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. Author manuscript; available in PMC: 2013 Oct 1.
Published in final edited form as: Mol Microbiol. 2012 Sep 7;86(2):246–257. doi: 10.1111/mmi.12002

The transferrin-iron import system from pathogenic Neisseria species

Nicholas Noinaj 1, Susan K Buchanan 1,*, Cynthia Nau Cornelissen 2,*
PMCID: PMC3468669  NIHMSID: NIHMS402530  PMID: 22957710

Summary

The two pathogenic species within the genus Neisseria cause the diseases gonorrhea and meningitis. While vaccines are available to protect against four N. meningitidis serogroups, there is currently no commercial vaccine to protect against serogroup B or against N. gonorrhoeae. Moreover, the available vaccines have significant limitations and with antibiotic resistance becoming an alarming issue, the search for effective vaccine targets to elicit long-lasting protection against Neisseria species is becoming more urgent. One strategy for vaccine development has targeted the neisserial iron import systems. Without iron, the Neisseriae cannot survive and therefore these iron import systems tend to be relatively well conserved and are promising vaccine targets, having the potential to offer broad protection against both gonococcal and meningococcal infections. These efforts have been boosted by recent reports of the crystal structures of the neisserial receptor proteins TbpA and TbpB, each solved in complex with human transferrin, an iron binding protein normally responsible for delivering iron to human cells. Here, we review the recent structural reports and put them into perspective with available functional studies in order to derive the mechanism(s) for how the pathogenic Neisseriae are able to hijack human iron transport systems for their own survival and pathogenesis.

Pathogenic Neisseria species

While at least ten Neisseria species are associated with humans, only N. gonorrhoeae and N. meningitidis are pathogenic to humans (Marri et al., 2010). N. gonorrhoeae causes the sexually-transmitted infection gonorrhea. By contrast N. meningitidis is a frequent colonizer of the human oropharynx, but can also cause invasive disease manifested as meningitis or septicemia. The reported incidence of gonorrhea in the United States is over 300,000 cases per year, in contrast to the incidence of invasive meningococcal disease, which has been decreasing and is currently below 1000 cases per year (CDC, 2012). N. meningitidis may be carried asymptomatically by up to 10% of healthy humans, but in rare cases, the pathogen can disseminate to cause rapidly-progressing septicemia as well as meningitis, both of which are potentially lethal infections (Stephens et al., 2007). In contrast, gonococcal infections are rarely life-threatening. Nonetheless, significant morbidity is associated with gonococcal infections as many are asymptomatic, particularly in women, which facilitates ascension into the upper reproductive tract, leading to salpingitis, pelvic inflammatory disease, infertility, and ectopic pregnancy (Sparling, 1990). Ascending gonococcal infections in men are uncommon but can lead to prostatitis, epididymitis and infertility (Sparling, 1990).

Despite the distinct diseases caused by the pathogenic Neisseria species, there are very few differences between the pathogens at the genomic level. The primary virulence factor employed by N. meningitidis, which is lacking in N. gonorrhoeae, is the polysaccharide capsule. This surface structure protects the meningococcus from desiccation, enhances serum resistance, and elicits a protective immune response (for review see Virji, 2009). The gonococcus, which lacks a polysaccharide capsule, is exquisitely sensitive to drying, leading to the necessity for intimate contact for transmission. While occasional dissemination to the bloodstream occurs as a consequence of gonococcal infections, serum resistance is mediated by factors other than encapsulation, including sialylation of the outer membrane-localized lipooligosaccharide (LOS) (Gulati et al., 2005). The polysaccharide capsule of N. meningitidis is a protective antigen; the efficacious vaccine that protects against meningococcal disease contains capsular material from four of the thirteen serogroups of N. meningitidis. The capsule from serogroup B N. meningitidis is a self-antigen, and thus not a component of the current vaccine. However, a vaccine against serogroup B N. meningitidis, employing sub-capsular protein antigens, is in development (Gossger et al., 2012). In stark contrast, N. gonorrhoeae lacks a capsule; therefore this structure cannot be utilized for vaccine development. Moreover, many surface antigens, including LOS, the proteinaceous pilus, and surface-deployed invasins called Opa proteins, are subject to high-frequency phase and antigenic variation, making these targets unacceptable vaccine antigens (Virji, 2009; Zhu et al., 2011). Even with many years of effort, no successful vaccine has yet been developed to prevent gonococcal infections.

