Meningococcal interactions with the host

Vaccine 27S (2009) B78–B89

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Meningococcal interactions with the host Etienne Carbonnelle a,1,2 , Darryl J. Hill c,1 , Philippe Morand a,2 , Natalie J. Griffiths c , Sandrine Bourdoulous b , Isabel Murillo c , Xavier Nassif a,2 , Mumtaz Virji c,∗ a

INSERM, unité 570, Université Paris Descartes, Faculté de Médecine, 156 rue de Vaugirard, 75015 Paris, France Institut Cochin, Département de Biologie Cellulaire, INSERM, Unité 567, CNRS, UMR8104, Université Paris Descartes, 22 rue Méchain, 75014 Paris, France c Department of Cellular and Molecular Medicine, School of Medical Sciences, University Walk, University of Bristol, Bristol BS8 1TD, UK b

a r t i c l e

i n f o

Keywords: Adhesion Receptors Pathogenic mechanisms

a b s t r a c t 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. © 2009 Elsevier Ltd. All rights reserved.

1. Introduction Neisseria meningitidis has evolved attributes that generally enable harmless colonisation of the human nasopharynx, its only known niche; and in most disease cases, asymptomatic colonisation of the nasopharynx precedes infection. Colonisation itself is achieved via versatile and dynamic adhesion and immune evasion mechanisms. Whilst antigenic and phase variations of surface structures, including the adhesins, enable bacteria to avoid immune detection, adhesion is maintained through redundancy, i.e. the expression of multiple adhesins, which also aids in colonisation of distinct niches. Although generally regarded as an extracellular pathogen, in vitro studies have shown that N. meningitidis entry into cultured human cells can occur via several distinct receptor–adhesin interactions [1–6]. Studies using nasopharyngeal epithelial organ cultures have also shown that meningococci are able to invade these tissues [8]. In addition, although not clearly demonstrated, meningococcal intracellular location has been implied within mucosal epithelial cells in one study that examined tonsillar biopsies in which meningococci were observed beneath epithelial surfaces [7]. Whether intracellular location in inflamed tissues often used for these investigations represents ‘the norm’ needs to be considered. Studies reported below suggest that

∗ Corresponding author. Tel.: +44 0 117 33 12035; fax: +44 0 117 33 12035. E-mail addresses: [email protected] (X. Nassif), [email protected] (M. Virji). 1 These authors have equally contributed to the work. 2 Tel.: +33 1 40 61 56 78; fax: +33 1 40 61 55 92. 0264-410X/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2009.04.069

inflammatory conditions of the host may influence the balance of the bacteria–host relationship that can change from large surface location to significant cellular penetration. Nonetheless, even low levels of internalisation in a healthy host could benefit bacterial survival through evasion of host phagocytic and antibody/complement mediated killing mechanisms. Internalisation may also provide access to a new source of nutrients and enable bacteria to interfere with host cell functions including innate response to the bacterial presence. While in vitro studies have helped unravel some of the complexities of meningococcal interactions with its unique niche, this specificity has prevented the use of animals as models and our understanding of in vivo situations remains unclear. To cause disease, the bacterium enters systemic circulation and multiplies within the host. For this, it requires a “permissive” often immunocompromised host [9]. During the passage across vascular endothelial and the blood–brain/blood–csf barriers, meningococci encounter additional human cells and serum factors with which they interact, often displaying specificity [10]. Since colonisation is the normal state of existence for meningococci, it may be surmised that the bacterial attributes which enable colonisation also aid their dissemination throughout the body. The major adhesins of N. meningitidis include the polymeric pili and the outer membrane opacity proteins, Opa and Opc [1,2,6,11,12]. In addition, a number of other adhesins have been recently identified (Table 1). There is a hierarchy in the utility of adhesins in the context of bacterial phenotype and location. Bacterial capsules tend to be highly hydrated and whilst it is suggested that they may help protect some air-borne bacterial species from desiccation, substantiation of this notion is required for N. meningitidis [13,14]. Interestingly, a significant number of mucosal N. meningi-

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Table 1 Neisseria meningitidis adhesions. Adhesin

Alternative name

Gene designation

Pili Opa

Fimbriae Class 5 outer membrane Proteins Class 5 C protein, OpcA

pilE1/pilE2 (pilin subunit) opa

Opc NhhA (Neisseria hia homolog A) App (adhesion and penetration protein) MspA (meningococcal serine protease A) NadA (neisserial adhesin A)

