One of the primary defense mechanisms of a host against invading bacterial pathogens is to deprive them from essential nutrients, such as iron, a defense mechanism that is known as nutritional immunity. In the human host, iron is bound by proteins, such as transferrin in serum, lactoferrin in secretions and mucosal surfaces, and hemoglobin and ferritin within cells. These proteins have very high affinities for iron, and, consequently, the concentration of free iron in the human fluids is too low to support microbial growth (Weinberg, 2009). Many bacteria produce siderophores, which are low-molecular-weight iron chelators that sequester otherwise inaccessible ferric iron from the environment. However, Neisseria spp. do not secrete siderophores; instead, they synthesize receptors that directly hijack iron sequestered in the iron-binding proteins of the host (Schryvers and Stojiljkovic, 1999). Several of these receptors consist of two proteins, a surface-exposed lipoprotein and an integral OMP. The integral OMP belongs to the TonB-dependent family (Tdf) of receptors, which includes also siderophore receptors. These receptors form 22-stranded β-barrels that are closed by an N-terminal plug. They interact with the TonB complex in the inner membrane, which deploys the proton-motive force to energize transport across the outer membrane (for a review, see Noinaj et al., 2010).

The transferrin receptor in Neisseria spp. consists of the Tdf member TbpA and the surface-exposed lipoprotein TbpB. The synthesis of these proteins is induced under iron limitation. In N. meningitidis, both proteins are essential for the acquisition of iron from human transferrin (Irwin et al., 1993), whilst TbpB is dispensable in N. gonorrhoeae, although its presence considerably enhances the efficiency of the process (Anderson et al., 1994). Both proteins can bind transferrin, but, in contrast to TbpA, TbpB preferably binds the iron-loaded form (Cornelissen and Sparling, 1996; Boulton et al., 1998). Thus, TbpB may enhance iron acquisition by selecting holo-transferrin to bind to the receptor. In addition, TbpB's affinity for the holo form may help in the release of the apo form from the receptor once iron is delivered. TbpB is a bilobed protein probably resulting from gene duplication. Each lobe consists of a β-barrel domain and an adjacent handle domain, which is also rich in β-sheet structure (Figure 5) (Calmettes et al., 2012; Noinaj et al., 2012). Human transferrin is also a bilobed protein with a Fe³⁺-binding site within each lobe (Hall et al., 2002). Structural analysis revealed that TbpA and TbpB both bind the C-lobe of transferrin using non-overlapping binding sites (Noinaj et al., 2012). The C-lobe of transferrin consists of two subdomains, C1 and C2, which are connected by a hinge at the base of a deep cleft that contains the Fe³⁺-binding site. TbpB binds both the C1 and C2 subdomains of holo-transferrin via the handle and β-barrel domains in its N-lobe, respectively (Calmettes et al., 2012). This explains the selectivity of TbpB for holo-transferrin as the apo-form shows a 51° rotation of the C2 domain which would drastically reduce the TbpB-transferrin interface (Calmettes et al., 2012). In the available structures, the C-lobe of TbpB is not involved in transferrin binding, and its function is unclear. An α-helix from extracellular loop 3 of TbpA intrudes into the cleft between the C1 and C2 subdomains of transferrin, which induces a partial opening of the cleft and destabilizes the coordination site of the ferric ion (Noinaj et al., 2012), which facilitates its release. Released iron would enter a closed chamber formed by TbpA, TbpB, and transferrin, which prevents its diffusion into the medium and positions it for further transport through the barrel of TbpA (Noinaj et al., 2012).

