Purification and identification of Haemophilus ducreyi cytotoxin by use of a neutralizing monoclonal antibody.

Haemophilus ducreyi produces a cytotoxin responsible for the killing of cultured human epithelial cells. Cytotoxin-neutralizing antibodies were detected in the majority of sera from patients with culture-proven chancroid, and a significantly higher level of such antibodies in patients than in blood donors was noted both in areas where the disease is endemic and those where it is not. We produced neutralizing monoclonal antibodies (MAbs) in mice with a crude osmotic preparation of the cytotoxin. These antibodies, with high capacity to neutralize cytotoxicity, were used for purification and identification of the cytotoxin. Purification was performed by a two-step procedure which included Sephacryl S-200 filtration followed by immunoaffinity chromatography. The purification resulted in poor cytotoxin protein recovery and contamination with MAbs from the affinity column. The results of the gel filtration experiments and immunoblotting indicate that the active cytotoxin consists of a single, small protein with an approximate molecular mass of 20 kDa. Cytotoxins from different strains seem to have the same or similar epitopes. The cytotoxin protein was not detected in preparations from nontoxic strains. The N-terminal amino acid sequence of the 20-kDa band was E-S-N-P-D-P-T-T-Y-P-D-V-E-L-S-P-P-P. This sequence does not resemble that of any currently known bacterial protein.

A diffusible cytotoxin of Haemophilus ducreyi.

Little is known about the virulence mechanisms employed by Haemophilus ducreyi in the production of genital ulcers. This Gram-negative bacterium previously has been shown to produce a soluble cytotoxic activity that kills HeLa and HEp-2 cells. We have now identified a cluster of three H. ducreyi genes that encode this cytotoxic activity. The predicted proteins encoded by these genes are most similar to the products of the Escherichia coli cdtABC genes that comprise the cytolethal distending toxin (CDT) of this enteric pathogen. Eleven of 12 H. ducreyi strains were shown to possess this gene cluster and culture supernatants from these strains readily killed HeLa cells. The culture supernatant from a single strain of H. ducreyi that lacked these genes was unable to kill HeLa cells. When the H. ducreyi cdtABC gene cluster was cloned into E. coli, culture supernatant from the recombinant E. coli clone killed HeLa cells. A monoclonal antibody that neutralized this soluble cytotoxic activity of H. ducreyi was shown to bind to the H. ducreyi cdtC gene product. This soluble H. ducreyi cytotoxin may play a role in the development or persistence of the ulcerative lesions characteristic of chancroid.

Cytotoxin production in 100 strains of Haemophilus ducreyi from different geographic locations.

One-hundred strains of Haemophilus ducreyi, representing isolates from different parts of the world, including the reference strains, were obtained from different collections and characterized with special reference to cytotoxin production in vitro. The cytotoxic activity on cultured epithelial cells (HEp-2) was examined with two methods. The activity in bacterial sonicates was tested on freshly trypsinated cells and strains manifesting little or no cytotoxic activity in sonicates were investigated using attached living bacteria on HEp-2 cell-monolayers. Sonicates from the majority of the H. ducreyi strains (89%) produced significant cytotoxic effects on HEp-2 cells. The reciprocal cytotoxic titers of the sonicates ranged from 2.4 x 10(2) to 5.3 x 10(5). Sonicates of 11 strains had low cytotoxic titers (< or = 1:3 to 1:81), eight of those originating from Asia and three from Africa. These 11 strains caused no damage to the cell monolayer, indicating that the 11 strains produce little or no cytotoxic activity in vitro. In summary, the majority of H. ducreyi isolates produce cytotoxic activity, which support the hypothesis that the cytotoxin may be an important virulence factor of this species.

Serum bactericidal activity and phagocytosis in host defence against Haemophilus ducreyi.

Serum bactericidal activity and phagocytic killing are two important mechanisms involved in the host defence against bacteria. Using some in vitro methods, serum bactericidal assay, phagocytic killing by polymorphonuclear leukocytes (PMN) and chemiluminescence, we have evaluated the significance of these mechanisms in the killing of Haemophilus ducreyi bacteria. Furthermore, induction of C3 conversion and deposition of immunoglobulins, C1q and C3, on the surface of bacteria was studied by crossed immunoelectrophoresis and ELISA, respectively. Transmission electron microscopy was employed to study internalization of bacteria by PMN. H. ducreyi and lipooligosaccharide preparations from these bacteria were able to induce conversion of complement factor C3 in normal human serum (NHS). Exposure of bacteria to NHS resulted in deposition of IgG, IgM and complement factors C1q and C3 on the surface of bacteria. H. ducreyi bacteria lost their viability when incubated with fresh but not inactivated normal serum at high concentrations, indicating that the bacteria are sensitive to the complement-dependent bactericidal activity of serum. There were some variations between different strains regarding their susceptibility to the bactericidal activity of NHS, but for eight strains tested, all of the bacteria exposed were not killed in medium containing up to 70% of fresh serum. Complement-mediated opsonophagocytic killing of H. ducreyi by PMN was more effective than complement-dependent bactericidal activity of fresh normal sera. Bacteria treated with heat inactivated immune sera, on the other hand, were as sensitive to the bactericidal effect of PMN as those treated with non-inactivated immune sera, indicating the role of antibodies in opsonophagocytosis. H. ducreyi bacteria were also killed by PMN in the absence of serum antibodies and complement. Using the chemiluminescence assay, H. ducreyi was shown to activate PMN in the absence of serum as well as after opsonization with complement and antibodies. Our results therefore indicate that both opsonic- and non-opsonic mechanisms are involved in the phagocytosis and the subsequent killing of H. ducreyi bacteria. Although both complement and antibodies enhance the ability of phagocytes to kill H. ducreyi, neither component is sufficient for effective killing of H. ducreyi.

