journa l homepage: www.e lsev ier .com/ locate /vacc ine
eningococcal interactions with the host
tienne Carbonnellea,1,2, Darryl J. Hill c,1, Philippe Moranda,2, Natalie J. Griffithsc,andrine Bourdoulousb, Isabel Murilloc, Xavier Nassif a,2, Mumtaz Virji c,∗
INSERM, unité 570, Université Paris Descartes, Faculté de Médecine, 156 rue de Vaugirard, 75015 Paris, FranceInstitut Cochin, Département de Biologie Cellulaire, INSERM, Unité 567, CNRS, UMR8104, Université Paris Descartes, 22 rue Méchain, 75014 Paris, FranceDepartment of Cellular and Molecular Medicine, School of Medical Sciences, University Walk, University of Bristol, Bristol BS8 1TD, UK
r t i c l e i n f o
eywords:dhesion
a b s t r a c t
Neisseria meningitidis interacts with host tissues through hierarchical, concerted and co-ordinated actions
eceptorsathogenic mechanisms
of a number of adhesins; many of which undergo antigenic and phase variation, a strategy that helpsimmune evasion. Three major structures, pili, Opa and Opc predominantly influence bacterial adhe-sion to host cells. Pili and Opa proteins also determine host and tissue specificity while Opa and Opcfacilitate efficient cellular invasion. Recent studies have also implied a role of certain adhesin–receptorpairs in determining increased host susceptibility to infection. This chapter examines our current knowl-edge of meningococcal adhesion and invasion mechanisms particularly related to human epithelial and
re of p
endothelial cells which a
. Introduction
Neisseria meningitidis has evolved attributes that generallynable harmless colonisation of the human nasopharynx, its onlynown niche; and in most disease cases, asymptomatic colonisa-ion of the nasopharynx precedes infection. Colonisation itself ischieved via versatile and dynamic adhesion and immune eva-ion mechanisms. Whilst antigenic and phase variations of surfacetructures, including the adhesins, enable bacteria to avoid immuneetection, adhesion is maintained through redundancy, i.e. thexpression of multiple adhesins, which also aids in colonisationf distinct niches. Although generally regarded as an extracel-ular pathogen, in vitro studies have shown that N. meningitidisntry into cultured human cells can occur via several distincteceptor–adhesin interactions [1–6]. Studies using nasopharyn-eal epithelial organ cultures have also shown that meningococcire able to invade these tissues [8]. In addition, although notlearly demonstrated, meningococcal intracellular location haseen implied within mucosal epithelial cells in one study that
xamined tonsillar biopsies in which meningococci were observedeneath epithelial surfaces [7]. Whether intracellular location in
nflamed tissues often used for these investigations represents ‘theorm’ needs to be considered. Studies reported below suggest that
inflammatory conditions of the host may influence the balance ofthe bacteria–host relationship that can change from large surfacelocation to significant cellular penetration. Nonetheless, even lowlevels of internalisation in a healthy host could benefit bacterial sur-vival through evasion of host phagocytic and antibody/complementmediated killing mechanisms. Internalisation may also provideaccess to a new source of nutrients and enable bacteria to interferewith host cell functions including innate response to the bacte-rial presence. While in vitro studies have helped unravel some ofthe complexities of meningococcal interactions with its uniqueniche, this specificity has prevented the use of animals as mod-els and our understanding of in vivo situations remains unclear. Tocause disease, the bacterium enters systemic circulation and mul-tiplies within the host. For this, it requires a “permissive” oftenimmunocompromised host [9]. During the passage across vascularendothelial and the blood–brain/blood–csf barriers, meningococciencounter additional human cells and serum factors with whichthey interact, often displaying specificity [10]. Since colonisation isthe normal state of existence for meningococci, it may be surmisedthat the bacterial attributes which enable colonisation also aid theirdissemination throughout the body.
The major adhesins of N. meningitidis include the polymeric piliand the outer membrane opacity proteins, Opa and Opc [1,2,6,11,12].In addition, a number of other adhesins have been recently iden-tified (Table 1). There is a hierarchy in the utility of adhesins in
the context of bacterial phenotype and location. Bacterial capsulestend to be highly hydrated and whilst it is suggested that theymay help protect some air-borne bacterial species from desicca-tion, substantiation of this notion is required for N. meningitidis[13,14]. Interestingly, a significant number of mucosal N. meningi-
E. Carbonnelle et al. / Vaccine 27S (2009) B78–B89 B79
Table 1Neisseria meningitidis adhesions.
