title: Activation and immunogenicity of Bordetella pertussis adenylate cyclase toxin
Creators: name: Hormozi, E. Kalantar
Publisher: name: University of Glasgow
Year: 1997

description: Adenylate cyclase toxin (CyaA) was produced and purified in the toxic CyaC-modified form and the unmodified non-toxic form from both B. pertussis and recombinant E. coli strains in sufficient quantity to allow large scale experimentation. Immunoblot analysis of crude and purified CyaA preparations revealed that the toxins were prepared as full length 200 kDa proteins with a small amount of degradation products of lower molecular weight protein in both toxic and non-toxic forms. The in vivo CyaC-modified CyaA toxin produced from recombinant E. coli showed comparable levels of cytotoxic and invasive activity to that produced from B. pertussis but its haemolytic activity was weaker. In addition, the leukotoxin (LktA) from Pasteurella haemolytica was also produced by expression from recombinant plasmids in E. coli, in both the LktC-modified toxic form and unmodified non-toxic form. The A and C proteins of both toxins were produced separately in E. coli and each could be co-expressed on compatible plasmids. This allowed heterologous activation of CyaA by LktC and LktA by CyaC. The LktC- or CyaC-modified LktA of 105 kDa protein was produced and partially purified from recombinant E. coli strains, but the yield of LktA production was low compared to that of CyaA in the same T7 expression system. The LktC-modified CyaA toxin was also produced but showed no cytotoxic or haemolytic activity. Heterologous activation of LktA by CyaC was successful and the toxin was almost as active against bovine lymphoma (BL3) cells as the LktC-modified toxin. However, the heterologous activation by CyaC did not change its specificity for ruminant cells, because no cytotoxic activity against mouse J774.2 cells or haemolytic activity against horse red blood cells was detected. The CyaC-modified LktA showed a greater haemolytic to cytotoxic ratio than LktC-modified LktA. Thus, LktA modified by CyaC was more haemolytic than the LktC-modified form, but nevertheless retained the specificity for ruminant cells which is a feature of the native toxin. Two hybrid toxins derived from CyaA and LktA were also produced and purified. Hybl contained the N-terminal enzymic domain and the pore forming domain from CyaA (amino acids [aa] 1-687), with the remainder of the protein derived from the C-terminal end of LktA (aa 379-953). Hyb2 was created from Hybl by replacement of the LktA C-terminal domain of Hybl with the C-terminal domain of CyaA (aa 919-1706 ). Part of the region concerned with C-modification site of CyaA (aa 688-918) was therefore replaced with the equivalent region from LktA. The Hybl toxin of 150 kDa protein had normal AC enzymic activity, but showed no toxic activity when modified in vivo by CyaC or LktC. In contrast to CyaA, the 200 kDa Hyb2 protein was activated more efficiently by LktC than by CyaC, although the cytotoxic and haemolytic activity of Hyb2 modified with LktC or CyaC was lower compared to recombinant active CyaA modified by CyaC. However, LktC-modified Hyb2 showed more toxic activity against ruminant than against murine nucleated cells, whereas CyaC-activated Hyb2 displayed a similar, but lower, activity against both cell types, indicating that LktC and the region with which it interacted had an influence on the target cell specificity of the activated toxin.