Marla K. Stevens

Characterization of Haemophilus ducreyi cdtA, cdtB, and cdtC Mutants in In Vitro and In Vivo Systems

ABSTRACT Haemophilus ducreyi expresses a soluble cytolethal distending toxin (CDT) that is encoded by the cdtABC gene cluster and can be detected in culture supernatant fluid by its ability to kill HeLa cells. The cdtA, cdtB, and cdtCgenes of H. ducreyi were cloned independently into plasmid vectors, and their encoded proteins expressed singly or in various combinations in an Escherichia coli background. All three gene products had to be expressed in order for E. coli-derived culture supernatant fluids to demonstrate cytotoxicity for HeLa cells. Isogenic H. ducreyi cdtA and cdtB mutants were constructed and used in combination with the wild-type parent strain and a previously describedH. ducreyi cdtC mutant (M. K. Stevens, J. L. Latimer, S. R. Lumbley, 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 exhibit cytotoxicity for HeLa cells. Whole-cell sonicates and periplasmic extracts from the cdtB and cdtC mutants had no effect on HeLa cells, whereas these 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 cdtAmutant and the cdtB mutant were found to be fully virulent in the temperature-dependent rabbit model for experimental chancroid.

Cloning and sequencing of the gene encoding the Cu,Zn-superoxide dismutase of Haemophilus ducreyi.

The sodC gene of Haemophilus ducreyi was cloned and sequenced. The deduced amino acid sequence of this protein exhibited 71.6% identity and 81.8% similarity to the H. influenzae and H. parainfluenzae copper (Cu), zinc (Zn)-superoxide dismutase (SOD) enzymes. This gene was localized to a 2.2-kb H. ducreyi chromosomal DNA insert in plasmid pHdSOD. SOD activity was expressed in cell-free extracts of Escherichia coli containing the recombinant plasmid pHdSOD and was localized to the periplasmic space. The Cu,Zn-SOD produced by the H. ducreyi sodC gene did not complement the aerobic growth defect of an E. coli SOD-deficient mutant.