INTRODUCTION

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Neisseria gonorrhoeae is the causative agent of the sexually transmitted disease gonorrhea, which has an annual incidence rate of more than 62 million cases worldwide (53). While frequently asymptomatic, gonococcal infection can be associated with serious sequelae, including salpingitis, endometritis, ectopic pregnancy, and infertility in women (49). In addition, gonococcal disease enhances the human immunodeficiency virus titer in seminal plasma of coinfected men, presumably increasing the likelihood of disease transmission (11). In light of these factors, compounded by increased antibiotic resistance (25) and the lack of an effective vaccine, gonorrhea remains a serious public health problem.

Iron is an essential nutrient for all microorganisms (21); however, free iron is rare in the human body, generally being complexed by a number of high-affinity binding proteins, including transferrin (Tf) in plasma and lactoferrin in secretions and macrophages (9). A total of 100% of clinical isolates of N. gonorrhoeae can obtain iron from human Tf (40), while approximately 50% of gonococcal isolates can obtain iron from human lactoferrin (39). The ability to use human proteins as iron sources may allow the gonococcus to overcome bacteriostatic iron limitation, which is typical in vivo (9). In contrast to other bacteria, the Tf-iron uptake system employed by pathogenic neisseriae is siderophore independent (3), relying instead on direct association between a surface-exposed bacterial receptor and ligand (3, 48). The receptor is specific for human Tf (31, 32), and Tf-iron internalization is energy (38) and Ton (5, 14) dependent.

The gonococcal Tf receptor is composed of two iron-regulated, Tf-binding proteins designated TbpA and TbpB (14). TbpA is highly conserved among the pathogenic neisseriae (12) and is a member of the TonB-dependent family of integral outer membrane proteins (16). Consistent with other proteins within this group (27, 34, 44), TbpA is likely to form a TonB-regulated transmembrane -barrel, through which iron can enter the periplasm. Recent crystallographic analyses of FhuA (35) and FepA (8) have demonstrated the involvement of surface-exposed loops in ligand binding. In addition, these studies demonstrated that FhuA and FepA each have an amino-terminal globular domain, which serves as a "plug" in the interior of the transmembrane pore (35). This domain was found to contain a conserved pentameric sequence referred to as the "TonB box" (46), a region that has been shown to interact with the energy transducer TonB (4, 10, 23, 30, 45). Thus, it is probable that the TonB box of TbpA likewise interacts with the periplasmic protein TonB, thereby energizing the Tf receptor and enabling iron internalization.

TbpB is more variable than TbpA, with molecular masses that range from 78 to 86 kDa in N. gonorrhoeae (2, 13) and from 65 to 85 kDa in N. meningitidis (33, 43, 47). TbpB is lipidated and exposed on the outer leaflet of the bacterial outer membrane (2, 33). Although both Tbps specifically and independently bind human Tf, TbpB selectively binds the ferrated form of this protein while TbpA binds both ferrated and apo Tf (7, 18).

Efficient utilization of Tf as a sole iron source requires expression of both gonococcal Tbps (2). A gonococcal mutant lacking TbpB exhibited decreased Tf-iron internalization relative to the wild-type strain, suggesting that while TbpB expression increases the efficiency of the receptor, it is not essential for Tf-iron internalization (2). In contrast, expression of TbpA is absolutely required for Tf-iron growth and Tf-iron internalization (16), consistent with its role as a transmembrane pore for Tf bound iron. Cornelissen and Sparling (18) demonstrated that TbpA and TbpB are in close proximity to one another and may interact, at least transiently, on the surface of intact gonococci. Surface exposure and Tf-binding characteristics of the cell-associated TbpA-TbpB complex are dependent upon a wild-type Ton system and TonB-box at the amino terminus of TbpA (14).

