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Infection and Immunity, July 2009, p. 3065-3074, Vol. 77, No. 7
0019-9567/09/$08.00+0     doi:10.1128/IAI.00034-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

An Immunogenic, Surface-Exposed Domain of Haemophilus ducreyi Outer Membrane Protein HgbA Is Involved in Hemoglobin Binding{triangledown} ,{dagger}

Igor Nepluev,1 Galyna Afonina,1 William G. Fusco,1 Isabelle Leduc,1 Bonnie Olsen,1 Brenda Temple,3 and Christopher Elkins1,2*

Division of Infectious Diseases, Departments of Medicine,1 Microbiology and Immunology,2 Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 275993

Received 9 January 2009/ Returned for modification 25 February 2009/ Accepted 5 May 2009

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.

* Corresponding author. Mailing address: Division of Infectious Diseases, Department of Medicine, University of North Carolina, 8341C MBRB, 111 Mason Farm Rd., Chapel Hill, NC 27599. Phone: (919) 843-5521. Fax: (919) 843-1015. E-mail: chriselk{at}med.unc.edu

{triangledown} Published ahead of print on 18 May 2009.

{dagger} Supplemental material for this article may be found at http://iai.asm.org/.

Editor: J. N. Weiser

Infection and Immunity, July 2009, p. 3065-3074, Vol. 77, No. 7
0019-9567/09/$08.00+0     doi:10.1128/IAI.00034-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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