The lactoferrin receptor is constituted by the Tdf member LbpA and the surface-exposed lipoprotein LbpB (Pettersson et al., 1994b, 1998; Prinz et al., 1999). Whilst the corresponding genes are ubiquitous in N. meningitidis, they are disrupted in about half of the N. gonorrhoeae isolates (Anderson et al., 2003). Nevertheless, expression of a functional lactoferrin receptor in N. gonorrhoeae provided a competitive advantage in a human infection model, and it was essential for infection by a strain lacking the transferrin receptor (Anderson et al., 2003). LbpA and LbpB both bind lactoferrin, but LbpB is not essential for its utilization as an iron source in both species (Pettersson et al., 1998; Biswas et al., 1999). Whilst lactoferrin and transferrin are homologous proteins, also LbpA and LbpB share extensive sequence similarity with TbpA and TbpB, respectively (Pettersson et al., 1994a, 1998). Crystallization of the N lobe of LbpB revealed a structure very similar to that of the corresponding part of TbpB. It also consists of two domains, a handle domain and a β-barrel domain with long loops projecting from both domains and forming an extended cap region (Figure 5) (Brooks et al., 2014). In silico docking experiments indicated that the cap region constitutes the binding site to which lactoferrin binds with its N-lobe (Brooks et al., 2014). However, recent biochemical analysis revealed that it preferentially binds the C-lobe of iron-loaded lactoferrin (Ostan et al., 2017), just like TbpB binds the C-lobe of iron-loaded transferrin (see above), suggesting a similar role for LbpB as for TbpB in selecting iron-loaded ligands. LbpB appears to have an additional role in protecting the bacteria against the bactericidal activity of lactoferricin (Morgenthau et al., 2012). Lactoferricin is a small cationic peptide, which is released from the N terminus of lactoferrin by proteolysis (Bellamy et al., 1992; Gifford et al., 2005). LbpB contains two highly variable stretches rich in negatively charged amino-acid residues in its C-lobe (Pettersson et al., 1999) that have been shown to mediate protection against this cationic peptide (Morgenthau et al., 2014). Thus, binding of the N-lobe of lactoferrin to the C-lobe of LbpB may prevent proteolysis events that generate lactoferricin. In addition or alternatively, the C-lobe may also be able to bind the free peptide, thus neutralizing its toxic effects (Ostan et al., 2017).

N. meningitidis can express two receptors for hemoglobin, HmbR and HpuAB, which enable it to extract heme from hemoglobin and use it as an iron source (Stojiljkovic et al., 1995; Lewis et al., 1997). Of these, the HpuAB system is also capable of extracting heme from hemoglobin-haptoglobin complexes (Lewis et al., 1997). Expression of the corresponding genes is controlled by iron availability and is prone to phase variation by slipped-strand mispairing (Lewis et al., 1999). HpuAB is a bipartite receptor, in which HpuB is a Tdf member and HpuA is a surface-exposed lipoprotein. In the absence of HpuA, HpuB still binds its ligands but with low affinity, and they cannot be used as an iron source (Rohde et al., 2002; Rohde and Dyer, 2004). Ligands did not bind to cells expressing only HpuA (Rohde and Dyer, 2004), but a direct interaction could be demonstrated in pull-down assays with purified HpuA (Wong et al., 2015). The crystal structure was solved of Kingella denitrificans HpuA alone and in complex with hemoglobin, and of a C-terminal fragment of N. gonorrhoeae HpuA (Wong et al., 2015). HpuA is about half the size of TbpB, and its structure shows a similar fold as a single lobe of TbpB (Figure 5). It consists of a compact C-terminal β-barrel and an N-terminal open β-sandwich domain. Two large loops, loops 1 and 5, extend from the core of the protein. These exposed loops show high sequence variability, suggesting they are under immune selection. In spite of their solvent exposure, these loops have a high content of hydrophobic amino-acid residues, and although they display high sequence variability, they are the major interaction sites of hemoglobin, with loops 1 and 5 contacting the β- and α-chains, respectively, of a hemoglobin dimer. Additional interactions involve residues on two other loops.

The complement system plays a key role in the host's defense against microbial invaders. Host cells are protected from complement activation, amongst others by binding the soluble factor H, a major negative regulator of the alternative complement pathway. By producing fHbp (a.k.a. GNA1870 or LP2086), N. meningitidis hijacks factor H and thereby limits complement activation at its surface (Madico et al., 2006; Schneider et al., 2006). Indeed, fHbp expression was found to improve bacterial survival in human blood and serum (Seib et al., 2009). Additionally, fHbp synthesis was found to protect the bacteria against the cationic antimicrobial peptide LL-37, a host defense peptide that destabilizes negatively charged bacterial membranes (Seib et al., 2009). Presumably, LL-37 interacts with fHbp also by electrostatic interaction.

fHbp is highly immunogenic and induces bactericidal antibodies that activate the classical complement pathway. In addition, antibodies may prevent binding of factor H to fHbp and thereby prevent inhibition of the alternative pathway. Based on sequence variation, two different subfamilies (Fletcher et al., 2004) or three variant groups (Masignani et al., 2003) were distinguished, with very limited immunological cross-reactivity between the groups (Masignani et al., 2003). One and two variants are included as components in the recently developed vaccines Bexsero® and Trumenba®, respectively.

