Characterization of an 18,000-molecular-weight outer membrane protein of Haemophilus ducreyi that contains a conserved surface-exposed epitope

Abstract

Identification of antigenically conserved surface components of Haemophilus ducreyi may facilitate the development of reagents to diagnose and prevent chancroid. A hybridoma derived from a mouse immunized with nontypeable Haemophilus influenzae produced a monoclonal antibody (MAb), designated 3B9, that bound to 35 of 35 H. ducreyi strains isolated from diverse geographic regions. The MAb 3B9 bound to a non-heat-modifiable H. ducreyi outer membrane protein (OMP) whose apparent molecular weight was 18,000 (the 18K OMP), and the 3B9 epitope did not phase vary at a rate of greater than 10(-3) in H. ducreyi. In immunoelectron microscopy, the 3B9 epitope was surface exposed, and there was intrastrain and interstrain variability in the amount of 3B9 labelling of whole cells. The MAb 3B9 cross-reacted with many species of the family Pasteurellaceae and bound to the 16.6K peptidoglycan-associated lipoprotein (P6 or PAL) of H. influenzae. Unlike P6, the 18K OMP did not copurify with peptidoglycan. In Western blots (immunoblots), five of seven serum samples obtained from patients with chancroid and four of five serum samples obtained from patients with other genital ulcer diseases at the time of presentation contained antibodies that bound to the 18K OMP. In a competition enzyme-linked immunosorbent assay, four of these serum samples inhibited the binding of 3B9 to H. ducreyi by more than 50%. We conclude that members of Pasteurellaceae expressed a conserved epitope on OMPs that sometimes had different physical characteristics. Patients with chancroid usually have antibodies to the 18K OMP and the 3B9 epitope that may have resulted from infection with H. ducreyi or previous exposure to other Haemophilus or Actinobacillus sp. strains.

Cytolethal Distending Toxin ofHaemophilus ducreyiInduces Apoptotic Death of Jurkat T Cells

“…Actinobacillus actinomycetemcomitans (ATCC 29522) was kindly provided by Dominique Galli of the Indiana University School of Dentistry. Haemophilus parahaemolyticus (ATCC 10014), Haemophilus paraphrohaemolyticus (ATCC 29237), Haemophilus parainfluenzae (ATCC 33392), and Haemophilus parainfluenzae (paraphrophilus) (ATCC 29242) were purchased from the American Type Culture Collection, Manassas, Va. Nontypeable Haemophilus influenzae 1479 and H. ducreyi 35000HP (HP stands for human passaged) and its isogenic hemolysin mutant, 35000HP-RSM1, were described previously (4,32,45). H. ducreyi 35000 and its isogenic cdtC mutant, designated 35000.303, were described previously (48).…”

Expression of Peptidoglycan-Associated Lipoprotein Is Required for Virulence in the Human Model of Haemophilus ducreyi Infection

“…Construction of an isogenic pal mutant in the H. ducreyi 35000HP background. The plasmid pHD18pal:mTn3(Cm) contains a 4.3-kb insert, which includes a 1.5-kb chloramphenicol acetyltransferase gene (cat) cassette located in the pal open reading frame (ORF) (44,46,47). The plasmid pRSM1791 utilizes lacZ as a counterselectable marker to facilitate allele exchange (12).…”

“…We recently characterized several H. ducreyi lipoproteins (25,46,47), one of which is the 18-kDa PAL. H. ducreyi PAL contains a conserved, surface-exposed epitope defined by monoclonal antibody (MAb) 3B9 (46).…”

“…We recently characterized several H. ducreyi lipoproteins (25,46,47), one of which is the 18-kDa PAL. H. ducreyi PAL contains a conserved, surface-exposed epitope defined by monoclonal antibody (MAb) 3B9 (46). MAb 3B9 cross-reacts with many proteins of similar molecular mass found in members of the family Pasteurellaceae and binds to the 16.6-kDa peptidoglycan-associated lipoprotein (P6) of Haemophilus influenzae (46).…”

“…H. ducreyi PAL contains a conserved, surface-exposed epitope defined by monoclonal antibody (MAb) 3B9 (46). MAb 3B9 cross-reacts with many proteins of similar molecular mass found in members of the family Pasteurellaceae and binds to the 16.6-kDa peptidoglycan-associated lipoprotein (P6) of Haemophilus influenzae (46). P6, a target of serum bactericidal antibodies, is being intensively studied as a candidate for a vaccine to prevent nontypeable H. influenzae infections, particularly otitis media (9,24,36).…”