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The lbgAB Gene Cluster of Haemophilus ducreyi Encodes a ß-1,4-Galactosyltransferase and an -1,6-DD-Heptosyltransferase Involved in Lipooligosaccharide Biosynthesis

Michael V. Tullius,^1, Nancy J. Phillips,¹ N. Karoline Scheffler,¹ Nicole M. Samuels,^1, Robert S. Munson, Jr.,² Eric J. Hansen,³ Marla Stevens-Riley,³ Anthony A. Campagnari,⁴ and Bradford W. Gibson^1,5*

Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143-0446,¹ Children's Research Institute and The Ohio State University, Columbus, Ohio 43205-2696,² Southwestern Medical Center, University of Texas, Dallas, Texas 75235-9048,³ State University of New York, Buffalo, New York 14214,⁴ Buck Institute for Age Research, Novato, California 94945⁵

Received 15 November 2001/ Returned for modification 19 December 2001/ Accepted 25 February 2002

All Haemophilus ducreyi strains examined contain a lipooligosaccharide (LOS) consisting of a single but variable branch oligosaccharide that emanates off the first heptose (Hep-I) of a conserved Hep₃-phosphorylated 3-deoxy-D-manno-octulosonic acid-lipid A core. In a previous report, identification of tandem genes, lbgA and lbgB, that are involved in LOS biosynthesis was described (Stevens et al., Infect. Immun. 65:651-660, 1997). In a separate study, the same gene cluster was identified and the lbgB (losB) gene was found to be required for transfer of the second sugar, D-glycero-D-manno-heptose (DD-Hep), of the major branch structure (Gibson et al., J. Bacteriol. 179:5062-5071, 1997). In this study, we identified the function of the neighboring upstream gene, lbgA, and found that it is necessary for addition of the third sugar in the dominant oligosaccharide branch, a galactose-linked ß14, to the DD-Hep. LOS from an lbgA mutant and an lbgAB double mutant were isolated and were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, carbohydrate analysis, mass spectrometry, and nuclear magnetic resonance spectroscopy. The results showed that the mutant strains synthesize truncated LOS glycoforms that terminate after addition of the first glucose (lbgAB) or the disaccharide DD-Hep16Glcß1 (lbgA) that is attached to the heptose core. Both mutants show a significant reduction in the ability to adhere to human keratinocytes. Although minor differences were observed after two-dimensional gel electrophoresis of total proteins from the wild-type and mutant strains, the expression levels of the vast majority of proteins were unchanged, suggesting that the differences in adherence and invasion are due to differences in LOS. These studies add to the mounting evidence for a role of full-length LOS structures in the pathophysiology of H. ducreyi infection.

Editor: B. B. Finlay

Present address: Department of Medicine, School of Medicine, University of California, Los Angeles, CA 90095-1688.

Present address: Department of Chemistry, University of California, Berkeley, CA 94720-1460.

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