The cytolethal distending toxin of Haemophilus ducreyi. Prevalence and effects on normal cells

Abstract: Haemophilus ducreyi is a fastidious Gram-negative bacterium that causes the sexually transmitted disease chancroid. The disease is endemic in many developing countries and occurs as restricted outbreaks in the industrialised part of the world. Chancroid and other STDs has been shown to increase the rate of transmission of HIV and this has given renewed attention to the disease. It is characterised by soft, slow healing ulcers on the external genitals, frequently accompanied by unilateral dissemination to local lymph nodes and the formation of buboes.The mechanism of pathogenesis in chancroid is not clear. H. ducreyi possesses several potential virulence factors, which could increase its persistence in the human host by avoiding host defence mechanisms and/or cause tissue damage. The focus of this thesis is the cytolethal distending toxin of H. ducreyi, HdCDT. It belongs to a family of toxins (CDT), which are all composed of three components CdtA, B and C and inhibit the proliferation of a variety of mammalian cells. The CdtB have homologies to deoxyribonuclease (DNase) and CDT evokes a cellular response characteristic for DNA-damage, which leads to cell cycle arrest. The aim of this thesis was to investigate the prevalence of cdtABC among H. ducreyi strains and to study the effect of HdCDT on normal human leukocytes and endothelial cells, including the formation of new blood vessels in vitro.The cytotoxic activity and the presence of cdtABC genes in H. ducreyi strains were studied with a toxicity assay on HEp-2 cells and polymerase chain reaction (PCR), with primers specific for the individual cdtA, cdtB and cdtC genes, respectively. The effects of HdCDT on peripheral blood leukocytes (T cells, B cells, monocytes/macrophages and polymorphonuclear cells) and on normal endothelial cells were investigated with the focus on cell proliferation and cell cycle progression. Continuous monocytic and T cell lines were included in the study. The influence of HdCDT on cells was studied with the thymidine incorporation assay, by measuring metabolic activity, by enumeration of immunoglobulin producing cells (ELISPOT) and by cell cycle analysis using flow cytometry. The presence of cell cycle regulatory proteins was investigated with SDS-PAGE/Western blotting. Moreover, the amount of IFN-g, IL-4, TNF-a and also immunoglobulins was measured. An in vitro model for angiogenesis was used to investigate the influence of HdCDT in the process of new blood vessel formation.We found that there was a strict correlation between the CDT activity and the presence of all three cdt genes. The prevalence of cdtABC genes in H. ducreyi was calculated to about 82 % in both culture collection and recently isolated strains. Exposure to HdCDT inhibited proliferation of B- and T cells, endothelial cells and a continuous monocytic cell line. There was, however, no effect on the amount of IFN-g or IL-4 in the culture supernatant of T cells, while the amount of B cell-produced immunoglobulins was reduced. Purified CD4+ and CD8+ T cells, the Jurkat T and monocytic continuous cell lines were arrested in the G2 phase of the cell cycle after exposure to the HdCDT. Furthermore, increased levels of the inactive cyclin-dependent kinase 1 (Cdc2) and the tumor suppressor p53 were detected in toxin-treated normal T cells. There were a certain proportion of T cells, which were not arrested in the G2 phase. These cells may be in either G1 or G0 (resting) phase. HdCDT inhibited endothelial cell proliferation, and the formation of tubuli in vitro. However, we were not able to detect an effect of HdCDT on normal monocytes, granulocytes or resting T cells with any of the methods employed. The anti-proliferative effects of HdCDT on a variety of normal human cells in vitro, indicate that CDT may also be important in the pathogenesis of chancroid.