Treatment of invasive meningococcal disease requires rapid parenteral administration of benzylpenicillin. N. meningitidis has yet to develop high-level resistance to this front-line, but still effective, antibiotic (Stephens et al., 2007). In contrast, N. gonorrhoeae has evolved resistance to every antimicrobial agent used to treat these infections. In 2007, ciprofloxacin was removed from the list of approved drugs for treatment of gonococcal infections (CDC, 2007), leaving only extended-spectrum cephalosporins as the treatment of choice. By 2011 however, resistance to the last line of defense, ceftriaxone, had emerged (CDC, 2011). N. gonorrhoeae is now recognized as a “superbug” with an enormous capacity for antigenic variation, against which there is no means of immunoprophylaxis.

A primary focus of current therapeutic design has been towards vaccine development to protect against infections by the pathogenic Neisseria species. Given the reported limitations of the existing vaccines, lack of a gonococcal vaccine, and the emergence of antibiotic resistant strains, there is an immediate need for rapid development of protective vaccines to protect against neisserial infections. Since Neisseria species cannot survive without iron, recent studies have targeted the iron import systems, which tend to be relatively well conserved and are promising vaccine targets, having the potential to offer broad protection against both species.

Iron import systems in pathogenic Neisseria

Most bacterial pathogens must compete with their hosts for iron, an essential nutrient for survival. For many pathogens, this process involves secretion of low-molecular weight chelators called siderophores, which sequester and solublize otherwise inaccessible ferric iron from the environment within the host (for recent review see Braun and Hantke, 2011). The ability to secrete siderophores and subsequently to internalize ferric-siderophore complexes is critical for the virulence of many bacterial pathogens (reviewed recently in Saha et al., 2012). In Gram-negative bacteria, ferric-siderophores are internalized in a conserved fashion utilizing a family of outer membrane transporters, which share sequence and structural similarity, called TonB-dependent transporters (TBDT) (Noinaj et al., 2011). The crystal structures of several of these transporters have been reported (reviewed in Noinaj et al., 2011), all sharing a TBDT fold characterized by an N-terminal plug domain of ~160 residues (plug domain) folded inside a C-terminal 22-stranded beta-barrel domain (beta-domain). The plug domain prevents entry of noxious substances into the periplasm until the appropriate ligand is bound; subsequently, the transporter is energized by TonB and the rest of the Ton system, which includes ExbB and ExbD (for a recent review, see Krewulak and Vogel, 2011). Although the precise details are not known, the plug is proposed to undergo a conformational change that leads to either partial or full ejection of the plug domain into the periplasm, thereby forming an entry pathway for the iron cargo directly through the outer membrane transporter.

The pathogenic Neisseria species are somewhat unusual in that they do not have the capacity to secrete siderophores. Despite this, they do express TBDTs of unknown function (TdfF, TdfG, TdfH and TdfJ; (Cornelissen and Hollander, 2011; Hagen and Cornelissen, 2006; Turner et al., 2001)) in addition to transporters such as FetA that enable the bacteria to utilize siderophores produced by neighboring bacteria (Carson et al., 1999; Hollander et al., 2011); however, the contribution of these transporters to neisserial pathogenesis has not been tested (Figure 1A). The pathogenic Neisseria species additionally express surface receptors that mediate direct extraction and import of iron from the human host iron-binding proteins hemoglobin, lactoferrin, and transferrin (Cornelissen and Hollander, 2011). Hemoglobin is predominantly sequestered within red blood cells and is a tetrameric protein with each subunit capable of binding one molecule of heme. Lactoferrin can be found in secretions, in milk, and in polymorphonuclear leukocytes and is a glycoprotein composed of two structurally similar domains (also called lobes), each of which has the capacity to bind a single iron atom. Transferrin can be found predominantly in serum and on inflamed mucosal surfaces and is structurally very similar to lactoferrin, binding one iron atom per lobe. All strains of N. meningitidis have the capacity to utilize hemoglobin, lactoferrin, and transferrin (Marri et al., 2010). In contrast, approximately half of gonococcal isolates have undergone a large deletion in the locus encoding the lactoferrin-iron internalization system, rendering this system inactive (Biswas et al., 1999). Further, engineered gonococcal mutants unable to utilize lactoferrin and transferrin as iron sources were found to be avirulent in a human male infection model of gonococcal disease (Cornelissen et al., 1998), attesting to the importance of these iron transport systems in initiating infection and proliferating in humans.

