Anthrax Toxin: Deadly Triumvirate of Anthrax

Jiri Patocka (1), Miroslav Splino (2), Roman Prymula (2), Roman Chlibek (2)

(1) Department of Toxicology, Military Medical Academy, Hradec Kralove and Department of Radiology, Faculty of Health and Social Studies, University of South Bohemia, Ceske Budejovice,
(2) Department of Epidemiology, Military Medical Academy, Hradec Kralove, Czech Republic

Summary
          Anthrax not a common disease for humans is caused by large bacterium, Bacillus anthracis, producing a deadly anthrax toxin. The toxin comprises three proteins, called protective antigen (PA), lethal factor (LF) and edema factor (EF). These three toxic proteins are critical for the deadly effect of the anthrax bacterium, B. anthracis. One of these toxins (PA) allows the other anthrax toxin components to enter cells. The second protein (LF) destroys immune system cells that normally defend the body. This process releases inflammatory molecules that can cause sepsis-related shock and death. The third protein (EF) causes potentially lethal swelling and fluid buildup in the body. By itself, edema factor can be deadly. It also makes lethal factor 10 to 100 times more potent. All these proteins create deadly triumvirate, miniature molecular machine which pierce into cells and kill them.

Introduction
          The anthrax bacillus (Bacillus anthracis), found in 1877 by Robert Koch, was the first bacterium shown to be the cause of a disease [1]. At that time, 126 years ago, anthrax was significant mainly as an economically damaging disease of domesticated animals and in humans was fairly rare; the risk of infection was about 1/100,000. Today anthrax is important agent of biological terrorism and threats to civilization [2].

Bacillus anthracis Characteristics
          B. anthracis is large, non-motile encapsulated gram-positive spore-forming rod, 1-1.2µm in width and 3-5µm in length with square or concave ends. Genotypically and phenotypically it is very similar to Bacillus cereus, which is found in soil habitats around the world, and to Bacillus thuringiensis, the pathogen for larvae of Lepidoptera. The three species have the same cellular size and morphology and form oval spores [3]. B. anthracis is capable of producing aerobic endospores that are highly resistant to dessication and capable of surviving for decades in soil. Soil is the natural reservoir for B. anthracis, but its mere presence in soil does not necessarily result in infection of grazing animals. Under favorable circumstances, spores enter a vegetative phase and multiply to levels high enough to infect grazing herbivores. The stability and infectivity of B. anthracis along with its ability to produce a rapidly lethal pneumonia has drawn attention to its potential as a weapon capable of mass casualties [4,5].

Anthrax Infection Course
          B. anthracis causes three forms of disease: cutaneous, pulmonary and gastro-intestinal [6] (Van Dissel et al., 2001). The pulmonary form is the most dangerous and may lead to death merely one to two days after onset of severe symptoms. Human infection is acquired from infected animals, and is therefore usually an occupational disease of farmers, slaughterers, woolworkers etc. In these cases the infectious spores in the animal's hair, meat, bones or secretions are the source of the disease. In over 95% of cases the infection is dermal, due to inoculation of spores into small abrasions on the skin. Anthrax of the skin is the commonest form of the disease.
          Although humans are less susceptible to anthrax than most herbivores, large inhaled doses of spores can produce serious pneumonia, and lead to devastating blood-borne infection. Particles between one and five microns in diameter can be inhaled and lodge in the lungs larger particles are trapped in the nose and pharynx, and smaller particles are expelled with the breath. Illness usually occurs two or three days after exposure, though longer incubation periods can follow mild degrees of exposure. For the first few hours there are influenza-like symptoms with aches and pains, fever and increasing cough and shortness of breath. The symptoms then progress rapidly to severe cough, collapse and respiratory failure, often with a fatal outcome in two or three days. A direct transmission of pulmonary anthrax from man to man has not been proved. At the beginning of the 1950s, an offensive research of B. anthracis was conducted together with preparation of possible use of anthrax spores as a biological weapon for its specific biological properties [7].

