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 Hep(3)-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 β1→4, 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 DDHep α1→6 Glcβ1 (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.
Haemophilus, lipopolysaccharides, Haemophilus ducreyi, Molecular Sequence Data, gene cluster, glycosyltransferases, Magnetic Resonance Spectroscopy, Bacterial Adhesion, Keratinocytes
NCBI PubMed ID: 12010972Journal NLM ID: 0246127Publisher: American Society for Microbiology
Correspondence: bgibson@buckinstitute.org
Institutions: Department of Pharmaceutical Chemistry, University of California, San Francisco, CA, USA, Children's Research Institute and The Ohio State University, Columbus, Ohio 43205-26962, Southwestern Medical Center, University of Texas, Dallas, Texas 75235-90483, State University of New York, Buffalo, New York 142144, and Buck Institute for Age Research, Novato, California 949455
Methods: NMR, MS, composition analysis, genetic methods, linkage analysis
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