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Transition metals are essential for metalloprotein function among all domains of life. Humans utilize nutritional immunity to limit bacterial infections, employing metalloproteins such as hemoglobin, transferrin, and lactoferrin across a variety of physiological niches to sequester iron from invading bacteria. Consequently, some bacteria have evolved mechanisms to pirate the sequestered metals and thrive in these metal-restricted environments.

Trace metals such as iron and zinc are vital nutrients known to play key roles in prokaryotic processes including gene regulation, catalysis, and protein structure. Metal sequestration by hosts often leads to metal limitation for the bacterium. This limitation induces bacterial gene expression whose protein products allow bacteria to overcome their metal-limited environment. Characterization of such genes is challenging. Bacteria must be grown in meticulously prepared media that allows sufficient access to nutritional metals to permit bacterial growth while maintaining a metal profile conducive to achieving expression of the aforementioned genes. As such, a delicate balance must be established for the concentrations of these metals. Growing a nutritionally fastidious organism such as Neisseria gonorrhoeae, which has evolved to survive only in the human host, adds an additional level of complexity. Here, we describe the preparation of a defined metal-limited medium sufficient to allow gonococcal growth and the desired gene expression. This method allows the investigator to chelate iron and zinc from undesired sources while supplementing the media with defined sources of iron or zinc, whose preparation is also described. Finally, we outline three experiments that utilize this media to help characterize the protein products of metal-regulated gonococcal genes.

Neisseria gonorrhoeae employs high-affinity metal acquisition systems to obtain necessary nutrients, such as iron (Fe) and zinc (Zn) from the environment. Because growth and replication depend upon successful metal acquisition, these high-affinity uptake systems are important virulence factors. Expression of metal acquisition systems is tightly controlled and preferentially expressed under low-metal conditions. Therefore, in order to optimally produce these transport proteins and study them in vitro, growth media must be deployed that mimic low-metal conditions. This chapter describes the chelators, media, and culturing conditions that can generate low-metal in vitro growth conditions.

The transferrin-binding proteins TbpA and TbpB enable Neisseria gonorrhoeae to obtain iron from human transferrin. The lipoprotein TbpB facilitates, but is not strictly required for, TbpA-mediated iron acquisition. The goal of the current study was to determine the contribution of two conserved regions within TbpB to the function of this protein. Using site-directed mutagenesis, the first mutation we constructed replaced the lipobox (LSAC) of TbpB with a signal I peptidase cleavage site (LAAA), while the second mutation deleted a conserved stretch of glycine residues immediately downstream of the lipobox. We then evaluated the resulting mutants for effects on TbpB expression, surface exposure, and transferrin iron utilization. Western blot analysis and palmitate labeling indicated that the lipobox, but not the glycine-rich motif, is required for lipidation of TbpB and tethering to the outer membrane. TbpB was released into the supernatant by the mutant that produces TbpB LSAC. Neither mutation disrupted the transport of TbpB across the bacterial cell envelope. When these mutant TbpB proteins were produced in a strain expressing a form of TbpA that requires TbpB for iron acquisition, growth on transferrin was either abrogated or dramatically diminished. We conclude that surface tethering of TbpB is required for optimal performance of the transferrin iron acquisition system, while the presence of the polyglycine stretch near the amino terminus of TbpB contributes significantly to transferrin iron transport function. Overall, these results provide important insights into the functional roles of two conserved motifs of TbpB, enhancing our understanding of this critical iron uptake system.

Neisseria gonorrhoeae produces two transferrin binding proteins, TbpA and TbpB, which together enable efficient iron transport from human transferrin. We demonstrate that expression of the tbp genes is controlled by MisR, a response regulator in the two-component regulatory system that also includes the sensor kinase MisS. The tbp genes were up-regulated in the misR mutant under iron-replete conditions but were conversely down-regulated in the misR mutant under iron-depleted conditions. The misR mutant was capable of transferrin-iron uptake at only 50% of wild-type levels, consistent with decreased tbp expression. We demonstrate that phosphorylated MisR specifically binds to the tbpBA promoter and that MisR interacts with five regions upstream of the tbpB start codon. These analyses confirm that MisR directly regulates tbpBA expression. The MisR binding sites in the gonococcus are only partially conserved in Neisseria meningitidis, which may explain why tbpBA was not MisR-regulated in previous studies using this related pathogen. This is the first report of a trans-acting protein factor other than Fur that can directly contribute to gonococcal tbpBA regulation.

