The cytolethal distending toxin of Haemophilus ducreyi. Purification and biological activity

Abstract: Haemophilus ducreyi is a Gram-negative bacterium that causes the sexually transmitted disease (STD) chancroid. The disease is endemic in many developing countries, and occurs as restricted outbreaks in the industrialized part of the world. Chancroid as other STDs represents a risk factor for the transmission of HIV and this has given renewed attention to this disease. The disease is characterised by soft, slow healing ulcers on the external genitals, frequently accompanied by unilateral dissemination to regional 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 novel family of toxins (CDTs), which are all composed of three components, CdtA, CdtB, and CdtC. The toxin has the ability to induce DNA double strand breaks, and thereby cause cell cycle arrest and death of target cells, a unique mechanism of action among bacterial toxins. The overall aim of the present thesis was to investigate the role of HdCDT in the pathogenesis of chancroid. The specific aims were; to purify and characterise the HdCDT components and the holotoxin, to study HdCDT intoxication and cell death of various human cells, and to study the pathologic activity of HdCDT in a rabbit model of H. ducreyi infection. The different HdCDT components and the holotoxin were purified from E. coli recombinants expressing the components individually. The purity of the protein preparations was evaluated by SDS-PAGE Coomassie blue staining and immunoblotting. The toxicity of the HdCDT components in different combinations was evaluated using a cytotoxicity assay and by cell cycle analysis using flow cytometry. The HdCDT-induced cell death was investigated using two apoptosis methods; a colorimetric caspase-3 assay and a method detecting phosphatidyl serine in the cell membrane using flow cytometry. The pathologic effects of the HdCDT components and the holotoxin in vivo were evaluated in rabbit skin. Animals were inoculated intradermally with a non-toxin producing H. ducreyi strain and for comparison a Haemophilus influenzae strain, with and without the addition of purified HdCDT. We found that HdCDT can be reconstituted from individually expressed HdCDT components. The reconstituted holotoxin was successfully purified and formed a three-component complex. The HdCdtB alone was shown to have deoxyribonuclease (DNase) activity in vitro. HdCDT induced apoptosis in more than 90% of T cells and monocytes, whereas epithelial cells, keratinocytes, and fibroblasts were less affected; apoptosis and necrosis were noted for about 26-32% and 6-12%, respectively. Purified HdCDT caused inflammation, but did not cause ulcer formation in rabbit skin alone. HdCDT delivered together with H. ducreyi bacteria resulted in significant aggravation of inflammatory lesions and development of ulcers in rabbit skin. A histological picture of chronic skin lesions was observed, which underlines the persistent nature of H. ducreyi infection. Production of HdCDT may be a strategy for H. ducreyi bacterium to hamper the immune response, and to delay the healing process, and thereby cause a persistent infection with characteristic ulcers.

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