Oxidized ATP Protection against Anthrax Lethal Toxin

Abstract

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.Anthrax toxin consists of three polypeptides that form two binary toxins. The lethal toxin (LT) and edema toxin (ET) each contain the receptor binding protein, protective antigen (PA), combined with an enzymatic cargo protein. Lethal factor (LF), a protease which cleaves members of the mitogen-activated protein kinase family (MEKs), and edema factor (EF), a calmodulin-dependent adenylate cyclase, are transported into the cell cytoplasm by PA oligomers. These oligomers can form only after binding and cleavage of PA from an 83-kDa protein (PA83) to its 63-kDa form (PA63) by furin at the cell surface. The heptameric PA63 oligomers carrying LF and/or EF are then conformationally altered in an intracellular acidic compartment to allow passage of their cargo to the cytosol through a pore (for a review, see reference 9).LT is present at high concentrations in the blood of infected animals and is sufficient to induce some symptoms of anthrax and lethality in animal models (4,21,41,11,29,30,56). Although many LT-mediated effects in cells and animals have been described over the years, the actual mechanism by which LT causes cell or animal death is unknown. Studying the toxin's unique induction of rapid lysis in murine macrophage lines (such as RAW264.7 and J774.A1 cells) or in primary macrophages from BALB/cJ mice may provide a better understanding of its function and cellular targets (23). The macrophage lysis assay also pro...

Amiodarone and Bepridil Inhibit Anthrax Toxin Entry into Host Cells

“…In contrast to their predicted actions as cardiac drugs, the most likely mechanism of toxin inhibition for both amiodarone and bepridil appears to be via neutralization of endosomal pH, thereby blocking toxin entry. These two drugs join the growing list of compounds that have been reported to block anthrax toxin activity in animal models via endosomal pH neutralization (2,36). We report that protection from LT in cell culture models correlates with IC 50 values of 3.5 M for amiodarone and 4.8 M for bepridil.…”

“…Lysosomotropic agents have previously been shown to block the acid-dependent entry of anthrax toxin (12,18,22,24,27,36). Recently, CQ was shown to be effective in reducing LTinduced mortality in animal models (2).…”

Anthrax lethal toxin and Salmonella elicit the common cell death pathway of caspase-1-dependent pyroptosis via distinct mechanisms

“…The route of potassium efflux during LT intoxication is unclear. Although potassium efflux can occur through the P2X 7 receptor ion channel, a role for this channel in LT intoxication was previously excluded (38).…”

A Heterodimer of a VHH (Variable Domains of Camelid Heavy Chain-only) Antibody That Inhibits Anthrax Toxin Cell Binding Linked to a VHH Antibody That Blocks Oligomer Formation Is Highly Protective in an Anthrax Spore Challenge Model

“…Toxin or antibody prebound toxin were prepared in serum-free DMEM and bound to cells for 1 h before washing and lysis in radioimmune precipitation assay buffer as previously described (26). In parallel experiments, trypsin (0.25% trypsin-EDTA, Invitrogen) was used to detach cells and strip cell surface proteins.…”

“…Trypsin was inactivated by the addition of 50% fetal calf serum, cells were centrifuged (1000 rpm, 10 min), washed twice with PBS, and lysed in radioimmune precipitation assay buffer. Western blot analyses were performed as previously described with loading of equal amounts of cell lysate protein in each lane (26).…”