Anthrax as a Weapon
          The tendency used of Bacillus anthracis as biological weapon is very old and fall with period between World War I and II. German agents attempted to disseminate B. anthracis among cattle and military horses and mules in Argentina, Romania, Spain and USA even during World War I [8]. Anthrax was in the biological arsenal of Japan Unit 731, headed by General Shiro Ishii, from 1932 to 1945 in China and first experiments with anthrax were carried out on Chinese prisoners in occupied Manchuria. But the Chinese civilian population was also involved. It is estimated that 2,000 prisoners and 700 civilians died from anthrax attacks [9]. In 1942 and 1943 the British tested anthrax bombs on Gruinard Island in Scotland [10]. In 1943 and 1944 U.S., Canadian, and British laboratories produced anthrax-filled cakes and bombs to infect livestock and humans, fortunately they were never used. The USA reaffirmed its position on biological weapons when Roosvelt stated that the U.S. would not use biological weapons, unless they were first used by its enemy [11]. Hitler's Germany conducted offensive anthrax rersearch in a secret laboratory near Poznan (Posen-Nesselstedt on the territory of occupied Poland) but the end of the war came earlier than results could be applied in practice [12].
          After World War II, anthrax research continued in several places. In 1979 an epidemic of pulmonary anthrax broke out in the Russian city Sverdlovsk (now Yekaterinburg). The Soviets claimed that the outbreak was caused by contaminated meat sold on the black marked and refused to allow an international inspection team into the area. Lastly was concluded that the outbreak resulted from an anthrax aerosol, which accidentally escaped from the top-secret military medical laboratory. In this outbreak at least 68 people died and about two hundred fell ill [13, 14]. It was reported that the first clinical symptoms appeared up to 43 days after the initial exposure. The exact date of exposure was only estimated and never exactly confirmed. The modal incubation period in that outbreak was reported to be 9 - 10 day [15]. After the Gulf War in 1995 Iraq was convicted of offensive research, production, weaponizing and storage of anthrax in rockets and bombs [16]. These facilities were destroyed after the Gulf War (1990-1991). Also Japanese religious sect Aum Shinrikyo, who attacked with sarin the Tokyo subway system and killed at least 12 people and injured about 5,500 others, attempted several times to kill people by anthrax aerosol in different places in Japan between 1990 and 1995. Fortunately, these attempts were without success [17]. In the USA, anthrax was misused by terrorists after 11 September 2001. The first inhalation case was reported on 4 October 2001, and the last one on 31 October, 2001 [18].

Hazardous Anthrax Spores
          A possibility of terrorist misuse of anthrax spores presupposes deliberate primary or secondary aerosolization of B. anthracis spores. The secondary aerosolization results from agitation of settled spores, e.g. by contaminated dust, human activities, handling animals, etc. The inhalation dose for anthrax spores in humans is not precisely known. The estimated infectious dose by the respiratory route required to cause inhalation anthrax in humans is 8,000 - 50,000 spores based on the data from studies on primates [19]. The aerosol particles larger than 5 microns quickly subside and form a possibility of a secondary aerosol formation. Smaller aerosol particles (1 - 5 microns in diameter) behave as gas and persist in the environment without settling. The reported incubation period for inhalation anthrax ranges from 1 to 43 days [13].
          The incubation period after inhalation of B. anthracis spores is related to the dose. A post-exposure prophylaxis during the outbreak may prolong the incubation period. The experimental results of laboratory animals suggest that B. anthracis spores convert into a vegetative form in the host for several weeks post-infection. The inhaled spores in the alveolar spaces of lungs are phagocyted by pulmonary macrophages. They gradually germinate, proliferate, and multiply. A prophylactic administration of antibiotics does not affect the germinating spores, but only the vegetative germs of B. anthracis. After discontinuation of antibiotics in the non-germinated spores (if sufficiently numerous) new germination occurs, gradual impairment of the immune mechanisms appears, and anthrax develops. The lethality in untreated inhalation anthrax reaches 86 - 97 % [19].

Anthrax Toxin Characterization
          The toxigenic properties of B. anthracis were discovered in 1954. Prior to that time, it was assumed that death was due to blockage of the capillaries by a large amount of spores. But experimentally it was shown that only about 3 x 106 cells.ml-1 is necessary to cause death of the animal. These observations led to the conviction that a specific anthrax exotoxin plays a major role in the pathogenesis of disease. Death from anthrax in humans or animals frequently occurs suddenly and unexpectedly. The level of lethal toxin in the circulation increases rapidly quite late in the disease, and it closely parallels the concentration of organisms in the blood.
          The production of anthrax toxin is mediated by plasmid pX01, of 110 MDa [20]. Anthrax toxin is not a single compound, but it is the complex substance composed of three antigenically distinct proteins. These are termed (for historic reasons) factor I, edema factor (EF), factor II, protective antigen (PA) and factor III, lethal factor (LF) [21] (Lacy and Collier, 2002). Each component of the toxin is a thermolabile protein with a molecular mass of approximately 80 kDa. The EF and the LF are internalized into the eukaryotic target cells via the protective antigen [22]. Apart from their antigenicity, each of the three factors exhibits no significant biological activity in an animal. However, combinations of two or three of the toxin components yield a lethal combination.
          The proteins attack human cells as a team. One protein - protective antigen - binds to a receptor on the cell surface where it is cleaved by enzymes. The part of PA that remains stuck, called PA63, provides a docking site for the other anthrax proteins - lethal factor and edema factor. Once assembled, the toxin enables lethal factor to enter the cell. There it chops up proteins, setting into motion the chain of events that leads to anthrax's symptoms. This triad makes blood pressure plummet, causes hemorrhaging, and can lead to coma and death.
          Toxic enzymes are compact cell-killing machines. Once inside the cell, they hop from molecule to molecule, destroying each in turn. These molecules are so effective that in some cases a single molecule can kill an entire cell.

