High-level synthesis of active adenylate cyclase toxin of Bordetella pertussis in a reconstructed Escherichia coli system.
The Bordetella pertussis adenylate cyclase(Cya) toxin-encoding locus (cya) is composed of five genes. The cyaA gene encodes a virulence factor (CyaA), exhibiting adenylate cyclase, hemolytic and invasive activities. The cyaB, D and E gene products are necessary for CyaA transport, and the cyaC gene product is required to activate CyaA. We reconstructed, in Escherichia coli, the cya locus of B. pertussis by cloning the different genes on appropriate vectors under the control of strong promoters and E. coli-specific translation initiation signals. We show that in the absence of additional gene products, CyaA is synthesized at high levels, is endowed with adenylate cyclase activity, but is devoid of invasive and hemolytic activities. CyaC is sufficient to confer upon the adenylate cyclase holotoxin full invasive and partial hemolytic activities. Coexpression of the cyaB, D and E genes neither stimulates nor potentiates the activation brought about by CyaC. This reconstructed system should help to elucidate both the mechanism and the structural requirements of holotoxin activation.
Sebo
P
P
Unité de Biochimie des Régulations Cellulaires, Institut Pasteur, Paris, France.
Glaser
P
P
Sakamoto
H
H
Ullmann
A
A
D037361:Adenylate Cyclase Toxin; D001483:Base Sequence; D001886:Bordetella pertussis / Q000201:enzymology / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial; D006461:Hemolysis; D008969:Molecular Sequence Data; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis; D010566:Virulence Factors, Bordetella / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification
D037361:Adenylate Cyclase Toxin; D011994:Recombinant Proteins; D010566:Virulence Factors, Bordetella