The role of different protein components from the Haemophilus ducreyi cytolethal distending toxin in the generation of cell toxicity

Cytolethal distending toxin of Haemophilus ducreyi (HdCDT) is a multicompon ent toxin, encoded by an operon consisting of three genes, cdtABC. To inves tigate the role of the individual products in generation of toxicity, recom binant plasmids were constructed allowing expression of each of the genes i ndividually or in different combinations in Escherichia coli and Vibrio cho lerae. Expression of all three genes (cdtABC) was necessary to generate tox icity on cells, and no activity was obtained using combinations in which on ly one or two of the genes were expressed. Of the individual gene products, the CdtA was shown to exist in two forms with an MW of 23 and 17 kDa, resp ectively The CdtB protein alone resulted in DNase activity. CdtC purified f rom both toxic and non-toxic extracts (from strains expressing cdtCAB and c dtC, respectively) had a molecular weight of about 20 kDa and reacted with a CdtC-specific monoclonal antibody. However, the protein isoelectric point (pl) of CdtC from toxic preparations was about 1.5 pH units more basic tha n from non-toxic ones. Both forms were immunogenic giving rise to toxin-neu tralizing antibodies. Toxicity was reconstructed by combining non-toxic cel l sonicates from E. coli, expressing CdtA, CdtB and CdtC proteins individua lly. Only combinations including all three products gave toxicity, indicati ng that all are actively involved in the generation of toxic activity on ce lls. The reconstruction resulted in a 1.5 pH unit shift in the PI of CdtC, making it identical to that of the protein isolated from bacteria expressin g cdtABC. The results showed that the CdtB component produces DNase activit y, but cell toxicity depends on the involvement of the other two components of CDT and is associated with absorption of all three proteins by HEp-2 ce lls. (C) Academic Press.