HrpA (haemagglutin/haemolysin-related protein A)

opcA nhha/GNA992 app NMB1985 NMB1998 NMB1994

TpsA (two partner secretion system protein A)

tidis isolates lack capsule genes [14–16]. However clinically, by far the most important meningococcal phenotype is capsulate which is required for survival in the bloodstream. Capsule, by its juxtaposition, also masks the outer membrane adhesins to some extent [2–4,17] and in capsulate meningococci, the polymeric pili, which traverse the capsule facilitate initial attachment to mucosal epithelial cells. In the respiratory tract, capsule becomes less important as is evident from frequent isolation of acapsulate N. meningitidis from the nasopharynx which may arise as a result of phase variation [18,19] or by down-modulation under the influence of environmental factors [20]. In such a phenotype, the outer membrane adhesins such as Opa and Opc become effective. The latter are also effective invasins and may synergise with pili resulting in enhanced infiltration of the target tissues [3,4]. Even though various regulatory pathways exist for gene expression, phase variation is the usual mechanism by which N. meningitidis controls the expression of the major adhesins. It is manifested via a process involving DNA slippage (commonly referred to as Slipped Strand Mispairing, SSM) engendered by repetitive sequences of nucleotides either within the open reading frame (leading to translational control of expression) or upstream of the gene (affecting the level of transcription) [21,22]. In the case of neisserial pili, several components involved in pilus biogenesis (see below) may affect pilus expression; but one major mechanism affecting the expression and antigenic variation of the major pilus subunit PilE, involves inter and intra-genomic RecA-dependent recombination events between one of several pilS (silent) pilin genes and pilE, the expressed pilin gene. This represents another example of remarkable similarities between N. meningitidis and N. gonorrhoeae [23–26] Below essential features of the currently known mechanisms of bacteria–host interactions, particularly at the human epithelial and endothelial barriers that may determine colonisation and dissemination are presented. In addition, the key surface protein structures that mediate these interactions are discussed in some depth. 2. Type IV pilus-mediated adhesion and signalling Type IV pili (Tfp) are widely found in Gram negative bacteria including pathogenic Neisseria and have been shown in some cases to be associated with diverse phenotypes or functions such as biofilm formation [27] and bacteriophage infection [28]. The latter two functions have been demonstrated in Pseudomonas spp. Tfp influence the dynamics of the adherence by powering a form of cell locomotion by crawling over a surface known as twitching motility [29] and by promoting the formation of bacterial aggregates [30]. In addition, Tfp are essential for the natural competence and DNA transformation, a property that contributes to their virulence by promoting exquisite genetic adaptability [31]. A remarkable

hrpA1(NMB0497), hrpA2(NMB1779) NMA0668, NMC0444, tpsA

Homology

Reference [157] [157]

H. influenzae Hsf/Hia E. coli AIDA-1 homolog H. influenzae Hap IgA1 protease Oligomeric coiled coil adhesin (Oca) family e.g., M. catarrhalis UspA B. pertussis FHA

[108] [141] [143] [145] [146] [143] [148]

[154] [158]

property of Tfp is their ability to retract into the bacterium from which they originate, via the action of the force-generating ATPase PilT [30,32]. Tfp retraction is essential to bacterial motility (twitching motility), competence for DNA transformation, and pilus-associated signalling to host cells [33]. 2.1. Pilus biogenesis Type IV pili are homopolymeric filaments found on N. meningitidis. Tfp are highly dynamic structures which undergo rapid cycles of extension and retraction. They extend from the inner membrane to the bacterial surface, passing through the outer membrane. They ´˚ and flexible filaments that can be up to several are thin (50–80 A) micrometers long and can sustain considerable mechanical stress. The major subunit of the fibre is pilin (PilE), the helical assembly of which leads to the formation of the fibre. The components involved in Tfp assembly, retraction and specific functions are distributed around the bacterial membranes, from the inner side of the cytoplasmic membrane to the outer membrane. Fifteen genes, scattered throughout the genome, are required for pilus biogenesis [34]. One set of genes, pilM, pilN, pilO, pilP, pilQ, is organised in an operon. It has been shown that Tfp biogenesis can be resolved into four genetically dissociable steps: assembly, functional maturation, emergence on the cell surface and counter-retraction. The protein products of some of these genes when mutated give rise to a nonpiliated phenotype which can be reversed in strain deficient for pilus retraction (Fig. 1). These proteins are therefore not required for the assembly of the fibre. When pili are retracted, the pilin subunits are stored within the cytoplasmic membrane. These different steps and the corresponding proteins are described in Fig. 1. 2.1.1. PilE and PilD The major neisserial pilus subunit, the pilin, is encoded by the pilE gene. Along with an intact copy of pilE, up to eight loci containing truncated copies of the pilin gene, named pilS (silent), are found on the chromosome. pilS copies are not expressed but serve as a source of variant pilin gene sequences which occasionally recombine with pilE, leading to the expression of novel PilE variants. Pilin is synthesised as a preprotein. A short leader sequence (5–6 residues) is cleaved by the prepilin peptidase PilD [35]. PilD is a leader peptidase localised to the inner membrane that specifically recognises the N-terminal part of prepilin and of prepilin-like molecules [36]. PilD also methylates the N-terminal phenylalanine of the mature protein product, which relies on the presence of a glutamic acid in position +5 of the substrate. However, the lack of methylation has little effects on Tfp assembly [37]. The mature protein is approximately 145–160 residues long after prepilin peptidase processing. It has a conserved hydrophobic N-terminal 25 residue and a carboxy-terminal disulphide bond.