Adhesin Alternative name Gene designation Homology Reference
Pili Fimbriae pilE1/pilE2 (pilin subunit) [157]Opa Class 5 outer membrane
Proteinsopa [157]
Opc Class 5 C protein, OpcA opcA [108]NhhA (Neisseria hia homolog A) nhha/GNA992 H. influenzae Hsf/Hia E. coli
AIDA-1 homolog[141] [143]
App (adhesion and penetration protein) app NMB1985 H. influenzae Hap [145]MspA (meningococcal serine protease A) NMB1998 IgA1 protease [146]NadA (neisserial adhesin A) NMB1994 Oligomeric coiled coil adhesin
(Oca) family e.g., M. catarrhalis[143] [148]
H hrphrpNM
ttip[tlaf[tsti
smiri(gsbasrgeg
bent
2
rcatili[Db
rpA (haemagglutin/haemolysin-relatedprotein A)
TpsA (two partner secretionsystem protein A)
idis isolates lack capsule genes [14–16]. However clinically, by farhe most important meningococcal phenotype is capsulate whichs required for survival in the bloodstream. Capsule, by its juxta-osition, also masks the outer membrane adhesins to some extent2–4,17] and in capsulate meningococci, the polymeric pili, whichraverse the capsule facilitate initial attachment to mucosal epithe-ial cells. In the respiratory tract, capsule becomes less importants is evident from frequent isolation of acapsulate N. meningitidisrom the nasopharynx which may arise as a result of phase variation18,19] or by down-modulation under the influence of environmen-al factors [20]. In such a phenotype, the outer membrane adhesinsuch as Opa and Opc become effective. The latter are also effec-ive invasins and may synergise with pili resulting in enhancednfiltration of the target tissues [3,4].
Even though various regulatory pathways exist for gene expres-ion, phase variation is the usual mechanism by which N.eningitidis controls the expression of the major adhesins. It
s manifested via a process involving DNA slippage (commonlyeferred to as Slipped Strand Mispairing, SSM) engendered by repet-tive sequences of nucleotides either within the open reading frameleading to translational control of expression) or upstream of theene (affecting the level of transcription) [21,22]. In the case of neis-erial pili, several components involved in pilus biogenesis (seeelow) may affect pilus expression; but one major mechanismffecting the expression and antigenic variation of the major pilusubunit PilE, involves inter and intra-genomic RecA-dependentecombination events between one of several pilS (silent) pilinenes and pilE, the expressed pilin gene. This represents anotherxample of remarkable similarities between N. meningitidis and N.onorrhoeae [23–26]
Below essential features of the currently known mechanisms ofacteria–host interactions, particularly at the human epithelial andndothelial barriers that may determine colonisation and dissemi-ation are presented. In addition, the key surface protein structureshat mediate these interactions are discussed in some depth.
. Type IV pilus-mediated adhesion and signalling
Type IV pili (Tfp) are widely found in Gram negative bacte-ia including pathogenic Neisseria and have been shown in someases to be associated with diverse phenotypes or functions suchs biofilm formation [27] and bacteriophage infection [28]. The lat-er two functions have been demonstrated in Pseudomonas spp. Tfpnfluence the dynamics of the adherence by powering a form of cell
ocomotion by crawling over a surface known as twitching motil-ty [29] and by promoting the formation of bacterial aggregates30]. In addition, Tfp are essential for the natural competence andNA transformation, a property that contributes to their virulencey promoting exquisite genetic adaptability [31]. A remarkable
UspAA1(NMB0497),A2(NMB1779)A0668, NMC0444, tpsA
B. pertussis FHA [154] [158]
property of Tfp is their ability to retract into the bacteriumfrom which they originate, via the action of the force-generatingATPase PilT [30,32]. Tfp retraction is essential to bacterial motil-ity (twitching motility), competence for DNA transformation, andpilus-associated signalling to host cells [33].
2.1. Pilus biogenesis
Type IV pili are homopolymeric filaments found on N. meningi-tidis. Tfp are highly dynamic structures which undergo rapid cyclesof extension and retraction. They extend from the inner membraneto the bacterial surface, passing through the outer membrane. They
are thin (50–80 ´̊A) and flexible filaments that can be up to severalmicrometers long and can sustain considerable mechanical stress.The major subunit of the fibre is pilin (PilE), the helical assem-bly of which leads to the formation of the fibre. The componentsinvolved in Tfp assembly, retraction and specific functions are dis-tributed around the bacterial membranes, from the inner side ofthe 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 anoperon. It has been shown that Tfp biogenesis can be resolved intofour genetically dissociable steps: assembly, functional maturation,emergence on the cell surface and counter-retraction. The proteinproducts of some of these genes when mutated give rise to a non-piliated phenotype which can be reversed in strain deficient forpilus retraction (Fig. 1). These proteins are therefore not requiredfor the assembly of the fibre. When pili are retracted, the pilin sub-units are stored within the cytoplasmic membrane. These differentsteps and the corresponding proteins are described in Fig. 1.
2.1.1. PilE and PilDThe major neisserial pilus subunit, the pilin, is encoded by the
pilE gene. Along with an intact copy of pilE, up to eight loci con-taining truncated copies of the pilin gene, named pilS (silent), arefound on the chromosome. pilS copies are not expressed but serveas a source of variant pilin gene sequences which occasionallyrecombine with pilE, leading to the expression of novel PilE vari-ants. Pilin is synthesised as a preprotein. A short leader sequence(5–6 residues) is cleaved by the prepilin peptidase PilD [35]. PilDis a leader peptidase localised to the inner membrane that specifi-cally recognises the N-terminal part of prepilin and of prepilin-likemolecules [36]. PilD also methylates the N-terminal phenylalanineof the mature protein product, which relies on the presence of a
glutamic acid in position +5 of the substrate. However, the lack ofmethylation has little effects on Tfp assembly [37].
