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Journal of Bacteriology, April 2000, p. 2292-2298, Vol. 182, No. 8
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Cloning and Characterization of the Lipooligosaccharide Galactosyltransferase II Gene of Haemophilus ducreyi

Shuhua Sun,1,2 Birgit Schilling,3 Laurie Tarantino,1 Michael V. Tullius,3 Bradford W. Gibson,3 and Robert S. Munson Jr.1,2,4,*

Children's Research Institute,1 Department of Pediatrics,4 and Department of Molecular Virology, Immunology and Medical Genetics,2 The Ohio State University, Columbus, Ohio 43205-2696, and Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143-04463

Received 29 July 1999/Accepted 27 January 2000

Haemophilus ducreyi is the etiologic agent of chancroid, a genital ulcer disease. The lipooligosaccharide (LOS) is considered to be a major virulence determinant and has been implicated in the adherence of H. ducreyi to keratinocytes. Strain A77, an isolate from the Paris collection, is serum sensitive, poorly adherent to fibroblasts, and deficient in microcolony formation. Structural analysis indicates that the LOS of strain A77 lacks the galactose residue found in the N-acetyllactosamine portion of the strain 35000HP LOS as well as the sialic acid substitution. From an H. ducreyi 35000HP genomic DNA library, a clone complementing the defect in A77 was identified by immunologic screening with monoclonal antibody (MAb) 3F11, a MAb which recognizes the N-acetyllactosamine portion of strain 35000HP LOS. The clone contained a 4-kb insert that was sequenced. One open reading frame which encodes a protein with a molecular weight of 33,400 was identified. This protein has homology to glycosyltransferases of Haemophilus influenzae, Haemophilus somnus, Neisseria species, and Pasteurella haemolytica. The putative H. ducreyi glycosyltransferase gene was insertionally inactivated, and an isogenic mutant of strain 35000HP was constructed. The most complex LOS glycoform produced by the mutant has a mobility on sodium dodecyl sulfate-polyacrylamide gel identical to that of the LOS of strain A77 and lacks the 3F11-binding epitope. Structural studies confirm that the most complex glycoform of the LOS isolated from the mutant lacks the galactose residue found in the N-acetyllactosamine portion of the strain 35000HP LOS. Although previously published data suggested that the serum-sensitive phenotype of A77 was due to the LOS mutation, we observed that the complemented A77 strain retained its serum-sensitive phenotype and that the galactosyltransferase mutant retained its serum-resistant phenotype. Thus, the serum sensitivity of strain A77 cannot be attributed to the galactosyltransferase mutation in strain A77.

* Corresponding author. Mailing address: Children's Research Institute, Room W402, 700 Children's Dr., Columbus, OH 43205. Phone: (614) 722-2680. Fax: (614) 722-3273. E-mail: munsonr{at}pediatrics.ohio-state.edu.

Journal of Bacteriology, April 2000, p. 2292-2298, Vol. 182, No. 8
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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