Anthrax toxin-mediated delivery of cholera toxin-A subunit into the cytosol of mammalian cells

The protective antigen (PA) component of anthrax toxin mediates delivery of either lethal factor (LF) or oedema factor into the cytosol of mammalian c ells. The N-terminal domain of LF1-254 (amino acids 1-254 of LF) binds to P A and, when fused to heterologous proteins, delivers such proteins into the cytosol. In the present study, we fused the catalytic subunit of cholera t oxin (CT-A) with LF1-254 and showed that the fusion protein LF1-254-CT-A re tains ADP-ribosylation activity in solution and increased intracellular cAM P levels in J774A.I macrophage cells when added together with PA. A mutant fusion protein, in which arginine-7 of CT-A was replaced with lysine, did n ot show ADP-ribosylation activity in solution and failed to increase cAMP l evels in macrophage cells, The data show that LF1-254-CT-A retains its cata lytic activity in solution as well as when translocated into the cytosol of eukaryotic cells via an alternative pathway to the GM(1) receptor used by CT.