The level of expression of fHbp varies considerably between different strains (Biagini et al., 2016), which may affect vaccine efficacy. Synthesis of the protein is induced under oxygen limitation (Oriente et al., 2010). Furthermore, fHbp synthesis is post-transcriptionally controlled by temperature due to the presence of a secondary structure at the 5′ end of its mRNA, which could function as a thermosensor; expression is considerably lower at 30°C than at 37°C (Loh et al., 2016). Thus, the synthesis of fHbp is presumably low on the mucosal surfaces of the nasopharynx, where the temperature is relatively low and the oxygen concentration is high. Consequently, vaccines solely based on fHbp will possibly not prevent carriage and spreading of the bacteria amongst the population and offer limited herd immunity (Loh et al., 2016). The synthesis of fHbp will increase as the bacteria reach the submucosal epithelial surfaces and the bloodstream, i.e., when protection against the host's defense mechanisms becomes essential. The synthesis of fHbp is also influenced by iron availability. However, whilst expression is reduced under iron limitation in most strains, it is increased in some lineages (Sanders et al., 2012), which makes it difficult to speculate about the biological significance of this phenomenon. Whether there is a relation with the reported capacity of fHbp to bind siderophores produced by other bacteria (Veggi et al., 2012) is questionable as fHbp synthesis is reduced in most strains under iron limitation when siderophore production is induced. A physiological role for the siderophore-binding capacity of fHbp has not been demonstrated.

The structure of fHbp has been determined by NMR and crystallography (Cantini et al., 2009; Schneider et al., 2009). The overall fold resembles those of HpuA and of the individual lobes of TbpB and LbpB and consists of a C-terminal β-barrel and an N-terminal taco-shaped barrel-like structure (Figure 5). Factor H consists of 20 complement control protein repeats, and of these, repeats 6 and 7 were found to bind fHbp with extensive interactions in co-crystals (Schneider et al., 2009). Interestingly, this binding site overlaps with the binding site for glycosaminoglycans to which factor H binds on mammalian cells to prevent complement activation.

Like fHbp, NHBA (a.k.a. GNA2132) is a component of the Bexsero® vaccine. In spite of considerable sequence variability, particularly near the N terminus (Bambini et al., 2009), NHBA elicits broadly cross-reactive bactericidal antibodies that conferred passive protection in a rat model (Welsch et al., 2003; Giuliani et al., 2010; Serruto et al., 2010).

NHBA was shown to bind heparin and heparan sulfate (Serruto et al., 2010). It consists of two domains. The structure of the conserved C-terminal domain has been determined by NMR and shows a similar 8-stranded β-barrel as those in the lipoproteins described above (Figure 5) (Esposito et al., 2011). The structure of the N-terminal domain has not been solved; it was predicted to be an intrinsically unfolded polypeptide (Esposito et al., 2011). In between these domains, NHBA contains an arginine-rich segment that binds heparin and heparan sulfate by electrostatic interaction (Serruto et al., 2010). The binding of heparin and heparin-like molecules has several functions. First, heparin binding was shown to correlate with increased survival of an unencapsulated N. meningitidis strain in human serum. In an encapsulated strain, such effect of NHBA synthesis was not observed, presumably because of the dominant role of the capsule in mediating serum resistance. As heparin is known to interact with complement proteins, including factor H, C4b-binding protein and C1 inhibitor (Yu et al., 2005), it was suggested that heparin bound at the cell surface might impart serum resistance by its capacity to bind such complement-regulatory molecules (Serruto et al., 2010). Alternatively, it might form a negatively charged capsule-like structure resembling the classical polysaccharide capsule (Serruto et al., 2010). NHBA has also been shown to function as an adhesin by binding to heparan sulfate proteoglycans that are present on surface of epithelial cells (Vacca et al., 2016).

Besides heparin and heparin-like molecules, NHBA also binds other polyanionic structures, including DNA (Arenas et al., 2013a). N. meningitidis forms microcolonies on the epithelial surfaces in the nasopharynx (Sim et al., 2000). Extracellular DNA (eDNA) is an important component of the extracellular matrix of these biofilm-like structures, and it is essential in the initiation of biofilm formation in most clonal lineages (Lappann et al., 2010). NHBA binds eDNA and inactivation of the nhbA gene in these lineages impairs biofilm formation (Arenas et al., 2013a).