In this study, we propose a model of gonococcal TbpA that combines computer analysis with observed sequence conservation among TonB-dependent receptors and sequence diversity among gonococcal TbpA sequences. We suggest that TbpA may form a -barrel composed of 22 transmembrane strands and probably has a periplasmic globular domain equivalent to that described for FhuA (35) and FepA (8). We have tested aspects of this model by individually deleting three regions of gonococcal TbpA, each predicted to form an extracellular, surface-exposed loop. We assessed the surface exposure and function of these deletion derivatives of TbpA and determined that, while expression of loops 4 and 5 of TbpA was critical for the Tf-binding function of this receptor, expression of loop 8 was required for optimal ligand interaction.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Bacterial strains, plasmids, and media. The bacterial strains and plasmids used in this study are described in Table 1. Unless otherwise stated, gonococci were grown on plates containing GC medium base (Difco) with Kellogg's supplement I (1%) (26) and 12 µM Fe(NO₃)₃ in a 5% CO₂ atmosphere at 35°C. For growth in iron-stressed conditions, gonococci were cultured in liquid CDM medium (51) which was pretreated with Chelex-100 (Bio-Rad) to remove residual iron. CDM-agarose plates containing 2.5 µM Tf (30% iron saturated) were used to assess the ability of gonococcal strains to utilize Tf-bound iron. Plasmids were propagated in Escherichia coli strains (Table 1) cultured on Luria-Bertani plates (37) containing appropriate antibiotics (50 µg of ampicillin or kanamycin per ml).

Standard recombinant DNA techniques and DNA sequencing. Isolation of plasmid DNA, digestion with restriction endonucleases and subsequent ligation were carried out according to manufacturer's recommendations. PCR amplification was carried in standard conditions (Gibco-BRL) with annealing temperatures optimized for each set of oligonucleotide primers. DNA was sequenced using a 377 DNA sequencer (Applied Biosystems) at the Virginia Commonwealth University Nucleic Acids Core Facility. The template was supplied as plasmid DNA, purified using the Wizard Mini-Prep system (Promega). Nucleotide positions are numbered in accordance with the gonococcal tbpA sequence from strain FA19 deposited in GenBank under the accession no. M96731 (16).

Gonococcal transformation. Mutated tbpA genes were transformed into gonococcal strains according to the method described by Gunn and Stein (22). Briefly, piliated gonococci were resuspended in GC broth to a density of approximately 2 × 10⁸ CFU/ml. Approximately 100 CFU were spotted onto GC agar plates, followed immediately by linearized plasmid preparations, which had been purified using the Wizard DNA clean-up kit (Promega). Following overnight growth, individual colonies were subcultured and then screened for the incorporation tbpA mutations into the chromosome. This analysis involved subjecting colony lysates to PCR using oligonucleotide primers designed to amplify a diagnostic product. Single colonies were passaged, and the genotype was confirmed by DNA sequencing.

Solid-phase binding assays. Whole-cell binding analyses were carried out using gonococci grown in iron-deficient, liquid CDM media as described above. After 3 h of iron-limited growth, normalized amounts of culture were applied to nitrocellulose filters. Blots were probed with horseradish peroxidase (HRP)-Tf (1 µg/ml) as described previously (6) and were developed with Opti-4-CN (Bio-Rad) according to manufacturer's recommendations. Duplicate blots were probed using an anti-TbpA polyclonal serum raised against recombinant meningococcal TbpA from strain SD (1), which cross-reacts with gonococcal TbpA. This sera was preabsorbed against whole, iron-stressed gonococcal strain FA6747 (Table 1) to remove nonspecific, cross-reacting antibodies against antigens other than TbpA. Dot blots were subsequently probed with a goat anti-rabbit immunoglobulin G (IgG)-HRP conjugate and developed with Opti-4-CN (Bio-Rad) according to the manufacturer's recommendations.

Liquid-phase Tf-binding assay. Liquid-phase Tf-binding assays were performed essentially as previously described (18). In these assays, iron-stressed gonococci were mixed with various amounts of iodinated, iron-saturated Tf in the presence or absence of unlabeled competitor Tf (iron saturated). After 20 min, the unbound, iodinated Tf was removed by filtration through 0.45-µm-pore-size nitrocellulose (Millipore Multi-Screen microtiter dish MAHV N45). The amount of bound Tf was quantitated with a gamma scintillation counter. The binding assays were standardized to whole-cell protein by performing protein assays (bicinchoninic acid assay; Pierce) on iron-stressed cultures. Three identical experiments, including growth and iron stress, were carried out; the datum points represent the mean of three independent experiments. Specific binding was calculated by subtracting nonspecific binding (detected in the presence of excess competitor) from total counts for each Tf concentration. The specific activity of the iodinated Tf was 1.61 × 10⁶ cpm/µg. Copy number and K_d estimates were obtained using Lundon software (Lundon Software) (36).

