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Infection and Immunity, July 2001, p. 4438-4446, Vol. 69, No. 7
0019-9567/01/$04.00+0   DOI: 10.1128/IAI.69.7.4438-4446.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Cloning, Overexpression, Purification, and Immunobiology of an 85-Kilodalton Outer Membrane Protein from Haemophilus ducreyi

Kevin L. Thomas,1,dagger Isabelle Leduc,2 Bonnie Olsen,1 Christopher E. Thomas,1 D. William Cameron,2,3 and Christopher Elkins1,4,*

Departments of Medicine1 and Microbiology and Immunology,4 School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599, and Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa,2 and Division of Infectious Diseases, The Ottawa Hospital,3 Ottawa, Ontario, Canada

Received 22 December 2000/Returned for modification 13 February 2001/Accepted 2 April 2001

We have identified an 85-kDa outer membrane protein that is expressed by all tested strains of Haemophilus ducreyi. Studies of related proteins from other pathogenic bacteria, including Haemophilus influenzae, Pasteurella multocida, Neisseria gonorrhoeae, Neisseria meningitidis, and Shigella dysenteriae, suggested a role for these proteins in pathogenesis and immunity. In keeping with the first such described protein from Haemophilus influenzae type B, we termed the H. ducreyi protein D15. The gene encoding the H. ducreyi D15 protein was cloned and sequenced, and the deduced amino acid sequence was found to be most similar to sequences of the D15-related proteins from other Pasteurella spp. The arrangement of the flanking genes was similar to that of H. influenzae Rd and suggested that D15 was part of a multigene operon. Attempts to make a null mutation of the D15 gene were unsuccessful, paralleling results in other D15 gene studies. Overexpression of H. ducreyi D15 in Escherichia coli resulted in a source of recombinant D15 (rD15) from which it was readily purified. rD15 was immunogenic, and it was found that immunization of rabbits with an rD15 vaccine preparation conferred partial protection against a virulent challenge infection. Antisera to an N-terminal peptide recognized all tested strains of H. ducreyi.

* Corresponding author. Mailing address: Departments of Medicine, and Microbiology and Immunology, School of Medicine, University of North Carolina, Chapel Hill, NC 27599. Phone: (919) 966-3661. Fax: (919) 966-6714. E-mail: chriselk{at}med.unc.edu.

dagger Present address: Duke University School of Medicine, Durham, N.C.

Infection and Immunity, July 2001, p. 4438-4446, Vol. 69, No. 7
0019-9567/01/$04.00+0   DOI: 10.1128/IAI.69.7.4438-4446.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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