NHBA can be cleaved immediately upstream of the arginine-rich segment by the autotransporter protease NalP (see below). Consequently, a C-terminal fragment including the arginine-rich segment is released into the milieu (Serruto et al., 2010). This fragment is taken up by endothelial cells and accumulates in mitochondria, where it induces the production of reactive oxygen species. The resulting phosphorylation of the adherens junction protein VE-cadherin and its subsequent internalization leads to increased endothelial permeability (Casellato et al., 2014). Thus, the released fragment of NHBA may contribute to the extensive vascular leakage that is typical for meningococcal sepsis. The arginine-rich segment was essential for this role of the NHBA C-terminal fragment presumably by functioning as a mitochondrial targeting signal. A slightly smaller fragment that is released from NHBA by lactoferrin-mediated cleavage and that lacks the arginine-rich segment did not induce endothelial leakage (Casellato et al., 2014).

IgA protease is produced by both N. meningitidis and N. gonorrhoeae. The passenger domain consists of two subdomains, an N-terminal domain containing the protease activity and a polypeptide called the α-peptide; these domains are connected via a small γ-peptide (Pohlner et al., 1987). The passenger is connected to the β-domain via a linker peptide. The protease domain can be released into the extracellular milieu by autocatalytic cleavage at sites (PAPSP, PPSP, or PPAP) located in between the protease domain and the γ-peptide, between the γ-peptide and the α-peptide, and between the α-peptide and the linker peptide. The presence of the latter processing site is strain dependent (Roussel-Jazédé et al., 2014). Alternatively, the entire passenger including the linker peptide can be released after cleavage by the autotransporter protease NalP (see below) (van Ulsen et al., 2003). IgA protease cleaves human IgA1 at a site (TPPTPSPS), which resembles the autocatalytic processing sites and is located in the hinge region between the Fab and the Fc domains (Plaut et al., 1975). IgA protease does not cleave IgA2, which lacks such a cleavage site (Plaut et al., 1975). Cleavage of IgA1 may inhibit IgA-mediated agglutination and subsequent mechanical clearance of the bacteria in the nasopharynx. IgA protease has also been shown to cleave the lysosome-associated membrane protein LAMP1 (Hauck and Meyer, 1997), which was reported to promote bacterial survival within epithelial cells (Lin et al., 1997) and transcytosis across polarized epithelia (Hopper et al., 2000). In addition, IgA protease cleaves the vesicular membrane protein synaptobrevin II in chromaffin cells (Binscheck et al., 1995) and the human chorionic gonadotropin hormone (Senior et al., 2001), but the physiological implications are not clear. All these alternative substrates have a target site resembling the autocatalytic cleavage sites.

The α-peptide contains a variable number of nuclear localization signals (NLS), which are arginine-rich peptide segments (Pohlner et al., 1995; Roussel-Jazédé et al., 2014), and it was indeed demonstrated to target an attached reporter protein to the nucleus of eukaryotic cells (Pohlner et al., 1995). When NalP releases IgA protease with attached α-peptide from the bacterial cell surface, the IgA protease is targeted to the nucleus where it cleaves the p65/RelA component of transcription factor NF-κB, thus silencing the expression of several NF-κB-responsive genes, including those encoding interleukin 8 and the anti-apoptotic protein cFLIP (Besbes et al., 2015). This altered gene expression results in sustained activation of c-Jun N-terminal kinase and apoptosis. Interestingly, purified IgA protease without attached α-peptide has been shown to inhibit TNFα-induced apoptosis of monocytes, presumably by the observed cleavage of the TNF receptor II (Beck and Meyer, 2000).

When NalP is not expressed and the autocleavage site between the α-peptide and the linker peptide is absent, the α-peptide is exposed at the bacterial cell surface covalently connected to the β-domain. Since NLS are positively charged protein segments, they bind polyanions like eDNA, thus stimulating biofilm formation (Arenas et al., 2013a). The α-peptide also binds heparin (Roussel-Jazédé et al., 2014) and may have similar functions in conferring serum resistance and mediating adhesion as discussed above for NHBA. Whether the α-peptide also directly affects gene expression by binding DNA after it has been targeted to the nucleus of eukaryotic cells has not been investigated yet.

The nalP gene is found in the vast majority of N. meningitidis isolates, with the notable exception of invasive isolates of clonal complexes ST-269 and ST-461 (Oldfield et al., 2013). Its expression is prone to phase variation by slipped-strand mispairing at a poly-C tract in the coding region (Turner et al., 2002; van Ulsen et al., 2003). An analysis of its phase variation status revealed that approximately half of the strains were phased on, and no significant differences were found in this respect between carriage and invasive strains (Oldfield et al., 2013).