The mature protein is approximately 145–160 residues longafter prepilin peptidase processing. It has a conserved hydropho-bic N-terminal 25 residue and a carboxy-terminal disulphide bond.
B80 E. Carbonnelle et al. / Vaccine 27S (2009) B78–B89
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, retractionpowered 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 platformcomplex (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, whichco-purifies with the inner membrane, interacts with PilQ. Two inner membrane ATPases, PilF and PilT, promote pilus elongation and pilus retraction respectively. Differentc J, PilKi 1, PilWP
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omponents are required to counteract pilus retraction (PilC1/C2, PilG, PilH, PilI, Pilnner membrane. Some components are important for the function of the pilus (PilCilJ, PilV, PilX and ComP), PilV, X and ComP are incorporated in the fibre [52].
he conserved hydrophobic N-terminal moiety that is embeddedn the core of the pilus is involved in the cohesion of the sub-nits as a fibre. The polymorphic C-terminus of pilin is partiallyxposed at the surface of the assembled pilus. The pilin also har-ours two cysteine residues that build a disulfide bridge, involved
n the formation of the surface accessible, variable and immuneominant part of the pilus. Pilins are therefore packed through
nternal hydrophobic interactions between conserved N-terminal �elices, leaving hypervariable C-terminal globular regions exposed38]. PilE undergoes several post translational modifications oferine residues including glycosylation at position 63 and an alpha-lycerophosphate at position 93 [39–42]. Whereas at position 68,he residue has been reported to be substituted with phosphate,hosphoethanolamine or phosphorylcholine [41,43]. The roles ofhese various modifications remain unclear. Concerning the gly-osylation, the structure of the sugar is different depending onhe strain [44,45]. Pilin glycosylation in N. meningitidis is underhe control of the pgl gene cluster [46–49], which encodes genesharacterised by length polymorphisms. The meningococcal pglA isubject to phase variation due to the presence of a poly-G stretch,hich is not the case for pgtA, the gonococcal homolog to pglA [50].
.1.2. PilGPilG is an inner membrane protein required for piliation [51].
t prevents pilus retraction, but does not seem to be required forilus biogenesis since apparently normal Tfp are seen in doubleilT pilG mutants [52]. The 3D reconstruction of PilG provides a clearvidence of a tetrameric quaternary arrangement [53].
.1.3. PilF and PilTTwo inner membrane-associated ATPases, PilF and PilT, are
hought to antagonistically promote extension and retraction of
fp; PilF being involved in elongation [36] and PilT in retraction30,54]. Nucleotide (NTP)-binding proteins are basic componentsf all Tfp machineries. They usually contain a “walker box” for theinding of ATP and belong to the family of AAA ATPases (ATPasesssociated with various cellular activities), which includes chap-
PilW). PilC1/C2 and PilW are located in the outer membrane whereas PilG is in the, PilI, PilJ, PilK, PilX, PilV and ComP). Among the minor pilin components (PilH, PilI,
erones and mechanoenzymes (for a review, see [55]). Unlike PilF[36], PilT is dispensable for assembly and expression of Tfp on thecell surface, but is required for their retraction. PilT was initiallyreported as an effector for transformation competence and twitch-ing motility [54], and plays a key role in the interaction with the hostcell [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 foundassociated with the inner membrane and in the cytoplasm [59,60].PilF is associated with the inner membrane, and its functionalityrelies on the integrity of the “walker box”.
2.1.4. PilQ and the secretin complexThe secretin PilQ supports Tfp extrusion and retraction, but
it also requires auxiliary proteins for its assembly and localisa-tion in the outer membrane. The secretin PilQ is a member ofa large family of integral outer membrane proteins with con-served C-terminal domains [61–63]. The meningococcal secretinPilQ oligomer consists of 12 identical monomers [64–68]. PilQ spon-taneously associates with Tfp when they are incubated together invitro; the PilQ oligomer binds at one end of the pilus fibre, whichpotentially fills the central chamber [67].
Involvement of PilP in gonococcal pilus biogenesis was firstreported by Drake et al. [69], they showed that pilP null mutantwere non-piliated. The pilP gene is located upstream of pilQ, in acluster with other pilus biogenesis genes: pilM, pilN, and pilO [70].The inactivation of the pilP gene in its 3′ region can therefore leadto a reduced quantity of pilQ transcript due to a polar effect. It hasbeen shown that PilP co-purifies with inner membrane componentsand that the N- and C-terminal regions of PilP recognise the centralpart of the PilQ monomer [71]. PilP is predicted to be a lipopro-tein, which most likely anchors it to the inner membrane [67,71].
It has now been shown that PilQ does not need PilP for its mem-brane localisation and/or stabilisation [34,71]. One candidate forthis function could be PilW, which is essential for Tfp biogenesisin N. meningitidis and PilW affects the stability of the PilQ complex[34], suggesting specific interactions between PilQ and PilW [72].
