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Characterization of a Haemophilus ducreyi mutant deficient in expression of cytolethal distending toxin
M K Stevens1, J L Latimer, S R Lumbley
1Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9048, USA.
Insights
Haemophilus ducreyi produces a cytolethal distending toxin (CDT) essential for killing human epithelial cells. A specific gene, cdtC, is crucial for CDT activity, but its absence doesn't impact H. ducreyi virulence in a rabbit model.
Area of Science:
- Microbiology
- Bacterial Pathogenesis
- Molecular Biology
Background:
- Haemophilus ducreyi causes chancroid, a sexually transmitted infection.
- The bacterium produces a cytolethal distending toxin (CDT) responsible for cytopathic effects.
- CDT activity is encoded by the cdtABC gene cluster, with antibodies to CdtC neutralizing the toxin.
Purpose of the Study:
- To investigate the role of the cdtC gene in H. ducreyi cytolethal distending toxin (CDT) activity.
- To determine if CDT production is essential for H. ducreyi virulence in a rabbit model of experimental chancroid.
Main Methods:
- Constructed a recombinant Escherichia coli strain expressing the H. ducreyi cdtABC gene cluster.
- Generated an isogenic H. ducreyi cdtC mutant through insertional inactivation.
- Assessed the cytotoxic activity of bacterial supernatant on human epithelial cells (HeLa, HaCaT, fibroblasts) in vitro.
- Evaluated the virulence of the cdtC mutant and wild-type strains in a rabbit model for experimental chancroid.
Main Results:
- Recombinant E. coli expressing cdtABC killed HeLa and HaCaT cells; inactivation of cdtC abolished this activity.
- The H. ducreyi cdtC mutant did not exhibit detectable cytotoxicity against human cells in vitro.
- Complementation with the wild-type cdtC gene restored cytotoxicity in the H. ducreyi mutant.
- The H. ducreyi cdtC mutant demonstrated similar virulence and survival in rabbit skin compared to the wild-type strain.
Conclusions:
- The cdtC gene product is essential for the cytotoxic activity of H. ducreyi cytolethal distending toxin (CDT) in vitro.
- CDT production is not required for H. ducreyi virulence or survival in the rabbit model of experimental chancroid.
Abstract:
Haemophilus ducreyi expresses a soluble cytolethal distending toxin (CDT) that kills HeLa, HEp-2, and other human epithelial cells in vitro. H. ducreyi CDT activity is encoded by a three-gene cluster (cdtABC), and antibody to the cdtC gene product can neutralize CDT activity in vitro (L. D. Cope, S. R. Lumbley, J. L. Latimer, J. Klesney-Tait, M. K. Stevens, L. S. Johnson, M. Purven, R. S. Munson, Jr., T. Lagergard, J. D. Radolf, and E. J. Hansen, Proc. Natl. Acad. Sci. USA 94:4056-4061, 1997). Culture supernatant fluid from a recombinant Escherichia coli strain containing the H. ducreyi cdtABC gene cluster readily killed both HeLa cells and HaCaT keratinocytes and had a modest inhibitory effect on the growth of human foreskin fibroblasts. Insertional inactivation of the cdtC gene in this recombinant E. coli strain eliminated the ability of this strain to kill HeLa cells and HaCaT keratinocytes. This mutated H. ducreyi cdtABC gene cluster was used to construct an isogenic H. ducreyi cdtC mutant. Monoclonal antibodies against the H. ducreyi CdtA, CdtB, and CdtC proteins were used to characterize protein expression by this cdtC mutant. Culture supernatant fluid from this H. ducreyi cdtC mutant did not detectably affect any of the human cells used in this study. The presence of the wild-type H. ducreyi cdtC gene in trans in this H. ducreyi mutant restored its ability to express a CDT that killed both HeLa cells and HaCaT keratinocytes. The isogenic H. ducreyi cdtC mutant was shown to be as virulent as its wild-type parent strain in the temperature-dependent rabbit model for experimental chancroid. Lack of expression of the H. ducreyi CdtC protein also did not affect the ability of this H. ducreyi mutant to survive in the skin of rabbits.