NalP is a subtilisin-like serine protease. Its passenger is released from the cell surface by autoproteolytic cleavage. However, after cleavage between the passenger and the β-domain, the passenger remains temporarily attached at the cell surface by an N-terminal lipid anchor (Roussel-Jazédé et al., 2013). In this position, it releases several autotransporters and lipoproteins from the bacterial cell surface. The release of these proteins has relevant consequences. As already discussed above, the NalP-mediated release of IgA protease with attached α-peptide and of the C-terminal part of NHBA from the cell surface stimulates apoptosis and increases the permeability of endothelial and epithelial cell layers. In contrast, when nalP is phased off, the α-peptide and complete NHBA are retained at the cell surface, which stimulates biofilm formation and adhesion and protects against host defenses. Thus, the absence of nalP expression appears to favor colonization and biofilm formation, whilst nalP expression may lead to dispersal of biofilms and invasion or spreading to a new host.

Another target of NalP is the LbpB component of the lactoferrin receptor (Roussel-Jazédé et al., 2010). LbpB is a very immunogenic protein, and its NalP-mediated release from the cell surface imparted protection against the complement-mediated killing by LbpB-specific bactericidal antibodies (Roussel-Jazédé et al., 2010). As discussed above, LbpB is not essential for the acquisition of iron from lactoferrin, although it may enhance the process by selecting holo-lactoferrin for binding to the receptor. The latter function may be lost when LbpB is cleaved from the cell surface by NalP, but this is compensated by protection against LbpB-specific bactericidal antibodies. Another function of LbpB discussed above, i.e., offering protection against the membrane-damaging effects of lactoferricin, is probably retained, even if the protein is released into the extracellular milieu. Thus, due to the phase-variable expression of nalP, a subpopulation of the bacterial cells will lose LbpB from the cell surface, and this subpopulation may be less efficient in iron acquisition but will have a competitive advantage when LbpB-specific antibodies are elicited in the host. It is noteworthy that in N. gonorrhoeae, which does not produce NalP because the gene is disrupted, the expression of lbpB itself is prone to phase variation (Anderson et al., 2003).

Besides bacterial proteins, NalP also cleaves at least one host protein, i.e., the complement factor C3. Thereby, it avoids complement activation and confers serum resistance (Del Tordello et al., 2014). Consistently, nalP expression was earlier reported to be upregulated during growth in an ex vivo human whole-blood model and to be essential for survival under these conditions (Echenique-Rivera et al., 2011b).

App and AusI (a.k.a. MspA) are both chemotrypsin-like serine proteases with homology to IgA protease (Abdel Hadi et al., 2001; van Ulsen et al., 2001, 2006; Turner et al., 2006). Whereas, an intact app gene is ubiquitous among N. meningitidis strains, the ausI gene is disrupted by premature stop codons in several strains, and its expression is prone to phase variation by the presence of a poly-C tract in the coding region (Martin et al., 2003; van Ulsen et al., 2006). Interestingly, an extensive study in mostly serogroup B and Y isolates revealed that the ausI gene was phased on in 90% of both invasive and carriage strains of serogroup B. As 33% phased on would be expected in the case of random on and off switching, this suggests an important role for AusI in the biology of serogroup B (Oldfield et al., 2013). Such a preference for the on phase of ausI was not found in the serogroup Y isolates.

App and AusI are both released from the bacterial cell surface by autoproteolytic cleavage. However, like for IgA protease, a larger form of these proteins with a C-terminal extension, called α-peptide, can be released by NalP-mediated cleavage (van Ulsen et al., 2003, 2006; Turner et al., 2006). The α-peptide of App contains two NLS similar to those in the α-peptide of IgA protease, but such sequences are not present in the α-peptide of AusI. Nevertheless, both proteins with attached α-peptides were shown to be targeted to the nucleus after being taken up by dendritic cells and to induce apoptosis, presumably by their capacity to bind histones and to cleave histone H3 (Khairalla et al., 2015). Uptake into the dendritic cells was shown to be mediated by the mannose receptor and the transferrin receptor 1 to which the proteases could be crosslinked.