The transferrin iron acquisition system of Neisseria gonorrhoeae consists of two dissimilar transferrin binding proteins (Tbp) A and B. TbpA is a TonB dependent transporter while TbpB is a lipoprotein that makes iron acquisition from transferrin (Tf) more efficient. In an attempt to further define the individual roles of these receptors in the process of Tf-iron acquisition, the kinetics of the receptor proteins in regards to ligand association and dissociation were evaluated. Tf association with TbpB was rapid as compared to TbpA. Tf dissociation from the wild-type receptor occurred in a biphasic manner; an initial rapid release was followed by a slower dissociation over time. Both TbpA and TbpB demonstrated a two-phase release pattern; however, TbpA required both TonB and TbpB for efficient Tf dissociation from the cell surface. The roles of TbpA and TbpB in Tf dissociation were further examined, utilizing previously created HA fusion proteins. Using a Tf-utilization deficient TbpA-HA mutant, we concluded that the slower rate of ligand dissociation demonstrated by the wild-type transporter was a function of successful iron internalization. Insertion into the C-terminus of TbpB decreased the rate of Tf dissociation, while insertion into the N-terminus had no effect on this process. From these studies, we propose that TbpA and TbpB function synergistically during the process of Tf iron acquisition and that TbpB makes the process of Tf-iron acquisition more efficient at least in part by affecting association and dissociation of Tf from the cell surface.

Iron is an essential nutrient for survival and establishment of infection by Neisseria gonorrhoeae . The neisserial transferrin binding proteins (Tbps) comprise a bipartite system for iron acquisition from human transferrin. TbpA is the TonB-dependent transporter that accomplishes iron internalization. TbpB is a surface-exposed lipoprotein that makes the iron uptake process more efficient. Previous studies have shown that the genes encoding these proteins are arranged in a bicistronic operon, with the tbpB gene located upstream of tbpA and separated from it by an inverted repeat. The operon is under the control of the ferric uptake regulator (Fur); however, promoter elements necessary for regulated expression of the genes have not been experimentally defined. In this study, putative regulatory motifs were identified and confirmed by mutagenesis. Further examination of the sequence upstream of these promoter/operator motifs led to the identification of several novel repeats. We hypothesized that these repeats are involved in additional regulation of the operon. Insertional mutagenesis of regions upstream of the characterized promoter region resulted in decreased tbpB and tbpA transcript levels but increased protein levels for both TbpA and TbpB. Using RNA sequencing (RNA-Seq) technology, we determined that a long RNA was produced from the region upstream of tbpB . We localized the 5′ endpoint of this transcript to between the two upstream insertions by qualitative RT-PCR. We propose that expression of this upstream RNA leads to optimized expression of the gene products from within the tbpBA operon.

The pathogenic Neisseria spp. are capable of iron utilization from host iron-binding proteins including transferrin and lactoferrin. Transferrin iron utilization is an energy-dependent, receptor-mediated event in which two identified transferrin-binding proteins participate. One of these proteins, TbpA, is homologous to the TonB-dependent family of outer membrane receptors that are required for high-affinity uptake of vitamin B12 and ferric siderophores. The 'TonB box' is a conserved domain near the amino-terminus of these proteins that has been implicated in interaction with TonB. Interaction between a periplasmic domain of TonB and the TonB box allows energy transduction to occur from the cytoplasmic membrane to the energy-dependent receptor in the outer membrane. We created a TonB box mutant of gonococcal TbpA and demonstrated that its binding and protease accessibility characteristics were indistinguishable from those of gonococcal Ton system mutants. The protease exposure of the second transferrin-binding protein, TbpB, was affected by the energization of TbpA, consistent with an interaction between these proteins. TbpB expressed by the de-energized mutants was readily accessible to protease, similar to TbpB expressed in the absence of TbpA. The de-energized mutants exhibited a marked decrease in transferrin diffusion rate, suggesting that receptor energization was necessary for ligand release. We propose a model to explain the observed Ton-dependent changes in the binding parameters and exposures of TbpA and TbpB.

