Surface antigens of Haemophilus ducreyi. Characterization, localization, role in adherence and mechanisms of host defense

Abstract

Haemophilus ducreyi is a gram-negative bacterium responsible for the ulcerogenital sexually transmitted disease chancroid (soft chancre). The disease is endemic in certain areas of Africa, Asia and Latin America and is regarded as a significant risk factor in the transmission of human immunodeficiency virus infection (HIV). Because of this risk, an effort has been made in recent years to increase our knowledge regarding the pathogenic mechanisms of H. ducreyi. The overall aim of the present thesis was to investigate surface antigens of H. ducreyi and to elucidate their potential role in the primary steps of the pathogenesis of the disease.The 24-kDa protein and the 58.5-kDa heat shock protein (Hsp) were purified and characterized using the methods of continuous-elution electrophoresis, ion-exchange and size-exclusion chromatography. Localization of these proteins on the bacterial surface was performed with two methods: immunoelectron microscopy and enzyme-linked immunosorbent assay (ELISA) with whole bacteria as antigen. Lipooligosaccharide (LOS) of H. ducreyi was characterized by ELISA and immunoblotting techniques with two monoclonal antibodies (MAbs) MAHD6 and MAHD7. Mechanisms involved in the adherence of H. ducreyi to eukaryotic cells were investigated by means of a micro-assay using radio-labeled bacteria. The ability of the 24-kDa protein and the 58.5-kDa Hsp to bind to eukaryotic cells was investigated by immunoblot. The significance of host defense mechanisms, such as bactericidal activity of serum and phagocytosis, on killing of H. ducreyi was evaluated. Accessibility of surface antigens to antibodies and complement was performed using whole-cell ELISA and immunoelectron microscopy.The 24-kDa protein was purified and an additional polymeric form of this protein was found, with a molecular mass of about 165 kDa. The molecular mass of the native 24-kDa protein was determined to 430 kDa. Following heat treatment of the native 430-kDa protein, one band of 24 kDa resulted on SDS-PAGE. The N-terminal amino acid sequence of 25 residues of the 24-kDa protein was M-R-S-K-T-I-T-F-P-V-L-K-L-T-G-Q-Q-Q-A-L-T-N-D-M-H and showed no homology with amino acid sequences of pili from other species. The 58.5-kDa Hsp was purified and visualized Coomassie stained with SDS-PAGE. The N-terminal amino acid sequence of 25 residues of the 58.5-kDa Hsp was A-I-K-D-V-K-F-G-N-D-A-R-V-K-M-L-K-G-V-N-I-L-A-D-A and proved identical to the previously deduced amino acid sequence of the cloned groEL gene of H. ducreyi. When bacteria were stained with gold anti-24-kDa antibodies, the 24-kDa protein was determined to be localized at the bacterial surface, comprised in a highly dense material surrounding the bacterium. All strains examined demonstrated the outer filamentous material, both as cell-associated and as free aggregates with gold incorporated. The 58.5-kDa Hsp was demonstrated to be associated with the cell surface using the MAb BB11. The MAb MAHD6 recognized an epitope present in the outer core region of LOS and the MAb MAHD7 recognized a conserved epitope of LOS, present on all strains of H. ducreyi. Two major structural groups of H. ducreyi LOS were distinguished with these two MAbs, with the majority of strains possessing a nonasaccharide LOS structure, and the minority a hexasaccharide structure. All LOS preparations from H. ducreyi were shown to contain sialic acid.A pronounced ability to adhere to different eukaryotic cells was manifested by H. ducreyi. No discrimination was evident between epithelial cells and fibroblasts of human and animal origin. The level of adherence was reduced in the presence of tetramethylurea, a hydrophobic bond-breaking agent, and H. ducreyi manifested a pronounced ability for binding Congo red to the surface, indicating that hydrophobic forces are involved. Since NaCl also decreased adherence, involvement of ionic forces was indicated as well. An EDTA extract (containing among other proteins the 58.5-kDa Hsp) and a purified 58.5-kDa Hsp preparation were found to partially inhibit adherence of H. ducreyi to Hep-2 cells, whereas neither the crude 24-kDa pili protein nor purified LOS affected adherence. Heparin and heparan sulphate were found to reduce adherence of H. ducreyi.H. ducreyi was demonstrated to be partially resistant to the bactericidal activity of human serum. Serum IgG antibodies specific to the 430-kDa protein and to LOS were present in the majority of sera from patients with chancroid as well as healthy individuals. However, no difference in the level of bactericidal activity between sera from chancroid patients and normal human serum (NHS) was demonstrated. A limited accessibility of LOS and of outer membrane proteins on the bacterial surface was demonstrated, reflecting a possible blocking of potential targets for antibodies and complement. Phagocytic killing was shown more effective than the bactericidal activity of serum. The ability of phagocytes to kill H. ducreyi was enhanced by both antibodies and complement, but neither component was shown sufficient for effective killing, indicating a relative resistance of H. ducreyi to some host defense mechanisms.