Identification by in vitro complementation of regions required for cell-invasive activity of Bordetella pertussis adenylate cyclase toxin.
The adenylate cyclase toxin (CyaA) of Bordetella pertussis is a 1706-residue protein composed of an amino-terminal adenylate cyclase (AC) domain linked to a 1300-residue channel-forming RTX (repeats in toxin) haemolysin. The toxin delivers its AC domain into a variety of eukaryotic cells and impairs cellular functions by catalysing unregulated synthesis of cAMP from intracellular ATP. We have examined toxin activities of a set of deletion derivatives of CyaA. The results indicate that CyaA does not have a dedicated target cell-binding domain and that structural integrity and co-operation of all domains, as well as the post-translational fatty acylation mediated by an accessory protein CyaC, are all essential for target cell association and toxin activity of CyaA. When tested individually, all toxin derivatives were inactive and impaired in the tight association with the target cell surface. However, pairs of constructs with nonoverlapping deletions complemented each other in vitro and exhibited a partially restored cytotoxic activity. This suggests that at least a part of the active toxin may act in the form of dimers or higher oligomers. The complementation analysis revealed that the last 217 residues of CyaA, containing the unprocessed secretion signal, form an autonomous domain essential for toxin activity, and that the region from residue 624 to 780 may be directly involved in delivery of the AC toxin into cells.
Iwaki
M
M
Unité de Biochemie des Régulations Cellulaires, Institut Pasteur, Paris, France.
Ullmann
A
A
Sebo
P
P
D000215:Acylation; D000255:Adenosine Triphosphate / Q000096:biosynthesis; D037361:Adenylate Cyclase Toxin; D000262:Adenylyl Cyclases / Q000737:chemistry* / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics; D001886:Bordetella pertussis / Q000235:genetics; D004912:Erythrocytes; D005816:Genetic Complementation Test; D006460:Hemolysin Proteins / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics; D010957:Plasmids; D011487:Protein Conformation; D011498:Protein Precursors / Q000737:chemistry* / Q000235:genetics; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D017384:Sequence Deletion; D012756:Sheep
D037361:Adenylate Cyclase Toxin; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D006460:Hemolysin Proteins; D010446:Peptide Fragments; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D000255:Adenosine Triphosphate; D000262:Adenylyl Cyclases