeted by bacteria, may encourage greater bacterial infiltration. In this study, human epithelial cells exposed to interferon‐gamma but not tumour necrosis factor‐alpha or interleukin 1‐beta supported increased meningococcal adhesion and invasion. The increase was related to Opa but not Opc or pili adhesin expression. De novo synthesis of carcinoembryonic antigen‐related cell adhesion molecule 1 (CEACAM1), a major Opa receptor, occurred in epithelial cells exposed to the cytokine, or when infected with Opa‐expressing bacteria. Cell line‐dependent differences in invasion that were observed could be correlated with CEACAM expression levels. There was also evidence for Opa/pili synergism leading to high levels of monolayer infiltration by capsulate bacteria. The use of nuclear factor‐kappa B (NFκB) inhibitors, diferuloylmethane (curcumin) and SN50, abrogated bacterial infiltration of both untreated and interferon‐gamma‐treated cells. The studies demonstrate the importance of CEACAMs as mediators of increased cellular invasion under conditions of inflammation and bring to light the potential role of NFκB pathway in Opa‐mediated invasion by meningococci. The data imply that cell‐surface remodelling by virally induced cytokines could be one factor that increases host susceptibility to bacterial infection.

Meningococcal surface fibril (Msf) binds to activated vitronectin and inhibits the terminal complement pathway to increase serum resistance

Complement evasion is an important survival strategy of Neisseria meningitidis (Nm) during colonization and infection. Previously, we have shown that Nm Opc binds to serum vitronectin to inhibit complement‐mediated killing. In this study, we demonstrate meningococcal interactions with vitronectin via a novel adhesin, Msf (meningococcal surface fibril, previously NhhA or Hsf). As with Opc, Msf binds preferentially to activated vitronectin (aVn), engaging at its N‐terminal region but the C‐terminal heparin binding domain may also participate. However, unlike Opc, the latter binding is not heparin‐mediated. By binding to aVn, Msf or Opc can impart serum resistance, which is further increased in coexpressers, a phenomenon dependent on serum aVn concentrations. The survival fitness of aVn‐binding derivatives was evident from mixed population studies, in which msf/opc mutants were preferentially depleted. In addition, using vitronectin peptides to block Msf–aVn interactions, aVn‐induced inhibition of lytic C5b‐9 formation and of serum killing could be reversed. As Msf‐encoding gene is ubiquitous in the meningococcal strains examined and is expressed in vivo, serum resistance via Msf may be of significance to meningococcal pathogenesis. The data imply that vitronectin binding may be an important strategy for the in vivo survival of Nm for which the bacterium has evolved redundant mechanisms.

Critical determinants of the interactions of capsule‐expressing Neisseria meningitidis with host cells: the role of receptor density in increased cellular targeting via the outer membrane Opa proteins

Neisseria meningitidis capsule is an important virulence determinant required for survival in the blood but is reportedly involved in inhibiting cellular interactions mediated by meningococcal outer membrane adhesins. However, evidence from our previous studies suggested that target receptor density on host cells may determine whether or not capsulate bacteria can adhere via outer membrane proteins such as Opa. To confirm this and evaluate the impact of capsulation on bacterial interactions, we used Opa+ and Opa– derivatives of capsulate and acapsulate meningococcal isolates and transfected cell lines expressing CEACAM1, a receptor targeted by Opa proteins. To assess the extent and rate of cell association, subpopulations of stably transfected Chinese hamster ovary cells with different receptor levels were derived. A quantitative correlation of CEACAM1 levels and Opa‐dependent binding of both capsulate and acapsulate bacteria was demonstrated, which was accelerated at high receptor densities. However, it appears that invasion by Opa+ capsulate bacteria only occurs when a threshold level of CEACAM density has been reached. Target cells expressing high levels of CEACAM1 (MFI c. 400) bound threefold more, but internalized 20‐fold more Opa+ capsulate bacteria than those with intermediate expression (MFI c. 100). No overall selection of acapsulate phenotype was observed in the internalized population. These observations confirm that capsule may not be an adequate barrier for cellular interactions and demonstrate the role of a host factor that may determine capsulate bacterial invasion potential. Upregulation of CEACAMs, which can occur in response to inflammatory cytokines, could lead to translocation of a small number of fully capsulate bacteria across mucosal epithelium into the bloodstream sufficient to cause a rapid onset of disseminated disease. Thus the data also suggest a novel rationale for the epidemiological observations that individuals with prior infectious/inflammatory conditions carry a high risk of invasive meningococcal disease.