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Fig. 1. Implication of the N. meningitidis Pil components in the different steps of Tfp biogenesis fibre assembly, functional maturation and counter retraction of fibre, retraction powered by the PilT protein and emergence of the fibres on the cell surface. At the inner membrane-periplasm interface, the pilin subunits (PilE) are assembled from a platform complex (PilD, PilF, PilM, PilN, PilO, PilP) and the growing fibre is translocated to the cell surface via the secretin (PilQ). It has been recently demonstrated that PilP, which co-purifies with the inner membrane, interacts with PilQ. Two inner membrane ATPases, PilF and PilT, promote pilus elongation and pilus retraction respectively. Different components are required to counteract pilus retraction (PilC1/C2, PilG, PilH, PilI, PilJ, PilK PilW). PilC1/C2 and PilW are located in the outer membrane whereas PilG is in the inner membrane. Some components are important for the function of the pilus (PilC1, PilW, PilI, PilJ, PilK, PilX, PilV and ComP). Among the minor pilin components (PilH, PilI, PilJ, PilV, PilX and ComP), PilV, X and ComP are incorporated in the fibre [52].

The conserved hydrophobic N-terminal moiety that is embedded in the core of the pilus is involved in the cohesion of the subunits as a fibre. The polymorphic C-terminus of pilin is partially exposed at the surface of the assembled pilus. The pilin also harbours two cysteine residues that build a disulfide bridge, involved in the formation of the surface accessible, variable and immune dominant part of the pilus. Pilins are therefore packed through internal hydrophobic interactions between conserved N-terminal ␣ helices, leaving hypervariable C-terminal globular regions exposed [38]. PilE undergoes several post translational modifications of serine residues including glycosylation at position 63 and an alphaglycerophosphate at position 93 [39–42]. Whereas at position 68, the residue has been reported to be substituted with phosphate, phosphoethanolamine or phosphorylcholine [41,43]. The roles of these various modifications remain unclear. Concerning the glycosylation, the structure of the sugar is different depending on the strain [44,45]. Pilin glycosylation in N. meningitidis is under the control of the pgl gene cluster [46–49], which encodes genes characterised by length polymorphisms. The meningococcal pglA is subject to phase variation due to the presence of a poly-G stretch, which is not the case for pgtA, the gonococcal homolog to pglA [50]. 2.1.2. PilG PilG is an inner membrane protein required for piliation [51]. It prevents pilus retraction, but does not seem to be required for pilus biogenesis since apparently normal Tfp are seen in double pilT pilG mutants [52]. The 3D reconstruction of PilG provides a clear evidence of a tetrameric quaternary arrangement [53]. 2.1.3. PilF and PilT Two inner membrane-associated ATPases, PilF and PilT, are thought to antagonistically promote extension and retraction of Tfp; PilF being involved in elongation [36] and PilT in retraction [30,54]. Nucleotide (NTP)-binding proteins are basic components of all Tfp machineries. They usually contain a “walker box” for the binding of ATP and belong to the family of AAA ATPases (ATPases associated with various cellular activities), which includes chap-

erones and mechanoenzymes (for a review, see [55]). Unlike PilF [36], PilT is dispensable for assembly and expression of Tfp on the cell surface, but is required for their retraction. PilT was initially reported as an effector for transformation competence and twitching motility [54], and plays a key role in the interaction with the host cell [56], since its absence prevents the onset of intimate adhesion. Like other AAA ATPases, PilT has a hexameric structure [57] and, at least in vitro, hydrolyzes ATP [58,59]. In vivo, PilT can be found associated with the inner membrane and in the cytoplasm [59,60]. PilF is associated with the inner membrane, and its functionality relies on the integrity of the “walker box”. 2.1.4. PilQ and the secretin complex The secretin PilQ supports Tfp extrusion and retraction, but it also requires auxiliary proteins for its assembly and localisation in the outer membrane. The secretin PilQ is a member of a large family of integral outer membrane proteins with conserved C-terminal domains [61–63]. The meningococcal secretin PilQ oligomer consists of 12 identical monomers [64–68]. PilQ spontaneously associates with Tfp when they are incubated together in vitro; the PilQ oligomer binds at one end of the pilus fibre, which potentially fills the central chamber [67]. Involvement of PilP in gonococcal pilus biogenesis was first reported by Drake et al. [69], they showed that pilP null mutant were non-piliated. The pilP gene is located upstream of pilQ, in a cluster with other pilus biogenesis genes: pilM, pilN, and pilO [70]. The inactivation of the pilP gene in its 3 region can therefore lead to a reduced quantity of pilQ transcript due to a polar effect. It has been shown that PilP co-purifies with inner membrane components and that the N- and C-terminal regions of PilP recognise the central part of the PilQ monomer [71]. PilP is predicted to be a lipoprotein, which most likely anchors it to the inner membrane [67,71]. It has now been shown that PilQ does not need PilP for its membrane localisation and/or stabilisation [34,71]. One candidate for this function could be PilW, which is essential for Tfp biogenesis in N. meningitidis and PilW affects the stability of the PilQ complex [34], suggesting specific interactions between PilQ and PilW [72].

