Anthrax toxin receptors and soluble receptor-based antitoxins

Bacillus anthracis secretes a tripartite toxin that contributes to bacterial virulence as well as causing disease symptoms. Previously, it was the belief in the field that anthrax toxin receptor/tumor endothelial marker 8 (ATR/TEM8) was the only host receptor that bound the protective antigen (PA) toxin subunit responsible for forming pores and translocating the 2 catalytic moieties into the host cytoplasm. We report here, the identification of a second class of toxin receptors encoded by capillary morphogenesis gene 2 (CMG2). PA domains 2 and 4 bind to an integrin-like inserted (I) domain at a metal ion dependent adhesion site (MIDAS) common to both receptors. The studies here distinguish PA binding to the two receptors by showing that the PA binding affinity of ATR/TEM8 is ∼1000-fold lower than that of CMG2 and that CMG2-PA binding is greatly reduced for dependence on the MIDAS site, as compared to ATR/TEM8-PA interactions. These different properties of receptor-PA binding have been exploited to develop CMG2-specific forms of PA, which have been used in vivo to demonstrate a major role for CMG2 in an established model system of anthrax intoxication. Additionally, structure-based mutagenesis of the receptor face that binds PA has allowed us to determine the molecular basis of PA binding and the receptor molecular clamp that prevents PA domain 2 pore formation and translocation until exposure to low pH. Our results suggest a model for pore formation where the major structural rearrangements that occur in the PA domain 2 membrane insertion loop require additional rearrangements in domain 4 and disruption of receptor contacts in this immediate region. In addition, a unique CMG2 surface region that contacts PA domain 2 is a major determinant for the higher CMG2 PA-affinity and the lower pH threshold for PA pore formation as compared to that of ATR/TEM8. Finally, I have demonstrated that soluble forms of CMG2 are potent inhibitors of anthrax intoxication both in vitro and in vivo.