Bacillus anthracis lethal toxin (LT) induces rapid lysis (<90 min) of murine macrophages from certain inbred strains. The mechanism for LT-induced cytolysis is currently unknown. We hypothesized that the
ATP-activated macrophage P2X7 receptors implicated in
nucleotide-mediated macrophage lysis could play a role in LT-mediated cytolysis and discovered that a potent P2X7 antagonist,
oxidized ATP (
o-ATP), protects macrophages against LT. Other
P2X7 receptor antagonists, however, had no effect on LT function, while oxidized
nucleotides, o-
ADP, o-
GTP, and o-
ITP, which did not act as receptor
ligands, provided protection. Cleavage of the LT substrates, the
mitogen-activated protein kinases, was inhibited by
o-ATP in RAW274.6 macrophages and CHO cells. We investigated the various steps in the intoxication pathway and found that binding of the protective-
antigen (PA) component of LT to cells and the enzymatic proteolytic ability of the lethal factor (LF) component of LT were unaffected by
o-ATP. Instead, the
drug inhibited formation of the
sodium dodecyl sulfate-resistant PA oligomer, which occurs in acidified endosomes, but did not prevent cell surface PA oligomerization, as evidenced by binding and translocation of LF to a
protease-resistant intracellular location. We found that
o-ATP also protected cells from
anthrax edema toxin and
diphtheria toxin, which also require an acidic environment for escape from endosomes. Confocal microscopy using pH-sensitive
fluorescent dyes showed that
o-ATP increased endosomal pH. Finally, BALB/cJ mice injected with
o-ATP and LT were completely protected against lethality.