Figure 1. Iron import systems in Neisseria species.

Figure 1

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A. Single component transporter systems contain only one surface protein, a TonB-dependent transporter (TBDT), which mediates iron-loaded siderophore transport across the outer membrane. Examples include FetA, HmbR, and TdfF. B. Two component transporter systems contain both a TBDT, which mediates iron transport, as well as, a lipoprotein co-receptor, which is anchored to the outer leaflet of the outer membrane (OM) and participates in capturing iron-containing substrates. Examples include HpuA/B (hemoglobin), LbpA/B (lactoferrin), and TbpA/B (transferrin). In both systems, energy for transport is supplied by the Ton system (TonB, ExbB, and ExbD) and substrates are then shuttled across the periplasm by periplasmic carrier proteins to an ATP-binding cassette (ABC) transporter to be transported across the inner membrane (IM) into the cytoplasm.

Unlike the siderophore transport system that contains only an outer membrane transporter, iron transport systems for the acquisition of iron from hemoglobin, lactoferrin, and transferrin, are comprised of a unique system containing two types of surface-exposed receptors having very different properties and roles in the iron acquisition process (Cornelissen and Hollander, 2011). In each case, the first receptor is a TonB-dependent transporter that serves as the pore through which the iron or heme is directly transported. The second protein is a co-receptor that is lipid-modified and entirely surface exposed (see Figure 1). The combined activities of these two proteins allow for species specific binding of human iron-binding proteins to the neisserial cell surface, followed by iron extraction and subsequent internalization of the iron cargo.

The neisserial iron import systems that utilize hemoglobin, lactoferrin and transferrin are believed to share many properties; however, the lack of structural information has hindered efforts to determine the exact mechanism for iron extraction and import. Recent studies (Calmettes et al., 2012; Moraes et al., 2009; Noinaj et al., 2012) have significantly advanced our understanding of the transferrin-iron import system, which gives clues to how other import systems may function. In this review, we will examine the functional aspects of the neisserial transferrin-iron acquisition system within the context of the newly elucidated structural details of the system.

Conservation of and immunity to the components of the transferrin iron acquisition system

The transferrin-iron import system consists of two transferrin binding proteins: a TonB dependent transporter (TbpA), and a lipoprotein co-receptor (TbpB). Both proteins work in concert to bind transferrin and then extract and import the iron across the outer membrane. The two proteins are coordinately expressed from a bicistronic operon, with the tbpB gene preceding the tbpA gene (Ronpirin et al., 2001). The tbpB transcript is approximately twice as prevalent as the tbpA transcript (Ronpirin et al., 2001). The promoter that drives expression of tbpBA operon is iron repressed by the regulatory protein, Fur, which transcriptionally silences the genes in the presence of iron. The sequence of TbpA is highly conserved among strains, and even between the two pathogenic species (Cornelissen et al., 2000). Antigenic and sequence variability of TbpB proteins is more extensive (Cornelissen et al., 1997a), but neither protein is subject to high-frequency phase or antigenic variation, as is the case with many other neisserial surface antigens. Both transferrin binding proteins are antigenic when animals are immunized with the purified proteins (Price et al., 2005; Price et al., 2007); however, natural gonococcal infections, which do not elicit protective immunity, also do not generate high titer anti-Tbp antibodies (Price et al., 2004). Meningococcal transferrin binding proteins are immunogenic in both animals (Rokbi et al., 1997) and humans (Gorringe et al., 1995), which is consistent with the hypothesis that gonococci are capable of immune suppression during infection (Liu et al., 2011). These observations suggest that vaccination with gonococcal Tbps, with an appropriate adjuvant, might be protective whereas natural infections are not. Given the sequence conservation between the species, it is also possible that immunization with gonococcal Tbps could additionally be protective against meningococcal infections.

The structure and function of the iron transporter TbpA

The structure-function relationships in the neisserial transferrin-iron acquisition system have been most thoroughly described, with less data available for those systems utilizing hemoglobin and lactoferrin. Similar studies on homologous transferrin-iron uptake systems from porcine pathogens have also significantly contributed to elucidating the mechanism of transferrin-iron import (Moraes et al., 2009). Neisserial mutants lacking TbpA are incapable of iron uptake from transferrin (Cornelissen et al., 1992; Irwin et al., 1993); however, isogenic mutants lacking TbpB are still able to utilize transferrin as a source of iron, albeit less efficiently (Anderson et al., 1994; Renauld-Mongenie et al., 2004b).

