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 translocate the catalytic moieties, lethal factor (LF), and edema factor (EF) into the cytosol of mammalian cells. Acidic pH triggers oligomerization and membrane insertion by PA63. A disordered amphipathic loop in domain II of PA (2beta2-2beta3 loop) is involved in membrane insertion by PA63. Because conditions required for membrane insertion coincide with those for oligomerization of PA63 in mammalian cells, residues constituting the 2beta2-2beta3 loop were replaced with the residues of the amphipathic membrane-inserting loop of its homologue iota-b toxin secreted by Clostridium perfringens. It was hypothesized that such a molecule might assemble into hetero-heptameric structures with wild-type PA ultimately leading to the inhibition of cellular intoxication. The mutation blocked the ability of PA to mediate membrane insertion and translocation of LF into the cytosol but had no effect on proteolytic activation, oligomerization, or binding LF. Moreover, an equimolar mixture of purified mutant PA (PA-I) and wild-type PA showed complete inhibition of toxin activity both in vitro on J774A.1 cells and in vivo in Fischer 344 rats thereby exhibiting a dominant negative effect. In addition, PA-I inhibited the channel-forming ability of wild-type PA on the plasma membrane of CHO-K1 cells thereby indicating protein-protein interactions between the two proteins resulting in the formation of mixed oligomers with defective functional activity. Our findings provide a basis for understanding the mechanism of translocation and exploring the possibility of the use of this PA molecule as a therapeutic agent against anthrax toxin action in vivo.
Centre for Biochemical Technology, Mall Road, Delhi-110007, India. ysingh@cbt.res.in
Y
Y
Singh
H
H
Khanna
A P
AP
Chopra
V
V
Mehra
D000818:Animals; D000942:Antigens, Bacterial; D001427:Bacterial Toxins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000276:immunology*; D016466:CHO Cells; D006224:Cricetinae; D005799:Genes, Dominant; D008854:Microscopy, Electron; D009154:Mutation
D000942:Antigens, Bacterial; D001427:Bacterial Toxins; C030325:anthrax toxin