Molecular characterization of outer membrane proteins of Haemophilus ducreyi as immune targets

Haemophilus ducreyi is the agent of chancroid, a genital ulcer disease that is a risk factor for human immunodeficiency virus transmission. H. ducreyi expresses several outer membrane proteins (OMPs), including a 37,000-42,000 molecular weight (37-42K) major OMP (MOMP) that has homology to OmpA of Escherichia coli, and a 28K protein that may be important for serodiagnosis of chancroid. The goal of this research was to characterize these H. ducreyi proteins, and to determine whether they, and other H. ducreyi antigens, play a role in bactericidal killing or serum resistance.; All isolates of H. ducreyi tested expressed a 28K or 28.5K OMP that contained an epitope defined by the monoclonal antibody (MAb) 5C9. MAb 5C9 bound to a conserved, surface-exposed epitope. The gene encoding the 28K OMP, designated hlp for H. ducreyi lipoprotein, was conserved among strains and had no observed homologies. Labeling of Hlp with ({dollar}sp3{dollar}H) palmitic acid and inhibition of Hlp processing by globomycin indicated that Hlp was a lipoprotein. Sera from individuals with or without chancroid contained antibodies to Hlp, suggesting that Hlp-like proteins may exist in other organisms.; MAbs 2C7 and 9D12 bound to surface-exposed epitopes on MOMP. The gene encoding MOMP was isolated by screening of expression libraries with MAbs 2C7, 9D12, and 3F12. MOMP was homologous to other OmpA proteins. A MOMP-deficient mutant did not bind MAb 3F12, but did bind MAbs 2C7 and 9D12, suggesting a second OmpA protein in H. ducreyi. Sequence of DNA downstream of momp indicated a second gene homologous to ompA that was designated ompA2.; High titer immune sera prepared from rabbits immunized with H. ducreyi antigens was tested in a bactericidal assay. The data indicated little to no killing of strain 35000 incubated in antisera to whole cells, total membrane proteins, OMP preparation, MOMP/OmpA2, Hlp, and the peptidoglycan-associated lipoprotein. A MOMP mutant, and not an LOS mutant, of H. ducreyi was sensitive to normal human serum, indicating a role for MOMP in serum resistance.