The Protective Antigen Component of Anthrax Toxin Forms Functional Octameric Complexes

Abstract

The assembly of bacterial toxins and virulence factors is critical to their function, but the regulation of assembly during infection has not been studied. We begin to address this question using anthrax toxin as a model. The protective antigen (PA) component of the toxin assembles into ring-shaped homooligomers that bind the two other enzyme components of the toxin, lethal factor (LF) and edema factor (EF), to form toxic complexes. To disrupt the host, these toxic complexes are endocytosed, such that the PA oligomer forms a membrane-spanning channel that LF and EF translocate through to enter the cytosol. We show using single-channel electrophysiology that PA channels contain two populations of conductance states, which correspond with two different PA pre-channel oligomers observed by electron microscopy—the well-described heptamer and a novel octamer. Mass spectrometry demonstrates that the PA octamer binds four LFs, and assembly routes leading to the octamer are populated with even-numbered, dimeric and tetrameric, PA intermediates. Both heptameric and octameric PA complexes can translocate LF and EF with similar rates and efficiencies. Here we also report a 3.2-Å crystal structure of the PA octamer. The octamer comprises ∼20−30% of the oligomers on cells, but outside of the cell, the octamer is more stable than the heptamer under physiological pH. Thus the PA octamer is a physiological, stable, and active assembly state capable of forming lethal toxins that may withstand the hostile conditions encountered in the bloodstream. This assembly mechanism may provide a novel means to control cytotoxicity.

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.…”

“…pH sensor | translocation | planar bilayer electrophysiology P athogenic bacteria secrete channel-forming toxins, often as monomeric protein subunits, which assemble at the host cell surface into non-membrane-inserted prechannel oligomers (1)(2)(3)(4)(5)(6). Conformational rearrangements triggered by changes in the local environment on or within the host cell allow the prechannel oligomers to obtain their final membrane-inserted channel state.…”

“…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.…”

“…Also shown is a record of channel insertion PA ch at pH 7.4. (B) Prechannel-to-channel transition mechanism depicted by low-resolution structures overlaid with a ribbons rendering of the PA 8 structure (3HVD) (2). Also shown is a hypothetical intermediate state and a channel model.…”

Symmetry Requirements for Effective Blocking of Pore-Forming Toxins: Comparative Study with α-, β-, and γ-Cyclodextrin Derivatives

“…The difference in the activities of ␥-CD derivatives against anthrax and staphylococcal toxins could be explained by the recent observation that in contrast to ␣-HL, PA presumably can form octameric pores in addition to heptameric ones (6). However, the currently available data are ambiguous.…”