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

Abstract

Many toxins assemble into oligomers on the surface of cells. Local chemical cues signal and trigger critical rearrangements of the oligomer, inducing the formation of a membrane-fused or channel state. Bacillus anthracis secretes two virulence factors: a tripartite toxin and a poly-γ-D-glutamic acid capsule (γ-DPGA). The toxin's channel-forming component, protective antigen (PA), oligomerizes to create a prechannel that forms toxic complexes upon binding the two other enzyme components, lethal factor (LF) and edema factor (EF). Following endocytosis into host cells, acidic pH signals the prechannel to form the channel state, which translocates LF and EF into the host cytosol. We report γ-DPGA binds to PA, LF, and EF, exhibiting nanomolar avidity for the PA prechannel oligomer. We show PA channel formation requires the pH-dependent disruption of the intra-PA domain-2-domain-4 (D2-D4) interface. γ-DPGA stabilizes the D2-D4 interface, preventing channel formation both in model membranes and cultured mammalian cells. A 1.9-Å resolution X-ray crystal structure of a D2-D4-interface mutant and corresponding functional studies reveal how stability at the intra-PA interface governs channel formation. We also pinpoint the kinetic pH trigger for channel formation to a residue within PA's membrane-insertion loop at the inter-PA D2-D4 interface. Thus, γ-DPGA may function as a chemical cue, signaling that the local environment is appropriate for toxin assembly but inappropriate for channel formation.pH sensor | translocation | planar bilayer electrophysiology

Circulating lethal toxin decreases the ability of neutrophils to respond toBacillus anthracis

“…Capsule components shed during infection can be found in circulation and may function to activate the complement cascade at sites remote from infection disrupting the chemoattractant gradient (Makino et al ., ). Circulating capsule may also increase the efficiency of cellular intoxication by stabilizing intracellular PA pore formation (Kintzer et al ., ).…”

Structure of a dominant negative mutant reveals a stalled intermediate state of anthrax protective antigen pore maturation

“…Despite the availability of two end-state structures for wild type (WT) PA prepore and pore (Jiang et al, 2015;, the extreme differences in their organization and molecular interactions has make it challenging to rationalize the sequential steps that are involved in the maturation process. Significant structural differences between the two end states include the formation of a 105 Å membrane-spanning channel, the nearly complete (Kintzer et al, 2012). (B) 2D schematic of the secondary structure organization of domain 2 (segment V262-N458) in the prepore (red dash box) and the pore (green dash box) states.…”

A Protective Antigen Mutation Increases the pH Threshold of Anthrax Toxin Receptor 2-Mediated Pore Formation

“…10−14 Studies have revealed that each PA63 monomer of the prepore binds a receptor through two domains. Interaction of PA domain 4 with the receptor is retained throughout the conversion process, whereas domain 2 contacts are lost.…”