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2.1.5. PilC proteins The PilC proteins play a crucial but still enigmatic role [12,73–76]. Two alleles were originally discovered [73]. Expression of both variants is subject to phase variation as a result of frameshift in homopolymeric “G” tracts located in the open reading frames [73]. Phase variation of PilC has also been shown in meningococcal clinical samples [77]. PilC-null strains show impaired pilus expression and lack the ability for transformation competence. In N. meningitidis, only PilC1 is required for adhesion. PilC2, which is expressed independently of PilC1, fails to promote adhesion despite identical functions in pilus expression and transformation competence [12,78]. Abolition of pilT in a PilC-null background restores piliation, confirming the hypothesis that PilC acts as an antagonist of PilT by preventing PilT-mediated retraction [54,79]. 2.1.6. Prepilin-like proteins Prepilin-like molecules, with pilin-like N-terminal sequences, play important roles in Tfp biology [80]. In pathogenic Neisseria, there are seven proteins cleaved by the prepilin-peptidase PilD: PilH, PilI, PilJ, PilK, ComP, PilV and PilX (PilL in the gonococcus). ComP, PilV and PilX, which have canonical PilD cleavage motifs and mature lengths similar to PilE, modulate Tfp-related functions. ComP and PilV are necessary for DNA transformation [81] and adhesion to human cells [82], respectively, whereas PilX in N. meningitidis is essential for bacterial aggregation and adhesion [83]. These proteins provide models to investigate the relation between the structure and the function of pilin-like proteins because the phenotypes associated with the corresponding mutants are not obscured by the absence of pili. The 3D structure of PilX shows that it closely resembles types IV pilins [84]. It has been proposed that when interacting with another PilX and under tension, PilX subunits may adopt a different conformation and prevent further slippage of pili. In the absence of PilX, pilus retraction allows the disruption of inter-bacterial Tfp-mediated interactions, and no aggregation is observed [85].

Fig. 2. Schematic representation of the signalling pathways triggered by N. meningitidis leading to the formation of the cortical plaque. Ezrin and moesin are key components of these complexes. These proteins are members of the ezrin/radixin/moesin (ERM) family that acts as a linker between the plasma membrane and the actin cytoskeleton [90,92,159]: Ezrin and moesin control the organization of the cortical plaques by interacting by their amino-terminal domain with the cytoplasmic domain of transmembrane proteins (so-called ERM binding proteins), such as CD44, ICAM-1, VCAM-1, E-selectin, and interact with F-actin by their carboxy-terminal domains. The recruitment and phosphorylation of cortactin at the N. meningitidis entry site is required for the formation of the actin-rich cell projections that promote efficient bacterial uptake [91]. Tyrosine phosphorylation of cortactin induced by N. meningitidis results from the clustering and activation of the host cell tyrosine kinase receptor ErbB2 and the downstream activation of the kinase src [89]. The interaction of N. meningitidis with human endothelial cells leads to the activation of ErbB2, indicating that N. meningitidis induces ErbB2 activation most likely via formation of ligand-independent ErbB2 homodimers, occurring by a mechanism which is still unresolved. In addition, the high forces that are generated by the retraction of the fibre are thought to assist close association of the pathogen with the host cell. Moreover, such forces are large enough to cause membrane protrusions [160,161].

2.2. Mechanism of pilus-mediated interaction Capsulate non-piliated bacteria interact with epithelial and endothelial cells inefficiently unless they express the type IV pili. The efficiency of the first step of adhesion of capsulate meningococci is dependent on the target cell type as well as the pilus structure [86]. Given the appropriate combination, pili mediate one of the most efficient meningococcal interactions observed in vitro [87]. The molecular mechanism responsible for the initial attachment of individual diplococci to the cells is still not fully understood. One report has suggested that the PilC1 protein could carry this cell binding domain [75], this hypothesis was based on inhibition of adhesion using purified PilC molecules. However, non-adhesive non-piliated isolates of serogroup B strain MC58 with high PilC expression, and piliated adhesive isolates with barely detectable PilC expression have been described [87]. In addition, another PilC+ /PilE- strain, in which PilC location has been demonstrated in the outer membrane, is unable to interact with eukaryotic cells (X. Nassif, unpublished observations), thus raising doubts on the role of PilC as an adhesin. As mentioned above, the minor pilin PilX is essential for promoting inter-bacterial interactions and pilX mutants, which are unable to from aggregates, have a phenotype similar to that of non-piliated strains even though they have bundled pili. It appears that these inter-bacterial interactions generated by the Tfp allow the bacteria, when multiplying, to spread onto the cells, which in turn relies on the ability of bacteria to retract their pili. Following the interactions between bacteria and target cells, in some systems, pili have been shown to retract over a period and eventually adherent

