Darran J. Wigelsworth

A Soluble Receptor Decoy Protects Rats against Anthrax Lethal Toxin Challenge

Successful postexposure treatment for inhalation anthrax is thought to include neutralization of anthrax toxin. The soluble anthrax toxin receptor/tumor endothelial marker 8 and capillary morphogenesis protein 2 (sATR/TEM8 and sCMG2, respectively) receptor decoys bind to anthrax toxin protective antigen (PA) and compete with cellular receptors for binding. Here, we show that, in a tissue-culture model of intoxication, sCMG2 is a 11.4-fold more potent antitoxin than sATR/TEM8 and that this increased activity corresponds to an approximately 1000-fold higher PA-binding affinity. Stoichiometric concentrations of sCMG2 protect rats against lethal toxin challenge, making sCMG2 one of the most effective anthrax antitoxins described to date.

Mapping dominant-negative mutations of anthrax protective antigen by scanning mutagenesis

The protective antigen (PA) moiety of anthrax toxin transports edema factor and lethal factor to the cytosol of mammalian cells by a mechanism that depends on its ability to oligomerize and form pores in the endosomal membrane. Previously, some mutated forms of PA, designated dominant negative (DN), were found to coassemble with wild-type PA and generate defective heptameric pore-precursors (prepores). Prepores containing DN–PA are impaired in pore formation and in translocating edema factor and lethal factor across the endosomal membrane. To create a more comprehensive map of sites within PA where a single amino acid replacement can give a DN phenotype, we used automated systems to generate a Cys-replacement mutation for each of the 568 residues of PA63, the active 63-kDa proteolytic fragment of PA. Thirty-three mutations that reduced PAs ability to mediate toxicity at least 100-fold were identified in all four domains of PA63. A majority (22) were in domain 2, the pore-forming domain. Seven of the domain-2 mutations, located in or adjacent to the 2β6 strand, the 2β7 strand, and the 2β10-2β11 loop, gave the DN phenotype. This study demonstrates the feasibility of high-throughput scanning mutagenesis of a moderate sized protein. The results show that DN mutations cluster in a single domain and implicate 2β6 and 2β7 strands and the 2β10–2β11 loop in the conformational rearrangement of the prepore to the pore. They also add to the repertoire of mutations available for structure–function studies and for designing new antitoxic agents for treatment of anthrax.

Anthrax Toxin Receptor 2-Dependent Lethal Toxin Killing In Vivo

Anthrax toxin receptors 1 and 2 (ANTXR1 and ANTXR2) have a related integrin-like inserted (I) domain which interacts with a metal cation that is coordinated by residue D683 of the protective antigen (PA) subunit of anthrax toxin. The receptor-bound metal ion and PA residue D683 are critical for ANTXR1-PA binding. Since PA can bind to ANTXR2 with reduced affinity in the absence of metal ions, we reasoned that D683 mutant forms of PA might specifically interact with ANTXR2. We show here that this is the case. The differential ability of ANTXR1 and ANTXR2 to bind D683 mutant PA proteins was mapped to nonconserved receptor residues at the binding interface with PA domain 2. Moreover, a D683K mutant form of PA that bound specifically to human and rat ANTXR2 mediated killing of rats by anthrax lethal toxin, providing strong evidence for the physiological importance of ANTXR2 in anthrax disease pathogenesis.

Insertion of Anthrax Protective Antigen into Liposomal Membranes EFFECTS OF A RECEPTOR

Protective antigen (PA), the receptor-binding component of anthrax toxin, heptamerizes and inserts into the endosomal membrane at acidic pH, forming a pore that mediates translocation of the enzymic components of the toxin to the cytosol. When the heptameric pre-insertion form of PA (the prepore) is acidified in solution, it rapidly loses the ability to insert into membranes. To maximize insertion into model membranes, we examined two ways to bind the protein to large unilamellar vesicles (LUV). One involved attaching a His tag to the von Willebrand factor A domain of one of the PA receptors, ANTXR2, and using this protein as a bridge to bind PA to LUV containing a nickel-chelating lipid. The other involved using a His tag fused to the C terminus of PA to bind the protein directly to LUV containing the same lipid. Both ways enhanced pore formation at pH 5.0 strongly and about equally, as measured by the release of K+. Controls showed that pore formation in this system faithfully reproduced that in vivo. We also showed that binding unmodified ANTXR2 von Willebrand factor A to the prepore in solution enhanced its pore forming activity by slowing its inactivation at acidic pH. These findings indicate that an important role of PA receptors is to promote partitioning of PA into the bilayer by maintaining the prepore close to the target membrane and presumably in the optimal orientation as it undergoes the acidic pH-dependent conformational transition to the pore.

Mutant Anthrax Toxin B Moiety (Protective Antigen) Inhibits Angiogenesis and Tumor Growth

Bacillus anthracis protective antigen (PA), the B subunit of the binary anthrax toxin, binds to the cellular receptors capillary morphogenesis gene 2 protein and tumor endothelial marker 8 with high affinity. Both receptors are expressed on endothelial cells during angiogenesis. We sought to determine whether one could inhibit angiogenesis by interfering with the binding of these receptors to their endogenous ligands. Here, we show that wild-type PA inhibits both vascular endothelial growth factor-induced and basic fibroblast growth factor-induced angiogenesis at moderate but statistically significant levels. Structure-activity studies identified a PA mutant that exhibited markedly enhanced inhibition of angiogenesis and also inhibited tumor growth in vivo. This mutant, PASSSR, is unable to undergo normal cellular processing and, thus, remains bound to the surface receptor. Further mutation of PASSSR so that it does not bind to these cell surface receptors abolished its ability to inhibit angiogenesis. We conclude that high-affinity anthrax toxin receptor (ATR) ligands, such as PA and PASSSR, are angiogenesis inhibitors and that ATRs are useful targets for antiangiogenic therapy. These results also suggest that endothelial cell-binding proteins from additional pathogens may inhibit angiogenesis and raise the question of the role of such inhibition in pathogenesis.