Intoxication of mammalian cells by the cytolethal distending toxin of Haemophilus ducreyi : a novel mechanism of action

The cytolethal distending toxins (CDTs) are a newly described family of bacterial protein toxins with a novel mechanism of action: DNA damage. Haemophilus ducreyi produces a cytotoxin that belongs to this family. This bacterium causes chancroid, a sexually transmitted disease, characterised by mucocutaneous, slowly healing genital ulcers. As all genital ulcerative diseases, chancroid is a predisposing factor in the transmission of HIV. Since ale pathogenesis of the disease is not known the Haemophilus ducreyi cytolethal distending toxin (HdCDT) represents a putative virulence factor.

At the beginning of our studies with HdCDT little was known about CDTs. To understand the mode of action of this toxin, we studied how it intoxicates mammalian cells. The morphological effect induced by the toxin was studied in epithelial-like cells and hamster fibroblasts. The intoxication was irreversible and appeared as a gradual cell distention, followed by cell death. A promotion of actin stress fibers was observed concomitantly with the cell enlargement. As shown for other CDTs, we found that HdCDT-intoxicated HEp-2 cells were arrested in the G2 phase of the cell cycle due to an accumulation of the tyrosine phosphorylated (inactive) form of the cyclin dependent kinase cdc2.

To characterize better the mode of action of HdCDT we tested a broad panel of human cell lines. We could demonstrate that the HdCDT effect is cell type specific and not exclusively related to G2 arrest. B cell lines underwent apoptosis, epithelial cells and keratinocytes arrested exclusively in G2 whereas normal fibroblasts arrested both in G1 and G2. Moreover, we showed that the response induced by HdCDT is similar to the checkpoint response activated by ionizing radiation (IR). Both responses were characterized by an early induction of the p53 gene and the cyclin dependent kinase inhibitor p21 in human fibroblasts and activation of chk2 kinase in HeLa cells. Our work also suggested that ATM, a key molecule in sensing DNA damage, is needed for the early response to HdCDT. However, in the absence of functional ATM the checkpoint was activated after a delay, probably by a homologue such as ATR. We also demonstrated that the promotion of actin stress fibers induced by HdCDT is dependent on Rho activation. Our observations made a link between Rho activation and DNA damage.

Finally, we demonstrated that these effects can occur only after cellular internalization of the toxin, and we have clarified some steps of the intracellular pathway followed by HdCDT. The toxin was found to enter HEp-2 cells via clathrin coated pits and to need an intact Golgi complex in order to induce intoxication.

In conclusion, this work, using HdCDT as a model, has improved our understanding of the mode of action of CDTs.

List of scientific papers

I. Cortes-Bratti X, Chaves-Olarte E, Lagergard T, Thelestam M (1999). "The cytolethal distending toxin from the chancroid bacterium Haemophilus ducreyi induces cell-cycle arrest in the G2 phase. " J Clin Invest 103(1): 107-15
https://pubmed.ncbi.nlm.nih.gov/9884340

II. Cortes-Bratti X, Karlsson C, Lagergard T, Thelestam M, Frisan T (2001). "The Haemophilus ducreyi cytolethal distending toxin induces cell cycle arrest and apoptosis via the DNA damage checkpoint pathways. " J Biol Chem 276(7): 5296-302
https://pubmed.ncbi.nlm.nih.gov/11076947

III. Cortes-Bratti X, Chaves-Olarte E, Lagergard T, Thelestam M (2000). "Cellular internalization of cytolethal distending toxin from Haemophilus ducreyi. " Infect Immun 68(12): 6903-11
https://pubmed.ncbi.nlm.nih.gov/11083812

IV. Cortes-Bratti X, Frisan T, Chaves-Olarte E, Thelestam M (2001). "Activation of Rho GTPase by the cytolethal distending toxin of Haemophilus ducreyi." (Manuscript)