Clostridium septicum alpha-toxin is proteolytically activated by furin

Clostridium septicum alpha-toxin is secreted as an inactive 46,450-Da protoxin. The protoxin is activated by proteolytic cleavage near the C terminus, which eventually causes the release of a 45-amino-acid fragment, Proteoytic activation and loss of the propeptide allow alpha-toxin to oligomerize and form pores on the plasma membrane, which results in colloidal-osmotic lysis, Activation may be accomplished in vitro by cleavage with trypsin at Arg(367) (J, Ballard, Y, Sokolov, W, L, Yuan, B, L, Kagan, and R Ii, Tweten, Mol, Microbiol. 10:627-634, 1993), which is located within the sequence KKRRGKR(367)S. A conspicuous feature of this site is a recognition site (RGKR) for the eukaryotic protease furin, Pro-alpha-toxin (AT(pro)) that was digested with trypsin or recombinant soluble furin yielded the 41,327-Da active form (AT(act)). A mutated alpha-toxin in which the furin consensus site was altered to KKRSGSRS at the cleavage site (AT(SGSR)) was cleaved and activated by trypsin but not by furin, In cytotoxicity assays, wild-type Chinese hamster ovary (CHO) and furin-deficient CHO (FD11) cells were killed by AT(pro) but not by AT(SGSR), Both cell types were killed by AT(SGSR) that was preactivated with trypsin, Propidium iodide uptake assays revealed that FD11 cells were approximately 22% less sensitive to AT(pro) than were CHO cells, AT(pro)-induced cell lysis of FD11 cells, assessed by propidium iodide uptake,,vas partially prevented by leupeptin (5 mM) and completely prevented by antipain (2.5 mM). The inhibition by antipain suggested the presence of cysteine or serine proteases that could also activate AT(pro), These findings demonstrate that furin is involved in the activation of C, septicum alpha-toxin on the cell surface but that alternate eukaryotic proteases can also activate the toxin, Regardless of the activating protease, the furin consensus site appears to be essential for the activation of alpha-toxin on the cell surface.