A new assay for the invasive adenylate cyclase toxin of Bordetella pertussis based on its morphological effects on the fibronectin-stimulated spreading of BHK21 cells.
When baby hamster kidney (BHK) cells are allowed to spread on fibronectin-coated substrata in the absence of serum and the presence of agents which elevate intracellular 3':5'-cyclic AMP (cAMP) levels they adopt an abnormal, stellated morphology. To determine whether the invasive adenylate cyclase (AC) toxin of Bordetella pertussis induced the same response, cell extracts were prepared from several B. pertussis strains. They were characterized for AC toxin production by enzymic assay and by immunoblotting with an AC-toxin-specific monoclonal antibody. Extracts of strains producing AC toxin induced elevated levels of intracellular cAMP in BHK cells and promoted a stellation response during cell spreading. Extracts prepared from strains defective in AC toxin production showed no effect. Using image analysis to quantify the morphological change, we have demonstrated that the effect of AC toxin on cell spreading is dose dependent. This technique is a rapid and sensitive assay for the invasive AC toxin.
Westrop
G D
GD
Department of Microbiology, University of Glasgow, UK.
Campbell
G
G
Kazi
Y
Y
Billcliffe
B
B
Coote
J G
JG
Parton
R
R
Freer
J H
JH
Edwards
J G
JG
D037361:Adenylate Cyclase Toxin; D000262:Adenylyl Cyclases / Q000032:analysis* / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D001426:Bacterial Proteins / Q000032:analysis* / Q000494:pharmacology; D001427:Bacterial Toxins / Q000032:analysis* / Q000494:pharmacology; D001681:Biological Assay; D001886:Bordetella pertussis / Q000201:enzymology*; D002448:Cell Adhesion / Q000187:drug effects; D002478:Cells, Cultured; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D005098:Exotoxins / Q000032:analysis* / Q000494:pharmacology; D005347:Fibroblasts / Q000187:drug effects* / Q000648:ultrastructure; D005353:Fibronectins / Q000494:pharmacology; D006460:Hemolysin Proteins / Q000032:analysis / Q000494:pharmacology; D008647:Mesocricetus; D011498:Protein Precursors / Q000032:analysis* / Q000494:pharmacology
D037361:Adenylate Cyclase Toxin; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D005098:Exotoxins; D005353:Fibronectins; D006460:Hemolysin Proteins; D011498:Protein Precursors; D000242:Cyclic AMP; D000262:Adenylyl Cyclases