Protease-accessibility assay. Protease accessibility experiments were performed as previously described (18). Briefly, iron-stressed whole gonococci (grown in CDM) were treated with trypsin (Sigma-Aldrich) at concentrations of 0.5 to 2.5 µg/ml for 0 to 30 min. Proteolysis was stopped by addition of 0.6 trypsin-inhibiting units of aprotinin (Sigma-Aldrich), and whole-cell lysates were prepared from samples taken at 10-min intervals. In some experiments, ferrated Tf was added prior to protease exposure, to a final concentration of 100 nM. Whole-cell proteins were resolved by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using 7.5% acrylamide gels (29). Western blots were probed with anti-TbpA polyclonal rabbit sera or with HRP-Tf (to detect TbpB) according to previously described methods (18).

Immunofluorescence microscopy. Immunofluorescence microscopy was used to confirm the surface exposure of TbpA on intact gonococci. Iron-stressed, whole cells from gonococcal strains FA19 (positive control), FA6747 (negative control), and MCV201 (Table 1) were mixed with dilutions anti-TbpA polyclonal rabbit sera (raised against meningococcal TbpA from strain SD) (1) in the presence of immunoglobulin-free bovine serum albumin (Sigma-Aldrich) as a blocking reagent. Preimmune rabbit sera were also used as a negative control. Unbound antibody was removed by centrifugation, and cells were then applied to glass microscope slides and fixed by immersion in 100% methanol. These slides were probed with goat anti-rabbit IgG conjugated to fluorescein isothiocyanate (Sigma-Aldrich), and cells were counterstained with Eriochrome Black (Integrated Diagnostics). Samples were visualized using an Olympus BHA microscope equipped with a BH2RFL reflected fluorescence attachment and a PM-10AD photomicrographic system (Olympus Corp).

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Development of a topology model of gonococcal TbpA and selection of loops for deletion analysis. We initially used the method of Klebba et al. (28) to convert the TbpA sequence into a two-dimensional topology model. We identified potential amphipathic -strands with the algorithm MOMENT (19) and putative -turns with the algorithm TURNPRED (52). In addition, our original model located most stretches of hydrophilic residues and all six cysteine residues within predicted surface-exposed loops. We modified this computer-generated model by superimposing on it observed sequence variation among TbpAs from the pathogenic neisseriae, Haemophilus influenzae, Actinobacillus pleuropneumoniae, Pasteurella haemolytica, and Moraxella catarrhalis (12). We reasoned that sequence diversity would be enriched in surface-exposed regions that were accessible to immune pressure and that conserved domains would be either inaccessible or functionally constrained. Figure 1 represents the latest version of our TbpA topology model and encompasses a further modification based upon pairwise comparisons between TbpA and the recently crystallized TonB-dependent receptor, FepA (8). Since the coordinates of all FepA residues have been elucidated in the crystal structure, we created a pairwise alignment between gonococcal TbpA and FepA and used this alignment to identify putative -strand endpoints. With this alignment, we also identified an amino-terminal, 162-residue domain of TbpA that was 29% identical and 44% similar to the analogous region of FepA. This amino-terminal region was shown in the FepA crystal structure to form an essentially globular "plug" that occludes the lumen of the -barrel (8). We have depicted the homologous region of TbpA (residues 1 to 162) in a similar position in Fig. 1. This region is very well conserved among the neisserial TbpAs (12), consistent with the suggestion that this domain is inaccessible from the outside and therefore not subject to immune pressure.

