STRUCTURE-FUNCTION STUDIES OF THE ADENYLATE-CYCLASE TOXIN OF BORDETELLA-PERTUSSIS AND THE LEUKOTOXIN OF PASTEURELLA-HAEMOLYTICA BY HETEROLOGOUS C-PROTEIN-ACTIVATION AND CONSTRUCTION OF HYBRID PROTEINS

The adenylate cyclase toxin (CyaA) from Bordetella pertussis and the l eukotoxin (LktA) from Pasteurella haemolytica are members of the RTX ( stands for repeats in toxin) family of cytolytic toxins. They have por e-forming activity and share significant amino acid homology but show marked differences in biological activity, CyaA is an invasive adenyla te cyclase and a weak hemolysin which is active on a wide range of mam malian cells, LktA is a cytolytic protein with a high target cell spec ificity and is able to lyse only leukocytes and platelets from ruminan ts. Each toxin is synthesized as an inactive protoxin encoded by the A gene, and the product of the accessory C gene is required for posttra nslational activation, Heterologous activation of LktA by CyaC did not result in a change in its specificity for nucleated cells, although t he toxin showed a greater hemolytic-to-cytotoxic ratio. LktC was unabl e to activate CyaA. A hybrid toxin (Hyb1), which contained the N-termi nal enzymic domain and the pore-forming domain from CyaA (amino acids [aa] 1 to 687), with the remainder of the protein derived from the C-t erminal end of LktA (aa 379 to 953), showed no toxic activity, Replace ment of part of the LktA C-terminal domain of Hyb1 by the CyaA C-termi nal domain (aa 919 to 1706) to create hybrid toxin 2 (Hyb2) partially restored toxic activity. In contrast to CyaA, Hyb2 was activated more efficiently by LktC than by CyaC, shelving the importance of the regio n between aa 379 and 616 of LktA for activation by LktC. LktC-activate d Hyb2 was more active against ruminant than murine nucleated cells, w hereas CyaC-activated Hyb2 displayed a similar, but lower, activity ag ainst both cell types, These data indicate that LktC and the region wi th which it interacts have an influence on the target cell specificity of the mature toxin.