Recently, the crystal structures of components of the meningococcal transferrin-iron uptake system were reported (Calmettes et al., 2012; Noinaj et al., 2012). The report of the crystal structure of neisserial TbpA in complex with apo-transferrin (Figure 2a) represents a significant advance in our understanding of TbpA structure/function relationships (Noinaj et al., 2012). This structure showed that, as predicted by a number of groups (Boulton et al., 2000; Oakhill et al., 2005), TbpA was indeed a TBDT, the largest with a defined structure, and had many unique features including very long extracellular loops that interact with transferrin, a helix finger at the apex of loop 3 that appears to be involved in catalyzing iron release, and an unusually long plug domain loop that may act as a sensor for ligand binding. TbpA was shown to bind transferrin at the very top of the beta-domain and exclusively along the C-lobe of transferrin, producing an extensive binding surface involving 81 residues of TbpA (~2,500 Å2 of buried surface area). This surface is largely electropositive, which complements the electronegative surface of transferrin. Despite TbpA having ligand bound, there were no obvious conformational changes within the plug domain compared to other TBDTs. The structure did, however, provide the precise locations and sequences of the long extracellular loops which could be important antigens for vaccine development.

Figure 2. Molecular details of the interactions between neisserial TbpA and human transferrin.

Figure 2

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A. The complex crystal structure of neisserial TbpA and human transferrin (apo form) (PDB code 3V8X) is shown in ribbon representation with the beta domain of TbpA in green, the plug domain in red, the helix finger of loop 3 (L3) in purple, and transferrin in gold (C-lobe) and light blue (N-lobe). The location of iron (red sphere) was modeled based on the diferric transferrin crystal structure (PDB code 3V83) and the putative docking site for FbpA along disordered periplasmic loop 8 (dashed green line) is indicated. B. 2D-topology diagram of TbpA highlighting selected mutations, regions of sequence conservation and sequence diversity (adapted from Boulton et al., 2000). Plug domain residues are highlighted in yellow, beta domain residues are in green, L3 helix finger residues are in cyan, residues that affect hTF binding when mutated are in orange, sites of HA tag insertions are in blue, and the iron binding motif EIEYE is shown as red squares. Deleted loops are shown in dark purple (loop 4 and loop 8) and in light blue (loop 5). The solid red bars indicate the start and end points for the loop 4+5 construct which retained hTF binding when individually expressed and purified, while the dashed red bar indicates the start point for the loop 5 only construct. Boldface circled residues are aromatics and boldface squared residues are those that are conserved among all TbpA proteins (even in other species). C. The structure of TbpA (green ribbon) depicting the locations of HA-insertions (blue spheres), and deletions (purple and light blue) and the resulting effect on transferrin binding and iron import. The locations of mutations that affected transferrin binding are indicated by orange spheres and the putative iron binding motif EIEYE is shown by red spheres.

In 2000, before the crystal structure of TbpA was reported, a hypothetical, yet remarkably accurate 2D topology model of gonococcal TbpA was generated based upon similarity with other TBDTs of known structure (Boulton et al., 2000). This 2D model (Figure 2b, updated to reflect the true structure of TbpA) was used to characterize the roles of the putative surface-exposed loops, transmembrane β-strands, and the N-terminal plug domain (Figure 2c). To determine the function of the putative surface-exposed loops, loops 4+5 and loop 5 alone were deleted leading to a loss of transferrin binding and iron uptake (Boulton et al., 2000). Deletion of loop 8 of TbpA resulted in reduced transferrin binding but no iron internalization from transferrin, consistent with this region serving a necessary docking region for human transferrin to enable iron extraction (Boulton et al., 2000). Further, individual loops of TbpA were expressed alone and tested to see whether any retained ligand binding function (Masri and Cornelissen, 2002). Surprisingly, loop 5 and the combination of loops 4+5 retained the ability to bind transferrin, despite the rest of the beta-barrel domain being absent. These observations are consistent with the crystal structure of TbpA in complex with transferrin, which demonstrates that loops 4 and 5 are in direct contact with transferrin.

