Cellular internalization of cytolethal distending toxin from Haemophilus ducreyi

The chancroid bacterium Haemophilus ducreyi produces a toxin (HdCDT) which is a member of the recently discovered family of cytolethal distending toxi ns (CDTs), These protein toxins prevent the cyclin dependent kinase cdc2 fr om being activated, thus blocking the transition of cells from the G(2) pha se into mitosis, with the consequent arrest of intoxicated cells in G(2). I t is not known whether these toxins act by signaling from the cell surface or intracellularly only. Here we report that HdCDT has to undergo at least internalization before being able to act, Cellular intoxication was inhibit ed (i) by removal of clathrin coats via K+ depletion, (ii) by treatment wit h drugs that inhibit receptor clustering into coated pits, and (iii) in cel ls genetically manipulated to fail in clathrin-dependent endocytosis. Intox ication was also completely inhibited in cells treated with bafilomycin Al or nocodazole and in cells incubated at 18 degreesC, i.e., under conditions known to block the fusion of early endosomes with downstream compartments. Moreover, disruption of the Golgi complex by treatment with brefeldin A or ilimaquinone blocked intoxication. In conclusion, our data indicate that H dCDT enters cells via clathrin-coated pits and has to be transported via th e Golgi complex in order to intoxicate cells. This is the first member of t he family of CDTs for which cellular internalization and some details of th e pathway have been demonstrated.