Neisseria are pathogenic bacteria that cause gonorrhea, septicemia, and meningitis. Like other pathogenic bacteria, Neisseria must acquire iron for survival from their local environment within the human host. Instead of secreting siderophores to scavenge iron, Neisseria steal iron from human iron binding proteins such as hemoglobin, transferrin and lactoferrin for survival. Recently we reported the crystal structures of the Neisseria meningitidis transferrin receptors TbpA and TbpB, as well as the structures of apo and holo human transferrin. We also analyzed these proteins using small angle X-ray scattering and electron microscopy to provide the molecular details explaining how Neisseria are able to interact with and extract iron from transferrin. Here, we utilize the structural reports, as well as the recently reported structure of the N-lobe of LbpB from Moraxella bovis, to assemble improved 3D homology models for the neisserial lactoferrin import receptors LbpA and LbpB, both of which are important vaccine targets against N. meningitidis. We then analyzed these models to gain structural insights into the lactoferrin-iron import system and form a mechanistic model fashioned in parallel to the homologous transferrin-iron import system.

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.

Neisseria gonorrhoeae causes the common sexually transmitted infection, gonorrhea. This microorganism is an obligate human pathogen, existing nowhere in nature except in association with humans. For growth and proliferation, N. gonorrhoeae requires iron and must acquire this nutrient from within its host. The gonococcus is well-adapted for growth in diverse niches within the human body because it expresses efficient transport systems enabling use of a diverse array of iron sources. Iron transport systems facilitating the use of transferrin, lactoferrin, and hemoglobin have two components: one TonB-dependent transporter and one lipoprotein. A single component TonB-dependent transporter also allows N. gonorrhoeae to avail itself of iron bound to heterologous siderophores produced by bacteria within the same ecological niche. Other TonB-dependent transporters are encoded by the gonococcus but have not been ascribed specific functions. The best characterized iron transport system expressed by N. gonorrhoeae enables the use of human transferrin as a sole iron source. This review summarizes the molecular mechanisms involved in gonococcal iron acquisition from human transferrin and also reviews what is currently known about the other TonB-dependent transport systems. No vaccine is available to prevent gonococcal infections and our options for treating this disease are compromised by the emergence of antibiotic resistance. Because iron transport systems are critical for the survival of the gonococcus in vivo, the surface-exposed components of these systems are attractive candidates for vaccine development or therapeutic intervention.

Neisseria gonorrhoeae produces no known siderophores but can employ host-derived, iron-binding proteins, including transferrin and lactoferrin, as iron sources. Given the propensity of this pathogen to hijack rather than synthesize iron-sequestering molecules, we hypothesized that the ability to use siderophores produced by other bacteria, or xenosiderophores, may also play a role in the survival of the gonococcus. Among a panel of diverse siderophores, only the catecholate xenosiderophores enterobactin and salmochelin promoted growth of gonococcal strain FA19. Surprisingly, the internalization pathway was independent of TonB or any of the TonB-dependent transporters. Xenosiderophore-mediated growth was similarly independent of the pilin-extruding secretin formed by PilQ and of the hydrophobic-agent efflux system composed of MtrCDE. The fbpABC operon encodes a periplasmic-binding-protein-dependent ABC transport system that enables the gonococcus to transport iron into the cell subsequent to outer membrane translocation. We hypothesized that the FbpABC proteins, required for ferric iron transport from transferrin and lactoferrin, might also contribute to the utilization of xenosiderophores as iron sources. We created mutants that conditionally expressed FbpABC from an IPTG-inducible promoter. We determined that expression of FbpABC was required for growth of gonococcal strain FA19 in the presence of enterobactin and salmochelin. The monomeric component of enterobactin, dihydroxybenzoylserine (DHBS), and the S2 form of salmochelin specifically promoted FbpABC-dependent growth of FA19. This study demonstrated that the gonococcal FbpABC transport system is required for utilization of some xenosiderophores as iron sources and that growth promotion by these ferric siderophores can occur in the absence of TonB or individual TonB-dependent transporters.