Earlier, both App and AusI were reported to function as adhesins (Serruto et al., 2003; Turner et al., 2006), a function which is difficult to reconcile with their cleavage from the cell surface. Perhaps, the proteins remain temporarily associated with the cell surface before they are released. Heterologous expression of App and AusI in E. coli mediated adhesion of the bacteria to Chang epithelial cells, whereas variable adhesion to other cell lines was observed (Serruto et al., 2003; Turner et al., 2006). App-mediated adhesion to Chang cells could also be demonstrated in N. meningitidis (Serruto et al., 2003). The presence of a capsule did not seem to interfere with the adhesin function, suggesting that this large protein (~160 kDa) extends beyond the capsule to interact with its receptor on the Chang cells. However, deletion analysis suggested that the α-peptide, which is located close to the bacterial cell surface in uncleaved App and is unlikely to extend beyond the capsule, is the main determinant for adhesion (Serruto et al., 2003). Protease treatment indicated the involvement of a proteinaceous receptor on Chang cells, but it is not known whether the transferrin receptor 1 or the mannose receptor, which mediate uptake of App and AusI in dendritic cells (see above), are involved. App was also shown to mediate interbacterial aggregation on cell layers (Serruto et al., 2003); possibly, this is mediated by the binding of the NLS in the α-peptide to eDNA, analogous to the role of the α-peptide of IgA protease in biofilm formation (see above).

AutA and AutB are two related, relatively small autotransporters of ~75 kDa including the β-domain. The autA gene is broadly distributed among Neisseria spp., whereas the presence of the autB gene is restricted to the pathogenic Neisseria spp., N. meningitidis and N. gonorrhoeae (Arenas et al., 2015a, 2016). Also some, but not all, strains of Haemophilus influenzae and closely related Haemophilus spp. contain an autB gene. Analysis of the autB-flanking sequences in both genera suggested that a common ancestor of the two pathogenic Neisseria spp. has acquired the gene from a H. influenzae strain by horizontal gene transfer (Davis et al., 2001; Arenas et al., 2016).

The expression of autA and autB is prone to phase variation by slipped-strand mispairing at AAGC nucleotide repeats located immediately downstream of the start codon (Peak et al., 1999). In addition, the autA gene was disrupted in 76% of the meningococcal genomes analyzed by premature stop codons, insertions and/or deletions (Arenas et al., 2015a) suggesting selection pressure against expression of the gene. However, the gene was phased on in ~33% of the strains with an intact autA, consistent with random on and off switching. In contrast to autA, autB was disrupted in only 8% of the N. meningitidis genomes analyzed (Arenas et al., 2016). Curiously, the gene is phased off in the vast majority of the genomes with an intact autB, and in only ~2% of the strains the gene could be expressed. This strongly suggests that, even if an intact autB is retained, its expression is under negative selection pressure probably already in the nasopharynx as no significant difference between invasive and carriage isolates was observed in this respect (Arenas et al., 2016).

The passenger domains of AutA and AutB are not proteolytically released, and they are exposed at the bacterial cell surface covalently attached to the β-domain (Arenas et al., 2015a, 2016). AutA expression was shown to cause bacterial autoaggregation (Arenas et al., 2015a), a trait associated with resistance to host immune defenses such as phagocytosis (Ochiai et al., 1993). Autoaggregation is mediated by mutual interaction of AutA proteins on neighboring cells. In addition, AutA was found to bind DNA, which acts as an adhesive between cells (Arenas et al., 2015a; Arenas and Tommassen, 2017). Autoaggregation also has consequences for the architecture of biofilms formed. Besides, AutA was reported to be a potent CD4⁺ T-cell and B-cell stimulating antigen (Ait-Tahar et al., 2000). Expression of autB was shown to enhance biofilm formation, and it retarded the passage of the bacteria through epithelial cell layers (Arenas et al., 2016). This suggests that AutB could have a role during host colonization.

NadA shows high sequence similarity to the well-known virulence factors YadA from Yersinia enterocolitica and UspA2 from Moraxella catarrhalis (van Ulsen et al., 2001). It is a component of the Bexsero® vaccine. However, the gene is present in only ~30% of N. meningitidis isolates with a lower prevalence in carriage strains (~15%) (Comanducci et al., 2002, 2004). Furthermore, based on sequence variability, two main groups of NadA variants can be discriminated (Bambini et al., 2014) with limited immunological cross-reactivity between the groups (Comanducci et al., 2004), and the expression levels are highly variable (Martin et al., 2003), which also may impact on vaccine efficacy (Comanducci et al., 2002). Expression levels are determined by phase variation at a TAAA tetranucleotide repeat region upstream of the nadA promoter (Martin et al., 2003), whilst also on/off switching can occur by phase variation at a poly-C stretch within the coding region of some nadA variants (Bambini et al., 2014). Expression of nadA is transcriptionally controlled by the repressor NadR (Schielke et al., 2009), which binds operator sequences upstream and downstream of the TAAA repeat region, the latter overlapping with the -10 region of the promoter (Metruccio et al., 2009). The metabolite 4-hydroxyphenylacetic acid was found to act as an inducer of nadA expression (Metruccio et al., 2009). This metabolite is secreted in human saliva, suggesting that nadA expression will be induced on the mucosal surfaces of the nasopharynx. Interestingly, upon derepression, the expression levels were similar independent of the number of TAAA repeats, indicating that the number of repeats determines the level of repression by NadR rather than the expression levels in the induced state.