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Fig. 2. Schematic representation of the signalling pathways triggered by N.meningitidis leading to the formation of the cortical plaque. Ezrin and moesinare key components of these complexes. These proteins are members of theezrin/radixin/moesin (ERM) family that acts as a linker between the plasmamembrane and the actin cytoskeleton [90,92,159]: Ezrin and moesin control theorganization of the cortical plaques by interacting by their amino-terminal domainwith the cytoplasmic domain of transmembrane proteins (so-called ERM bindingproteins), such as CD44, ICAM-1, VCAM-1, E-selectin, and interact with F-actin bytheir carboxy-terminal domains. The recruitment and phosphorylation of cortactinat the N. meningitidis entry site is required for the formation of the actin-rich cellprojections that promote efficient bacterial uptake [91]. Tyrosine phosphorylation ofcortactin induced by N. meningitidis results from the clustering and activation of thehost cell tyrosine kinase receptor ErbB2 and the downstream activation of the kinasesrc [89]. The interaction of N. meningitidis with human endothelial cells leads to theactivation of ErbB2, indicating that N. meningitidis induces ErbB2 activation mostlikely via formation of ligand-independent ErbB2 homodimers, occurring by a mech-
E. Carbonnelle et al. / V
.1.5. PilC proteinsThe PilC proteins play a crucial but still enigmatic role
12,73–76]. Two alleles were originally discovered [73]. Expres-ion of both variants is subject to phase variation as a result oframeshift in homopolymeric “G” tracts located in the open readingrames [73]. Phase variation of PilC has also been shown in meningo-occal clinical samples [77]. PilC-null strains show impaired pilusxpression and lack the ability for transformation competence. In. meningitidis, only PilC1 is required for adhesion. PilC2, which isxpressed independently of PilC1, fails to promote adhesion despitedentical functions in pilus expression and transformation compe-ence [12,78]. Abolition of pilT in a PilC-null background restoresiliation, confirming the hypothesis that PilC acts as an antagonistf PilT by preventing PilT-mediated retraction [54,79].
.1.6. Prepilin-like proteinsPrepilin-like molecules, with pilin-like N-terminal sequences,
lay important roles in Tfp biology [80]. In pathogenic Neisseria,here are seven proteins cleaved by the prepilin-peptidase PilD:ilH, PilI, PilJ, PilK, ComP, PilV and PilX (PilL in the gonococcus).omP, PilV and PilX, which have canonical PilD cleavage motifsnd mature lengths similar to PilE, modulate Tfp-related func-ions. ComP and PilV are necessary for DNA transformation [81]nd adhesion to human cells [82], respectively, whereas PilX in N.eningitidis is essential for bacterial aggregation and adhesion [83].
hese proteins provide models to investigate the relation betweenhe structure and the function of pilin-like proteins because thehenotypes associated with the corresponding mutants are notbscured by the absence of pili. The 3D structure of PilX shows thatt closely resembles types IV pilins [84]. It has been proposed that
hen interacting with another PilX and under tension, PilX subunitsay adopt a different conformation and prevent further slippage
f pili. In the absence of PilX, pilus retraction allows the disruptionf inter-bacterial Tfp-mediated interactions, and no aggregation isbserved [85].
.2. Mechanism of pilus-mediated interaction
Capsulate non-piliated bacteria interact with epithelial andndothelial cells inefficiently unless they express the type IV pili.he efficiency of the first step of adhesion of capsulate meningo-occi is dependent on the target cell type as well as the pilustructure [86]. Given the appropriate combination, pili mediate onef the most efficient meningococcal interactions observed in vitro87].
The molecular mechanism responsible for the initial attachmentf individual diplococci to the cells is still not fully understood.ne report has suggested that the PilC1 protein could carry thisell binding domain [75], this hypothesis was based on inhibitionf adhesion using purified PilC molecules. However, non-adhesiveon-piliated isolates of serogroup B strain MC58 with high PilCxpression, and piliated adhesive isolates with barely detectableilC expression have been described [87]. In addition, anotherilC+/PilE- strain, in which PilC location has been demonstrated inhe outer membrane, is unable to interact with eukaryotic cells (X.assif, unpublished observations), thus raising doubts on the rolef PilC as an adhesin.
As mentioned above, the minor pilin PilX is essential for promot-ng inter-bacterial interactions and pilX mutants, which are unableo from aggregates, have a phenotype similar to that of non-piliatedtrains even though they have bundled pili. It appears that these
nter-bacterial interactions generated by the Tfp allow the bacte-ia, when multiplying, to spread onto the cells, which in turn reliesn the ability of bacteria to retract their pili. Following the inter-ctions between bacteria and target cells, in some systems, piliave been shown to retract over a period and eventually adherent
anism which is still unresolved. In addition, the high forces that are generated by theretraction of the fibre are thought to assist close association of the pathogen withthe host cell. Moreover, such forces are large enough to cause membrane protrusions[160,161].