Survival of Neisseria gonorrhoeae within host epithelial cells is expected to be important in the pathogenesis of gonococcal disease. We previously demonstrated that strain FA1090 derives iron from a host cell in a process that requires the Ton complex and a putative TonB-dependent transporter, TdfF. FA1090, however, lacks the gonococcal genetic island (GGI) that is present in the majority of strains. The GGI in strain MS11 has been partially characterized, and it encodes a type IV secretion system (T4SS) involved in DNA release. In this study we investigated the role of iron acquisition and GGI-encoded gene products in gonococcal survival within cervical epithelial cells. We demonstrated that intracellular survival of MS11 was dependent on acquisition of iron from the host cell, but unlike the findings for FA1090, expression of the Ton complex was not required. Survival was not dependent on a putative TonB-like protein encoded in the GGI but instead was directly linked to T4SS structural components in a manner independent of the ability to release or internalize DNA. These data suggest that expression of selected GGI-encoded open reading frames confers an advantage during cervical cell infection. This study provides the first link between expression of the T4SS apparatus and intracellular survival of gonococci.

The pathogenic Neisseria species are capable of utilizing transferrin as their sole source of iron. A neisserial transferrin receptor has been identified and its characteristics defined; however, the biochemical identities of proteins which are required for transferrin receptor function have not yet been determined. We identified two iron-repressible transferrin-binding proteins in Neisseria gonorrhoeae, TBP1 and TBP2. Two approaches were taken to clone genes required for gonococcal transferrin receptor function. First, polyclonal antiserum raised against TBP1 was used to identify clones expressing TBP1 epitopes. Second, a wild-type gene copy was cloned that repaired the defect in a transferrin receptor function (trf) mutant. The clones obtained by these two approaches were shown to overlap by DNA sequencing. Transposon mutagenesis of both clones and recombination of mutagenized fragments into the gonococcal chromosome generated mutants that showed reduced binding of transferrin to whole cells and that were incapable of growth on transferrin. No TBP1 was produced in these mutants, but TBP2 expression was normal. The DNA sequence of the gene encoding gonococcal TBP1 (tbpA) predicted a protein sequence homologous to the Escherichia coli and Pseudomonas putida TonB-dependent outer membrane receptors. Thus, both the function and the predicted protein sequence of TBP1 were consistent with this protein serving as a transferrin receptor.

Neisseria gonorrhoeae requires iron for survival in the human host and therefore expresses high-affinity receptors for iron acquisition from host iron-binding proteins. The gonococcal transferrin-iron uptake system is composed of two transferrin binding proteins, TbpA and TbpB. TbpA is a TonB-dependent, outer membrane transporter critical for iron acquisition, while TbpB is a surface-exposed lipoprotein that increases the efficiency of iron uptake. The precise mechanism by which TbpA mediates iron acquisition has not been elucidated; however, the process is distinct from those of characterized siderophore transporters. Similar to these TonB-dependent transporters, TbpA is proposed to have two distinct domains, a β-barrel and a plug domain. We hypothesize that the TbpA plug coordinates iron and therefore potentially functions in multiple steps of transferrin-mediated iron acquisition. To test this hypothesis, we targeted a conserved motif within the TbpA plug domain and generated single, double, and triple alanine substitution mutants. Mutagenized TbpAs were expressed on the gonococcal cell surface and maintained wild-type transferrin binding affinity. Single alanine substitution mutants internalized iron at wild-type levels, while the double and triple mutants showed a significant decrease in iron uptake. Moreover, the triple alanine substitution mutant was unable to grow on transferrin as a sole iron source; however, expression of TbpB compensated for this defect. These data indicate that the conserved motif between residues 120 and 122 of the TbpA plug domain is critical for transferrin-iron utilization, suggesting that this region plays a role in iron acquisition that is shared by both TbpA and TbpB.

The transferrin iron acquisition system of Neisseria gonorrhoeae is necessary for iron uptake from transferrin in the human host and requires the participation of two distinct proteins: TbpA and TbpB. TbpA is a TonB-dependent outer membrane transporter responsible for the transport of iron into the cell. TbpB is a lipid-modified protein, for which a precise role in receptor function has not yet been elucidated. These receptor complex proteins show promise as vaccine candidates; therefore, it is important to identify surface-exposed regions of the proteins required for wild-type functions. In this study we examined TbpB, which has been reported to be surface exposed in its entirety; however, this hypothesis has never been tested experimentally. We placed the hemagglutinin (HA) epitope into TbpB with the dual purpose of examining the surface exposure of particular epitopes as well as their impact on receptor function. Nine insertion mutants were created, placing the epitope downstream of the signal peptidase II cleavage site. We report that the HA epitope is surface accessible in all mutants, indicating that the full-length TbpB is completely surface exposed. By expressing the TbpB-HA fusion proteins in N. gonorrhoeae , we were able to examine the impact of each insertion on the function of TbpB and the transferrin acquisition process. We propose that TbpB is comprised of two transferrin-binding-competent lobes, both of which are critical for efficient iron uptake from human transferrin.

