Characterization ofHaemophilus ducreyi cdtA, cdtB, andcdtCMutants in In Vitro and In Vivo Systems

Publisher Website
Google Scholar
Abstract
Haemophilus ducreyiexpresses a soluble cytolethal distending toxin (CDT) that is encoded by thecdtABCgene cluster and can be detected in culture supernatant fluid by its ability to kill HeLa cells. ThecdtA, cdtB, andcdtCgenes ofH. ducreyiwere cloned independently into plasmid vectors, and their encoded proteins expressed singly or in various combinations in anEscherichia colibackground. All three gene products had to be expressed in order forE. coli-derived culture supernatant fluids to demonstrate cytotoxicity for HeLa cells. IsogenicH. ducreyi cdtAandcdtBmutants were constructed and used in combination with the wild-type parent strain and a previously describedH. ducreyi cdtCmutant (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 forH. ducreyi-derived culture supernatant fluid to exhibit cytotoxicity for HeLa cells. Whole-cell sonicates and periplasmic extracts from thecdtBandcdtCmutants had no effect on HeLa cells, whereas these same fractions from acdtAmutant had a very modest cytotoxic effect on these same human cells. CdtA appeared to be primarily associated with theH. ducreyicell envelope, whereas both CdtB and CdtC were present primarily in the soluble fraction from sonicated cells. Both thecdtAmutant and thecdtBmutant were found to be fully virulent in the temperature-dependent rabbit model for experimental chancroid.