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Characterization of a novel lipoprotein expressed by Haemophilus ducreyi.

ABSTRACT: Pooled sera from patients with chancroid contain antibodies to a Haemophilus ducreyi antigen with an approximate molecular weight of 28,000 (28K). Rabbit polyclonal serum that reacts to a 28K protein can be used to detect H. ducreyi in clinical samples. A monoclonal antibody, designated 5C9, bound to a 28K outer membrane protein and to 35 of 35 H. ducreyi isolates with diverse geographic origins and did not bind to many species of the families Pasteurellaceae, Neisseriaceae, and Enterobacteriaceae or to Corynebacterium and Candida species strains. A 5C9-reactive phage was recovered from a genomic library, and the gene encoding the 28K protein was localized to a 626-bp open reading frame, designated hlp, for H. ducreyi lipoprotein. Translation of hlp predicted a 23K polypeptide that contained a lipoprotein processing site. Escherichia coli transformed with a plasmid containing hlp expressed a novel, membrane-associated protein that could be labeled with [3H]palmitic acid. In H. ducreyi, processing of Hlp was inhibited by globomycin. Database searches found no homologies to hlp or to the predicted Hlp amino acid sequence. Restriction enzyme analysis indicated that hlp was conserved among H. ducreyi isolates. Serum samples from patients with chancroid and other genital ulcer diseases and from normal subjects contained antibodies that bound to purified, recombinant Hlp. Although monoclonal antibody 5C9 recognizes a species-specific epitope of a unique H. ducreyi lipoprotein, the presence of serum antibodies to Hlp may not indicate previous infection with H. ducreyi.

SUBMITTER: Hiltke TJ

PROVIDER: S-EPMC174487 | biostudies-other | 1996 Dec

REPOSITORIES: biostudies-other

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Characterization of a novel lipoprotein expressed by Haemophilus ducreyi.

Hiltke T J TJ Campagnari A A AA Spinola S M SM

Infection and immunity 19961201 12

Pooled sera from patients with chancroid contain antibodies to a Haemophilus ducreyi antigen with an approximate molecular weight of 28,000 (28K). Rabbit polyclonal serum that reacts to a 28K protein can be used to detect H. ducreyi in clinical samples. A monoclonal antibody, designated 5C9, bound to a 28K outer membrane protein and to 35 of 35 H. ducreyi isolates with diverse geographic origins and did not bind to many species of the families Pasteurellaceae, Neisseriaceae, and Enterobacteriace ...[more]

PMID: 8945545

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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.

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Characterization of a novel lipoprotein expressed by Haemophilus ducreyi.

Publications

Characterization of a novel lipoprotein expressed by Haemophilus ducreyi.

Similar Datasets

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