Neisseria gonorrhoeae has evolved a repertoire of iron acquisition systems that facilitate essential iron uptake in the human host. Acquisition of iron requires both the energy-harnessing cytoplasmic membrane protein, TonB, as well as specific outer membrane TonB-dependent transporters (TdTs.) Survival within host epithelial cells is important to the pathogenesis of gonococcal disease and may contribute to the persistence of infection. However, the mechanisms by which gonococci acquire iron within this intracellular niche are not currently understood. In this study, we investigated the survival of gonococcal strain FA1090 within ME180 human cervical epithelial cells with respect to high affinity iron acquisition. Intracellular survival was dependent upon iron supplied by the host cell. TonB was expressed in the host cell environment and this protein was critical to gonococcal intracellular survival. Furthermore, expression of the characterized outer membrane transporters TbpA, FetA and LbpA and putative transporters TdfG, TdfH and TdfJ were not necessary for intracellular survival. Conversely, intracellular survival was dependent on expression of the putative transporter, TdfF. Expression of TdfF was detected in the presence of epithelial cell culture media containing fetal bovine serum. Expression was further modulated by iron availability. To our knowledge, this study is the first to demonstrate the specific requirement for a single iron transporter in the survival of a bacterial pathogen within host epithelial cells.

Members of the families Neisseriaceae, Pasteurellaceae and Moraxellaceae are capable of transferrin-iron acquisition in the absence of siderophore production. They do so via expression of a bi-partite receptor composed of two dissimilar proteins, TbpA and TbpB. Both proteins are surface exposed, iron-regulated and capable of binding transferrin. However, other physiochemical, antigenic, and immunogenic characteristics of the proteins are quite distinct. TbpB is a lipoprotein, which like the mammalian transferrin receptor is capable if discriminating between apo- and holo-transferrin. Expression of TbpB is not essential for transferrin-iron uptake, and in rare situations, the gene that encodes this protein is not linked to the gene encoding the second component. TbpA is a member of a family of TonB-dependent transporters, others of which accomplish ferric-siderophore and vitamin B12 uptake at the expense of a proton gradient across the cytoplasmic membrane. However, unlike the other TonB-dependent receptors where vitamins or ferric-siderophores are wholly internalized, the bacterial transferrin receptor must remove iron from transferrin at the cell surface. This review focuses on the structure-function relationships in the transferrin-binding proteins, their sequence and antigenic diversity, and the mechanisms by which they accomplish transferrin-iron uptake. The contribution of these proteins to pathogenesis and vaccine development based on TbpA and TbpB are also discussed.

Iron scavenging byNeisseria gonorrhoeaeis accomplished by the expression of receptors that are specific for host iron-binding proteins, such as transferrin and lactoferrin. Efficient transferrin-iron acquisition is dependent on the combined action of two proteins, designated TbpA and TbpB. TbpA is a TonB-dependent outer membrane receptor, whereas TbpB is lipid modified and serves to increase the efficiency of transferrin-iron uptake. Both proteins, together or separately, can be isolated from the gonococcal outer membrane by using affinity chromatography techniques. In the present study, we identified an additional protein in transferrin-affinity preparations, which had an apparent molecular mass of 45 kDa. The ability to copurify this protein by transferrin affinity was dependent upon the presence of TbpA and not TbpB. The amino-terminal sequence of the 45-kDa protein was identical to the amino terminus of gonococcal TonB, indicating that TbpA stably interacted with TonB, without the addition of chemical cross-linkers. Using immunoprecipitation, we could recover TbpA-TonB complexes without the addition of transferrin, suggesting that ligand binding was not a necessary prerequisite for TonB interaction. In contrast, a characterized TonB box mutant of TbpA did not facilitate interaction between these two proteins such that complexes could be isolated. We generated an in-frame deletion of gonococcal TonB, which removed 35 amino acids, including aNeisseria-specific, glycine-rich domain. This mutant protein, like the parental TonB, energized TbpA to enable growth on transferrin. Consistent with the functionality of this deletion derivative, TbpA-TonB complexes could be recovered from this strain. The results of the present study thus begin to define the requirements for a functional interaction between gonococcal TbpA and TonB.

