Determination of surface-exposed, functional domains of gonococcal transferrin-binding protein A

Abstract

The gonococcal transferrin receptor is composed of two distinct proteins, TbpA and TbpB. TbpA is a member of the TonB-dependent family of integral outer membrane transporters, while TbpB is lipid modified and thought to be peripherally surface exposed. We previously proposed a hypothetical topology model for gonococcal TbpA that was based upon computer predictions and similarity with other TonB-dependent transporters for which crystal structures have been determined. In the present study, the hemagglutinin epitope was inserted into TbpA to probe the surface topology of this protein and secondarily to test the functional impacts of site-specific mutagenesis. Twelve epitope insertion mutants were constructed, five of which allowed us to confirm the surface exposure of loops 2, 3, 5, 7, and 10. In contrast to the predictions set forth by the hypothetical model, insertion into the plug region resulted in an epitope that was surface accessible, while epitope insertions into two putative loops (9 and 11) were not surface accessible. Insertions into putative loop 3 and beta strand 9 abolished transferrin binding and utilization, and the plug insertion mutant exhibited decreased transferrin-binding affinity concomitant with an inability to utilize it. Insertion into putative beta strand 16 generated a mutant that was able to bind transferrin normally but that was unable to mediate utilization. Mutants with insertions into putative loops 2, 9, and 11 maintained wild-type binding affinity but could utilize only transferrin in the presence of TbpB. This is the first demonstration of the ability of TbpB to compensate for a mutation in TbpA.

FIG. 1.

Hypothetical topological model of gonococcal TbpA. Schematic representation of the hypothetical two-dimensional model of gonococcal TbpA proposed previously (5). The horizontal dashed lines represent the boundaries of the outer membrane. The protein sequence is shown as the solid black line, with putative transmembrane β strands shown as vertical boxes. Putative extracellular loops are designated L1 through L11. The open circles represent six conserved cysteine residues. The locations of the HA epitopes inserted during the present study are indicated by dark gray boxes. The previously proposed trypsin cleavage sites are indicated with asterisks. The modified positions of accessible trypsin cleavage sites demonstrated by the present study are shown as black bars.

FIG. 2.

Expression of epitope-tagged TbpA proteins. Iron-stressed gonococci were lysed, and whole-cell proteins were separated by SDS-PAGE. Following protein transfer to solid support, blots were probed with one of the following antibody preparations: polyclonal anti-TbpA serum (A), polyclonal serum specific for epitopes within putative loops 4 and 5 of TbpA (B), and high-affinity, anti-HA monoclonal antibody conjugated to peroxidase (C and D). Relative to the blot in panel C, the blot represented in panel D was prepared with twofold more protein antigen, as described in Materials and Methods. Each lane is labeled above according to the strain from which the whole-cell proteins were derived. Those lanes containing proteins isolated from the TbpB⁻ derivatives are highlighted with the bracket. The approximate positions of molecular mass markers (MWM), in kilodaltons, are located to the left of each blot.

FIG. 3.

Solid-phase, anti-HA antibody binding. Whole, iron-stressed gonococcal cells were applied to a nitrocellulose membrane and probed with a high-affinity monoclonal anti-HA antibody. Dots are labeled to the left and right according to the strain that was applied to the membrane. The wild-type (FA19), TbpB⁻ (FA6905), and TbpA⁻ TbpB⁻ (FA6815) strains served as negative controls since they do not contain the HA epitope.

FIG. 4.

¹²⁵I-transferrin binding isotherms. Whole, iron-stressed gonococci were mixed with various amounts of ¹²⁵I-labeled human transferrin, and specific binding of the ligand was calculated by subtracting nonspecific binding (with excess competitor) from total binding. The amount of specific transferrin bound is reported on the y axis as nanograms of transferrin (Tf) bound per microgram of total cell protein (TCP). Isotherms are grouped for display according to the putative location of the epitope tag within TbpA: plug (A), extracellular loops (B), and periplasmic turns and β strands (C). Representative samples from each group are shown. The data shown represent the means and standard deviations generated from three independent binding experiments.

FIG. 5.

Transferrin utilization growth assay. The ability of mutants to utilize transferrin as the sole iron source was evaluated by 24-h growth on CDM plates supplemented with 5 μM human transferrin (ca. 30% saturated with iron). The growth phenotype of each TbpA epitope insertion mutant was evaluated in a TbpB⁺ (shown as +) and in a TbpB⁻ (shown as −) background. Each plate growth assay included a positive control strain expressing TbpA (TbpA⁺) and a negative control strain which did not express TbpA (TbpA⁻); neither control strain expressed TbpB.

FIG. 6.

Trypsin accessibility of epitope-tagged TbpA mutants. Iron-stressed gonococci were incubated with trypsin for 0, 10, 20, or 30 min before the protease was inactivated by the addition of aprotinin. The trypsinized cells were pelleted, lysed, and separated by SDS-PAGE. After transfer, the blots were probed with a polyclonal anti-TbpA serum. The duration of the trypsin incubation (in minutes) is shown above each lane; lanes labeled NT contain proteins that were not treated with trypsin before the addition of aprotinin. The approximate positions of molecular mass markers (MW), shown in kilodaltons, are located to the left of the blots. The previously characterized TbpA-derived trypsin digestion products, T1 (95.2 kDa) and T2 (54.9 kDa), are labeled on the right.

FIG. 7.

Mapping of trypsin cleavage sites with the HA epitope tags. Iron-stressed gonococci were incubated with trypsin for 0, 10, 20, or 30 min before the protease was inactivated by the addition of aprotinin. The trypsinized cells were pelleted, lysed, and separated by SDS-PAGE. After transfer, the blots were probed with a monoclonal anti-HA antibody conjugated to peroxidase. The length of the trypsin incubation (in minutes) is shown above each lane; lanes labeled NT contain proteins that were not treated with trypsin before the addition of aprotinin. The approximate positions of molecular mass markers (MW), in kilodaltons, are located to the left of the blots. The previously characterized TbpA-derived trypsin digestion products, T1 (95.2 kDa) and T2 (54.9 kDa), are labeled on the right.

FIG. 8.

Comparison of the three-dimensional structure of the plug region of FepA with the predicted structure of the TbpA plug. The amino termini of the proteins are indicated by N. The carboxy termini are represented by C. The asterisk in the TbpA plug panel indicates the approximate position of Ala110 after the HA epitope was fused in the PHA mutant. β-Strand structure is indicated in yellow, α-helical structure is indicated in red, β-turn structure is indicated in blue, and unstructured sequence is shown in white. A sequence consisting of amino acids 1 to 162 of the processed form of gonococcal TbpA was aligned with the analogous sequence of FepA using the 3D-JIGSAW comparative modeling server.