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.
Highlights
Anthrax toxin consists of three polypeptides that form two binary toxins
We investigated the various steps in the intoxication pathway and found that binding of the protective-antigen (PA) component of lethal toxin (LT) to cells and the enzymatic proteolytic ability of the lethal factor (LF) component of LT were unaffected by oxidized ATP (o-ATP)
LT-sensitive RAW264.7 macrophages treated with o-ATP for 10 min prior to the addition of LT were protected from lysis in a dose-dependent manner at concentrations of 200 M
Summary
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. P2X7 (previously known as P2Z) receptors are members of the P2 purinergic receptor family responsible for cell responses to extracellular nucleotides These receptors play many roles in immunomodulation and signaling through binding of extracellular ATP, which is often released from cells in a controlled manner via nonlytic events or more rapidly due to cell damage (for reviews, see references 15 and 16). This ubiquitous receptor is itself an ATP-gated ion channel, the prolonged activation of which leads to progressive increase in the size of a nonselective membrane pore, allowing passage of molecules of up to 900 Da [10, 16, 50]. We present data on the effectiveness of this novel LT inhibitor in protecting against toxin-induced lethality in mice
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