Using the 2D topological model of TbpA, surface exposure was accurately determined for loops 2, 3, 5, 7 and 10, and even for the extended plug domain loop (Yost-Daljev and Cornelissen, 2004). The function of the HA-epitope insertion mutants were further investigated by measuring transferrin binding and iron uptake. Surprisingly, most of the insertion mutants retained function (Yost-Daljev and Cornelissen, 2004), indicating that the insertions did not significantly affect the overall structure of TbpA. Exceptions were insertions into loop 3 and into beta-strand 9, which resulted in the loss of transferrin binding and iron import. Insertions into the plug domain and into β-strand 16 resulted in decreased iron uptake capacity, consistent with these regions playing important roles in iron internalization. Interestingly, insertions into loops 2, 9 and 11 resulted in a loss of iron uptake that could be rescued by co-expression of TbpB, indicating that these regions are required for some aspect of iron internalization that can be duplicated by TbpB (Yost-Daljev and Cornelissen, 2004). A summary of these studies is depicted in Figure 2c.

Antibodies were designed to target specific surface-exposed loops of TbpA to determine if they could block the interactions with human transferrin (Masri and Cornelissen, 2002). Early attempts using the 2D model for TbpA to target gonococcal loops 2, 5 and 4+5 for their ability to produce antibodies that could interfere with ligand binding were unsuccessful (Masri, 2003). However, with the benefit of the TbpA-transferrin complex structure, it was demonstrated that antibodies against meningococcal loops 3, 7 and 11 and the long extended plug loop (Figure 2) were able to block interactions with transferrin using in vitro ELISA assays (Noinaj et al., 2012). However, more studies are needed to determine the usefulness of the TbpA structure for designing vaccines to protect against neisserial infections.

The structure and function of the co-receptor TbpB

Unlike TbpA, which is required to mediate the transport of iron across the outer membrane, TbpB is not required; however, it does significantly increase the efficiency of the import system in a number of ways. First, while TbpA binds both apo and holo transferrin with similar affinity, TbpB preferentially binds holo transferrin (Cornelissen and Sparling, 1996; Retzer et al., 1998), thereby saturating iron-loaded substrate on the neisserial cell surface. It was found that an engineered gonococcal mutant lacking TbpB internalizes approximately half of the wild-type amount of iron from transferrin (Anderson et al., 1994). Second, while the exact mechanism remains unknown, it has been proposed that TbpB may participate in the extraction of iron from transferrin (Siburt et al., 2009), either by direct removal or in concert with TbpA. Third, the presence of TbpB on the cell surface facilitates release of transferrin (DeRocco et al., 2008); presumably after iron has been extracted and transported. As for association with the neisserial cell surface, enhanced dissociation from the cell when TbpB is expressed is likely the result of TbpB’s strict specificity for holo-transferrin (DeRocco et al., 2008).

A number of studies have used various techniques to probe the interactions of TbpB with transferrin and have found that the N-lobe of TbpB is the primary domain responsible for the interaction (Calmettes et al., 2011; Cornelissen et al., 1997a; Moraes et al., 2009; Renauld-Mongenie et al., 2004a; Sims and Schryvers, 2003). The first glimpse of the fold in this co-receptor was reported in 2009 with the report of the crystal structure of TbpB from the porcine pathogen Actinobacillus pleuropneumoniae (Moraes et al., 2009). This study also demonstrated that a single mutation within the N-lobe was sufficient to eliminate in vitro transferrin binding (Moraes et al., 2009). Recently, the crystal structures of TbpB from two N. meningitidis strains were reported (Calmettes et al., 2012; Noinaj et al., 2012) and found to be very similar to those previously reported for the porcine pathogen with the major differences being among several loops including the predicted transferrin binding regions. The structure of TbpB consists of two structurally similar domains or lobes, an N-terminal lobe and a C-terminal lobe, with each lobe containing an 8-stranded beta-barrel flanked by a beta-rich handle domain. Several large portions of the structure were found disordered (indicating conformational flexibility), including the N-terminal region, the linker region between the N-lobe and the C-lobe, and several loops along the C-lobe. Interestingly, while the C-lobe and the core of the N-lobe are the most conserved, the most variable region of neisserial TbpB was mapped to the distal face of the N-lobe which is responsible for binding transferrin (Calmettes et al., 2012; Noinaj et al., 2012). A significant contribution to our understanding of the role of TbpB also came recently with the report (Noinaj et al., 2012) of the crystal structure of the neisserial TbpB-human transferrin complex (Figure 3a). This structure provided the molecular details showing exactly how TbpB interacts with transferrin and suggested a novel role in iron acquisition, whereby TbpB increases the fidelity of the neisserial import system by actively locking transferrin in an iron-bound state for delivery to TbpA and preventing premature iron release (Figure 3b).

