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The lbgAB gene cluster of Haemophilus ducreyi encodes a beta-1,4-galactosyltransferase and an alpha-1,6-DD-heptosyltransferase involved in lipooligosaccharide biosynthesis.

ABSTRACT: 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 beta1-->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 DD-Hepalpha1-->6Glcbeta1 (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.

SUBMITTER: Tullius MV

PROVIDER: S-EPMC128009 | biostudies-literature | 2002 Jun

REPOSITORIES: biostudies-literature

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The lbgAB gene cluster of Haemophilus ducreyi encodes a beta-1,4-galactosyltransferase and an alpha-1,6-DD-heptosyltransferase involved in lipooligosaccharide biosynthesis.

Tullius Michael V MV Phillips Nancy J NJ Scheffler N Karoline NK Samuels Nicole M NM Munson Jr Robert S RS Hansen Eric J EJ Stevens-Riley Marla M Campagnari Anthony A AA Gibson Bradford W BW

Infection and immunity 20020601 6

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 th ...[more]

PMID: 12010972

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Project description: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.

Project description:To define the role of the surface lipooligosaccharide (LOS) of Haemophilus ducreyi in the pathogenesis of chancroid, Tn916 mutants of H. ducreyi 35000 defective in expression of the murine monoclonal antibody (MAb) 3F11 epitope on H. ducreyi LOS were identified by immunologic screening. One mutant, designated 1381, has an LOS which lacks the MAb 3F11 epitope and migrates with an increased mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The gene disrupted by the Tn916 element in strain 1381 was identified by cloning the sequences flanking the Tn916 element. The sequences were then used to probe a lambda DASHII genomic library. In strain 1381, Tn916 interrupts a gene which encodes an open reading frame (ORF) with an Mr of 40,246. This ORF has homology to the product of the rfaK gene of Escherichia coli. The major LOS glycoform produced by strain 1381 was analyzed by using a combination of mass spectrometry, linkage and composition analysis, and 1H nuclear magnetic resonance spectroscopy. The major LOS species was found to terminate in a single glucose attached to the heptose (L-glycero-D-manno-heptose, or Hep) trisaccharide core. In the wild-type strain 35000, glucose serves as the acceptor for the addition of the D-glycero-D-manno-heptose (or DDHep), which extends to form the mature branch of the H. ducreyi LOS. This mature oligosaccharide is in turn partially capped by the addition of sialic acid (NeuAc), i.e., NeuAc2 alpha-->3Gal beta1-->4GlcNAc beta1-->3Gal beta1-->4DDHep alpha1-->6Glc beta1 (W. Melaugh et al., Biochemistry 33:13070-13078, 1994). Since this LOS terminates prior to the addition of the branch DD-heptose, this gene is likely to encode the D-glycero-D-manno-heptosyltransferase. Strain 1381 exhibits a significant reduction in adherence to and invasion of primary human keratinocytes. This defect was complemented by the cloned heptosyltransferase gene, indicating that the terminal portion of the LOS oligosaccharide plays an important role in adherence to human keratinocytes.

Dataset Information

The lbgAB gene cluster of Haemophilus ducreyi encodes a beta-1,4-galactosyltransferase and an alpha-1,6-DD-heptosyltransferase involved in lipooligosaccharide biosynthesis.

Publications

The lbgAB gene cluster of Haemophilus ducreyi encodes a beta-1,4-galactosyltransferase and an alpha-1,6-DD-heptosyltransferase involved in lipooligosaccharide biosynthesis.

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