title: 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.
authors.affiliation: Department of Medicine, Division of Infectious Diseases, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
authors.forename: Igor
authors.initials: I
authors.lastname: Nepluev
authors.forename: Galyna
authors.initials: G
authors.lastname: Afonina
authors.forename: William G
authors.initials: WG
authors.lastname: Fusco
authors.forename: Isabelle
authors.initials: I
authors.lastname: Leduc
authors.forename: Bonnie
authors.initials: B
authors.lastname: Olsen
authors.forename: Brenda
authors.initials: B
authors.lastname: Temple
authors.forename: Christopher
authors.initials: C
authors.lastname: Elkins
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000276:immunology; D001425:Bacterial Outer Membrane Proteins / Q000276:immunology / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D018604:Epitope Mapping; D006191:Haemophilus ducreyi / Q000378:metabolism*; D006454:Hemoglobins / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D017384:Sequence Deletion; D013552:Swine
chemical_list: D000907:Antibodies, Bacterial; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D002352:Carrier Proteins; D006454:Hemoglobins; C017206:hemoglobin-binding protein, bacteria