Anthrax toxin: channel-forming activity of protective antigen in planar phospholipid bilayers.

Abstract

The three separate proteins that make up anthrax toxin-protective antigen (PA), edema factor (EF), and lethal factor (LF)-act in binary combinations to produce two distinct reactions in experimental animals: edema (PA + EF) and death (PA + LF). PA is believed to interact with a membrane receptor, and after proteolytic processing, to mediate endocytosis and subsequent translocation ofEF or LF into the cytosol. PA can be separated, after mild trypsinolysis, into two fragments, PA6s (65 kDa) and PA" (20 kDa). We demonstrate that trypsin-cleaved PA is capable of forming cationselective channels in planar phospholipid bilayer membranes and that this activity is confined to the PAO1 fragment; PA20, LF, and EF are devoid of channel-forming activity. These PA65 channels exhibit pH-dependent and voltage-dependent activity-a property reminiscent of the channels formed by the two-chain proteins diphtheria, tetanus, and botulinum toxins.The pathogenesis ofBacillus anthracis, the causative agent of anthrax, depends on two important virulence factors: an antiphagocytic poly(D-glutamic acid) capsule and "anthrax toxin." The latter term refers collectively to three proteins encoded by plasmid pXO1 (1): edema factor (EF; 89 kDa), lethal factor (LF; 83 kDa), and protective antigen (PA; 85 kDa), the last so named because of its use in vaccines to generate antibodies which protect against anthrax infection. These three toxin components have no known biological effect when administered individually to experimental animals, but they act in binary combinations to produce two distinct reactions. Intradermal coinjection of PA and EF (a combination termed "edema toxin") produces edema, while coinjection of PA and LF (a combination termed "lethal toxin") causes death in susceptible animals. EF has been shown to be a calmodulin-dependent adenylate cyclase (2), whereas the molecular action of LF remains unknown. EF must penetrate to the cytosolic compartment to contact calmodulin and substrate ATP, and it is generally assumed that LF also penetrates to the cytosol, where it is believed to inactivate an essential cellular component. Although the mechanism(s) by which EF and LF are translocated to the cytoplasm is poorly understood, PA clearly plays a central role. Leppla and coworkers (4, 5) have proposed a mechanism of toxicity in which (i) PA binds to a receptor on the cell surface; (ii) a small N-terminal piece of PA (PA20; 20 kDa) is removed proteolytically, leaving the larger C-terminal piece (PA65; 65 kDa) bound to the receptor; (iii) EF or LF binds to PA65; (iv) the receptor-PA-EF or receptor-PA-LF complex undergoes receptor-mediated endocytosis; and finally, (v) EF or LF (within an endocytic or derivative vesicle) undergoes membrane translocation and is released into the cytosol.Recent studies on LF (6) suggest that toxicity requires passage through an acidic vesicle, a step reminiscent of the pathway leading to intoxication by diphtheria, tetanus, and botulinum toxins. Indeed, the entire sequence of events is quite analogous...

Alternative Splicing in Class V Myosins Determines Association with Rab10

“…Channels formed by toxin A in lipid bilayers had a single-channel conductance of about 1 to 2 nanosiemens in 1 M KCl. This result suggests that toxin A-induced channels have approximately the same dimensions as channels formed by porins (57) or by the binding components of C2 and anthrax toxins (53,58). It is noteworthy that toxin A and toxin B insert into artificial bilayers in the absence of any specific receptors.…”

Evidence that translocation of anthrax toxin's lethal factor is initiated by entry of its N terminus into the protective antigen channel

“…The fact that the translocation and channel-blocking activities occur in a pH range in which the side chain of His is expected to be protonated suggests a role for electrostatic potential. The lumen of the prepore is negatively charged, and presumably so is the cation-selective channel (8,9). A positively charged N terminus might be attracted into the channel by electrostatics, even in the absence of an applied voltage, and indeed we have shown this (A.F., unpublished results).…”

“…The assembled complexes are endocytosed and trafficked to an acidic compartment, believed to be the endosome. There, the acidic pH induces a conformational rearrangement in (PA 63 ) 7 , allowing it to form an aqueous transmembrane channel that mediates translocation of EF and LF to the cytosol (8).…”

Immunogenicity of Bacillus anthracis Protective Antigen Domains and Efficacy of Elicited Antibody Responses Depend on Host Genetic Background

“…The spot ELISA was performed as reported previously (51), with some minor modifications. Briefly, polystyrene plates were coated with 1 g of rPA domain proteins in coating buffer (20 mM K 2 HPO 4 , 10 mM KH 2 PO 4, 1 mM sodium EDTA, 0.8% NaCl, 0.01% NaN 3 ) for 2 h at 37°C in 10% CO 2 and blocked overnight at 4°C with 2% BSA in Tris-buffered saline (TBS). Control wells were coated without rPA.…”