The effect of temperature on the interaction of Haemophilus ducreyi with human epithelial cells.

To investigate if temperature affects the interaction of Haemophilus ducreyi with human epithelial cells, nine strains were used to evaluate the adhesion kinetics of the organism at 33 degrees C and 37 degrees C. The effect of the free toxin on the epithelial cells at those temperatures was also assessed. The cyto-adherence kinetics of H. ducreyi to the epithelial cells was significantly greater at 33 degrees C (10 times more) than at 37 degrees C in all seven clinical isolates tested. There was a significant difference in cell-associated H. ducreyi at 33 degrees C as compared with 37 degrees C. Control strains showed similar adhesion properties at both temperatures. However, the virulent strain CIP542 adhered in larger amounts than the avirulent strain A77. Electron microscopy revealed that there was more tissue necrosis at the lower than the higher temperature. The effect of the free toxin was the same at each temperature. However, strain A77 had significantly lower toxicity than strain CIP542 and the clinical isolates. These results suggest that H. ducreyi displays a temperature-dependent interaction with human epithelial cells, and this feature may play a role in the virulence of the organism in vivo. While the overall toxic effect of viable bacteria depends on the metabolic activity of the bacteria and is, therefore, higher at 33 degrees C than at 37 degrees C withthe same initial inoculum, the effect of the extracted toxin at molecular level with fixed concentrations is a temperature-independent event.

Localisation and immunological properties of a 24-kDa surface protein of Haemophilus ducreyi.

The cell wall and outer structures of Haemophilus ducreyi bacteria were investigated. The 24-kDa outer protein from two strains was purified with an SDS-PAGE preparative continuous-elution electrophoresis cell. The protein was further characterised by SDS-PAGE and immunoblotting, and the immunological properties were investigated by ELISA. Localisation on the bacterial surface was investigated by immuno-electron-microscopy with a polyclonal antiserum raised against the purified protein. A triple-laminar cell wall typical of gram-negative bacteria, close cellular contact between bacterial cells and outer blebs were seen on thin sections. An additional high mol. wt band of c. 165 kDa was seen when not treated by heating to 100 degrees C. A high density fibrilla-like material was detected on the bacterial cell and in the environment by negative staining and immuno-electron-microscopy with antisera specific for the 24-kDa protein. The surface localisation of the 24-kDa protein was confirmed by an ELISA technique with the specific antiserum and whole bacterial cells as antigen. The presence of antibodies to the 24-kDa protein was demonstrated in antisera to 13 strains of H. ducreyi, indicating antigenic identity or within-species cross-reactivity. Low titres of antibodies to this protein were also detected in 19 antisera raised against different strains of gram-negative bacteria, indicating cross-reactivity with other species. Antibody response to the 24-kDa protein in rabbits immunised subcutaneously with live bacteria resulted in a secondary IgG response. Of 28 sera from patients with culture-verified chancroid, 26 manifested high titres of IgG antibodies to the 24-kDa protein, thus indicating the involvement of this antigen in the disease process in man.

Haemophilus ducreyi: pathogenesis and protective immunity.

Haemophilus ducreyi is the etiological agent of chancroid, a sexually transmitted disease that is common in developing countries and that has characteristic genital mucocutaneous lesions. The adherence and growth of bacteria on the surface of eukaryotic cells, and the production of cytotoxin(s) result in cell damage that may be responsible for the development of ulcers. The mechanisms for protective immunity in chancroid are unclear, but both humoral and cell-mediated mechanisms may be involved.

Haemophilus ducreyi attaches to and invades human epithelial cells in vitro.

Haemophilus ducreyi is a sexually transmitted pathogen that causes genital ulcers and inguinal adenopathy. Because chancroidal ulcers are most commonly located on the foreskins of uncircumcised males, we utilized human foreskin epithelial cells (HFECs) to investigate the initial interaction of H. ducreyi with its host. The eight different strains of H. ducreyi that were studied varied in their abilities to attach to these epithelial cells, with six strains consistently attaching to > or = 90% of HFECs and two strains attaching to < 25% of HFECs. The strains with low levels of adherence also failed to exhibit chaining in broth culture and were avirulent in the rabbit model, suggesting that virulence in this model and attachment may be linked. The most adherent strain, LA228R, was further evaluated for its ability to invade HFECs and HEp-2 cells. Scanning electron microscopy and transmission electron microscopy of HFECs after interaction with LA228R produced images consistent with attachment, ingestion into vesicles, and escape from the vesicles into the cytoplasm. In addition, the gentamicin protection assay and inhibition of invasion by cytochalasin B and D indicated that LA228R was able to invade both HFECs and HEp-2 cells. Further examination of the mechanisms involved in the adherence and invasion of H. ducreyi into epithelial cells and their correlation with virulence will provide a better understanding of the pathogenesis of the disease caused by this important pathogen.

In vitro model of Haemophilus ducreyi adherence to and entry into eukaryotic cells of genital origin.