Figure 3. Molecular details of the interactions between neisserial TbpB and human transferrin.

Figure 3

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A. The complex crystal structure of neisserial TbpB and human transferrin (PDB code 3VE1) is shown in ribbon representation with TbpB in light purple (N-lobe) and dark purple (C-lobe), and transferrin in gold (C-lobe) and light blue (N-lobe). B. Mechanism for how TbpB locks transferrin in a closed conformation, by drastically reducing the pKa of the transferrin H349, a residue important for the pH regulated release of iron. C. The modeled structure of gonococcal TbpB (pink and purple) depicting the locations of HA-insertions (blue spheres) and the resulting effect on transferrin binding and iron import. Mutations that affected transferrin binding are shown as orange spheres and the location of loops that were found disordered in the meningococcal crystal structures are indicated in gray ribbon.

Studies using an HA epitope tagging approach had previously been reported to probe the regions of TbpB that are important for transferrin binding and transferrin-iron uptake in gonococci (DeRocco and Cornelissen, 2007). As shown in Figure 3c, an HA epitope was placed at nine distinct locations in TbpB, five within the N-lobe and four within the C-lobe. All HA epitopes were found surface exposed in gonococci, consistent with this protein being tethered by the lipidated amino-terminus to the outer leaflet of the outer membrane. HA epitopes 3, 4, 5 and 8 individually interfered with transferrin binding and iron import, while the combined insertions 4+8 and 5+8 completely abrogated transferrin binding, in agreement with the recent structural reports. Importantly though, these results indicated that not only does the N-lobe of TbpB interact with transferrin, but that in vivo, the C-lobe may also participate in transferrin binding and iron import as well. This notion is further supported by other studies which have shown that the C-lobe of TbpB can interact with transferrin with low affinity (Renault-Montgenie et al., 1997). While it is possible that some of the HA epitope insertions may disrupt the structure of TbpB, these results, along with other studies, indicate that both lobes of TbpB may be important for optimal transferrin binding and subsequent iron import through TbpA.

Interactions between TbpA and TbpB

The current model for transferrin-iron import involves initial binding of transferrin to TbpB followed by TbpB escorting transferrin to TbpA for iron extraction and import. Using the recently reported complex crystal structures and electron microscopy analysis of the purified complex, a model for the quaternary structure of the fully assembled iron import complex (TbpA-TbpB-transferrin) was formulated (Figure 4a) and further supports this mechanistic model for iron import and reveals that while TbpA and TbpB both interact the C-lobe of transferrin, they do so with no overlap of their binding sites and have no obvious interactions with one another. However, several studies have reported a direct interaction and even in our own unpublished work, purified TbpB (in the absence of transferrin) can be pulled down using purified TbpA as bait despite not observing any interaction on a gel filtration column, suggesting the interaction may be low affinity or transient, yet nevertheless important. Other studies have also shown that the exposure of TbpB depends on TbpA, and whether or not TbpA can be energized by TonB (Cornelissen and Sparling, 1996; Cornelissen et al., 1997b). Mutations in the TonB-box of TbpA demonstrated that the protease accessibility of TbpB at the gonococcal cell surface was distinct from that of the wild type strain and that the interaction between transferrin and the gonococcal cell surface receptors depends on the presence of both proteins (Cornelissen et al., 1997b). The individual affinities of the receptor proteins for transferrin are distinct from one another (TbpA, TbpB) and from the affinity of the combined receptor (TbpA/B) (Cornelissen and Sparling, 1996), suggesting that TbpA and TbpB may interact with one another on the cell surface resulting in unique characteristics including conformation, exposure, and transferrin interaction. Using TbpA-specific antibodies, TbpB can be co-immunoprecipitated from outer membrane fractions, even in the absence of transferrin (Kenney, 2002). However, the reverse is not true. TbpB-specific antibodies do not co-immunoprecipitate TbpA, suggesting that there is a population of ‘free’ TbpB that does not interact with TbpA on the cell surface and that is conformationally distinct from that which is complexed with TbpA (Kenney, 2002).

Figure 4. The role of TbpA in iron extraction from transferrin and import across the outer membrane.