Evidence of Haemophilus ducreyi adherence to and cytotoxin destruction of human epithelial cells.

The adherence of ten different Haemophilus ducreyi strains to cultured human epithelial cells and the subsequent destruction of these cells was investigated in vitro using HEp-2 and HeLa cells. Bacterial adherence was measured with two assays, one employing viable bacteria and the other radiolabeled bacteria. In addition, the capacity of H. ducreyi to invade/penetrate the HEp-2 cells was examined. Differential interference contrast and transmission electron microscopy techniques were also used. In both cell lines, all ten strains of H. ducreyi manifested substantial adherence (the rates being 4-20% of the inoculum), irrespective of whether the bacteria were cultivated on solid or liquid media. Bacterial adherence reached a peak after about 2-3 h of incubation, though it was already manifest after only 15 min, a finding suggesting constitutive rather than inducible properties of H. ducreyi adhesins to be involved. The adherence capacity was diminished, but not totally abolished, when bacteria were heat-treated at 100 degrees C for 30 min, indicating the adhesins to be fairly stable. On the other hand, treatment of HEp-2 cells with methanol, glutaraldehyde and emetine dichloride significantly reduced the adherence, indicating viable eukaryotic cells with native surface structures to be involved in bacterial adherence. This capacity of H. ducreyi to adhere to HEp-2 cells was confirmed both by electron microscopy and by differential interference microscopy. Some adherent bacteria were also capable of penetrating epithelial cells, as observed with an invasion assay and confirmed by transmission electron microscopy. Further incubation of the cell monolayers with the ten strains resulted in the cell-death and total damage of monolayers for seven cytotoxin-producing strains, indicating cytotoxin action to be responsible for the destruction of the monolayer. All strains manifested capacity to survive and multiply on the cell monolayer. We propose the first step in the pathogenesis of chancroid to be the adherence of bacteria to epithelial cells, followed by the action of cytotoxin and further bacterial proliferation. This sequence of events is suggested to result in the production of genital ulcers by H. ducreyi organisms.

Neutralizing antibodies to Haemophilus ducreyi cytotoxin.

Neutralizing antibodies against cytotoxin produced by Haemophilus ducreyi bacteria were studied in rabbits by an assay employing HEp-2 cells and diluted crude cytotoxin preparations from the organism. Antisera to 12 different H. ducreyi strains were prepared by immunization of rabbits with bacterial sonicates combined with Freund's adjuvant. The antibody response during infection with H. ducreyi was studied in two groups of rabbits which were infected with five live strains by either single or multiple intradermal injections. Neutralizing antibodies in hyperimmune sera to sonicates from 12 H. ducreyi strains tested against their homologous cytotoxin preparations were present with titers ranging from 1:80 to 1:640. Similar antibody titers against heterologous cytotoxin preparations were recorded, indicating immunological similarity or identity between cytotoxins from the various H. ducreyi strains. Three strains did not produce cytotoxin, and these strains did not induce toxin-neutralizing antibodies. Hyperimmune sera to other gram-negative bacteria had no detectable neutralizing capacity, indicating species specificity of the H. ducreyi cytotoxin. Cytotoxin-neutralizing antibodies were not detectable in rabbit sera before infection with H. ducreyi. Repeated single injections with live bacteria resulted in development of low levels of neutralizing antibodies. Multiple primary injection of live bacteria of the cytotoxin-producing strain CCUG 7470 resulted in a low immune response to the cytotoxin preparation from the same strain. A booster infection resulted in the development of neutralizing antibodies in all rabbits infected with cytotoxin-producing strains. The antibody titers determined against the homologous cytotoxin preparation were similar to those recorded for two heterologous cytotoxin preparations.

Haemophilus ducreyi, a cytotoxin-producing bacterium.

An extra- and an intracellular product from the bacterium Haemophilus ducreyi were shown to have a cytotoxic effect on cell lines of epithelial origin, e.g., HEp-2 and HeLa cells. The cytotoxic effect appeared on cells within 24 h and resulted in cell death and morphologically changed cells. The cytotoxic activity was heat and pronase sensitive. The activity could be removed by incubation with the target cells, suggesting attachment of the agent to the cells. The cytotoxic products were secreted into the environment during exponential growth of bacteria and produced by most of the H. ducreyi strains tested. The activity was neutralized by homologous rabbit immune serum but not by the corresponding preimmune serum. The results indicate that strains of H. ducreyi produce a cytotoxin which may be of importance to the pathogenesis of chancroid.