An immunogenic, surface-exposed domain of Haemophilus ducreyi outer membrane protein HgbA is involved in hemoglobin binding

Abstract

HgbA is the sole TonB-dependent receptor for hemoglobin (Hb) acquisition of Haemophilus ducreyi. Binding of Hb to HgbA is the initial step in heme acquisition from Hb. To better understand this step, we mutagenized hgbA by deletion of each of the 11 putative surface-exposed loops and expressed each of the mutant proteins in trans in host strain H. ducreyi FX547 hgbA. All mutant proteins were expressed, exported, and detected on the surface by anti-HgbA immunoglobulin G (IgG). Deletion of sequences in loops 5 and 7 of HgbA abolished Hb binding in two different formats. In contrast, HgbA proteins containing deletions in the other nine loops retained the ability to bind Hb. None of the clones expressing mutant proteins were able to grow on plates containing low concentrations of Hb. Previously we demonstrated in a swine model of chancroid infection that an HgbA vaccine conferred complete protection from a challenge infection. Using anti-HgbA IgG from this study and the above deletion mutants, we show that loops 4, 5, and 7 of HgbA were immunogenic and surface exposed and that IgG directed against loops 4 and 5 blocked Hb binding. Furthermore, loop 6 was cleaved by protease on intact H. ducreyi, suggesting surface exposure. These data implicate a central domain of HgbA (in respect to the primary amino acid sequence) as important in Hb binding and suggest that this region of the molecule might have potential as a subunit vaccine.

FIG. 1.

Two-dimensional model of the Hb receptor of H. ducreyi HgbA. This model was derived by in silico comparison of the primary sequence of HgbA with that of FepA, whose crystal structure has been solved. The letters represent the amino acid sequence in one-letter code. The 11 putative surface-exposed loops of HgbA are numbered 1 through 11. Amino acids predicted to be outside the membrane shown to vary between H. ducreyi strains are orange. Yellow residues were deleted in first-generation loop mutants, with the exception that the first residue missing in the loop 4 deletion, an A, is orange, since it was variable between strains. For the second-generation loop 5 and 7 deletions, a red line shows the boundaries of the deleted amino acids in loops. The amino acid residues predicted to form β strands are boxed. Loops were formed to fit the page, and no loop structure is proposed or implied. The N-terminal plug (155 amino acids missing), believed to be located in the periplasm and within the pore, is not shown. Bold residues at the bottom of the figure are proposed to be located in the periplasmic space.

FIG. 2.

Strategy used to construct loop deletions by PCR. Steps are numbered 1 through 5 in this example of a loop 4 deletion mutant. In step 1, pUNCH555 was restricted with ScaI and HpaI to generate a 3.1-kb hgbA fragment with blunt ends as shown at the top. This product was self-ligated under dilute conditions to form the circle shown in step 1. This circle contains the entire hgbA open reading frame and is the template for the generation of all 11 loop mutants. In step 2, inverse PCR was done with primers containing XmaI sites at their ends (Loop4F and Loop4R) to generate a 2.7-kb fragment in the example for the loop 4 mutant construction shown here. These XmaI sites are at the desired point of deletion in loop 4 (Pro330 to Gly455 of the parent protein). In step 3, the 2.7-kb PCR product was restricted with XmaI, self-ligated under dilute conditions, and then PCR amplified with hgbA primers flanking the deletion (Hgb1.05 and Hgb4) to form the product shown in step 4. In step 5, the PCR product was digested with StyI and SwaI, which cut at sites that flank the deletion, generating a 293-bp product that was cloned into the same sites in pUNCH555 to create pUNCH555 Δloop4. The arrows indicate the primers used for PCR. The SwaI-to-StyI insert of pUNCH555 Δloop4 was moved into the same sites of pUNCH1261 to create pUNCH1261Δloop4. a.a., amino acids.

FIG. 3.

