Anthrax toxin (AT) is a tripartite toxin and has three components: edema factor (EF), lethal factor (LF) and protective antigen (PA). Recent studies have demonstrated that AT binds through its PA component to cells through its unique and widely expressed transmembrane receptor termed (ATR). After binding to ATR, PA is cleaved by furin-like proteases and carboxyl (PA63) terminal of this molecule heptamerizes with EF and/or LF. The complex is then taken up by cells by receptor-mediated endocytosis in low pH endosomal compartment. Once inside the cell, EF mediates its enzymatic activity (adenylate cyclase) and inhibits professional phagocytosic activity. LF is a zinc-dependent protease that cleaves MAP kinases and causes lysis of macrophages. In addition, it has been recently shown that the extracellular domain (ec) of ATR is cleaved which may bind and inhibit interaction of PA and thus LF and EF to ATR. In this study we propose to express and purify recombinant extracellular domain of ATR from E. coli and test its protective activity in human and murine cell lines and primary cell cultures. Neutralization of both AT and Bacillus anthracis induced cytotoxicity will be tested in vitro and in animal models. For in vivo testing, rat intoxication model will be utilized.