Figure 4

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A. Model for the neisserial TbpA-TbpB- transferrin triple complex in the outer membrane (OM). TbpA is shown in green surface representation, TbpB in purple and human transferrin in gold. Dashed circles indicate the approximate location of the enclosed chamber (~1000 Å3) formed by the union of the triple complex and black arrows indicate regions of putative interactions between TbpA and TbpB. B. Proposed mechanism for TbpA-mediated iron release from transferrin, where K359 of TbpA neutralizes the negative charge of D634 of transferrin, producing a charge-charge repulsion of K534 and R632 and leading to a conformational change in transferrin and iron release. C. A model for the role of the TbpA plug domain (green) in serving as a transient docking site during iron transport. Inset shows a top down view of the iron coordination. D. Molecular dynamics simulations designed to mimic the role of the Ton system demonstrated a systematic unfolding of the plug domain and formation of a pore (gray surface) that could facilitate iron passage.

It is also conceivable that only high affinity interactions are easily observed in vitro while in vivo, lower affinity interactions between the components of the import system are preserved and play a critical functional role. While the model for the fully assembled iron import complex (Figure 4a) indicates unique and non-overlapping binding sites for TbpA and TbpB on the C-lobe of transferrin, this model does not account for the large disordered loops found within the C-lobe of TbpB (Figure 3c) which could mediate a direct interaction with TbpA either in the presence or absence of transferrin. Additionally, these loops in TbpB could further assist in assembly of the enclosed chamber that is formed by the union of the TbpA-TbpB-transferrin triple complex which has been proposed to steer iron through the beta-domain of TbpA and prevent diffusion. While the structures have significantly advanced our understanding of the role of TbpB, more studies are needed to fully elucidate the putative role of the C-lobe of TbpB and its interactions with TbpA.

Iron extraction and import by TbpA

The mechanism iron extraction by TbpA at neutral pH remains elusive; however, the recent crystal structures provide valuable clues. The most likely mechanism involves a conserved lysine residue within the helix finger of loop 3 (Figure 2a) which may serve to catalyze the release of iron by hijacking the pH sensing triad of the C-lobe of transferrin. Here, K359 of meningococcal TbpA may serve to sequester the negative charge of D634 of transferrin, leading to charge repulsion between K534 and R632 that mediates a conformation change and eventual iron release (Figure 4b). D634 typically serves the role of neutralizing this positive charge and when this transferrin residue is mutated to alanine, iron release increases by ~100 fold (Steere et al., 2011). Other studies have shown that mutating either K534 or R632 to alanine renders the C-lobe of transferrin locked in an iron-bound state and insensitive to pH changes (Noinaj, Buchanan and Mason, unpublished).

While it was anticipated that the loops of neisserial TbpA would be critical for the interaction with human transferrin on the cell surface, it was hypothesized that the plug domain may participate in removal of iron from transferrin and its transport into the periplasm. Because iron is insoluble and potentially toxic, it was proposed that conserved residues within the plug domain could transiently coordinate the iron during the transport process. This led to the identification of a highly conserved stretch of residues containing the putative iron-binding motif EIEYE (Noto and Cornelissen, 2008). Mutating residues within this motif did not affect transferrin binding, however, these mutations were sufficient to prevent iron transport by TbpA (Noto and Cornelissen, 2008). These results are consistent with the importance of this sequence for iron removal from transferrin and/or iron transport into the cell. Further evidence was reported recently in which it was determined that the conserved EIEYE sequence is able to coordinate iron directly in an in vitro system (Figure 4c) (Banerjee et al., 2012). This putative iron-binding motif was mapped to the crystal structure of meningococcal TbpA and is located within a potential iron transport pathway through the beta-domain (Figure 2c). Together, these results suggest that the EIEYE motif participates in iron coordination and is critical to the process of iron extraction and iron transport into the periplasm.

Presumably, TonB provides the energy to extricate the plug from the beta-domain, which may then lead to iron release into the periplasm. To investigate this further, recent studies (Noinaj et al., 2012) reported molecular dynamics simulations that mimic the role of TonB and found that upon pulling on the N-terminus, that the plug domain was systematically unfolded to expose a pore through TbpA that could serve as a pathway for iron transport (Figure 4d). Then, as iron is presented at the inner orifice of the beta-domain, apo FbpA, which has been proposed to dock at the periplasmic side of TbpA (Siburt et al., 2009), would be able to immediately bind the iron with high affinity to enable subsequent transport across the periplasm.