meningococci appear non-piliated [30,56]. Measurements using optical tweezers showed that retraction of a single Tfp generates forces up to 110 pN, in a transient manner for each fibre. Bundles of Tfp, which result from the association of 8–10 pili, act as coordinated retractable units. Filament bundles produce retraction forces that generate forces in the nanonewton range [88]. The successive extension, binding and retraction of Tfp enable bacteria to move by twitching motility and spread on the apical surface of the host cells. 2.3. Host cell rearrangements mediated by Tfp Several relatively recent studies have lead to a much greater understanding of the cellular events involved in N. meningitidis invasion processes of human endothelial cells, and of the complex molecular signalling pathways induced by the pathogen which lead to its uptake in non phagocytic cells [89–92] (Fig. 2). Adhesion of N. meningitidis to endothelial cells promotes the local formation of membrane protrusions reminiscent of epithelial microvilli structures that surround bacteria. The formation of these membrane protrusions by capsulate N. meningitidis results from the organization of specific molecular complexes, referred to as cortical plaques, beneath bacterial colonies [90,93,94] (Fig. 2). While only a small fraction of bacteria colonising the apical surface are internalised [1,90], it is believed that the microvillilike structures induced by the signalling linked to pilus-mediated adhesion initiate internalisation of these bacteria within intracellular vacuoles [90]. Interestingly, the formation of such protrusions was also observed ex vivo, by transmission electron microscopy

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Fig. 3. An overview of known epithelial receptors of non-pilus adhesins of N. meningitidis. Although receptors have been reported for Opa, Opc and NhhA as indicated in the diagram, those of the minor adhesins, App, NadA, MspA and Tps (HrpA), remain to be determined. HSPG: heparan sulphate proteoglycans.

analysis of brain sections from a child who died from fulminant meningitis [10], suggesting that such morphological modifications of the host cell membrane may be responsible for the internalisation of a small fraction of adhesive bacteria. These observations therefore raised the hypothesis that N. meningitidis could cross the endothelial barrier by a transcytosis pathway. In addition, recent studies have provided evidence that N. meningitidis induce host cell surface reorganisation by promoting invagination within cellular interstices. These rearrangements confer to bacterial colonies formed at the apical surface of host cells the ability to resist the high mechanical forces exerted by the blood flow [95,96]. Exploitation of the host-cell signalling pathways could therefore be pivotal in promoting intimate attachment to the cell surface, avoiding bacterial detachment under flow conditions in the blood as well as in the nasopharynx. Considering that at least in vitro, both on epithelial and endothelial cells, most of the bacteria interacting with cells are found localised at the apical surface, it is likely that the signalling induced by pilus-mediated adhesion has been selected during the evolution to allow intimate attachment and therefore bacterial colonisation of its only niche, the human nasopharynx. Regarding the meningeal invasion, one hypothesis is that the small fraction of internalised bacteria transcytose through the monolayer of endothelial cells, alternatively the signalling induced by pili may lead to disruption of the intercellular junctions. In addition, when large numbers of piliated bacteria are present, meningococci cause damage to human cells via LPS-mediated cytopathic damage, a process that has been shown to be enhanced with piliated bacteria [97]. 2.4. Host cell receptors for meningococcal pili CD46, a transmembrane glycoprotein, which regulates complement activation was described as a cellular receptor for Type IV pili of Neisseriae (N. meningitidis and N. gonorrhoea) [98]. Subsequently, a transgenic CD46 mouse was developed to model the interaction of N. meningitidis with endothelial cells of the blood brain barrier, and meningitis [99]. It has however been demonstrated that pilus-

mediated adherence of N. gonorrhoea does not correlate with CD46 expression [100,101] and is unaffected by exogenous CD46 expression or by depletion of CD46 on epithelial cells [101]. It is therefore likely that one or several cellular components beside CD46 is/are responsible for the interactions observed between pili and human barrier cells. In conclusion, pili are one of the major attributes allowing interaction of N. meningitidis with human mucosal epithelial cells and necessary for initial colonisation. The ability of pili to aggregate bacterial cells and to induce twitching motility via pilus retraction appears to be important processes in the colonisation of the apical surfaces of the cells. On the other hand, the nature of the pilus component/s that induce/s the initial interaction remains unclear. The signalling events stimulated by pili allow intimate interactions between the host cells and bacteria, enabling attached bacteria also to resist the various fluid flows in the extracellular environment. This property is likely to be required for the bacteria not only to colonise the nasopharynx; but once in the bloodstream, it may also help prevent bacterial detachment from the vessel walls. The importance of pili in meningococcal colonisation may suggest their potential as vaccine antigens but the frequency of their variation hampers any such utility.

3. Non-pilus adhesins Besides pili, at least seven other N. meningitidis adhesins have been described (Table 1). The most studied of these adhesins are the opacity proteins Opa and Opc, for which several host cell targeting mechanisms are known (Fig. 3). The available in vitro studies also show the other more recently identified adhesins to be several orders of magnitude less effective in mediating interactions with target cells compared with the opacity proteins or pili; although, their functions in vivo may possibly be more efficient. However, the lack of adequate animal models for the human-specific bacterium has hampered studies to describe in vivo functions of adhesins in general.

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4. The Opacity proteins Both Opa and Opc (previously class 5 proteins) are expressed in N. meningitidis; the term ‘opacity’ was first used for gonococcal Opa proteins as colonies of bacteria expressing the proteins appeared opaque under specific sub-stage lighting conditions [102]. In meningococci, such opacities are only clearly discernible in acapsulate phenotypes [17].