View larger version (56K): [in this window] [in a new window]   FIG. 1.   Putative topology model of gonococcal TbpA. Horizontal dotted lines represent the planes of the gonococcal outer membrane. The predicted amphipathic -strands (22 in total) are shown in gray boxes. Putative surface exposed loops are numbered L1 to L11. Loops 4, 5, and 8 are indicated by red boxes, and the endpoints of the L4, L5, and L8 deletions are indicated by short horizontal black bars. Residues that are conserved among all 17 TbpAs sequenced to date (12) are indicated by the squares. Residues that fall within previously characterized TonB homology domains (16) are shaded yellow. Residues that are variant among a panel of 5 gonococcal TbpAs (12) are shaded blue. Aromatic residues are circled. Cysteine residues are shaded orange. Two predicted trypsin cleavage sites are shaded pink. Although the TbpA sequence contains many other predicted trypsin cleavage sites, cleavage at these two sites would generate proteolytic cleavage products that most closely match the observed sizes of trypsin digest products T1 and T2 (see text). The sequences of three peptides used to generate antipeptide antibodies (12) are highlighted in green.

Construction of L4TbpA, L5TbpA, and L8TbpA mutants. The method for the construction of TbpA-encoding plasmids from which loop 4 was deleted is schematically represented in Fig. 2. To delete loop 4 from TbpA of strain FA19 (Table 1), we used the oligonucleotide pair HIND and oVCU-16 to amplify the 5' half of tbpA; to amplify the 3' half of tbpA, we used oVCU-17 and oVCU-7 (Table 2 and Fig. 2). These oligonucleotides introduced novel BamHI sites flanking the sequence that encodes loop 4. The separate PCR fragments were cloned independently into pCR2.1, yielding pVCU206 and pVCU207 (Table 1 and Fig. 2). Subsequently, the 5' and 3' halves of tbpA were excised and cloned into the pET21 expression vector in a single three-part ligation, producing plasmid pVCU208 (Table 1 and Fig. 2). Sequence analysis confirmed the deletion of 144 bp of gonococcal tbpA in this plasmid. The deleted region encoded 48 amino acids, which were replaced by two residues: glycine and serine. However, two additional single-base mutations were noted in the mutated tbpA. The first was a silent mutation, and the second exchanged an alanine for a glycine. Given the conservative nature of these base changes, no corrective mutagenesis was undertaken.

View larger version (23K): [in this window] [in a new window]   FIG. 2.   Construction of L4TbpA. The 5' (striped) and 3' (gray) PCR-amplified halves of tbpA were individually cloned into pCR2.1, which contains a gene encoding ampicillin resistance (stippled bar). This procedure resulted in plasmids pVCU206 and pVCU207, respectively. These inserts were excised and ligated together in pET21 producing pVCU208, which contained L4tbpA, downstream of a T7 promoter (black bar). The position of the TbpA start codon is shown as an open arrow, and that of the TbpA stop codon is marked with an asterisk. Approximate positions of oligonucleotides are indicated by closed arrows, with their respective names indicated in parentheses.

Solid-phase Tf binding by gonococcal TbpA deletion mutants. To determine the functionality of the TbpA loop-deletion mutants, we first tested the ability of strains expressing deleted TbpAs to bind Tf in a solid-phase binding assay. As shown in Fig. 3A, strains expressing TbpA loop deletions and a functional TbpB (MCV201, MCV203, and MCV205) retained Tf-binding capability as expected. In the absence of a functional TbpB, strains MCV202 (L5TbpA, TbpB) and MCV204 (L4TbpA, TbpB) were no longer competent for Tf binding unlike their isogenic parent FA6905, which expresses a full-length TbpA (Fig. 3A). MCV206 (L8TbpA, TbpB) bound somewhat less Tf than did the wild-type strain in the same assay (Fig. 3A), suggesting that this strain, while capable of Tf binding, did so with diminished affinity or capacity. Thus, deletion of either loop 4 or loop 5 prevented ligand binding by TbpA and indicated that these regions contain residues that are directly or indirectly required for Tf binding. The apparent reduction of Tf binding to L8TbpA suggested that the presence loop 8 could be important for high-affinity interactions between TbpA and its ligand.