meningococci appear non-piliated [30,56]. Measurements usingoptical tweezers showed that retraction of a single Tfp generatesforces up to 110 pN, in a transient manner for each fibre. Bundlesof Tfp, which result from the association of 8–10 pili, act as coordi-nated retractable units. Filament bundles produce retraction forcesthat generate forces in the nanonewton range [88]. The successiveextension, binding and retraction of Tfp enable bacteria to move bytwitching 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 greaterunderstanding of the cellular events involved in N. meningitidisinvasion processes of human endothelial cells, and of the complexmolecular signalling pathways induced by the pathogen which leadto its uptake in non phagocytic cells [89–92] (Fig. 2). Adhesion ofN. meningitidis to endothelial cells promotes the local formation ofmembrane protrusions reminiscent of epithelial microvilli struc-tures that surround bacteria. The formation of these membraneprotrusions by capsulate N. meningitidis results from the organiza-tion 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 microvilli-like structures induced by the signalling linked to pilus-mediatedadhesion initiate internalisation of these bacteria within intracel-lular vacuoles [90]. Interestingly, the formation of such protrusionswas also observed ex vivo, by transmission electron microscopy
B82 E. Carbonnelle et al. / Vaccine 27S (2009) B78–B89
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ig. 3. An overview of known epithelial receptors of non-pilus adhesins of N. meniniagram, those of the minor adhesins, App, NadA, MspA and Tps (HrpA), remain to
nalysis of brain sections from a child who died from fulminanteningitis [10], suggesting that such morphological modifications
f the host cell membrane may be responsible for the internali-ation of a small fraction of adhesive bacteria. These observationsherefore raised the hypothesis that N. meningitidis could cross thendothelial barrier by a transcytosis pathway. In addition, recenttudies have provided evidence that N. meningitidis induce hostell surface reorganisation by promoting invagination within cel-ular interstices. These rearrangements confer to bacterial coloniesormed at the apical surface of host cells the ability to resist theigh mechanical forces exerted by the blood flow [95,96]. Exploita-ion of the host-cell signalling pathways could therefore be pivotaln promoting intimate attachment to the cell surface, avoiding bac-erial detachment under flow conditions in the blood as well as inhe nasopharynx. Considering that at least in vitro, both on epithe-ial and endothelial cells, most of the bacteria interacting withells are found localised at the apical surface, it is likely that theignalling induced by pilus-mediated adhesion has been selecteduring the evolution to allow intimate attachment and thereforeacterial colonisation of its only niche, the human nasopharynx.egarding the meningeal invasion, one hypothesis is that the smallraction of internalised bacteria transcytose through the monolayerf endothelial cells, alternatively the signalling induced by pili mayead to disruption of the intercellular junctions. In addition, whenarge numbers of piliated bacteria are present, meningococci causeamage to human cells via LPS-mediated cytopathic damage, a pro-ess that has been shown to be enhanced with piliated bacteria97].
.4. Host cell receptors for meningococcal pili
CD46, a transmembrane glycoprotein, which regulates comple-
ent activation was described as a cellular receptor for Type IV pili
f Neisseriae (N. meningitidis and N. gonorrhoea) [98]. Subsequently,transgenic CD46 mouse was developed to model the interactionf N. meningitidis with endothelial cells of the blood brain barrier,nd meningitis [99]. It has however been demonstrated that pilus-
. Although receptors have been reported for Opa, Opc and NhhA as indicated in theermined. HSPG: heparan sulphate proteoglycans.
mediated adherence of N. gonorrhoea does not correlate with CD46expression [100,101] and is unaffected by exogenous CD46 expres-sion or by depletion of CD46 on epithelial cells [101]. It is thereforelikely that one or several cellular components beside CD46 is/areresponsible for the interactions observed between pili and humanbarrier cells.
In conclusion, pili are one of the major attributes allowing inter-action of N. meningitidis with human mucosal epithelial cells andnecessary for initial colonisation. The ability of pili to aggregatebacterial cells and to induce twitching motility via pilus retractionappears to be important processes in the colonisation of the api-cal surfaces of the cells. On the other hand, the nature of the piluscomponent/s that induce/s the initial interaction remains unclear.The signalling events stimulated by pili allow intimate interactionsbetween the host cells and bacteria, enabling attached bacteria alsoto resist the various fluid flows in the extracellular environment.This property is likely to be required for the bacteria not only tocolonise the nasopharynx; but once in the bloodstream, it mayalso help prevent bacterial detachment from the vessel walls. Theimportance of pili in meningococcal colonisation may suggest theirpotential as vaccine antigens but the frequency of their variationhampers any such utility.
3. Non-pilus adhesins
Besides pili, at least seven other N. meningitidis adhesins havebeen described (Table 1). The most studied of these adhesins arethe opacity proteins Opa and Opc, for which several host cell tar-geting mechanisms are known (Fig. 3). The available in vitro studiesalso show the other more recently identified adhesins to be several
target cells compared with the opacity proteins or pili; although,their functions in vivo may possibly be more efficient. However, thelack of adequate animal models for the human-specific bacteriumhas hampered studies to describe in vivo functions of adhesins ingeneral.
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. The Opacity proteins
Both Opa and Opc (previously class 5 proteins) are expressedn N. meningitidis; the term ‘opacity’ was first used for gonococ-al Opa proteins as colonies of bacteria expressing the proteinsppeared opaque under specific sub-stage lighting conditions [102].n meningococci, such opacities are only clearly discernible in acap-ulate phenotypes [17].
