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Identification of tandem genes involved in lipooligosaccharide expression by Haemophilus ducreyi.

ABSTRACT: A transposon insertion mutant of Haemophilus ducreyi 35000 possessing a truncated lipooligosaccharide (LOS) failed to bind the LOS-specific monoclonal antibody 3E6 (M. K. Stevens, L. D. Cope, J. D. Radolf, and E. J. Hansen, Infect. Immun. 63:2976-2982, 1995). This transposon was found to have inserted into the first of two tandem genes and also caused a deletion of chromosomal DNA upstream of this gene. These two genes, designated lbgA and lbgB, encoded predicted proteins with molecular masses of 25,788 and 40,236 Da which showed homology with proteins which function in lipopolysaccharide biosynthetic in other gram-negative bacteria. The tandem arrangement of the lbgA and lbgB genes was found to be conserved among H. ducreyi strains. Isogenic LOS mutants, constructed by the insertion of a cat cartridge into either the lbgA or the lbgB gene, expressed an LOS phenotype indistinguishable from that of the original transposon-derived LOS mutant. The wild-type LOS phenotype could be restored by complementation with the appropriate wild-type allele. These two LOS mutants proved to be as virulent as the wild-type parent strain in an animal model. A double mutant with a deletion of the lbgA and lbgB genes yielded equivocal results when its virulence was tested in an animal model.

SUBMITTER: Stevens MK

PROVIDER: S-EPMC176110 | biostudies-other | 1997 Feb

REPOSITORIES: biostudies-other

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Identification of tandem genes involved in lipooligosaccharide expression by Haemophilus ducreyi.

Stevens M K MK Klesney-Tait J J Lumbley S S Walters K A KA Joffe A M AM Radolf J D JD Hansen E J EJ

Infection and immunity 19970201 2

A transposon insertion mutant of Haemophilus ducreyi 35000 possessing a truncated lipooligosaccharide (LOS) failed to bind the LOS-specific monoclonal antibody 3E6 (M. K. Stevens, L. D. Cope, J. D. Radolf, and E. J. Hansen, Infect. Immun. 63:2976-2982, 1995). This transposon was found to have inserted into the first of two tandem genes and also caused a deletion of chromosomal DNA upstream of this gene. These two genes, designated lbgA and lbgB, encoded predicted proteins with molecular masses o ...[more]

PMID: 9009327

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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

Identification of tandem genes involved in lipooligosaccharide expression by Haemophilus ducreyi.

Publications

Identification of tandem genes involved in lipooligosaccharide expression by Haemophilus ducreyi.

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