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Cytolethal distending toxin of Haemophilus ducreyi (HdCDT) is a multicomponent toxin, encoded by an operon consisting of three genes, cdtABC . To investigate the role of the individual products in generation of toxicity, recombinant plasmids were constructed allowing expression of each of the genes individually or in different combinations in Escherichia coli and Vibrio cholerae . Expression of all three genes ( cdtABC ) was necessary to generate toxicity on cells, and no activity was obtained using combinations in which only 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, respectively. The CdtB protein alone resulted in DNase activity. CdtC purified from both toxic and non-toxic extracts (from strains expressing cdtCAB and cdtC , respectively) had a molecular weight of about 20 kDa and reacted with a CdtC-specific monoclonal antibody. However, the protein isoelectric point (pI) of CdtC from toxic preparations was about 1.5 pH units more basic than from non-toxic ones. Both forms were immunogenic giving rise to toxin-neutralizing antibodies. Toxicity was reconstructed by combining non-toxic cell sonicates from E. coli , expressing CdtA, CdtB and CdtC proteins individually. Only combinations including all three products gave toxicity, indicating that all are actively involved in the generation of toxic activity on cells. 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 expressing cdtABC . The results showed that the CdtB component produces DNase activity, 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 cells. |
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