NadA functions as an adhesin and invasin. Its heterologous expression in E. coli promoted adherence of the bacteria to and invasion of Chang epithelial cells, and this function was confirmed in N. meningitidis (Capecchi et al., 2005). NadA function could also be reconstituted in a yadA mutant of Y. enterocolitica, and it was shown that β1 integrins function as its receptor on host cells (Nägele et al., 2011). Curiously, NadA was also shown to bind the heat shock protein Hsp90, a normally cytoplasmic chaperone, and purified Hsp90 was shown to inhibit NadA-mediated adhesion and invasion in vitro (Montanari et al., 2012). The physiological significance of these observations is not clear.

Whilst the passenger domains of classical autotransporters usually form a long β-helical structure that displays globular functional domains, trimeric autotransporters have a completely different architecture consisting of a stalk domain that displays one or several head domains (Grijpstra et al., 2013). The stalk is a coiled-coil structure that extends from the α-helices that occupy the membrane-embedded β-barrel (Figure 1D). NadA has a similar overall architecture but is somewhat atypical in that a separate globular head domain is lacking (Malito et al., 2014). Instead, the coiled coils extend up to the N terminus and the head is formed by insertions of 36 residues that protrude from the stalk and form wing-like structures (Figure 6). The adhesin activity was mapped to the head region (Capecchi et al., 2005; Tavano et al., 2011), which is apparently sufficiently exposed from the bacterial cell surface to be functional also in encapsulated strains (Capecchi et al., 2005).

NhhA (a.k.a. Msf) was identified as a homolog of the adhesin Hia of non-typeable H. influenzae (Peak et al., 2000; van Ulsen et al., 2001), and the nhhA gene was found to be ubiquitously present in all N. meningitidis strains examined, although disrupted in some of them (Peak et al., 2000). Expression levels varied widely and were affected in one particular lineage, i.e., ST41/44, by an amino-acid substitution in the β-domain, which prevents trimerization and surface exposition of the passenger domain (Echenique-Rivera et al., 2011a). The function of NhhA as an adhesin was demonstrated after its heterologous production in E. coli, which mediated adherence of the bacteria to epithelial cell lines, and was confirmed after constructing an nhhA mutant in an encapsulated N. meningitidis strain (Scarselli et al., 2006). It was demonstrated that purified NhhA and NhhA-producing cells bind the extracellular matrix components laminin and heparan sulfate (Scarselli et al., 2006). In addition, NhhA was shown to bind activated vitronectin, a complement regulatory protein, and, thus, to impart serum resistance to the bacteria by inhibiting the formation of the membrane-attack complex (Griffiths et al., 2011). Consistently, an nhhA mutant was less virulent and showed reduced survival in blood after intraperitoneal challenge of mice (Sjölinder et al., 2008). Expression of the protein appeared to be essential also for the colonization of the nasopharyngeal mucosa and for disease development after intranasal challenge in a murine model of meningococcal disease. The reduced colonization levels are probably related to the in vitro observations that wild-type bacteria are more resistant than the nhhA mutant to phagocytosis (Sjölinder et al., 2008) and that NhhA induces apoptosis of macrophages (Sjölinder et al., 2012). In addition, immune modulatory activities of NhhA were reported (Wang et al., 2016). NhhA blocked differentiation of monocytes into dendritic cells and induced their differentiation into macrophages that failed to produce proinflammatory mediators but produced anti-inflammatory responses. These differentiated macrophages were highly efficient in eliminating the bacteria. Hence, this response prevents dissemination and sustains asymptomatic colonization (Wang et al., 2016).