4.1. Opa proteins Opa proteins are eight-stranded transmembrane beta-barrel structures with four surface-exposed loops. The first loop (proximal to the N-terminus) is semivariable (SV), whereas loops 2 and 3 are hypervariable (HV1 and HV2) and the fourth loop is invariant. In N. meningitidis, 3-4 complete copies of opa genes are found. The expression Opa protein is translationally controlled whereby the number of repeats (CTCTT) within the opa gene determines whether the gene is in frame. As each opa gene is expressed independently, this process is also linked to Opa antigenic variation [103,104]. It is clear that this generates a vast array of Opa variants, which while enabling bacteria to evade host immune mechanisms, poses the problem of maintaining their functional role as adhesins. How the surface-exposed variable domains of Opa affect their interactions with target cells has been a subject of several studies and those of N. meningitidis in the context of CEACAM receptor targeting are described below. The repertoire of Opa structures in a population may also be enhanced by inter or intra-genomic recombination [103]. However, recent surveys of clinical and carriage isolates have shown that despite the presence of a wide range of possible alleles, specific arrays of Opa variants are prevalent in N. meningitidis isolates [105–107]. This is very likely a result of immunological selection, but in addition, functional constraints driven by their ability to interact efficiently with certain target host receptors such as CEACAMs (4.3.4) may enhance bacterial survival in vivo, thus selecting functionally important opa alleles.

4.2. Opc protein N. meningitidis Opc (OpcA), a 10-stranded ␤-barrel molecule with five surface exposed loops, is encoded by a single gene (opcA) [108–110]. Opc was the first neisserial integral outer membrane protein to be crystallised [111]. Although structurally relatively invariant, the levels of Opc expressed by a strain may vary in vivo and in vitro by a transcriptional control mechanism [22]. The expression of Opc protein has not been demonstrated in gonococci or certain clonal lineages of N. meningitidis e.g. ET37 [112]. Interestingly, the strains belonging to ET37 complex apparently have a tendency to cause severe septicaemia and are less associated with meningitis [113–115]. It is possible that Opc imparts N. meningitidis with the ability to cause meningitis [113].

4.3. Opacity protein-host cell interactions in colonisation and pathogenesis Opa and Opc, both basic in nature, bind to at least two common negatively charged molecules: heparan sulphate proteoglycans (HSPGs) and sialic acids [6,109,116] but they also display a degree of receptor specificity [116]. Opacity proteins may also engage in cis interactions with LPS sialic acids, which may be responsible for inefficient Opa-mediated adhesion of bacteria with sialylated LPS compared with those lacking the sialylation [3,116].

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4.3.1. Proteoglycan and extracellular matrix interactions It appears that meningococcal Opa and Opc can interact directly with HSPGs [6,111,117]. As Opc can also interact directly with extracellular matrix (ECM) proteins such as vitronectin and fibronectin, and as ECM proteins contain HSPGs binding sites, a range of complex interactions become possible via Opc [6,113,118]. HSPG interaction is generally of low affinity, and does not result in efficient uptake but co-ligation of secondary receptors such as integrins via ECM proteins leads to increased cellular invasion. The modes of Opa and Opc interactions are apparently determined by the target cell type [6,111,117]. 4.3.2. Serum proteins and integrins as Opc receptors N. meningitidis interactions with endothelial cells are important from the point of view that Opc may increase the propensity of N. meningitidis strains to cause meningitis as has been suggested [113]. In vitro studies have shown that Opc targets endothelial integrins ␣V␤3 (vitronectin receptor) and ␣5␤1 (fibronectin receptor) by interacting with the serum proteins vitronectin and/or fibronectin [3,113,118]. Current studies are suggesting that vitronectin may be the major serum target for N. meningitidis and it the activated form of vitronectin that is required for efficient interactions [119]. It is noteworthy that Opc expression in heterologous strains (E. coli) does not support adhesion of E. coli to endothelial cell integrins. It is possible that further factors, perhaps meningococcal LPS, may be required as integrins often interact with proteins and some glycans simultaneously [17,120]. However, it is also possible that the level of Opc expressed by E. coli is not optimum since efficient interactions of N. meningitidis via Opc require the protein to be expressed at a high density on the bacterial surface [3]. 4.3.3. Cytoskeletal molecules as targets In a recent study, internalised Opc-expressing N. meningitidis were found to interact with the cytoskeletal protein, ␣-actinin, within endothelial cells [121]. ␣-actinins interact with a number of molecules, and regulate receptor activities as well as serve as scaffolds to connect the cytoskeleton to a variety of signalling pathways [122]. Their involvement in linking the cytoskeleton and adhesive receptors, such as integrins [123] and syndecans [124], is of interest as both are receptors for Opc. The true significance such interactions to bacterial pathogenic potential remains to be investigated. 4.3.4. Opa and CEACAMs CEACAMs (carcinoembryonic antigen-related cell adhesion molecules) are members of the Immunoglobulin superfamily and comprise several important receptors including CEACAM1, CEACAM3, CEA (the product of ceacam5 gene) and CEACAM6 [125] (Fig. 4). As the expression of some of the receptors is restricted to particular cells or tissues, the choice of CEACAM member for adhesion imparts tissue tropism to the bacteria. CEACAM1 is the most widely expressed member, and it is also the most frequently targeted molecule by Opa-expressing meningococci. Previous studies on meningococcal serogroup A and B strains have demonstrated that Opa proteins make up distinct structures through various combinations of their HV domains, and regions within HV1 and HV2 appear to be involved in tropism for distinct CEACAMs [117,126]. Overall, the receptor specificity of Opa proteins is achieved through targeting a set of conserved residues on the Nterminal domain of the receptor [117]. For the vast majority of the Opa types that are generated and maintained in neisserial genomic pool (presumably as they impart survival advantage to the bacteria), the property of targeting CEACAM1 is maintained. In addition, a number of Opa types also compensate for the heterogeneity in their target receptor family, thus also increasing their host tissue range.