Edema factor (EF)
          EF is necessary for the edema causing activity of the toxin. EF from B. anthracis is 800 amino acids (minus 33 signal sequence) in size, 89 kDa secreted adenylate cyclase exotoxin and is activated by the host -resident protein calmodulin [23]. A truncated form of the enzyme lacking the aminoterminal 290 amino acids is sufficient to reproduce the calmodulin-activated adenylate-cyclase activity. The activated EF is able to generate cyclic AMP. This messenger quickly reaches abnormally high levels, blocking them from performing their usual role of sending out cytokines to alert the immune system to a bacterial invasion [24]. Calmodulin is a ubiquitous intracellular calcium sensor in eukaryotes and activates edema factor nearly 1000-fold upon binding [25]. EF is known to be an inherent adenylate cyclase, which leads to an impairment of host defenses, similar to the Bordetella pertussis adenylate cyclase toxin. The edema toxin rises by binding of PA to EF (PA+EF) and this complex induces edema [26].

Protective antigen (PA)
          PA is produced in a precursor form as a protein of 764 amino acids, 85.8 kDa protein, and induces protective antitoxic antibodies in guinea pigs [27]. Cleavage of a 29 residue signal sequence releases the mature 83 kDa protein (PA83). In this state PA83 is inactive and binds to specific cell surface receptors. From the 83 kD PA molecule a domain 20 kD in size is cleaved off by a host protease, furin, to a 63-kDa fragment forms (PA63) [28]. As shown in Fig.1, seven molecules of the PA toxin subunit bind to one another to form a pre-pore on the cell's surface [29]. The other toxin subunits, LF and EF, bind to the pre-pore with high affinity (Kd 1 nM). At the same time PA form a barrel-shaped structure with a docking site for either EF or LF, creating channel in the cell's membranes which allows these two factors to enter the cytoplasm of the target cell [30]. After internalization by receptor-mediated endocytosis, the complexes are transported to the endosome. In the acid environment of the endosome, the pre-pore inserts into the membrane and mediates translocation of EF and LF to the cytosol, where they destroy the cell [31]. EF is an adenylate cyclase that has an inhibitory effect on professional phagocytes, and LF is a protease that acts specifically on macrophages, causing their death and the death of the host. PA and LF act together to form lethal toxin (LT) which is primarily responsible for the death of infected animals.
          Anthrax LT is a virulence factor responsible for the major pathologies seen during systemic anthrax infections. Injection of sterile LT into test animals mimics the shock and sudden death seen during active bacterial infections. Once large levels of LT are produced within the body, destruction of bacteria by administration of antibiotics is usually unsuccessful. About the LT is believed to be secreted into the bloodstream where it circulates freely throughout the body, and binds and enters host cells. Once in the cytoplasm, the lethal factor acts as a zinc-metallo-protease disrupting normal homoeostatic functions. Low levels of LT induce macrophage production, in vitro, of the shock-inducing cytokines TNF and Il-1beta. Higher levels of LT cause over-production of reactive oxygen intermediates, bursting of macrophages and release of shock mediators [32].
          The LF acts as a protease, which inactivates the mitogen-activated protein kinase (MAPK) pathway used by cells to control cell growth, maturation, and development. LF appears to proteolytically cleave MAPK-activating enzymes (MAPK kinase 1, MAPKK1 and MAPKK2) leading to inhibition of the MAPK pathway.

Lethal factor (LF)
          LF is essential for the lethal effects of the anthrax toxin. LF is a protein of 809 amino acids (minus 33 signal sequence), relative molecular mass 90 kDa, that is critical in the pathogenesis of anthrax [33]. This protein is a highly specific zinc dependent metalloprotease that cleaves members of the mitogen-activated protein kinase kinase (MAPKK) family near their amino termini, leading to the inhibition of one or more signaling pathways. The cleavage of this class of proteins occurs in the N-terminal proline-rich region preceding the kinase domain. Protein-protein interactions neccessary for the assembly of signaling complexes are thus disrupted. LF comprises four domains:

• PA+LF combine to produce lethal toxin
• EF+PA combine to produce edma toxin
• EF+LF is inactive
• PA+LF+EF produces edema and necrosis and is lethal

          These experiments suggest that the anthrax toxin have the familiar A-B enzymatic-binding structure of bacterial exotoxins with PA acting as the B fragment, and either EF or LF acting as the active A fragment [34].
          Combination EF+PA has been shown to elevate cyclic AMP to extraordinary levels in susceptible cells. Changes in intracellular cAMP are known to affect changes in membrane permeability and may account for edema. In macrophages and neutrophils, an additional effect is the depletion of ATP reserves that are needed for the engulfment process. Hence, one effect of the toxin may be to impair the activity of regional phagocytes during the infectious process [35]. It is not yet clear how macrophages trench anthrax toxicity. One idea is that the macrophages are abruptly killed, releasing a flood of cytokines. The cytokines, which cause a useful inflammatory response in small doses, are lethal in giant doses when liable for septic shock. However, the production of cytokines by macrophages after anthrax toxin treatment is not verified enough, and anthrax might kill by some other means.

