Identification of discrete domains within gonococcal transferrin-binding protein A that are necessary for ligand binding and iron uptake functions

Abstract

The availability of free iron in vivo is strictly limited, in part by the iron-binding protein transferrin. The pathogenic Neisseria spp. 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 beta-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 DeltaL8TbpA mutant suggests that high-affinity ligand interaction is required for transferrin-iron internalization.

FIG. 1

Putative topology model of gonococcal TbpA. Horizontal dotted lines represent the planes of the gonococcal outer membrane. The predicted amphipathic β-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 ΔL4, ΔL5, and ΔL8 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.

FIG. 2

Construction of ΔL4TbpA. 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 ΔL4tbpA, 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.

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 (ΔL5TbpA, TbpB⁺); 6, MCV202 (ΔL5TbpA, TbpB⁻); 7, MCV203 (ΔL4TbpA, TbpB⁺); 8, MCV204 (ΔL4TbpA, TbpB⁻); 9, MCV205 (ΔL8TbpA, TbpB⁺); and 10, MCV 206 (ΔL8TbpA, TbpB⁻). Images were scanned using a Hewlett-Packard ScanJet 6300c. Final images were annotated using Adobe Photoshop 4.0.

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 (ΔL8TbpA, TbpB⁻); ▴, binding to MCV202 (ΔL5TbpA, TbpB⁻); ●, binding to MCV204 (ΔL4TbpA, TbpB⁻). The datum points are the means of three independent experiments. Error bars indicate the standard deviations within each triplicate set.

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 (ΔL5TbpA, TbpB⁺); F, MCV202 (ΔL5TbpA, TbpB⁻); G, MCV203 (ΔL4TbpA, TbpB⁺); H, MCV204 (ΔL4TbpA, TbpB⁻); I, MCV205 (ΔL8TbpA, TbpB⁺); and J, MCV 206 (ΔL8TbpA, TbpB⁻). Images were scanned using a Hewlett-Packard ScanJet 6300c. The final images were annotated using Adobe Photoshop 4.0.

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.