Anthrax
The bacterium Bacillus anthracis produces the three components of anthrax toxin It is a gram positive spore forming rod, meaning that it can survive for long periods of time in more extreme conditions than non-spore forming bacteria.  Furthermore, it can survive in aerobic or anaerobic conditions.  Spores of Bacillus a. can often be found in the soil, even in geographical locations where anthrax is not a widespread problem.
 The incidence of anthrax in humans is quite rare, although the awareness of anthrax as an agent of biological terrorism has been boosted with the recent anthrax scare in the United States.  The most affected organisms are mammals but not humans; rather, the natural occurrences of anthrax are in wild and domestic herbivorous animals such as sheep, cattle, horses, mules, and goats.
 Anthrax can be introduced into the affected organism by three main routes.  Cutaneous anthrax is an infection resulting from the inoculation of an injured area or mucous membrane by anthrax spores.  Inhalation anthrax, the so-called “woolsorters disease” is contracted by the inhalation of spores.  The name is a reference to the susceptibility of those who used to comb sheep wool to anthrax from inhaling spore filled dust.  Gastrointestinal anthrax begins in the intestinal mucosa after ingestion of infected food (e.g. poorly cooked meat).  All of the conditions outlined above often prove fatal, especially if only sub-standard treatment is available.
 
 

The anthrax toxin can more precisely be described as “toxins” (plural) since there are 3 antigenic components: edema factor, protective antigen, and lethal factor.

Lethal factor (LF)

   LF is a 4-domain protein with a molecular mass of 90 kD; it is 100 Å tall and 70 Å wide at the base.  Domain I resides in top of the other three domains and consists of a 12 helix bundle pushed against the face of a four-stranded b-sheet. The only other domain having any contact with domain I is domain II; the interactions between domain I and II can be described as primarily charged polar and water mediated.
Domain II comprises residues 263-297 and 385-550. The structure of this domain resembles VIP2, a domain of a close relative, B. cereus, but it lacks some of the structural features necessary for any catalytic activity (ADP ribosylation in B. cereus).
Domain 3 is composed of a helices arranged in a bundle to create a hydrophobic core.  Five tandem repeats can be found from amino acid residues 282-382.  Domain III makes a hydrophobic contact with domain IV, but makes very little contact with domain II.  Point mutations studies carried out to probe the action of LF indicate that domain III is necessary for catalytic function.
Domain IV (residues 552-576)  also consists of a four-stranded b-sheet pushed against a helix bundle.  In domain IV, however, the bundle contains a Zn2+ ion that is coordinated by a water molecule, 2 His side chains, and a Glu side chain.  A broad, deep groove leading to the Zn2+ catalytic site and formed by the contact between domain II, III, and IV is thought to bind 16 residues of one of its substrates MAPKK-2 (mitogen activated protein kinase 2).  See below for stereoview of active site.

 

Edema Factor (EF)

Edema factor is an approximately 80 kD protein made up of 4 domains; it binds the enzyme CaM (calmodulin) in the host cell.  A chime structure here shows the three globular domains making up the catalytic site of EF.  CA and CB are the names of the first two domains; domain 3 is a helical domain which moves away from CA and CB upon binding of CaM.  The 3 domains making up the catalytic portion of EF have a molecular mass of 58 kD and make up residues 291-800.  Three segments called switches A, B, and C also change conformation drastically when CaM binds.

Protective Antigen (PA)

Protective antigen has a molecular mass of 83 kD.  The monomeric form of PA consists mostly of antiparallel b-sheets.  The four domains of the monomer are domain 1, an amino terminal domain responsible for activation of proteases; domain 2, a heptamerization domain; domain 3, the function of which is unknown; and domain 4, a receptor bindin domain.  The conversion of the monomer to heptamer and the resulting form of the heptamer is described in the next section.

Mechanism of Action

    EF and LF are inactive in the cell unless PA is present, indicating that they cannot enter into the cell to produce any adverse effects without PA.  Indeed, PA is a delvery system for anthrax LF and EF.  The way in which it works is somewhat analogous to that of a drug delivery system which relies on a large ring forming molecule to deliver a drug into the cell.  Once LF and EF are inside a host cell, EF binds CaM and begins to catalyze the conversion of ATP to cyclic AMP, a second messenger normally controlled in a very stringent manner by hormones or other signalling molecules secreted by the host cell; LF is a protease which can bind and cut several members of the MAP kinase family.  The rise in cAMP destroys the delicate balance normally controlled by the cellular machinery, and the cleavage of the MAPKs disables much of the signalling capability of the cell.
    The mechanism by which PA works is basically this: The monmer form of PA undergoes proteolytic activation on the host cell surface, losing a portion called PA20 (20 kD) from the N-terminus and leaving PA63(63 kD).  This loss of PA20 results in the rearrangement into a heptamer of 7 PA63 fragments.  The heptamer form is water soluble and capable of translocating LF and EF across the cell membrane.