Analysis of first-generation (left panel) and second-generation (right panel) HgbA Δ loop mutant proteins and strains. FX547 hgbA containing pUNCH1261 (expressing full-length hgbA), empty vector pLSKS, or each of the indicated Δ loop deletion plasmids was analyzed for expression and cellular localization of HgbA and the ability of each HgbA protein to bind Hb. In panels A to C, Western blot assays with polyclonal rabbit anti-rHgbA antibodies were used to visualize the presence of HgbA from total cellular proteins, whole-cell immunoprecipitation, and Sarkosyl-insoluble OMP, respectively. In panel D, the ability of HgbA mutant proteins to bind Hb was examined. OMPs were solubilized in Zw and bound to Hb-agarose. After washing and elution, OMPs bound to Hb-agarose from first-generation mutants were analyzed by SDS-PAGE and Coomassie staining (*, left panel). Alternatively, proteins bound to Hb from second-generation mutants were analyzed by Western blotting with rabbit anti-rHgbA antibodies. In panels E and F, dot blots were blocked and probed with iodinated pig anti-nHgbA IgG (E) or iodinated human Hb (F) and subjected to autoradiography. Whole cells of loop mutant strains were immobilized and analyzed by using a dot blot assay. #aa, number of amino acids.

FIG. 4.

Use of loop mutants to identify immunogenic surface-exposed loops of HgbA. (A) Loop mutants bind less IgG. Unabsorbed IgG was bound to the indicated H. ducreyi strains, and antibody binding was measured as described in Materials and Methods. A decrease in antibody binding in Δ loop clones was interpreted as the possible loss of immunogenic epitopes. p1261, FX547/pUNCH1261 expressing full-length HgbA protein; pLSKS, FX547 containing empty vector pLSKS; Δ4, Δ5, Δ6, and Δ7, clones containing deletions in loops 4, 5, 6, and 7, respectively (Fig. 1). Data represent the mean values from four separate experiments. (B) Absorption of anti-HgbA IgG with certain loop mutants identifies immunogenic exposed loops. Unabsorbed or absorbed pig anti-HgbA IgG, as indicated, was tested for binding to FX547/pUNCH1261 (upper panel) or FX547/pLSKS (lower panel). Shown is the percent IgG binding of the indicated Δ loop-absorbed IgG compared to that of unabsorbed FX547/pUNCH1261, which was defined as 100%. P values indicate comparison of the IgG absorbed against the indicated mutant versus IgG absorbed against FX547/pUNCH1261. Data represent the mean values from three separate experiments. (C) Evidence that IgG was exhaustively absorbed and additional evidence that mutant proteins were surface exposed. To show that absorption was complete, we examined the binding of absorbed IgG to the absorbing mutant, selected other mutants, and controls. Note that anti-HgbA IgG absorbed against deletion clones Δ4, Δ5, and Δ7 lost binding against the homologous mutants, respectively, indicating complete absorption. In contrast, IgG absorbed against deletion clones Δ4, Δ5, and Δ7 retained binding to heterologous mutants that contained those loops, respectively, indicating that these mutant proteins were surface exposed. Data represent the mean values from three separate experiments.

FIG. 5.

Ability of anti-loop IgG to inhibit binding of Hb to HgbA in an ELISA format. Native HgbA purified from H. ducreyi under nondenaturing conditions was immobilized on an ELISA plate, the plate was blocked, and the indicated IgGs were added. Digoxigenin-labeled human Hb was then added, and binding was monitored with an alkaline phosphatase-conjugated anti-digoxigenin secondary antibody. Values obtained from wells that received no antibody were defined as 100% Hb binding. Data are mean values compiled from five separate experiments. Statistical comparisons were done between each IgG absorbed on loop mutants and absorption on the FX547/pUNCH1261 control expressing full-length HgbA protein. *, P = 0.008 for both Δ loop 4 and 5 IgGs (Mann-Whitney rank sum t test).