Anthrax toxin refers to three proteins secreted by virulent strains of the bacterium Bacillus anthracis. These three proteins act together in a synergistic way in which they are endocytosed and translocated into the cytoplasm of a macrophage, where it disrupts cellular signaling and induces cell death, allowing the bacteria to evade the immune system.
The disease known as anthrax is caused by Bacillus anthracis, a spore-forming bacterium whose pathogenesis is primarily the result of a tripartite toxin. This toxin is composed of three proteins: the protective antigen (PA), the edema factor (EF) and the lethal factor (LF). These proteins work together to enter a cell and disrupt the signaling pathways, eventually leading to apoptosis. The molecular actions of PA, EF, and LF also provide a model biochemical system that demonstrates a variety of structure-function relationships seen in biochemistry.
Subscribe in a readerMolecular action of anthrax toxin
The three proteins of the anthrax toxin depend on each other for their toxic effect. Each protein is nontoxic on its own, but when combined, these proteins produce the lethal symptoms of anthrax. When injected in laboratory animals, the combination of EF and LF shows no effect. The combination of PA and EF, however, cause local edema, and PA mixed with LF rapidly leads to death. These studies show that these proteins work synergistically, where EF and LF depend on the presence of PA for the toxic effect.
Shuttle into cell
PA is necessary because both LF and EF function inside cells, but they are too large (90.2 kDa and 88.9 kDa, respectively) to enter via existing channels. Through a series of steps, PA helps to shuttle EF and LF into the cell (. This process starts when the 83 kDa PA (PA83) monomers bind to the largely ubiquitous human tumor endothelium marker-8 (TEM8) or capillary morphogenesis protein 2 (CMG2) receptors. Once bound, a 20 kDa N-terminal fragment (PA20) is cleaved off of PA83 by membrane endoproteases from the furin family, exposing binding sites for LF, EF, and other molecules of cleaved PA. Because of this cleavage the remaining 63 kDa portion (PA63) rapidly oligomerizes to form a heptamer pre-pore, which then associates with up to three molecules of EF and/or LF.The cell then endocytoses the complex and carries it to an acidic compartment, where the low pH causes a conformational change in the PA63 pre-pore that forms a cation-specific channel and allows the EF and LF to enter into the cytosol.
Extracellular toxin structure-function relationship
The mechanism of anthrax toxin action is the result of the molecular structures of the three toxin proteins in combination with biomolecules of the host cell. The molecular interactions are apparent upon performing a detailed analysis of the structures of PA, EF, LF, and the cellular receptors (ANTXR1 and ANTXR2). Structures for the toxin molecules , the receptor,, and for the complexes of the molecules all provided insight on the synergistic actions of these proteins. Analyses on binding sites and conformational changes augmented the structural studies, elucidating the functions of each domain of PA, LF, and EF.
The structure of PA was the first to be determined (Fig. 3).This structure and that of its cellular receptor shed much light on the specificity of recognition and binding. This specificity of PA and the receptor CMG2 (similar to type I integins) is due to interactions through a metal ion dependent adhesion site (MIDAS), a hydrophobic groove, and a β-hairpin projection. These all contribute to a tight interaction in which much protein surface area on CGM2 (and TEM8) is buried.
Petosa et al.solved the structure of a PA63 heptamer at 4.5 Å (0.45 nm). The structure they solved was of a non-membrane bound pre-pore, the conformation of the heptamer before the complex extends a β-barrel through the plasma membrane to shuttle the LF and EF into the cytosol.
Heptamerization and pore formation is sterically hindered by the PA20 fragment, but when it is removed from the top of the monomer, the pre-pore is quickly formed. The heptamer formation causes no major changes in the conformation of each individual monomer, but by coming together, more than 15400 Ã…² (154 nm²) of protein surface is buried. This buried surface consists mostly of polar or charged side groups from domains 1 and 2.
During the heptamerization of PA63, molecules of EF and/or LF rapidly and simultaneously bind to the heptamer pre-pore. This binding occurs because after removing the PA20 domain, a large site is uncovered on domain 1 of PA63. Domain 1 provides a large surface that the interacts with the N-terminus of EF and LF, which is almost completely homologous for the first ~36 residues and similar in tertiary structure for the first ~250 residues. Studies on the binding region of LF and EF demonstrated that a large surface area contacts with domain 1 of two adjacent PA63 molecules when in the heptamer conformation. This large binding area explains why previous studies could only bind up to three molecules on a PA63 heptamer. The LF/EF binding site is now being utilized for delivery of therapeutics via fusion proteins.
Upon formation of the prepore and attachment of LF and/or EF, the heptamer migrates to a lipid raft where it is rapidly endocytosed. Endocytosis occurs as a result of a series of events. This begins when CGM2 or TEM8 is palmitoylated, which inhibits the association of the receptor with lipid rafts. This inhibits the receptor from being endocytosed before PA83 is cleaved and before LF or EF can associate with the heptamer. Reassociation of the receptor with the cholesterol and glycosphigolipid-rich microdomains (lipid rafts) occurs when PA63 binds to the receptor and heptamerizes. Once the receptor and PA returns to the lipid raft, E3 ubiquitin ligase Cb1 ubiquitinates the cytoplasmic tail of the receptor, signaling the receptor and associated toxin proteins for endocytosis. Dynamin and Eps15 are required for this endocytosis to occur, indicating that anthrax toxin enters the cell via the clathrin-dependent pathway.
As discussed, each molecule interacts with several others in order to induce the endocytosis of the anthrax toxin. Once inside, the complex is transferred to an acidic compartment, where the heptamer, still in the non-membrane-spanning pre-pore conformation, is prepared for translocation of EF and LF into the cytosol
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