View larger version (19K): [in this window] [in a new window]   FIG. 3.   Whole-cell, solid-phase Tf-binding assay. Whole, iron-stressed gonococci were spotted onto nitrocellulose filters and probed with HRP-Tf (A) or anti-TbpA polyclonal serum (B). Numbering refers to the following gonococcal strains; 1, FA19 (wild type); 2, FA6905 (TbpA+ TbpB); 3, FA6747 (TbpA TbpB+); 4, FA6815 (TbpA TbpB); 5, MCV201 (L5TbpA, TbpB+); 6, MCV202 (L5TbpA, TbpB); 7, MCV203 (L4TbpA, TbpB+); 8, MCV204 (L4TbpA, TbpB); 9, MCV205 (L8TbpA, TbpB+); and 10, MCV 206 (L8TbpA, TbpB). Images were scanned using a Hewlett-Packard ScanJet 6300c. Final images were annotated using Adobe Photoshop 4.0.

Localization of gonococcal TbpA deletion mutants. The inability of the loop 4 and loop 5 deletion mutants to bind Tf in the solid-phase assay could have been the result of inappropriate processing or localization of the mutant proteins. To address this possibility, duplicate whole-cell dot blots were probed with a polyclonal anti-TbpA serum that recognized surface-exposed TbpA. As shown in Fig. 3B, this serum bound to all strains expressing either wild-type TbpA (FA19 and FA6905) or mutated TbpAs (strains MCV201 to MCV206). Strains FA6747 and FA6815 (both TbpA) were not recognized by this serum (Fig. 3B). These observations indicated that wild-type TbpA and L4TbpA, L5TbpA, and L8TbpA were each surface exposed and accessible to antibody under these conditions.

Liquid-phase Tf binding by gonococcal TbpA deletion mutants. We used a liquid-phase Tf-binding assay to quantitate the binding phenomena associated with the TbpA deletion derivatives in the solid-phase binding assay. The curves shown in Fig. 4 represent the amount of specific Tf bound as a function of Tf concentration, which ranged from 1.2 to 160 nM. As in the solid-phase Tf binding assay, neither MCV204 (L4TbpA, TbpB) nor MCV202 (L5TbpA, TbpB) bound any detectable Tf in this assay. In contrast, MCV206 (L8TbpA, TbpB) bound Tf with nearly the same capacity as the wild-type strain but with a much lower affinity. Whereas the wild-type TbpA (expressed by FA6905 in the absence of TbpB) bound Tf with a K_d of approximately 2.8 nM (Fig. 4 and reference 18), the L8TbpA mutant bound Tf with a K_d of 22 nM. The capacity of the wild-type and L8TbpA mutant strain were virtually indistinguishable, with copy numbers of 8.2 × 10⁸ and 8.3 × 10⁸ receptors/µg of total cell protein, respectively. These data confirmed and extended the results of the solid-phase Tf binding analysis, indicating that deletion of loop 4 or loop 5 prevented ligand binding completely, whereas deletion of loop 8 resulted in a 10-fold decrease in the affinity of TbpA for Tf without affecting the copy number.

View larger version (16K): [in this window] [in a new window]   FIG. 4.   Equilibrium-phase Tf binding by TbpA deletion mutants. The plot shows the amount of specifically bound Tf (in picograms bound per microgram of total cell protein [TCP]) as a function of Tf concentration. , Binding to FA6905 (TbpA+ TbpB); , binding to MCV206 (L8TbpA, TbpB); , binding to MCV202 (L5TbpA, TbpB); , binding to MCV204 (L4TbpA, TbpB). The datum points are the means of three independent experiments. Error bars indicate the standard deviations within each triplicate set.

Growth of TbpA deletion mutants with transferrin as a sole iron source. To assess the impact of TbpA mutagenesis on Tf-iron utilization, strains MCV201 to MCV206 were plated on minimal medium containing partially saturated Tf as the sole iron source. As shown previously, the presence of TbpB increases the efficiency of Tf-iron uptake but is not required for growth on Tf as a sole iron source (2). All of the TbpA deletion derivatives failed to grow on Tf, regardless of the presence of a functional TbpB (Fig. 5), indicating that deletion of putative loop 4 or 5 rendered TbpA unable to bind Tf and, despite apparent surface exposure, unable to facilitate the uptake of iron from Tf. Interestingly, L8TbpA, which bound Tf with lower affinity but equal capacity, did not facilitate iron internalization. All strains grew equally well on GC base medium supplemented with Fe(NO₃)₃.