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Fig. 4. Opa protein interactions with different CEACAM family members. CEACAM3 and CEA are specific for neutrophils and epithelial cells respectively. CEACAM6 is expressed on both cell types, whereas CEACAM1 is ubiquitous on epithelial and endothelial cells as well as cells of the immune system [125]. Engagement of different CEACAMs results in a range of cellular responses through which N. meningitidis may be able to invade cells and gain access to deeper tissues and blood vessels. The epithelial cell shown on the right depicts inflammatory responses to prior infections that may lead to increased CEACAM1 density on epithelial cells enabling encapsulated N. meningitidis to be internalised, a process involving NF␬B pathway [4]. Bacterial interactions may also lead to de novo CEACAM1 synthesis through the action on NF-␬B [4]. In addition, binding to neutrophil-expressed CEACAMs results in bacterial internalisation by the phagocytic cells [162].

4.3.5. CEACAM1 in health and disease From studies to date, it can be concluded that the interactions of Opa proteins with CEACAM1 result in cellular invasion [4,117,127], CEACAM1 is normally expressed at low levels but its expression is known to be upregulated significantly under the influence of inflammatory cytokines. In this context, in an in vitro study with A549 lung carcinoma cells, CEACAM1 expression was shown to be upregulated considerably in response to IFN␥, which resulted in infiltration of the target cells not only by acapsulate but also by fully capsulate serum resistant N. meningitidis [4]. The efficiency of this process was enhanced in piliated bacteria but required Opa expression and high levels of target cell CEACAMs [4]. Perhaps the Opa-mediated invasion process is aided by the pilus retraction forces, although this has not been demonstrated. Such blood invasion by capsulate serum resistant bacteria would increase the chances of widespread dissemination especially in an immunocompromised host. Thus changes in adhesin-receptor dynamics may be an additional host factor determining an individuals susceptibility to N. meningitidis infection [4,128,129]. The data also suggest that receptors such as CEACAMs could be at the cross-roads of colonisation and pathogenesis. Such cell adhesion molecules expressed at low levels on mucosal epithelial cells may support attachment without significant invasion, favouring a colonisation state. However, when upregulated during inflammation, the same receptors may become the portal of tissue entry. In addition, increased interactions through

CEACAMs with phagocytic cells could result in incomplete elimination of bacteria and the possibility of transmission within them. 4.3.6. CEACAMs and T cells A variety of CEACAMs are expressed on human immune cells but stimulated CD4+ T cells express only CEACAM1 [130]. Studies with gonococci have suggested that Opa-CEACAM1 interactions suppress CD4+ T cell proliferative responses [131]. A similar suppressive effect on T cell activation and proliferative response was reported for meningococcal Opa-containing outer membrane vesicles [132]. Notably however, in other systems, CEACAM1 ligation has been shown to increase the proliferation of T cells [130,133]. In addition, in one study on peripheral blood mononuclear cells, a high proliferative response to meningococcal Opa protein was observed [134]. Our current studies using live or killed meningococci have not shown any Opa-dependent immunosuppressive effects on CD4+ T cell proliferation [163]. 5. Targeting invasins for the intervention in meningococcal infectivity Opc is highly immunogenic in humans and elicits serum bactericidal and opsonic antibodies [135,136]. Moreover, cross-reactive anti-Opc monoclonal antibodies can be generated [109]. However, it has been surmised that their efficacy in vivo might be limited