.1. Opa proteins
Opa proteins are eight-stranded transmembrane beta-barreltructures with four surface-exposed loops. The first loop (proxi-al to the N-terminus) is semivariable (SV), whereas loops 2 and 3
re hypervariable (HV1 and HV2) and the fourth loop is invariant.n N. meningitidis, 3-4 complete copies of opa genes are found. Thexpression Opa protein is translationally controlled whereby theumber of repeats (CTCTT) within the opa gene determines whetherhe gene is in frame. As each opa gene is expressed independently,his process is also linked to Opa antigenic variation [103,104]. It islear that this generates a vast array of Opa variants, which whilenabling bacteria to evade host immune mechanisms, poses theroblem of maintaining their functional role as adhesins. How theurface-exposed variable domains of Opa affect their interactionsith target cells has been a subject of several studies and those
f N. meningitidis in the context of CEACAM receptor targeting areescribed below.
The repertoire of Opa structures in a population may also benhanced by inter or intra-genomic recombination [103]. How-ver, recent surveys of clinical and carriage isolates have shownhat despite the presence of a wide range of possible alleles, spe-ific arrays of Opa variants are prevalent in N. meningitidis isolates105–107]. This is very likely a result of immunological selection,ut in addition, functional constraints driven by their ability to
nteract efficiently with certain target host receptors such as CEA-AMs (4.3.4) may enhance bacterial survival in vivo, thus selectingunctionally important opa alleles.
.2. Opc protein
N. meningitidis Opc (OpcA), a 10-stranded �-barrel moleculeith five surface exposed loops, is encoded by a single gene (opcA)
108–110]. Opc was the first neisserial integral outer membranerotein to be crystallised [111]. Although structurally relatively
nvariant, the levels of Opc expressed by a strain may vary in vivo andn vitro by a transcriptional control mechanism [22]. The expressionf Opc protein has not been demonstrated in gonococci or certainlonal lineages of N. meningitidis e.g. ET37 [112]. Interestingly, thetrains belonging to ET37 complex apparently have a tendency toause severe septicaemia and are less associated with meningitis113–115]. It is possible that Opc imparts N. meningitidis with thebility to cause meningitis [113].
.3. Opacity protein-host cell interactions in colonisation andathogenesis
Opa and Opc, both basic in nature, bind to at least two commonegatively charged molecules: heparan sulphate proteoglycans
HSPGs) and sialic acids [6,109,116] but they also display a degreef receptor specificity [116]. Opacity proteins may also engage inis interactions with LPS sialic acids, which may be responsible fornefficient Opa-mediated adhesion of bacteria with sialylated LPSompared with those lacking the sialylation [3,116].
27S (2009) B78–B89 B83
4.3.1. Proteoglycan and extracellular matrix interactionsIt appears that meningococcal Opa and Opc can interact directly
with HSPGs [6,111,117]. As Opc can also interact directly with extra-cellular matrix (ECM) proteins such as vitronectin and fibronectin,and as ECM proteins contain HSPGs binding sites, a range of complexinteractions become possible via Opc [6,113,118]. HSPG interactionis generally of low affinity, and does not result in efficient uptakebut co-ligation of secondary receptors such as integrins via ECMproteins leads to increased cellular invasion. The modes of Opa andOpc interactions are apparently determined by the target cell type[6,111,117].
4.3.2. Serum proteins and integrins as Opc receptorsN. 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) byinteracting with the serum proteins vitronectin and/or fibronectin[3,113,118]. Current studies are suggesting that vitronectin maybe the major serum target for N. meningitidis and it the acti-vated 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, maybe required as integrins often interact with proteins and some gly-cans simultaneously [17,120]. However, it is also possible that thelevel of Opc expressed by E. coli is not optimum since efficient inter-actions of N. meningitidis via Opc require the protein to be expressedat a high density on the bacterial surface [3].
4.3.3. Cytoskeletal molecules as targetsIn 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 ofmolecules, and regulate receptor activities as well as serve as scaf-folds to connect the cytoskeleton to a variety of signalling pathways[122]. Their involvement in linking the cytoskeleton and adhesivereceptors, such as integrins [123] and syndecans [124], is of interestas both are receptors for Opc. The true significance such interactionsto bacterial pathogenic potential remains to be investigated.
4.3.4. Opa and CEACAMsCEACAMs (carcinoembryonic antigen-related cell adhesion
molecules) are members of the Immunoglobulin superfamily andcomprise several important receptors including CEACAM1, CEA-CAM3, CEA (the product of ceacam5 gene) and CEACAM6 [125](Fig. 4). As the expression of some of the receptors is restrictedto particular cells or tissues, the choice of CEACAM member foradhesion imparts tissue tropism to the bacteria. CEACAM1 is themost widely expressed member, and it is also the most frequentlytargeted molecule by Opa-expressing meningococci.
