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 leukotoxin (LktA) from Pasteurella haemolytica are members of the RTX (stands for repeats in toxin) family of cytolytic toxins. They have pore-forming activity and share significant amino acid homology but show marked differences in biological activity. CyaA is an invasive adenylate cyclase and a weak hemolysin which is active on a wide range of mammalian cells. LktA is a cytolytic protein with a high target cell specificity and is able to lyse only leukocytes and platelets from ruminants. Each toxin is synthesized as an inactive protoxin encoded by the A gene, and the product of the accessory C gene is required for posttranslational activation. Heterologous activation of LktA by CyaC did not result in a change in its specificity for nucleated cells, although the toxin showed a greater hemolytic-to-cytotoxic ratio. LktC was unable to activate CyaA. A hybrid toxin (Hyb1), which contained the N-terminal 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-terminal end of LktA (aa 379 to 953), showed no toxic activity. Replacement of part of the LktA C-terminal domain of Hyb1 by the CyaA C-terminal 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, showing the importance of the region between aa 379 and 616 of LktA for activation by LktC. LktC-activated Hyb2 was more active against ruminant than murine nucleated cells, whereas CyaC-activated Hyb2 displayed a similar, but lower, activity against both cell types. These data indicate that LktC and the region with which it interacts have an influence on the target cell specificity of the mature toxin.
Westrop
G
G
Division of Infection and Immunity, IBLS, Glasgow University, United Kingdom.
Hormozi
K
K
da Costa
N
N
Parton
R
R
Coote
J
J
D037361:Adenylate Cyclase Toxin; D000262:Adenylyl Cyclases / Q000235:genetics / Q000633:toxicity*; D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000633:toxicity*; D001427:Bacterial Toxins / Q000235:genetics / Q000633:toxicity*; D001886:Bordetella pertussis; D002417:Cattle; D003603:Cytotoxins / Q000235:genetics / Q000633:toxicity*; D004926:Escherichia coli / Q000235:genetics; D005098:Exotoxins / Q000235:genetics / Q000633:toxicity*; D005816:Genetic Complementation Test; D006460:Hemolysin Proteins / Q000235:genetics / Q000633:toxicity*; D006461:Hemolysis; D016978:Mannheimia haemolytica; D051379:Mice; D008969:Molecular Sequence Data; D011498:Protein Precursors / Q000235:genetics / Q000633:toxicity*; D011993:Recombinant Fusion Proteins / Q000633:toxicity; D012756:Sheep; D013329:Structure-Activity Relationship
D037361:Adenylate Cyclase Toxin; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D003603:Cytotoxins; D005098:Exotoxins; D006460:Hemolysin Proteins; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; C102677:lktA protein, bacteria; D000262:Adenylyl Cyclases