Neisseria gonorrhoeaeis capable of utilizing host iron-binding proteins, such as transferrin, lactoferrin, and hemoglobin, as the sole source of iron. The receptor involved in transferrin iron acquisition is composed of two distinct transferrin-binding proteins, TbpA and TbpB. The genes that encode these proteins are linked on the chromosome in the ordertbpB-tbpAbut are separated by an inverted repeat of unknown function. In this study, we sought to understand the transcriptional organization and regulation of thetbpgenes, using a combination oflacZtranscriptional fusion analysis and reverse transcriptase PCR (RT-PCR). First, we demonstrated thattbpBandtbpAare cotranscribed and coregulated from the common upstream promoter that precedestbpB. Using β-galactosidase activity as a surrogate fortbp-specific transcription, we found thattbpB-specific transcripts were more prevalent thantbpA-specific transcripts after 2 h of growth under iron stress conditions. We confirmed the results obtained by fusion analysis by using RT-PCR applied to native RNA isolated from wild-type gonococci. Three different varieties of RT-PCR were employed: relative, competitive, and real time quantitative. The results of all analyses indicated thattbpB-specific transcripts were approximately twofold more prevalent thantbpA-specific transcripts at steady state. In iron-stressed cultures, the ratio oftbpB- totbpA-specific message was approximately 2; however, in iron-replete cultures, this ratio dropped to 1. Using these techniques, we also quantitated the effects of iron, external pH, and presence of ligand ontbpmRNA levels.

The availability of free iron in vivo is strictly limited, in part by the iron-binding protein transferrin. The pathogenicNeisseriaspp. can sequester iron from this protein, dependent upon two iron-repressible, transferrin-binding proteins (TbpA and TbpB). TbpA is a TonB-dependent, integral, outer membrane protein that may form a β-barrel exposing multiple surface loops, some of which are likely to contain ligand-binding motifs. In this study we propose a topological model of gonococcal TbpA and then test some of the hypotheses set forth by the model by individually deleting three putative loops (designated loops 4, 5, and 8). Each mutant TbpA could be expressed without toxicity and was surface exposed as assessed by immunoblotting, transferrin binding, and protease accessibility. Deletion of loop 4 or loop 5 abolished transferrin binding to whole cells in solid- and liquid-phase assays, while deletion of loop 8 decreased the affinity of the receptor for transferrin without affecting the copy number. Strains expressing any of the three mutated TbpAs were incapable of growth on transferrin as a sole iron source. These data implicate putative loops 4 and 5 as critical determinants for receptor function and transferrin-iron uptake by gonococcal TbpA. The phenotype of the ΔL8TbpA mutant suggests that high-affinity ligand interaction is required for transferrin-iron internalization.

We cloned lbpB , encoding a predicted 80-kDa lipoprotein, upstream of lbpA . A nonpolar mutant (LbpB − LbpA + ) had normal lactoferrin (LF) binding and grew normally with LF as an iron source, whereas LbpB − LbpA − and LbpB + LbpA − strains had reduced binding of LF and did not grow with LF as an iron source. LbpB bound LF directly in an affinity purification, suggesting that LbpB might play a still-uncharacterized role in the LF iron utilization.

Iron, an essential nutrient for most microorganisms, is sequestered by the host to decrease the concentration of iron available to bacterial pathogens. Neisseria gonorrhoeae, the causative agent of gonorrhoea, can acquire iron by direct interaction with human iron-binding proteins, including the serum glycoprotein, transferrin. Iron internalization from host transferrin requires the expression of a bacterial receptor, which specifically recognizes the human form of transferrin. Two gonococcal transferrin-binding proteins have been implicated in transferrin receptor function, TbpA and TbpB. We constructed a gonococcal transferrin receptor mutant without the introduction of additional antibiotic resistance markers and tested its ability to cause experimental urethritis in human male volunteers. The transferrin receptor mutant was incapable of initiating urethritis, although the same inoculum size of the wild-type parent strain, FA1090, causes urethritis in >90% of inoculated volunteers. To our knowledge, this is the first experimental demonstration that a bacterial iron acquisition system is an essential virulence factor for human infection.

