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Characterization of adenylate cyclase toxin from a mutant of Bordetella pertussis defective in the activator gene, cyaC.
- Source :
-
The Journal of biological chemistry [J Biol Chem] 1993 Apr 15; Vol. 268 (11), pp. 7842-8. - Publication Year :
- 1993
-
Abstract
- Bordetella pertussis adenylate cyclase (AC) toxin has the abilities to 1) enter target cells where it catalyzes cyclic AMP production and 2) lyse sheep erythrocytes, and these abilities require post-translational modification by the product of an accessory gene cyaC (Barry, E. M., Weiss, A. A., Ehrmann, E. E., Gray, M. C., Hewlett, E. L., and Goodwin, M. St. M. (1991) J. Bacteriol. 173, 720-726). In the present study, AC toxin has been purified from an organism with a mutation in cyaC, BPDE386, and evaluated for its physical and functional properties in order to determine the basis for its lack of toxin and hemolytic activities. AC toxin from BPDE386 is indistinguishable from wild-type toxin in enzymatic activity, migration on SDS-polyacrylamide gel electrophoresis, ability to bind calcium, and calcium-dependent conformational change. Although unable to elicit cAMP accumulation, AC toxin from BPDE386 exhibits binding to the surface of Jurkat cells which is comparable to that of wild-type toxin. This target cell interaction is qualitatively different, however, in that 99% of the mutant toxin remains sensitive to trypsin, whereas approximately 20% of cell-associated wild-type toxin enters a trypsin-resistant compartment. To evaluate the ability of this mutant AC toxin to function at its intracellular site of action, the cAMP-stimulated L-type calcium current in frog atrial myocytes was used. Extracellular addition of wild-type toxin results in cAMP-dependent events that include activation of calcium channels and enhancement of calcium current. In contrast, there is no response to externally applied toxin from BPDE386. When injected into the cell interior, however, the AC toxin from BPDE386 is able to produce increases in the calcium current comparable to those observed with wild-type toxin. Although AC toxin from BPDE386 is unaffected in its enzymatic activity, calcium binding, and calcium-dependent conformational change, the mutation in cyaC does result in a toxin which is able to bind to target cells but unable to elicit cAMP accumulation. In that AC toxin from BPDE386 is able to function normally when injected artificially to an intracellular site, we conclude that the disruption of cyaC produces a defect in insertion and transmembrane delivery of the catalytic domain.
- Subjects :
- Adenylyl Cyclases genetics
Adenylyl Cyclases isolation & purification
Animals
Calcium metabolism
Calcium Channels drug effects
Calcium Channels physiology
Cell Line
Cells, Cultured
Cyclic AMP metabolism
Electrophysiology methods
Heart drug effects
Heart physiology
Hemolysis
Humans
In Vitro Techniques
Models, Biological
Protein Conformation
Protein Processing, Post-Translational
Rana catesbeiana
Recombinant Proteins isolation & purification
Recombinant Proteins metabolism
Recombinant Proteins pharmacology
Spectrometry, Fluorescence
T-Lymphocytes, Helper-Inducer
Virulence Factors, Bordetella isolation & purification
Virulence Factors, Bordetella pharmacology
Adenylate Cyclase Toxin
Adenylyl Cyclases metabolism
Bordetella pertussis enzymology
Bordetella pertussis genetics
Genes, Bacterial
Genes, Regulator
Virulence Factors, Bordetella genetics
Subjects
Details
- Language :
- English
- ISSN :
- 0021-9258
- Volume :
- 268
- Issue :
- 11
- Database :
- MEDLINE
- Journal :
- The Journal of biological chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 8385122