View larger version (132K): [in this window] [in a new window]   FIG. 5.   Growth of gonococcal strains using Tf as a sole iron source. Gonococcal strains were grown on CDM-agarose plates, supplemented with Tf (30% iron saturated) as an iron source. Sector labeling refers to the following gonococcal strains: A, FA19 (wild type); B, FA6747 (TbpA TbpB+); C, FA6905 (TbpA+ TbpB); D, FA6815 (TbpA TbpB); E, MCV201 (L5TbpA, TbpB+); F, MCV202 (L5TbpA, TbpB); G, MCV203 (L4TbpA, TbpB+); H, MCV204 (L4TbpA, TbpB); I, MCV205 (L8TbpA, TbpB+); and J, MCV 206 (L8TbpA, TbpB). Images were scanned using a Hewlett-Packard ScanJet 6300c. The final images were annotated using Adobe Photoshop 4.0.

Protease accessibility of TbpA in deletion mutants. We previously identified energy-dependent conformational changes in the Tf receptor based partly on the results of limited proteolysis of whole, iron-stressed gonococci (14). Thus, we used trypsin accessibility of the TbpA deletion derivatives as a measure of wild-type surface exposure and overall conformation. When wild-type TbpA was exposed to low concentrations of trypsin, two diagnostic products were generated, one with a molecular mass of 95.2 kDa (T1) and the other with a molecular mass of 54.9 kDa (T2) (Fig. 6A and reference 18). A mutant TbpA lacking a functional TonB box (14) likewise demonstrated a trypsin accessibility pattern characterized by wild-type-sized T1 and T2 products (Fig. 6A). The deletion derivatives of TbpA were universally accessible to exogenous trypsin, a finding consistent with normal processing and surface exposure of these proteins. The proteolytic fragments generated by trypsin digestion of the deletion derivatives did not differ greatly in size from those generated by cleavage of the wild-type protein, with T1 and T2 equivalents apparent in two out of the three deletion mutants (Fig. 6A). The sizes of T1 and T2 equivalents in these mutants were consistent with the number of amino acids that had been deleted in each construct. In addition to T1 and T2, L4TbpA spontaneously proteolyzed, producing a detectable breakdown product at 0 min of trypsin exposure (Fig. 6A). This slight instability could have resulted from deletion of most of a proposed membrane-spanning -strand (see Fig. 1) in this construct.

View larger version (20K): [in this window] [in a new window]   FIG. 6.   Trypsin accessibility of mutant TbpAs in the presence and absence of ligand. (A) Whole, iron-stressed cells from the indicated gonococcal strains were treated with trypsin (0.5 µg/ml) for 0 min (lane 1), 10 min (lane 2), 20 min (lane 3), or 30 min (lane 4) as indicated above each panel. Whole-cell lysates were prepared from the trypsinized cells, and the proteins were separated by SDS-PAGE. The resulting Western blots were probed with polyclonal anti-TbpA serum (16). (B) Whole, iron-stressed cells from the indicated gonococcal strains were exposed to 100 nM iron-saturated Tf and then treated with trypsin (0.5 µg/ml) for 0 min (lane 1), 10 min (lane 2), 20 min (lane 3), or 30 min (lane 4) as indicated above each panel. The resulting Western blots were probed with polyclonal anti-TbpA serum (16). The labels T1 and T2 on the left of each panel indicate the positions of two characteristic products that resulted from limited proteolysis of TbpA. Approximate positions of molecular mass standards are indicated in kilodaltons on the right of each panel. Images were scanned using a Hewlett-Packard ScanJet 6300c. The final images were annotated using Adobe Photoshop 4.0.

Protease accessibility of TbpA in the presence of transferrin. We have previously demonstrated that TbpA to which Tf is bound is less accessible to exogenously added trypsin than unliganded TbpA (18), while a de-energized, TonB-box mutant of TbpA was almost completely inaccessible to trypsin in the presence of ligand (14). As shown in Fig. 6B, the proteolytic digestion patterns of L4TbpA and L5TbpA were unchanged in the presence of Tf, whereas proteolysis of L8TbpA was inhibited in the presence of Tf, similar to wild-type TbpA. These observations are consistent with our assertion that neither L4TbpA nor L5TbpA bound Tf because the presence of ligand did not effect the extent of proteolysis. In contrast, since trypsin accessibility was inhibited by ligand addition to L8TbpA, we conclude that this derivative retained Tf binding capabilities, an idea consistent with the results of Tf-binding assays (Fig. 3 and 4).

