Characterization of Haemophilus ducreyi cdtA, cdtB, and cdtC mutants in invitro and in vivo systems

Haemophilus ducreyi expresses a soluble cytolethal distending toxin (CDT) t hat is encoded by the cdtABC gene cluster and can be detected in culture su pernatant fluid by its ability to kill HeLa cells. The cdtA, cdtB, and cdtC genes of H. ducreyi were cloned independently into plasmid vectors, and th eir encoded proteins expressed singly or in various combinations in an Esch erichia coli background. All three gene products had to be expressed in ord er for E. coli-derived culture supernatant fluids to demonstrate cytotoxici ty for HeLa cells. Isogenic H. ducreyi cdtA and cdtB mutants were construct ed and used in combination with the wild-type parent strain and a previousl y described H. ducreyi cdtC mutant (M. K. Stevens, J. L. Latimer, S. R. Lum bley, C. K. Ward, L. D. Cope, T. Lagergard, and E. J. Hansen, Infect. Immun . 67:3900-3908, 1999) to determine the relative contributions of the CdtA, CdtB, and CdtC proteins to CDT activity. Expression of CdtA, CdtB, and CdtC appeared necessary for H. ducreyi-derived culture supernatant fluid to exh ibit cytotoxicity for HeLa cells. Whole-cell sonicates and periplasmic extr acts from the cdtB and cdtC mutants had no effect on HeLa cells, whereas th ese same fractions from a cdtA mutant had a very modest cytotoxic effect on these same human cells. CdtA appeared to be primarily associated with the H. ducreyi cell envelope, whereas both CdtB and CdtC were present primarily in the soluble fraction from sonicated cells. Both the cdtA mutant and the cdtB mutant were found to be fully virulent in the temperature-dependent r abbit model for experimental chancroid.