Identification of TbpA residues required for transferrin-iron utilization by Neisseria gonorrhoeae

Abstract

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 beta-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.

FIG. 1.

Sequence alignment of TbpA plug domains from bacterial pathogens. The first two letters preceding the amino acid sequences represent the genus and species name of the bacterial pathogen: Ng, Neisseria gonorrhoeae; Nm, Neisseria meningitidis; Hi, Haemophilus influenzae; Ap, Actinobacillus pleuropneumoniae; Mc, Moraxella catarrhalis. The subsequent numbers or letters represent the bacterial strain selected for analysis. The sequences represent mature TbpA plug domains, and the amino acids are numbered accordingly to the right of the sequence. Dots indicate identical amino acids, dashes represent positions in which gaps were introduced in the alignment, and letters indicate the specific amino acid changes. Amino acids in bold type and underlined represent potential iron-coordinating residues. These amino acids coordinate iron in human transferrin (YHD) (32) and bacterial ferric binding protein A (YHE) (54). The box indicates the conserved sequence motif selected for site-directed, alanine substitution mutagenesis at amino acids 118 (E), 120 (E), 121 (Y), and 122 (E).

FIG. 2.

Alanine substitution mutants expressed wild-type levels of TbpA and TbpB. Iron-stressed gonococci were lysed and standardized to a constant cell density. Whole-cell lysates were separated by SDS-PAGE and then transferred to nitrocellulose membranes. Blots were probed with anti-TbpA (α-TbpA) or anti-TbpB (α-TbpB) polyclonal antibodies as labeled on the left. Each lane is labeled according to the strain name with amino acid substitutions in parentheses. (A) Alanine substitution mutants in the FA19 (TbpB⁺) background; (B) alanine substitution mutants in the FA6905 (TbpB⁻) background. Controls include FA19 (positive control, TbpA⁺ TbpB⁺), FA6905 (TbpA⁺ TbpB⁻), and FA6815 (negative control, TbpA⁻ TbpB⁻).

FIG. 3.

Alanine substitution mutants bound transferrin at wild-type levels in equilibrium-phase transferrin binding assays. Whole, iron-stressed gonococci were mixed with various concentrations of ¹²⁵I-labeled human transferrin (0 to 100 nM). Specific binding of transferrin was determined by subtracting nonspecific binding (with excess competing unlabeled human transferrin) from total binding. Specific transferrin binding is reported on the y axis as nanograms of transferrin bound per microgram of total cell protein (ng Tf/μg TCP). Only strains in the FA6905 (TbpB⁻) background are shown in order to evaluate specific transferrin binding attributable to TbpA. Each curve is labeled according to the strain name with amino acid substitutions shown. Each point represents an average of at least three independent experiments. (A) Single alanine substitution mutants; (B) double and triple alanine substitution mutants in comparison to the appropriate controls. Controls include FA6905 (positive control, TbpA⁺ TbpB⁻) and FA6815 (negative control, TbpA⁻ TbpB⁻). Standard deviations are represented by error bars.

FIG. 4.

Double and triple alanine substitution mutants internalized less iron in transferrin-iron uptake assays. Iron-stressed gonococci were incubated with ⁵⁵Fe-labeled human transferrin. ⁵⁵Fe internalization was measured as picomoles of iron internalized after 30 min. Each bar represents the mean of at least six independent experiments and is labeled according to the strain name with amino acid substitutions in parentheses. (A) Alanine substitution mutants in the FA6905 (TbpB⁻) background; (B) alanine substitution mutants in the FA19 (TbpB⁺) background. Controls include FA19 (positive control, TbpA⁺ TbpB⁺), FA6905 (TbpA⁺ TbpB⁻), and FA6815 (negative control, TbpA⁻ TbpB⁻). Standard deviations are represented by error bars. The asterisk indicates P ≤ 0.01 in comparison to both FA19 and FA6905.

FIG. 5.

The triple alanine substitution mutant was unable to utilize transferrin as a sole iron source in the absence of TbpB in transferrin-iron utilization growth assays. Gonococcal strains were grown on CDM plates containing 30% iron-saturated human transferrin as a sole iron source. The ability of mutants to utilize transferrin as a sole source of iron was evaluated by growth at 37°C with 5% CO₂ for 24 h. Strains are labeled according to the strain name with specific amino acid substitutions in parentheses. Controls include FA19 (positive control, TbpA⁺ TbpB⁺), FA6905 (TbpA⁺ TbpB⁻), FA6747 (negative control, TbpA⁻ TbpB⁺), and FA6815 (negative control, TbpA⁻ TbpB⁻).

FIG. 6.

Predicted structural model of N. gonorrhoeae TbpA plug domain. The mature N. gonorrhoeae TbpA plug domain (amino acids 1 to 162) was aligned with the homologous sequence of the E. coli FepA plug domain using the 3D-Jigsaw comparative modeling program. The modeled region of TbpA spans from Thr₂₅ (N terminus) to Thr₁₅₁ (C terminus). The resulting output file was visualized with First Glance in Jmol. The image shown was captured and imported into Adobe Photoshop. The amino acids 120 (E), 121 (Y), and 122 (E) are represented by balls and sticks. Red indicates hydrophobic atoms, while blue indicates hydrophilic atoms. Alpha helices and beta strands are shown as ribbons, with arrowheads pointing toward the C terminus. Random coils are shown as smooth backbone traces.