Previous studies on meningococcal serogroup A and B strainshave demonstrated that Opa proteins make up distinct structuresthrough various combinations of their HV domains, and regionswithin HV1 and HV2 appear to be involved in tropism for distinctCEACAMs [117,126]. Overall, the receptor specificity of Opa proteinsis achieved through targeting a set of conserved residues on the N-terminal domain of the receptor [117]. For the vast majority of the
Opa types that are generated and maintained in neisserial genomicpool (presumably as they impart survival advantage to the bacteria),the property of targeting CEACAM1 is maintained. In addition, anumber of Opa types also compensate for the heterogeneity in theirtarget receptor family, thus also increasing their host tissue range.
B84 E. Carbonnelle et al. / Vaccine 27S (2009) B78–B89
Fig. 4. Opa protein interactions with different CEACAM family members. CEACAM3 and CEA are specific for neutrophils and epithelial cells respectively. CEACAM6 is expressedon 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 resultsin 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 showno creasei lead tot ytic ce
4
t[eivw�agbcadbtam[CSesdt
n the right depicts inflammatory responses to prior infections that may lead to innternalised, a process involving NF�B pathway [4]. Bacterial interactions may alsoo neutrophil-expressed CEACAMs results in bacterial internalisation by the phagoc
.3.5. CEACAM1 in health and diseaseFrom studies to date, it can be concluded that the interac-
ions of Opa proteins with CEACAM1 result in cellular invasion4,117,127], CEACAM1 is normally expressed at low levels but itsxpression is known to be upregulated significantly under thenfluence of inflammatory cytokines. In this context, in an initro study with A549 lung carcinoma cells, CEACAM1 expressionas shown to be upregulated considerably in response to IFN-, which resulted in infiltration of the target cells not only bycapsulate but also by fully capsulate serum resistant N. menin-itidis [4]. The efficiency of this process was enhanced in piliatedacteria but required Opa expression and high levels of targetell CEACAMs [4]. Perhaps the Opa-mediated invasion process isided by the pilus retraction forces, although this has not beenemonstrated. Such blood invasion by capsulate serum resistantacteria would increase the chances of widespread dissemina-ion especially in an immunocompromised host. Thus changes indhesin-receptor dynamics may be an additional host factor deter-ining an individuals susceptibility to N. meningitidis infection
4,128,129]. The data also suggest that receptors such as CEA-AMs could be at the cross-roads of colonisation and pathogenesis.
uch cell adhesion molecules expressed at low levels on mucosalpithelial cells may support attachment without significant inva-ion, favouring a colonisation state. However, when upregulateduring inflammation, the same receptors may become the por-al of tissue entry. In addition, increased interactions through
d CEACAM1 density on epithelial cells enabling encapsulated N. meningitidis to bede novo CEACAM1 synthesis through the action on NF-�B [4]. In addition, binding
lls [162].
CEACAMs with phagocytic cells could result in incomplete elim-ination of bacteria and the possibility of transmission withinthem.
4.3.6. CEACAMs and T cellsA variety of CEACAMs are expressed on human immune cells
but stimulated CD4+ T cells express only CEACAM1 [130]. Stud-ies with gonococci have suggested that Opa-CEACAM1 interactionssuppress CD4+ T cell proliferative responses [131]. A similar sup-pressive effect on T cell activation and proliferative response wasreported for meningococcal Opa-containing outer membrane vesi-cles [132]. Notably however, in other systems, CEACAM1 ligationhas been shown to increase the proliferation of T cells [130,133]. Inaddition, in one study on peripheral blood mononuclear cells, a highproliferative response to meningococcal Opa protein was observed[134]. Our current studies using live or killed meningococci have notshown any Opa-dependent immunosuppressive effects on CD4+ Tcell proliferation [163].
5. Targeting invasins for the intervention in meningococcalinfectivity
Opc is highly immunogenic in humans and elicits serum bacte-ricidal and opsonic antibodies [135,136]. Moreover, cross-reactiveanti-Opc monoclonal antibodies can be generated [109]. However,it has been surmised that their efficacy in vivo might be limited
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ue to the small heterogeneity observed in Opc between strainsnd due to variable levels of its expression [137]. In this context,n alternate view might also be considered. While as a sole vac-ine antigen, Opc may be of limited value, as a component of aomplex vaccine, it may prove to be of importance. First, in vitrotudies have shown that the loop 2 of Opc, which is the mostmmunogenic and prominent [138], is also involved in influenc-ng bacterial interactions in a number of model systems, includingnteractions with host cell receptors and serum proteins [118]. Sec-nd, it seems that Opc may enhance bacterial serum resistance byinding to serum vitronectin [119], a well known modulator of com-lement action. As such, antibodies that eliminate any circulatingpc+ phenotypes or reduce their virulence by blocking interac-
ions with complement modulators would be beneficial to theost.
