BORDETELLA-PERTUSSIS ADENYLATE-CYCLASE TOXIN - PROCYAA AND CYAC PROTEINS SYNTHESIZED SEPARATELY IN ESCHERICHIA-COLI PRODUCE ACTIVE TOXIN IN-VITRO

Bordetella pertussis produces a cell-invasive adenylate cyclase toxin (CyaA) which is related to the RTX family of pore-forming toxins. Like all RTX toxins, CyaA is synthesised as a protoxin (proCyaA), encoded by the cyaA gene. Activation to the mature cell-invasive toxin involve s palmitoylation of lysine 983 and is dependent on co-expression of cy aC. The role of the cyaC gene product in the acylation reaction has no t been determined. We have developed an efficient T7 RNA polymerase sy stem for overexpression of cyaA and cyaC separately in Escherichia col i. Each protein accumulated intracellularly in an insoluble form and c ould be collected by centrifugation of lysed cells. A single-step puri fication was achieved by extraction of the aggregated material with 8 M urea. Active cell-invasive CyaA was produced in vitro when the proCy aA and CyaC proteins were mixed with a cytosolic extract of either E. coli or B. pertussis. Activation was assumed to occur by an acylation reaction requiring acyl carrier protein (ACP) as cofactor, as the cyto solic factor required for toxin activation was lost if the S100 extrac t was dialysed before use and the cytosolic factor could be replaced i n the in vitro reaction by ACP charged separately in vitro with palmit ic acid, as reported previously for activation of the homologous E. co li haemolysin (HlyA). The in vitro activation system may be used to in vestigate the mechanism of the CyaC-dependent acylation of proCyaA and the effect of variation of the modifying fatty acyl group on target c ell specificity and toxic activity of CyaA.