Co-ordinate action of bacterial adhesins and human carcinoembryonic antigen receptors in enhanced cellular invasion by capsulate serum resistant Neisseria meningitidis

Neisseria meningitidis (Nm) is a human specific opportunistic pathogen that occasionally penetrates mucosal barriers via the action of adhesins and invasins and evades host immune mechanisms during further dissemination via capsule expression. From in vitro studies, the primary adhesion of capsulate bacteria is believed to be mediated by polymeric pili, followed by invasion via outer membrane adhesins such as Opa proteins. As the latter requires the surface capsule to be down‐modulated, invading bacteria would be serum sensitive and thus avirulent. However, there is recent evidence that capsulate bacteria may interact via Opa proteins when host cells express high levels of carcinoembryonic antigen‐related cell adhesion molecules (CEACAMs), their target receptors. Such a situation may arise following increased circulation of inflammatory cytokines that upregulate certain adhesion molecules on host cells. In this study, using a tetracycline controlled expression system, we have developed cell lines with inducible CEACAM expression to mimic post‐inflammation state of target tissues and analysed the interplay between the three surface components capsule, pili and Opa proteins in cellular interactions. With two distinct cell lines, not only the level but also the rate of adhesion of capsulate Opa‐expressing Nm increased concurrently with CEACAM density. Moreover, when threshold levels of receptor were reached, cellular invasion ensued in an Opa‐dependent manner. In studies with cell lines intrinsically expressing pilus receptors, notable synergism in cellular interactions between pili and Opa of several meningococcal strains was observed and was independent of capsule type. A number of internalized bacteria were shown to express capsule and when directly isolated from host cells, these bacteria were as serum resistant as the inoculated phenotype. Furthermore, we observed that agents that block Opa–CEACAM binding substantially reduced cellular invasion, while maintaining a low level of cellular adhesion. These studies highlight some of the factors that may determine increased host susceptibility to infection by serum resistant phenotypes; and demonstrate the potential of selective inhibition of key interactions in preventing target tissue penetration while maintaining a level of colonization.

Neisseria meningitidis Opc invasin binds to the cytoskeletal protein alpha-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.

Identification and Therapeutic Potential of a Vitronectin Binding Region of Meningococcal Msf

The human pathogen Neisseria meningitides (Nm) attains serum resistance via a number of mechanisms, one of which involves binding to the host complement regulator protein vitronectin. We have shown previously that the Meningococcal surface fibril (Msf), a trimeric autotransporter, binds to the activated form of vitronectin (aVn) to increase Nm survival in human serum. In this study, we aimed to identify the aVn-binding region of Msf to assess its potential as an antigen which can elicit antibodies that block aVn binding and/or possess bactericidal properties. Using several recombinant Msf fragments spanning its surface-exposed region, the smallest aVn-binding recombinants were found to span residues 1-86 and 39-124. The use of further deletion constructs and overlapping recombinant Msf fragments suggested that a region of Msf comprising residues 39-82 may be primarily important for aVn binding and that other regions may also be involved but to a lesser extent. Molecular modelling implicated K66 and K68, conserved in all available Msf sequences, to be involved in the interaction. Recombinant fragments which bound to aVn were able to reduce the survival advantage conveyed by aVn-interaction in serum bactericidal assays. Antibodies raised against one such fragment inhibited aVn binding to Msf. In addition, the antibodies enhanced specific killing of Msf-expressing Nm in a dose-dependent manner. Overall, this study identifies an aVn-binding region of Msf, an adhesin known to impart serum resistance properties to the pathogen; and shows that this region of Msf can elicit antibodies with dual properties which reduce pathogen survival within the host and thus has potential as a vaccine antigen.