In the case of Opa, in vitro transfected or cytokine-treated cellsused as post-inflammation model systems) in which increasedellular invasion by meningococci could be observed, served asplatform to assess the efficacy of intervention in Opa-CEACAM
nteractions in a setting of high receptor density. With the use ofrecombinant CEACAM-binding molecule (which was based on
he Moraxella catarrhalis receptor binding adhesin UspA1) able tonhibit all Opa-mediated interactions [139], selective cellular infil-ration could be avoided, whilst maintaining adhesion of capsulateiliated bacteria [4]. This suggests the possibility that, if anti-pa antibodies could be generated that block its interactions withEACAMs, it may be possible to interfere selectively with cellular
nvasion, while maintaining colonisation, a state believed to boostmmunity and provide long-term protection of the host. Further, its noteworthy that polymorphisms in CEACAM structure can sig-ificantly influence meningococcal interactions as demonstratedy in vitro mutagenesis studies of CEACAM1 [140]. In other studies,ertain CEACAM haplotypes have been found to be associated withncreased host susceptibility, and certain meningococcal Opa reper-oires with hyper-invasiveness and disease [105,106]. Association ofimited numbers of Opa repertoires with disease also suggests thatpa proteins could be candidate vaccine antigens in their own right.herefore, understanding their full potential as virulence factors ismportant for future approaches to control meningococcal infec-ion. Integral to this aim is the identification of the salient featuresf the Opa proteins that must be retained for the receptor targeting.f identified, it may be possible to interfere selectively with cellularnvasion.
. Recently identified adhesins and related proteins
Following genome sequencing of N. meningitidis strains, a num-er of novel adhesins (Table 1) and related proteins have come to
ight. They can be broadly grouped in four structural categories.heir known properties are described below.
.1. The autotransporters
NhhA (Neisseria hia homolog A, Mr 57 kDa) and App (Adhe-ion and penetration protein, Mr 160 kDa) are homologous tohe autotransporter proteins Hsf/Hia and Hap of H. influenzae,espectively. NhhA is widely expressed in virulent N. meningi-idis strains [141–143]. NhhA-mediated low levels of adhesion topithelial cells, heparan sulphate proteoglycans (HSPG) and lamininave been described [142]. App, which may be processed andeleased, is present in both pathogenic and commensal neisserial
pp. [144,145]. It is suggested that it may aid bacterial colonisa-ion by increasing adhesion before autocleavage and detachmentfter cleavage, thus facilitating their spread [144]. The presencef capsule apparently does not interfere with NhhA/App-mediateddhesion [142]. MspA (Meningococcal serine protease, Mr 157 kDa)
27S (2009) B78–B89 B85
is homologous to App and IgA1 protease; it is cleaved and secreted.It is expressed by several but not all virulent meningococcal lin-eages. It is reported to mediate binding of E. coli expressing theprotein to both epithelial and endothelial cells and elicit bacterici-dal antibodies [146].
6.2. The Oca-type adhesin
NadA (neisserial adhesin A, Mr 38 kDa) is a member of theoligomeric coiled coil family of adhesins (Oca), that interacts withan uncharacterised protein receptor on epithelial cells via its N-terminal globular region [147]. Three out of four hyper-virulentN. meningitidis lineages express distinct alleles of the gene. Itsexpression is phase variable and may be growth-phase dependent[148]. NadA mutation decreases epithelial adhesion of capsu-late N. meningitidis and invasion by acapsulate N. meningitidis. Asanti-NadA antibodies are bactericidal, it may be a potential vac-cine 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 proteinwith four surface exposed loops and a homolog of Opa adhesins,although it is not phase variable. The crystal structure of NspA hasbeen described [150,151]. Structural analyses suggest a potentialsite for binding to hydrophobic ligands such as lipids [151], althoughthis remains unexplored. Protective bactericidal antibodies to therecombinant NspA can be elicited [150] but the accessibility of pro-tein 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 bac-teria is a widespread route for the secretion of very large proteinsof over 1000 amino acid residues. TPS systems are encoded bytwo genes, tpsA and tpsB. The TpsB inserts into the outer mem-brane and facilitates transport of the TpsA to the cell surface. Threedistinct TPS systems were identified among meningococci [153].System 1 is ubiquitous, while systems 2 and 3 were significantlymore prevalent among hyper-invasive isolates than among isolatesof low invasive capacity. Recently the role of these TPS proteinsin bacterial interactions with target cells has been suggested. Itseems that these systems (also designated HrpA/HrpB in anal-ogy with haemagglutin/haemolysin-related proteins) may favourbacterial adhesion and/or are essential for intracellular survival[154,155].
7. Conclusions
This review has considered in some depth the interactions orlikely interactions of N. meningitidis with human barrier cells viaprotein adhesins. Numerous other interactions are also known tooccur, notably those via LPS which involve multiple host compo-nents and receptors such as CD14, TLR4, Siglecs and others. It isclear that when present in large numbers, meningococci causecytotoxic damage either directly or indirectly to cultured humancells, especially endothelial cells [156,97]. However, at non-toxicdoses, bacterial direct interactions with barrier cells via numer-ous protein adhesins may enable penetration into deeper tissues
by transcellular or paracellular routes without discernible cellu-lar damage. Additional considerations include interactions withphagocytic cells through for example pairing of LPS and Siglecsor Opa and CEACAMs. As these can also result in cellular infiltra-tion, they may constitute additional mechanisms of meningococcal
B accine
tlm
A
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issue invasion; in this case by being carried across epithe-ial and endothelial barriers inside phagocytes in a Trojan-horse
anner.
cknowledgement
Conflicts of interest: The authors declare no potential conflicts ofnterest.
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