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due to the small heterogeneity observed in Opc between strains and due to variable levels of its expression [137]. In this context, an alternate view might also be considered. While as a sole vaccine antigen, Opc may be of limited value, as a component of a complex vaccine, it may prove to be of importance. First, in vitro studies have shown that the loop 2 of Opc, which is the most immunogenic and prominent [138], is also involved in influencing bacterial interactions in a number of model systems, including interactions with host cell receptors and serum proteins [118]. Second, it seems that Opc may enhance bacterial serum resistance by binding to serum vitronectin [119], a well known modulator of complement action. As such, antibodies that eliminate any circulating Opc+ phenotypes or reduce their virulence by blocking interactions with complement modulators would be beneficial to the host. In the case of Opa, in vitro transfected or cytokine-treated cells (used as post-inflammation model systems) in which increased cellular invasion by meningococci could be observed, served as a platform to assess the efficacy of intervention in Opa-CEACAM interactions in a setting of high receptor density. With the use of a recombinant CEACAM-binding molecule (which was based on the Moraxella catarrhalis receptor binding adhesin UspA1) able to inhibit all Opa-mediated interactions [139], selective cellular infiltration could be avoided, whilst maintaining adhesion of capsulate piliated bacteria [4]. This suggests the possibility that, if antiOpa antibodies could be generated that block its interactions with CEACAMs, it may be possible to interfere selectively with cellular invasion, while maintaining colonisation, a state believed to boost immunity and provide long-term protection of the host. Further, it is noteworthy that polymorphisms in CEACAM structure can significantly influence meningococcal interactions as demonstrated by in vitro mutagenesis studies of CEACAM1 [140]. In other studies, certain CEACAM haplotypes have been found to be associated with increased host susceptibility, and certain meningococcal Opa repertoires with hyper-invasiveness and disease [105,106]. Association of limited numbers of Opa repertoires with disease also suggests that Opa proteins could be candidate vaccine antigens in their own right. Therefore, understanding their full potential as virulence factors is important for future approaches to control meningococcal infection. Integral to this aim is the identification of the salient features of the Opa proteins that must be retained for the receptor targeting. If identified, it may be possible to interfere selectively with cellular invasion. 6. Recently identified adhesins and related proteins Following genome sequencing of N. meningitidis strains, a number of novel adhesins (Table 1) and related proteins have come to light. They can be broadly grouped in four structural categories. Their known properties are described below. 6.1. The autotransporters NhhA (Neisseria hia homolog A, Mr 57 kDa) and App (Adhesion and penetration protein, Mr 160 kDa) are homologous to the autotransporter proteins Hsf/Hia and Hap of H. influenzae, respectively. NhhA is widely expressed in virulent N. meningitidis strains [141–143]. NhhA-mediated low levels of adhesion to epithelial cells, heparan sulphate proteoglycans (HSPG) and laminin have been described [142]. App, which may be processed and released, is present in both pathogenic and commensal neisserial spp. [144,145]. It is suggested that it may aid bacterial colonisation by increasing adhesion before autocleavage and detachment after cleavage, thus facilitating their spread [144]. The presence of capsule apparently does not interfere with NhhA/App-mediated adhesion [142]. MspA (Meningococcal serine protease, Mr 157 kDa)

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is homologous to App and IgA1 protease; it is cleaved and secreted. It is expressed by several but not all virulent meningococcal lineages. It is reported to mediate binding of E. coli expressing the protein to both epithelial and endothelial cells and elicit bactericidal antibodies [146]. 6.2. The Oca-type adhesin NadA (neisserial adhesin A, Mr 38 kDa) is a member of the oligomeric coiled coil family of adhesins (Oca), that interacts with an uncharacterised protein receptor on epithelial cells via its Nterminal globular region [147]. Three out of four hyper-virulent N. meningitidis lineages express distinct alleles of the gene. Its expression is phase variable and may be growth-phase dependent [148]. NadA mutation decreases epithelial adhesion of capsulate N. meningitidis and invasion by acapsulate N. meningitidis. As anti-NadA antibodies are bactericidal, it may be a potential vaccine candidate for use against meningococcal serogroup B strains [149]. 6.3. The ˇ-barrel protein NspA (Mr 18 kDa) is a highly conserved basic ␤-barrel protein with four surface exposed loops and a homolog of Opa adhesins, although it is not phase variable. The crystal structure of NspA has been described [150,151]. Structural analyses suggest a potential site for binding to hydrophobic ligands such as lipids [151], although this remains unexplored. Protective bactericidal antibodies to the recombinant NspA can be elicited [150] but the accessibility of protein to such antibodies has not been universally observed [152]. 6.4. The two partner secretion systems The two-partner secretion (TPS) pathway of Gram-negative bacteria is a widespread route for the secretion of very large proteins of over 1000 amino acid residues. TPS systems are encoded by two genes, tpsA and tpsB. The TpsB inserts into the outer membrane and facilitates transport of the TpsA to the cell surface. Three distinct TPS systems were identified among meningococci [153]. System 1 is ubiquitous, while systems 2 and 3 were significantly more prevalent among hyper-invasive isolates than among isolates of low invasive capacity. Recently the role of these TPS proteins in bacterial interactions with target cells has been suggested. It seems that these systems (also designated HrpA/HrpB in analogy with haemagglutin/haemolysin-related proteins) may favour bacterial adhesion and/or are essential for intracellular survival [154,155]. 7. Conclusions This review has considered in some depth the interactions or likely interactions of N. meningitidis with human barrier cells via protein adhesins. Numerous other interactions are also known to occur, notably those via LPS which involve multiple host components and receptors such as CD14, TLR4, Siglecs and others. It is clear that when present in large numbers, meningococci cause cytotoxic damage either directly or indirectly to cultured human cells, especially endothelial cells [156,97]. However, at non-toxic doses, bacterial direct interactions with barrier cells via numerous protein adhesins may enable penetration into deeper tissues by transcellular or paracellular routes without discernible cellular damage. Additional considerations include interactions with phagocytic cells through for example pairing of LPS and Siglecs or Opa and CEACAMs. As these can also result in cellular infiltration, they may constitute additional mechanisms of meningococcal

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