Mechanism of Anthrax Toxins
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Mechanism of Anthrax Toxins

Anthrax is an acute infectious disease caused by the spore-forming gram positive, aerobic bacterium Bacillus anthracis. Anthrax most commonly occurs in wild and domestic lower vertebrates (cattle, sheep, goats, camels, antelopes, and other herbivores), but it can also occur in humans when they are exposed to infected animals or tissue from infected animals. The disease is initiated by the entry of spores into the host body. This can occur via a minor abrasion, via an insect bite, by eating contaminated meat or inhaling airborne spores. When the bacterium is inhaled, its spores are surrounded by alveolar macrophages in the lung, the beginning stage of normal immune response. But instead of succumbing to the defensive assault, they survive and germinate within the cells, traveling with the macrophages in their normal sentinel duty throughout the body to the lymph nodes, and eventually into the bloodstream, ultimately leading to fatal systemic shock (Ref.1).

There are three types of human infection: cutaneous, gastrointestinal, and inhalational (pulmonary). Each form can progress to fatal systemic anthrax. The cutaneous form, the most common, is first manifested as a small pimple that develops, within a few days, into a black eschar, characteristic of anthrax (Ref.2). The major virulence factors of B. anthracis are a poly-D glutamic acid capsule and a three-component protein exotoxin. The genes coding for the toxin and the enzymes responsible for capsule production are carried on plasmid pXO1 and pXO2, respectively. The three proteins of the exotoxin are PA (Protective Antigen, 83 kDa), LF (Lethal Factor, 90 kDa), and EF (Edema Factor, 89 kDa). Secreted from the bacteria as nontoxic monomers, these proteins assemble on the surface of receptor-bearing eukaryotic cells to form toxic noncovalent complexes (Ref.3). Assembly of the toxic complexes begins when PA binds to a cellular receptor; a widely expressed type 1-membrane protein termed Anthrax Toxin Receptor. Anthrax Toxin Receptor is conserved among many different species and is found in a variety of cell types including neural, cardiac, pulmonary, and lymphocytes. Anthrax Toxin Receptor is a membrane protein with an extracellular Von Willebrand factor A domain that binds directly to PA. Von Willebrand factor is an adhesive protein, which is present in blood and in endothelium. It is responsible for adhesion of platelets to damaged vascular endothelium; these interactions are essential for hemostasis. The factor’s A domain is responsible for adhesion to glycoprotein Ib. It has been observed that addition of the soluble version of this domain can protect cells from the action of the PA toxin (Ref.4). After PA binds to Anthrax Toxin Receptor, it is cleaved by a member of the Furin class of proteases, causing removal of an amino-terminal 20-kDa segment of the protein (PA20), and leaving the carboxyl-terminal 63-kDa fragment (PA63) bound to Anthrax Toxin Receptor. Unlike native PA, PA63 oligomerizes to form a ring-shaped heptamer with Anthrax Toxin Receptor. The heptamer binds up to three copies of EF and/or LF competitively and with high affinity (Ref.5). Whereas native PA persists on the cell surface, the heptamer is endocytosed, presumably because of oligomerization aggregates Anthrax Toxin Receptor. The endocytosed toxic complexes are trafficked to endosomal compartments. In the endosomal compartment, the acidic pH causes a conformational change that inserts PA fragments and releases LF and EF into the cytoplasm. Delivery of EF and LF to the cytosol occurs in concert with channel formation and may involve passage of these proteins through the channel.

EF is a calmodulin-dependent AC (Adenylate Cyclase), the activity of which protects the bacteria from phagocytic destruction. EF combines with PA to form edema toxin (coded by the cya gene of plasmid pX01). EF takes ATP and cleaves two phosphates, reconnecting the remaining one back in a small loop to form cAMP (cyclic AMP). cAMP is an important messenger in cells, often used to relay messages that are sent by hormones. High intracellular levels of cAMP lead to impaired maintenance of water homeostasis and characteristic edema. Edema toxin also inhibits neutrophil function (Ref.6).

LF and PA together form a toxin referred to as lethal toxin (coded by the lef gene on pX01). Lethal toxin is the dominant virulence factor produced by B. anthracis and is the major cause of death of infected animals. LF is composed of four domains: domain I II, III and IV. Domain I binds the membrane-translocating component of anthrax toxin to the PA. LF floods the cell with cAMP, destroying the careful balance normally achieved by hormones (Ref.7). The LF attacks at another sensitive spot. It is a Zn2+-dependent protease that cleaves members of the MAPKK (Mitogen-Activated Protein Kinase Kinase) family near to their amino termini, leading to the inhibition of one or more signaling pathways. Domains II, III and IV, which make up the LF molecule, together form a long deep groove that holds the 16-residue N-terminal tail of MAPKK2 before cleavage. Cleavage and inhibition of the MAPKKs by LF, interferes with MAPK signaling and induces apoptosis. The MAPK pathway serves a very important function in cellular signaling. Members of the MAPK family include the ERKs (Extracellular Signal Regulated Kinase), the JNKs (Jun N-terminal Kinases) and the p38 protein whose enzymatic activity is essential for survival of the macrophage. They also activate many signaling cascades, resulting in the activation of transcription factors and regulate genes involved in inflammation, Cytokineproduction, cell growth, and cell differentiation (Ref.8). Lethal toxin is also thought to stimulate overproduction of Cytokine (e.g., tumor necrosis factor TNF-Alpha and IL-1Beta (Interleukin-1Beta), which lead to lysis of macrophages. Rapid release of inflammatory mediators also may contribute to the sudden death that can occur with anthrax. Several potential mechanisms could be used to block anthrax toxin action, one of which was demonstrated by the design of a multivalent protein inhibitor of toxin interaction with PA.