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Characterization of the hgbA locus encoding a hemoglobin receptor from Haemophilus ducreyi.

ABSTRACT: Haemophilus ducreyi can bind hemoglobin and use it as a source of heme, for which it has an obligate requirement. We previously identified and purified HgbA, a hemoglobin-binding outer membrane protein from H. ducreyi. In this report, we describe the molecular cloning, expression, DNA sequence, and mutagenesis of the structural gene for HgbA, hgbA. H. ducreyi and recombinant Escherichia coli expressing hgbA bound [125I]hemoglobin, establishing HgbA as a receptor. Insertions or deletions in the cloned hgbA gene abolished expression of HgbA and hemoglobin binding in E. coli. Mutagenesis of H. ducreyi by allelic exchange of insertions into hgbA abolished its ability to bind [125I]hemoglobin or utilize hemoglobin as a source of heme. The deduced protein sequence was similar to those of the TonB-dependent family of outer membrane receptors. The most similar member was HutA (heme receptor) from Vibrio cholerae. Tbp1 and Lbp1 (transferrin and lactoferrin receptors, respectively, from pathogenic Neisseria spp.) also showed very significant homology. Thus, by characterizing the hgbA locus, this work elucidates a potentially important role of HgbA in obtaining heme and/or iron from the host.

SUBMITTER: Elkins C

PROVIDER: S-EPMC173285 | biostudies-other | 1995 Jun

REPOSITORIES: biostudies-other

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Characterization of the hgbA locus encoding a hemoglobin receptor from Haemophilus ducreyi.

Elkins C C Chen C J CJ Thomas C E CE

Infection and immunity 19950601 6

Haemophilus ducreyi can bind hemoglobin and use it as a source of heme, for which it has an obligate requirement. We previously identified and purified HgbA, a hemoglobin-binding outer membrane protein from H. ducreyi. In this report, we describe the molecular cloning, expression, DNA sequence, and mutagenesis of the structural gene for HgbA, hgbA. H. ducreyi and recombinant Escherichia coli expressing hgbA bound [125I]hemoglobin, establishing HgbA as a receptor. Insertions or deletions in the c ...[more]

PMID: 7768598

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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 the hgbA locus encoding a hemoglobin receptor from Haemophilus ducreyi.

Publications

Characterization of the hgbA locus encoding a hemoglobin receptor from Haemophilus ducreyi.

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