Like the TpsA proteins, the MafB proteins are polymorphic toxins that display a small variable toxic domain at their C terminus (Arenas et al., 2015b; Jamet et al., 2015). The mafB genes are localized on the chromosome on genomic islands designated Maf Genomic Islands (MGI) (Jamet et al., 2015). Up to three MGI can be present in a single meningococcal strain. The gene organization on the MGI is reminiscent of the loci encoding the TPS systems (Figure 7). The mafB genes are usually flanked by a mafA and a mafI gene. The mafI gene encodes a small immunity protein, which has been shown to interact with the toxic domain of MafB and to neutralize its toxic activity (Arenas et al., 2015b; Jamet et al., 2015). Indeed, MafB producers were shown to inhibit the growth of congeners that do not produce the cognate immunity protein. Downstream of the mafI gene, a variable number of 5′ truncated mafB-related genes are located, called mafB-CT cassettes (Figure 7). Like the tpsC cassettes, these mafB-CT cassettes offer a reservoir of alternative toxic domains, which could potentially be displayed at the C terminus of MafB after a genetic recombination event.

Apart from the presence of a toxic domain, MafB proteins do not show any similarity with TpsA proteins. They are synthesized with a signal sequence for transport across the inner membrane via the Sec machinery. Immediately after the signal sequence, all MafB proteins contain a conserved domain of unknown function, DUF1020, followed by the C-terminal toxic domain (Figure 7). In some MafB proteins, a Hint domain, which is expected to mediate protein splicing (Amitai et al., 2003), separates the DUF1020 from the toxic domain. How MafB is translocated across the outer membrane is unclear. Considering the parallelism with the TPS loci (Figure 7), MafA would be an obvious candidate for a protein involved in the secretion process. Earlier studies in N. gonorrhoeae indicated that MafA is an adhesin that binds host glycolipids (Paruchuri et al., 1990). Accordingly, these proteins were designated members of a multiple adhesin family (maf). MafA doesn't show any similarity with TpsB. It is produced with a signal sequence containing a lipobox motif, suggesting it's a lipoprotein, and secondary structure predictions didn't suggest a β-barrel structure, which is difficult to reconcile with its possible role as a transporter (Arenas et al., 2015b). Indeed, MafB was found in the extracellular medium when expressed in a Neisseria cinerea mutant lacking MafA (Jamet et al., 2015), suggesting that MafB secretion is independent of MafA. Furthermore, MafB was not secreted when produced in E. coli suggesting that it is not a novel type of autotransporter and that the transport mechanism involved is not present in E. coli (Jamet et al., 2015). It was suggested that MafB may be released with OMVs, which are abundantly shed from the surface in N. meningitidis (Jamet et al., 2015).

The name TspB, for T- and B-cell-stimulating protein B, may be a misnomer as evidence for such a function has, to the best of our knowledge, never been published. TspB was identified as a protein that binds immunoglobulins (Ig) (Müller et al., 2013). The protein is encoded by a prophage that is associated with invasive-disease isolates (Bille et al., 2005; Müller et al., 2013), and its synthesis is induced in the presence of human serum (Müller et al., 2013). Up to four copies of the tspB gene can be found in a genome.

The TspB proteins contain variable sequences at the N- and C-terminal ends and a conserved core region followed by a variable proline-rich segment (Müller et al., 2013). The core region of TspB binds the Fcγ region of IgG2. Ig-binding proteins are produced by several human pathogens, and they prevent the activation of complement. Indeed, TspB was shown to contribute to the survival of N. meningitidis in normal human serum by inhibiting IgM-mediated activation of the classical complement pathway (Müller et al., 2015). Since TspB binds IgM only poorly (Müller et al., 2013), this effect is probably indirect. TspB was found to bind DNA and to the form a biofilm-like matrix consisting of TspB, IgG, and DNA surrounding bacterial aggregates (Müller et al., 2013, 2015). Possibly, the presence of IgG in the matrix stimulates the non-productive activation of complement far away from the bacterial cell surface.

The secretion mechanism of TspB remains unclear. It could be released as a minor coat protein of filamentous phage particles, which was demonstrated to be mediated by the secretin PilQ, a protein also responsible for extrusion of type IV pili from the bacteria (Bille et al., 2005). However, in the studies of Müller et al. (2013), TspB appeared not to be associated with phage particles. The protein is produced with an N-terminal signal sequence for Sec-mediated transport across the inner membrane and a hydrophobic region near the C terminus that should act as a stop-transfer signal and anchor the protein in the membrane. Thus, possibly, the protein is only released after cell lysis.