Protease accessibility of TbpB in gonococcal deletion mutants. Previously, we demonstrated that TbpB is protected from limited proteolysis only in the presence of an energized TbpA (14). Thus, we analyzed the accessibility of TbpB to trypsin in presence of the TbpA deletion derivatives to access their ability to interact with and functionally protect TbpB from trypsin. When coexpressed with L4TbpA, L5TbpA, or L8TbpA TbpB was protected at levels equivalent to those observed with wild-type TbpA (data not shown), indicating that the mutant TbpAs could interact with TbpB such that a portion of the TbpB population was resistant to trypsin, similar to the situation seen with the wild-type TbpA.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

In this study, we propose a topological model of gonococcal TbpA in which this protein forms an outer membrane pore composed of 22 transmembrane -strands. In addition, we suggest that gonococcal TbpA has a periplasmic globular domain, one similar to that identified in the crystal structures of two other TonB-dependent receptors, FepA (8) and FhuA (35). The authors of those studies hypothesized that the globular domain serves as a "plug" (35) or "hatch" (8) that occludes the lumen of the -barrel formed by other transmembrane -strands. Given the extent of conservation between TbpA and the TonB-dependent receptors and also the relative lack of divergence among neisserial TbpA sequences in this region (12), we suggest that this domain may have a similar location and function in gonococcal TbpA. The large size, structural complexity, and dynamic nature of both TbpA (14) and Tf (50), suggest that multiple surface-exposed epitopes may determine the ligand-binding characteristics of this receptor. Thus, our proposed topological model includes 11 putative loops, any or all of which could include ligand-binding domains.

In the current study, we determined the phenotypes of mutant gonococcal strains expressing TbpAs deleted of putative loop 4, 5, or 8. These mutant proteins were expressed from the gonococcal chromosome without apparent toxicity or inhibition of bacterial growth. Using solid- and liquid-phase Tf-binding assays, immunoblots, protease accessibility, and TbpB interaction, we confirmed the outer membrane localization of each mutant protein. Extreme structural perturbation of outer membrane proteins typically results in aberrant localization, spontaneous proteolysis and, in some cases, inviability (41, 42). In the absence of these effects, we conclude that the native conformation adopted by wild-type TbpA was broadly conserved among the mutants characterized in this study. The sole exception to this overall conclusion is L4TbpA, which exhibited slight proteolytic instability and therefore native folding might have been effected by deletion of most of proposed -strand 8 in addition to loop 4. However, as evaluated by the trypsin accessibility pattern of TbpA expressed by this mutant, a large portion of the deleted protein was exported to the surface in a conformation similar to that of wild-type TbpA. The L5TbpA protein was apparently surface exposed and resulted in no spontaneous breakdown products, suggesting that deletion of this putative loop did not negatively impact protein export. Likewise, the observation of nearly identical numbers of L8TbpA receptors relative to the wild-type receptor in whole-cell, liquid-phase Tf-binding assays indicates that deletion of these loops had no discernible deleterious effects on protein export or surface exposure. Therefore, we suggest that the alteration in Tf binding by these mutants resulted from the deletion of functionally significant epitopes rather than from gross conformational disruption or reduced surface exposure.

Deletion of loop 4 or loop 5 resulted in mutant versions of TbpA that were incapable of Tf binding and that did not support growth on Tf-bound iron, suggesting that these putative loops are critical for ligand interaction and subsequent iron internalization. Even in the presence of a functional TbpB, iron utilization was prevented by deletion of loop 4 or loop 5 from TbpA, indicating that the mere proximity of ferrated Tf (bound to TbpB) was insufficient to allow iron uptake. Deletion of loop 8 from TbpA resulted in a mutant that was capable of binding Tf with a 10-fold decreased affinity relative to wild-type TbpA. However, this deletion mutant was rendered completely incompetent for Tf-iron internalization. These observations suggest that intimate, high-affinity interactions between TbpA and Tf are required for iron removal and subsequent iron transport through the outer membrane.