Electron microscopy was used to examine Haemophilus ducreyi adherence to and entry into eukaryotic cells of genital origin. A clinical H. ducreyi isolate (90-244) adhered in snake-like whorls to the surfaces of cervical carcinoma cells (HeLa 229), endometrial adenocarcinoma cells (HEC-1-B), and human neonatal foreskin fibroblast (HFF) cells. A prototype strain of H. ducreyi (CIP542) adhered in randomly organized clumps on the surfaces of HFF. Strain 90-244 entered HFF and HEC-1-B cells but did not enter HeLa cells. The H. ducreyi in the HFF cells at 2 h were partly surrounded by a membrane consistent with that of a phagocytic vacuole. At 2 h, strain CIP542 was found in interstitial spaces between the HFF cells and also in the cytoplasm of the cells. After 7 and 24 h, both strains of H. ducreyi were found in the large interstitial spaces between the HFF cells, in the cytoplasm, and extracellularly. This model of in vitro H. ducreyi infection of eukaryotic cells will allow for more specific study of factors that determine the virulence of H. ducreyi.

Association of Haemophilus ducreyi with cell-culture lines.

The association of Haemophilus ducreyi with epithelial cell cultures was studied by light microscopy, electronmicroscopy and viable counts. Associated organisms were engulfed by epithelial cells and sequestered from the cell-surface environment. Large numbers of organisms within epithelial cells appeared to induce cell lysis and release of H. ducreyi. Such a mechanism occurring in vivo may assist H. ducreyi to evade the bactericidal action of polymorphonuclear leucocytes and may explain some of the tissue damage seen in genital ulcers caused by H. ducreyi.

Cytopathic effect of Haemophilus ducreyi for human foreskin cell culture.

An explant adult foreskin cell culture (FS2-3) was compared with human lung carcinoma cell culture (A549) with regard to the ability of Haemophilus ducreyi to produce a cytopathic effect. The survival of H. ducreyi for up to 26 days in FS2-3 cells was far greater than in any previously described in-vitro culture system. H. ducreyi survived for up to 7 days in A549 cells. The H. ducreyi cells grew and formed "fungal-like" microcolonies on the eukaryotic monolayer. Portions of the microcolonies remained attached despite extensive washing. Transmission electronmicroscopy indicated that, at 48 h after infection, the H. ducreyi cells did not penetrate the FS2-3 cells but they were closely associated with them; there was only a 2-5 nm gap between the H. ducreyi cell wall and the FS2-3 membrane. The virulent H. ducreyi strains RO18 and 35,000 produced a cytopathic effect on FS2-3 cells that did not appear to be due to a soluble toxin. These strains did not produce any CPE on A549 cells. H. influenzae and the avirulent H. ducreyi strain CIP542, inoculated in the same concentration and incubated for the same length of time, did not produce CPE on FS2-3 cells. This study demonstrated that the use of FS2-3 foreskin cell culture provided an in-vitro approach for evaluating the cytopathic effect of virulent H. ducreyi whereby, unlike in other in-vitro systems, viability of the micro-organism could be readily maintained.

Characterization of pili expressed by Haemophilus ducreyi.

Twelve strains of Haemophilus ducreyi isolated primarily from chancroid outbreaks in North America were examined for the presence of pili by transmission electron microscopy. We identified piliated cells in 10 of the 12 strains. Pilin extracts were prepared from the mechanically sheared cells of the 12 H. ducreyi strains as well as the stably piliated H. influenzae strain R890 and its non-piliated parent R906. Pili were present in 12 out of 12 H. ducreyi extracts and in the R890 extract but not in the R906 preparation. Pili were purified by cycles of differential pH solubilization and crystallization. In SDS-PAGE, the preparation consisted predominantly of a protein whose apparent relative molecular mass was 24,000 (24 k), and an electron micrograph showed that the preparation contained pili. Three H. ducreyi strains were passed 52 times on agar plates, and extracts prepared from these strains contained pili. There was no evidence of binding of erythrocytes obtained from nine mammalian and avian species to colonies of one of the stably piliated H. ducreyi strains. We conclude that H. ducreyi expressed pili, that the relative molecular mass of the pilin monomer was 24 k, that pilus expression was not readily lost in passage and that H. ducreyi pili may not bind to an erythrocyte receptor.

Effect of iron limitation on protein composition and ultrastructure of Haemophilus ducreyi.

Protein profiles of whole cells of Haemophilus ducreyi grown in the presence or absence of the iron chelator desferal, were compared by polyacrylamide gel electrophoresis. Each of four strains produced novel proteins in the range 43-160 kDa when cultured under conditions of reduced iron availability. At some sub-inhibitory concentrations, desferal produced enhanced growth, possibly due to it functioning as an exogenous siderophore. Organisms grown under conditions of reduced iron availability ultrastructurally showed also large periplasmic spaces between cytoplasm and outer membrane.

Biology of Haemophilus ducreyi.

The etiological agent of the sexually transmitted genital ulcer disease chancroid was first described in 1889 by Auguste Ducrey following repeated autoinoculation of purulent ulcer material from a series of patients. The organism was isolated on artificial media a decade later but has remained difficult to isolate consistently, resulting in controversy over its characteristics and role as the causative agent of chancroid. Because of its fastidious growth requirements, including unknown components in blood, the organism was included in the original description of the genus Haemophilus. Requirement for exogenous hemin and limited phenotypic characteristics, including structural and antigenic properties, suggested that Haemophilus ducreyi was a valid member of the genus Haemophilus. Recent studies of respiratory quinones, deoxyribonucleic acid hybridization, and competition for homologous transformation of the type species, H. influenzae, suggest that H. ducreyi is unrelated to any of the present species of the family Pasteurellaceae, which includes members of the genera Haemophilus, Actinobacillus, and Pasteurella. This review summarizes the early studies with H. ducreyi and our current knowledge of the microbiology of this important human pathogen.