A dominant negative mutant of Bacillus anthracis protective antigen inhibits anthrax toxin action in vivo

PA63, a proteolytically activated 63-kDa form of anthrax protective antigen (PA), forms heptameric oligomers and has the ability to bind and transloca te the catalytic moieties, lethal factor (LF), and edema factor (EF) into t he cytosol of mammalian cells. Acidic pH triggers oligomerization and membr ane insertion by PA63, A disordered amphipathic loop in domain II of PA (2 beta2-2 beta3 loop) is involved in membrane insertion by PA63. Because cond itions required for membrane insertion coincide with those for oligomerizat ion of PA63 in mammalian cells, residues constituting the 2 beta2-2 beta3 l oop were replaced with the residues of the amphipathic membrane inserting l oop of its homologue iota-b toxin secreted by Clostridium perfringens. It w as hypothesized that such a molecule might assemble into heteroheptameric s tructures with wild-type PA ultimately leading to the inhibition of cellula r intoxication, The mutation blocked the ability of PA to mediate membrane insertion and translocation of Lf into the cytosol but had no effect on pro teolytic activation, oligomerization, or binding LF. Moreover, an equimolar mixture of purified mutant PA (PA-I) and wild-type PA showed complete inhi bition of toxin activity both in vitro on J774A.1 cells and in vivo in Fisc her 344 rats thereby exhibiting a dominant negative effect. In addition, PA -I inhibited the channel-forming ability of wild-type PA on the plasma memb rane of CHO-K1 cells thereby indicating protein-protein interactions betwee n the two proteins resulting in the formation of mixed oligomers with defec tive functional activity. Our findings provide a basis for understanding th e mechanism of translocation and exploring the possibility of the use of th is PA molecule as a therapeutic agent against anthrax toxin action in vivo.