    More specifically, the cell membrane protease responsible for cleaving PA20 away from PA63 is called furin; this cleavage occurs after the binding of PA to a cell surface receptor via a 19 residue loop on domain 4.  The formation of the hepatamer occurs with domains 1' (domain 1 after cleavage) and domain 2 on the inside and domains 3 and 4 on the outside of a donut shaped structure.  A large exposed hydrophobic surface on the top of the heptamer provides a sit to which LF and EF could bind.  The mechanism by which PA inserts into the cell membrane has not been fully elucidated.  However, it has been speculated that two B- loops on domain 2 of each monomer assemble into a membrane spanning, hydrophobic B- barrel, allowing for the translocation of LF and EF into the cell.
    Once EF is in the host cell, it binds to CaM and begins to catalyze the reaction of ATP to cAMP.  EF has a catalytic core consisting of 3 domains.  When CaM, a controler of many intracellular processes, binds EF, domain 3 translates 15 Å and rotates about 30o to encircle CaM.  In addition, segments of PA named Switches 1, 2, and 3 change conformation greatly upon CaM binding; these can be viewed as activating EF to catalyze the cyclization of ATP.

    The translocation of LF into the cell leads to the cleavage of MAPKKs (Mitogen Activated Protein Kinase Kinases) near their amino termini.  Although the action of LF in cleaving MAPKKs has been documented by several independant researchers, the cellular effects are still not fully understood.  The cell type most affected by LF is the macrophage, which is a leukocyte responsible for engulfing and destroying bacteria and other foreign organisms or debris.  The result of high levels of B. anthracis in the bloodstream is macrophage lysis and the release of large amounts of NO and TNF-a (Tumor Necrosis Factor-alpha).  Yet, the same investigators have found that the eventual lysis of macrophages is preceded by inhibition of the release of NO and TNF-a at low concentration of B. anthracis in the bloodstream.

Effects
        The effects of anthrax, as you may have guessed, heavily depend on the mode of infection; the common denominator is that all lead to death if untreated.  Cutaneous anthrax is preceded by an incubation period of 2-5 days.  A small papule rapidly develops into a vesicle about 1 cm in diameter and leads to edema in the surrounding tissue.  After about a week, the lesion breaks open and turns into an eschar, the typical black sore associated with anthrax.
    Inhalational anthrax has an incubation period of 1-6 days associated with it.  The movement of B. anthracis spores to the lymph nodes is followed by a rapid decrease in their functional capability.  The symptoms felt before the overwhelming of the lymph nodes are quite nonspecific and includes such things as fever, cough, and fatigue lasting a few days.  When the bacteria have spread throughout the body, more severe symptoms including acute respiratory distress, shock, and hypoxemia.  The second stage in which the more severe symptoms develop lasts about 24 hours and usually results in death.
    Gastrointesinal anthrax is the most rare form of infection.  The first symptoms-nausea, vomiting, and diarrhea, and fever-show up a few days after infection.  The bacteria gather in the lymph nodes and eventually invade the whole circualtory system.  Clinical symptoms include GI hemorrhage, renal failure, edema of the stomach/intestine, and eventually death.  A more common form of GI anthrax is oropharyngeal anthrax in which fever and swelling of the neck-among many other symptoms-occurs.
Treatments
        Vaccination against anthrax is a preventative measure that can be taken but has many adverse effects associated with it.  For GI anthrax, large volumes of fluids must be administered in addition to antibiotic treatment.  Normally, anthrax is treated with antibiotics and sometimes with a antibiotic/vaccine combination.  Fluoroquinolines are the preferred method of treatment although penicillins, ciprofloxacin, streptamycin, and tetracycline may be used depending on the application.  For strains of anthrax suspected of being genetically engineered, the preferred method is treatment with ciprofloxacin.  The survival rate of those infected with cutaneous anthrax rather than inhalational or GI anthrax is much higher in the presence of proper treatment, mostly due to the fact that cutaneous anthrax often remains localized.