Role of the Protective Antigen Octamer in the Molecular Mechanism of Anthrax Lethal Toxin Stabilization in Plasma

Abstract

Anthrax is caused by strains of Bacillus anthracis that produce two key virulence factors, anthrax toxin (Atx) and a poly-γ-D-glutamic acid capsule. Atx is comprised of three-proteins: protective antigen (PA) and two enzymes, lethal factor (LF) and edema factor (EF). To disrupt cell function, these components must assemble into holotoxin complexes, which contain either a ring-shaped homooctameric or homoheptameric PA oligomer bound to multiple copies of either LF and/or EF, producing lethal toxin (LT), edema toxin, or mixtures thereof. Once a host cell endocytoses these complexes, PA converts into a membrane-inserted channel that translocates LF and EF into the cytosol. LT may assemble on host cell surfaces or extracellularly in plasma. We show that under physiological conditions in bovine plasma that LT complexes containing heptameric PA aggregate and inactivate more readily than LT complexes containing octameric PA. LT complexes containing octameric PA possess enhanced stability, channel forming activity, and macrophage cytotoxicity relative to those containing heptameric PA. Under physiological conditions, multiple biophysical probes reveal that heptameric PA can prematurely adopt the channel conformation, but octameric PA complexes remain in their soluble prechannel configuration allowing them to resist aggregation and inactivation. We conclude that PA may form an octameric oligomeric state as a means to produce a more stable and active LT complex that may circulate freely in the blood.

Anthrax toxin protective antigen integrates poly-γ- d -glutamate and pH signals to sense the optimal environment for channel formation

“…S5A), and we presume structural changes observed in the D2-D4 interface (Fig. S5B) explain why PA 8 is more pH stable than PA 7 (2,10,15). Thus, interfacedisrupting D2-D4 mutations accelerate channel formation, and γ-DPGA inhibits channel formation by modulating a rate-limiting, pH-dependent barrier to channel formation.…”

“…Western blots of J774A.1 extracts following exposure to preassembled PA 7 LF 3 and unassembled LT (PA + LF) were used to determine whether PA channels formed in the presence of γ-DPGA. This assay works because the PA 7 channel is an SDS-stable complex (10,15). We found that full-length γ-DPGA, but not CapD-or HCl-hydrolyzed γ-DPGA, blocks proteolysis for monomeric PA 83 and blocks channel formation for assembled PA 7 complexes (Fig.…”

“…Recombinant PA, LF, EF, sANTXR2, and CapD were purified from Escherichia coli overexpression (2,10,11,16). PA 7 and PA 7 LF 3 prechannel complexes were produced as described (2,10).…”

“…Substructures within individual monomer subunits may unfold in response to ligand binding or changes in pH and then refold to form the transmembrane domain (7)(8)(9). Local sensing of protons and other key ligands provide critical signals to a toxin complex (2)(3)(4)(5)(6)(7)(8)(9)(10)(11) or virus (12), indicating that the environment is appropriate for membrane fusion.…”

“…Prechannel PA binds LF and EF to make lethal toxin (LT) and edema toxin (ET), respectively (1, 2). For (ii), plasma proteases process PA, and LF/EF and PA coassemble to form LT and ET (10). Here, the major functional prechannel oligomer is PA 8 because of the inactivity of PA 7 , which prematurely converts to insoluble channel complexes as a result of its relative instability (10).…”