Mechanism for iron acquisition in pathogenic Neisseria

As discussed throughout this review, a number of recent studies have together contributed to elucidating the mechanism by which Neisseria species are able to utilize iron from human transferrin. With the crystal structures of both TbpA and TbpB now known and with the interaction of these receptors with transferrin described at the molecular level, we can now merge this new information with the current functional work to present an improved model for iron import in Neisseria species involving both TbpA and TbpB (Figure 5). At the cell surface, initially, TbpB would preferentially bind iron-loaded transferrin locking it in a closed state to ensure the iron is not prematurely released. Then TbpB would transfer transferrin to TbpA where a transient triple complex would be formed until TbpA catalyzes a conformational change in transferrin, likely in a TonB-dependent manner. This conformational change would lead to iron release from transferrin and subsequent dissociation of apo-transferrin, facilitated by TbpB and its specificity for the ferrated ligand. TonB interacts with a defined sequence of the extreme amino-terminus of TbpA called the TonB-box. This high affinity interaction is expected to lead to systematic TonB-dependent conformational changes within the entire plug domain, which would then allow the formation of a transient docking site for iron. Further unraveling of the plug domain would eventually allow the iron to become exposed to the periplasm and then be transferred to FbpA, which is expected to be docked along the periplasmic face of TbpA. FbpA would then shuttle the iron across the periplasm where it would eventually be transported into the cytoplasm to be used for essential cellular functions.

Figure 5. Stepwise mechanism for the acquisition of iron from human transferrin by Neisseria species.

Figure 5

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The first step involves the outer membrane (OM) co-receptor TbpB (pink) preferentially binding iron-loaded transferrin (iron shown as red sphere, hTF is depicted in gold), which is then escorted to TbpA (green). Binding of transferrin leads to a Ton system-mediated conformational change within the plug domain of TbpA, forming a transient iron binding site where subsequent transport of iron is accomplished through the beta-domain. As the iron passes into the periplasm, it immediately binds to ferric binding protein A (FbpA, cyan) which in its apo-form is docked to the periplasmic face of TbpA. Upon iron binding, FbpA is released and shuttles the iron across the periplasm where the iron is eventually transported into the cytoplasm. After iron has been extracted from transferrin, TbpB may facilitate removal of apo-transferrin from TbpA by virtue of its specificity for the ferrated form of transferrin. Subsequently, TbpB dissociates from TbpA, which then returns to its ground state conformation, primed for another cycle of iron acquisition.

While this mechanistic model represents the majority of what is currently known, there are still some observations that cannot be accounted for and many details that remain to be determined. For example, what regions of TbpA and TbpB interact with each other and do these proteins only form a complex under some conditions? Does the conformation of TbpB change as a consequence of ligand binding, delivery to TbpA and transferrin release? After iron is released from transferrin, how then is the apo-transferrin molecule removed to allow another cycle of iron import? Does TbpB actively participate in this process? Another interesting question is why only the C-lobe of transferrin is bound by both TbpA and TbpB, why not the N-lobe as well? It can be rationalized that utilizing iron from both the N- and C-lobes of transferrin would be beneficial for Neisseria species by accessing all available iron sources for survival. Studies have already shown that apo FbpA can dock along TbpA, but it remains to be determined where this may occur. The crystal structure of TbpA will surely assist in finally elucidating the site of this interaction. Using the structures now available, we can further probe the iron import pathway through the TbpA beta-barrel and assess the function of the helix finger in loop 3 in iron extraction from transferrin. Lastly, defined TbpA peptides elicited antibodies that were capable of blocking transferrin binding in vitro. Will these anti-peptide antibodies block the function of the receptor in vivo and would blocking antibodies be protective? With the availability of a transgenic mouse that expresses human transferrin (Szatanik et al., 2011), this question can effectively be addressed using a female genital tract mouse model of gonococcal infection (Jerse et al., 2011). While it remains to be determined whether these studies can be translated to the human host, the recent structural and functional work has significantly contributed to these efforts, offering promise to speed vaccine development against Neisseria species.

Acknowledgments

N.N. and S.K.B. are supported by the Intramural Program of the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health. Funding was provided to C.N.C. by U.S. Public Health Service grant numbers AI065555 and AI084400 from the National Institute of Allergy and Infectious Diseases at the National Institutes of Health. C.N.C. is also supported by the SE STI Center grant (U19 AI31496) from the National Institute of Allergy and Infectious Diseases.

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