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

D. A. Lewis, M. K. Stevens, J. L. Latimer, C. K. Ward, K. Deng, R. Blick, S. R. Lumbley, C. A. Ison, E. J. Hansen

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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 cdtC genes 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 described H. 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 cdtA mutant and the cdtB mutant were found to be fully virulent in the temperature-dependent rabbit model for experimental chancroid.

Original languageEnglish (US)
Pages (from-to)5626-5634
Number of pages9
JournalInfection and Immunity
Volume69
Issue number9
DOIs
StatePublished - 2001

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Haemophilus ducreyi
HeLa Cells
Chancroid
Escherichia coli
Multigene Family
Genes
In Vitro Techniques
Plasmids
Theoretical Models
cytolethal distending toxin
Rabbits
Temperature

ASJC Scopus subject areas

  • Immunology

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Lewis, D. A., Stevens, M. K., Latimer, J. L., Ward, C. K., Deng, K., Blick, R., ... Hansen, E. J. (2001). Characterization of Haemophilus ducreyi cdtA, cdtB, and cdtC mutants in in vitro and in vivo systems. Infection and Immunity, 69(9), 5626-5634. https://doi.org/10.1128/IAI.69.9.5626-5634.2001

Characterization of Haemophilus ducreyi cdtA, cdtB, and cdtC mutants in in vitro and in vivo systems. / Lewis, D. A.; Stevens, M. K.; Latimer, J. L.; Ward, C. K.; Deng, K.; Blick, R.; Lumbley, S. R.; Ison, C. A.; Hansen, E. J.

In: Infection and Immunity, Vol. 69, No. 9, 2001, p. 5626-5634.

Research output: Contribution to journalArticle

Lewis, DA, Stevens, MK, Latimer, JL, Ward, CK, Deng, K, Blick, R, Lumbley, SR, Ison, CA & Hansen, EJ 2001, 'Characterization of Haemophilus ducreyi cdtA, cdtB, and cdtC mutants in in vitro and in vivo systems', Infection and Immunity, vol. 69, no. 9, pp. 5626-5634. https://doi.org/10.1128/IAI.69.9.5626-5634.2001
Lewis, D. A. ; Stevens, M. K. ; Latimer, J. L. ; Ward, C. K. ; Deng, K. ; Blick, R. ; Lumbley, S. R. ; Ison, C. A. ; Hansen, E. J. / Characterization of Haemophilus ducreyi cdtA, cdtB, and cdtC mutants in in vitro and in vivo systems. In: Infection and Immunity. 2001 ; Vol. 69, No. 9. pp. 5626-5634.
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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 cdtC genes 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 described H. 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 cdtA mutant and the cdtB mutant were found to be fully virulent in the temperature-dependent rabbit model for experimental chancroid.",
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