title: 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.
authors.affiliation: Microbial Pathogenesis Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Mahtab
authors.initials: M
authors.lastname: Moayeri
authors.forename: Katherine E
authors.initials: KE
authors.lastname: Wickliffe
authors.forename: Jason F
authors.initials: JF
authors.lastname: Wiggins
authors.forename: Stephen H
authors.initials: SH
authors.lastname: Leppla
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000502:physiology; D000818:Animals; D000942:Antigens, Bacterial / Q000633:toxicity*; D001427:Bacterial Toxins / Q000037:antagonists & inhibitors* / Q000633:toxicity*; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D051379:Mice; D008807:Mice, Inbred BALB C; D010084:Oxidation-Reduction
chemical_list: D000942:Antigens, Bacterial; D001427:Bacterial Toxins; C030325:anthrax toxin; C017199:2',3'-dialdehyde ATP; D000255:Adenosine Triphosphate