Limited proteolysis has been used to identify flexible domains in globular enzymes (24) and as a probe of conformational change in soluble proteins (20). In this study, we used accessibility to exogenous protease as a measure of surface exposure and overall membrane topology in the TbpA deletion derivatives. We have previously shown that exposure of surface-exposed TbpA to low concentrations of trypsin produced two diagnostic proteolytic products (18). Although mature TbpA contains 129 predicted trypsin cleavage sites, only two are readily accessible in the membrane-bound, surface-exposed protein. Since protease accessibility is correlated with unstructured, highly flexible, hinge regions (24), we suggest that the trypsin cleavage sites in TbpA might reflect structurally important features of the receptor. The measured size of the largest wild-type proteolytic digestion product (T1) most closely corresponds to a peptide that spans from the mature amino terminus to Arg857, located in putative loop 11. The size of the T2 proteolytic product corresponds in size to a peptide that spans from the mature amino terminus to Arg498, located in putative loop 5. Thus, these regions are predicted to be sufficiently flexible to allow protease cleavage, and might represent hinge regions separating structurally constrained domains.

When we applied the protease susceptibility analysis to the deletion derivatives of TbpA, similarly sized proteolytic products were generated, indicating that the gross topological features of the mutant TbpAs were similar to those expressed by the wild-type strain. Both T1 and T2 equivalents produced by trypsin cleavage of L4TbpA were proportionally smaller than their wild-type counterparts, which is consistent with deletion of loop 4. Only a T1 equivalent was generated by trypsin digestion of L5TbpA, which is consistent with the concomitant deletion of the accessible trypsin site (Arg498) in loop 5. Of the two proteolytic digestion products generated from L8TbpA, the T1 equivalent was proportionally smaller by the size of the loop 8 deletion, but the T2 product was wild-type sized. From this analysis, we conclude that putative loop 8 is located between the two accessible trypsin sites that generated T1 and T2.

We have previously shown that by binding Tf prior to trypsin treatment, the accessibility of TbpA to the protease is inhibited (14, 18). In mutants in which TbpA is de-energized by Ton system defects, TbpA is almost completely protected from proteolysis, suggesting that TonB-derived energy is required for Tf release from the receptor (14). We analyzed the loop deletion mutants for their trypsin accessibility following ligand binding and found that, unlike a de-energized TbpA, all were accessible to trypsin. However, L8TbpA was protected by the presence of ligand to an extent similar to that in wild-type TbpA, a finding consistent with the Tf-binding capabilities of this mutant. Unlike L8TbpA, mutants L4TbpA and L5TbpA were equally susceptible to trypsin cleavage in the presence or absence of ligand, a result consistent with their complete inability to bind Tf.

In a similar analysis, we found that TbpB was protected from exogenous trypsin when coexpressed with either wild-type (14, 18) or mutant TbpA. Using this protease-resistant characteristic of TbpB, we developed a model of energy-dependent, conformational change or complex formation between TbpA and TbpB (14). Because the deletion mutants described in this study protect coexpressed TbpB from trypsin in a manner similar to that seen with wild-type TbpA, we conclude that on whole, iron-stressed gonococci, TbpA and TbpB formed a transient, energy-dependent complex similar to that detected in the wild-type strain. This implies that, under these conditions, the deleted regions were not essential for the preservation of physical proximity between TbpA and TbpB or for their proposed energy-dependent interaction.

Since expression of the Tf receptor is required to initiate infection in a human challenge model of gonococcal infection (17), identification of domains of TbpA that are necessary for the optimal function of this receptor could lead to therapies or prevention strategies aimed at abrogating its function. Thus, loops 4 and 5, which are essential for optimal ligand binding, could constitute novel targets for immunoprophylaxis against gonococcal disease. Alternatively, inhibitors of transferrin binding by these loops might serve as a mode of therapy against an already established infection. Loop 8, which appears to play a more subtle role in Tf binding but nevertheless critically impacts Tf-iron utilization, may also warrant assessment as a candidate vaccine antigen or target of inhibition. We are currently constructing smaller deletions and point mutations to further characterize the complex ligand binding and interactive properties of gonococcal TbpA.