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Characterization of a WaaF (RfaF) homolog expressed by Haemophilus ducreyi.

ABSTRACT: Haemophilus ducreyi lipooligosaccharide (LOS) is capable of inducing an inflammatory response in skin (A. A. Campagnari, L. M. Wild, G. Griffiths, R. J. Karalus, M. A. Wirth, and S. M. Spinola, Infect. Immun. 59:2601-2608, 1991) and likely contributes to the virulence of this sexually transmitted pathogen (B. A. Bauer, M. K. Stevens, and E. J. Hansen, Infect. Immun. 68:4290-4298, 1998). An open reading frame in H. ducreyi 35000 was found to encode a predicted protein that was 59% identical to the protein product of the rfaF (waaF) gene of Salmonella typhimurium. The H. ducreyi waaF gene was able to complement an S. typhimurium rfaF (waaF) mutant, a result which confirmed the identity of this gene. In contrast to the rfaF (waaF) gene of enteric bacteria, the H. ducreyi waaF gene was not located adjacent to other genes involved in lipopolysaccharide expression. Inactivation of the H. ducreyi waaF gene by insertion mutagenesis resulted in expression of a LOS that migrated much faster than wild-type LOS in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The LOS of this mutant also did not bind a monoclonal antibody directed against a cell surface-exposed epitope of wild-type H. ducreyi LOS. Testing of the wild-type H. ducreyi strain and its isogenic waaF mutant in the temperature-dependent rabbit model for dermal lesion production by H. ducreyi revealed that this waaF mutant was less virulent than the wild-type parent strain. Complementation of the H. ducreyi waaF mutant with the wild-type H. ducreyi waaF gene resulted in expression of both wild-type LOS and wild-type virulence by this mutant.

SUBMITTER: Bauer BA

PROVIDER: S-EPMC96402 | biostudies-literature | 1999 Feb

REPOSITORIES: biostudies-literature

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Characterization of a WaaF (RfaF) homolog expressed by Haemophilus ducreyi.

Bauer B A BA Lumbley S R SR Hansen E J EJ

Infection and immunity 19990201 2

Haemophilus ducreyi lipooligosaccharide (LOS) is capable of inducing an inflammatory response in skin (A. A. Campagnari, L. M. Wild, G. Griffiths, R. J. Karalus, M. A. Wirth, and S. M. Spinola, Infect. Immun. 59:2601-2608, 1991) and likely contributes to the virulence of this sexually transmitted pathogen (B. A. Bauer, M. K. Stevens, and E. J. Hansen, Infect. Immun. 68:4290-4298, 1998). An open reading frame in H. ducreyi 35000 was found to encode a predicted protein that was 59% identical to th ...[more]

PMID: 9916106

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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.

Dataset Information

Characterization of a WaaF (RfaF) homolog expressed by Haemophilus ducreyi.

Publications

Characterization of a WaaF (RfaF) homolog expressed by Haemophilus ducreyi.

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