Neisseria gonorrhoeae is an obligate pathogen that hijacks iron from the human iron transport protein, holo-transferrin (Fe(2)-Tf), by expressing TonB-dependent outer membrane receptor proteins, TbpA and TbpB. Homologous to other TonB-dependent outer membrane transporters, TbpA is thought to consist of a β-barrel with an N-terminal plug domain. Previous reports by our laboratories show that the sequence EIEYE in the plug domain is highly conserved among various bacterial species that express TbpA and plays a crucial role in iron utilization for gonococci. We hypothesize that this highly conserved EIEYE sequence in the TbpA plug, rich in hard oxygen donor groups, binds with Fe(3+) through the transport process across the outer membrane through the β-barrel. Sequestration of Fe(3+) by the TbpA-plug supports the paradigm that the ferric iron must always remain chelated and controlled throughout the transport process. In order to test this hypothesis here we describe the ability of both the recombinant wild-type plug, and three small peptides that encompass the sequence EIEYE of the plug, to bind Fe(3+). This is the first report of the expression/isolation of the recombinant wild-type TbpA plug. Although CD and SUPREX spectroscopies suggest that a non-native structure is observed for the recombinant plug, fluorescence quenching titrations indicate that the wild-type recombinant TbpA plug binds Fe (3+) with a conditional log K(d) = 7 at pH 7.5, with no evidence of binding at pH 6.3. A recombinant TbpA plug with mutated sequence (NEIEYEN → NEIAAAN) shows no evidence of Fe(3+) binding under our experimental set up. Interestingly, in silico modeling with the wild-type plug also predicts a flexible loop structure for the EIEYE sequence under native conditions which once again supports the Fe(3+) binding hypothesis. These in vitro observations are consistent with the hypothesis that the EIEYE sequence in the wild-type TbpA plug binds Fe(3+) during the outer membrane transport process in vivo.

Pathogenic Neisseria species have been shown to scavenge iron from transferrin (Tf), although the mechanism is not yet fully understood. Two iron-repressible proteins that exhibit Tf-binding activity have been identified. This work describes the cloning and sequencing of tbpB, a 2.1-kb gene in N. gonorrhoeae that encodes Tbp2, an 85-kDa iron-repressible lipoprotein. Transcriptional interruption of tbpB had a strong polar effect on tbpA, the structural gene for Tbp1 that is located immediately downstream from tbpB. Such tbpB mutants did not express either Tbp2 or Tbp1, did not bind Tf to whole cells, did not grow on Tf plates, and did not take up iron from Tf. A mutant in which most of tbpB was deleted, presumably leaving tbpA under transcriptional control of the tbpB promoter, was constructed. This mutant did not express Tbp2 but expressed wild-type levels of Tbp1 and possessed the phenotype of reduced binding of Tf, decreased iron uptake from Tf, but normal growth on Tf plates. Mutants expressing Tbp2 and not Tbp1 bound less Tf, did not grow on Tf plates, and did not take up iron from Tf. These results suggest that tbpB and tbpA are polycistronic. Tbp2 apparently facilitates binding of Tf but is not essential for acquisition of iron from Tf under these in vitro conditions.

Neisseria gonorrhoeae has the capacity to acquire iron from its human host by removing this essential nutrient from serum transferrin. The transferrin binding proteins, TbpA and TbpB constitute the outer membrane receptor complex responsible for binding transferrin, extracting the tightly bound iron from the host-derived molecule, and transporting iron into the periplasmic space of this Gram-negative bacterium. Once iron is transported across the outer membrane, ferric binding protein A (FbpA) moves the iron across the periplasmic space and initiates the process of transport into the bacterial cytosol. The results of the studies reported here define the multiple steps in the iron transport process in which TbpA and TbpB participate. Using the SUPREX technique for assessing the thermodynamic stability of protein-ligand complexes, we report herein the first direct measurement of periplasmic FbpA binding to the outer membrane protein TbpA. We also show that TbpA discriminates between apo- and holo-FbpA; i.e. the TbpA interaction with apo-FbpA is higher affinity than the TbpA interaction with holo-FbpA. Further, we demonstrate that both TbpA and TbpB individually can deferrate transferrin and ferrate FbpA without energy supplied from TonB resulting in sequestration by apo-FbpA.