Haemophilus ducreyi is the causative agent of chancroid, a disease characterized by genital ulcers and, in 50% of the cases, inflammation of the regional lymph nodes. The occurrence of chancroid outbreaks in the United States coupled with its association with the heterosexual transmission of HIV in Africa makes understanding the pathogenesis of this disease imperative so that rational intervention strategies can be devised. Little is known about the pathogenesis of H. ducreyi but the necrosis seen in genital ulcers suggests that these organisms produce toxins. Potential targets of these toxins are both epithelial and inflammatory cells, resulting in destruction of epithelial tissues and impairment of the immune response. The primary goal of these proposed studies is the identification of cytolysins produced by H. ducreyi and elucidation of their role in the pathogenesis of chancroid. I have detected a cytolytic activity in H. ducreyi by its ability to lyse red blood cells. Further characterization of this activity revealed that it is expressed optimally in late logarithmic growth phase, is inhibited by phosphate and EGTA, and is enhanced by calcium. Transposon mutagenesis of cytolytic strain 35000 resulted in nine transposon mutants with three phenotypes: two with no cytolysin expression, one with reduced cytolysin expression, and six with enhanced cytolysin expression. Using Southern blots of whole cell DNA from strain 35000 and radiolabelled probes, I showed homology with both the RTX class of hemolysins and a Serratia-like hemolysin. The restriction fragment containing the Serratia-like toxin is disrupted in the cytolysin- negative mutants but not in the mutants with reduced or enhanced expression of cytolysin. These results suggest that the mutation is in the structural gene for a Serratia-like cytolysin in the cytolysin- negative mutants, and in genes that are regulators or modulators of cytolysin expression in the other mutants. In the studies outlined in this proposal, I plan to characterize the H. ducreyi cytolysin using genetic and biochemical techniques. The contribution of the cytolysin to the pathogenesis of chancroid will be determined using rabbit and primate models for chancroid. The range of cells the toxin affects in vivo will be studied by analyzing the toxin's effect on different cell types, including inflammatory cells and primary cultures of human foreskin epithelial and fibroblast cells. These studies will provide a better understanding of the virulence determinants of H. ducreyi and will provide a groundwork on which to base future strategies for disease intervention.
| Wood, G E; Dutro, S M; Totten, P A (2001) Haemophilus ducreyi inhibits phagocytosis by U-937 cells, a human macrophage-like cell line. Infect Immun 69:4726-33 |
| Wood, G E; Dutro, S M; Totten, P A (1999) Target cell range of Haemophilus ducreyi hemolysin and its involvement in invasion of human epithelial cells. Infect Immun 67:3740-9 |
| Alfa, M J; DeGagne, P; Totten, P A (1996) Haemophilus ducreyi hemolysin acts as a contact cytotoxin and damages human foreskin fibroblasts in cell culture. Infect Immun 64:2349-52 |