Anthrax Toxin Receptor (ATR)
          Recently, the human cellular receptor for PA has been identified [36]. It occurs in a considerable amount on the surface of macrophage cell lines cells. When inhaled, B. anthracis spores are ingested by immune-system macrophages in the lungs. The macrophages normally destroy such invaders, but in this case the spores kill the macrophages instead. The bacterium then finds its way into the bloodstream, where it secretes the three-component anthrax toxin. The ATR protein is thought to be the first point of contact between the toxin and the cell. From the discovery of ATR, it is expected to lead to a clearer understanding of how the anthrax toxin enters the cell where it works its destruction and, perhaps, to a strategy for fighting infection in people even after symptoms develop. ATR was identified recently as a type I transmembrane protein with unknown function that contains an extracellular integrin-like inserted (I) domain. Integrin a2ß1 is the collagen receptor on platelets and fibroblasts as well as the receptor for echovirus-1, a human pathogen. ATR is supposed to exhibit the same folding pattern as the integrin a2 I domain [37].
          The ATR I domain contains the toxin binding site, and a soluble form of this domain was shown to serve as an effective antitoxin to protect cultured cells from toxin action. ATR is encoded by the tumor endothelial marker 8 (TEM8) gene, which is selectively up-regulated during blood vessel formation and in tumor vasculature, raising the possibility that this protein normally functions in angiogenesis. Therefore, identification of the cellular receptor for anthrax toxin has made possible new avenues of research in the areas of anthrax pathogenesis, antitoxin development, and cancer biology [38].
          ATR and TEM8 are supposed to originate from the same gene by alternative splicing [37]. A truncated, soluble form of ATR is able to protect cell cultures against the lethal action of anthrax toxin. ATR is expressed in a variety of tissues including the central nervous system, heart, lungs, and lymphocytes. The ATR cDNA codes a protein of 368 amino acids. It is predicted to have a 27 amino acid leader sequence, an extracellular domain of 293 amino acids, a 23 residue transmembrane region, and a short cytoplasmic tail at the C-terminus. It was achieved that 321-343 residues are needed for cell surface anchoring. Further evidence that the cytosolic domain plays no essential role in anthrax toxin action was obtained by showing that TEM8 anchored by a glycosylphosphatidylinositol tail also functioned as a PA receptor [39].
          The virulence of B. anthracis is attributable to three bacterial components: EF component of exotoxin, LF component of exotoxin and capsule component . The EF and LF components have been already characterized. As for capsular material, it is known that B. anthracis forms a single antigenic type of capsule consisting of a poly-D-glutamate polypeptide. All virulent strains of B. anthracis form this capsule [40]. The capsule production depends on a 60 MDa plasmid pX02 [41]. The capsule of B. anthracis, composed of poly-D-glutamic acid, serves as one of the principal virulence factors during anthrax infection. Both the capsule and the anthrax toxin can play a role in the early stages of infection, through their direct effects on phagocytes.

Anthrax Toxin Inhibitors
          Although a vaccine against anthrax exists [42, 43] various factors make mass vaccination impractical [44]. The bacteria can be eradicated from the host by treatment with antibiotics, but because of the continuing action of the toxin, such therapy is of little value once symptoms have become evident. Thus, a specific inhibitor of the toxin's action might prove a valuable adjunct to antibiotic therapy. Mourez et al. [45] (2001) reported the designing and testing of a polyvalent inhibitor of anthrax toxin that binds to heptameric PA63 and blocks its interaction with EF and LF. They identified from a phage-display library [46] a dodecameric peptide that binds to PA63 and weakly inhibits its interaction with the enzymatic moieties of the toxin. When they linked covalently multiple copies of this peptide to a polyacrylamide backbone, they demonstrated that the resulting polyvalent molecule strongly inhibited ligand binding by PA63 and toxin action in cell culture and in an animal intoxication model.

Conclusion

          Bacillus anthracis, the microbe that causes anthrax, produce three proteins that combine to form a toxin. The proteins attack human cells as a triumvirate team. One protein-protective antigen (PA) - binds to a receptor on the cell surface and is cleaved by enzymes there. The part of PA that remains stuck, called PA63, provides a docking site for the other anthrax proteins - lethal factor and edema factor. Once assembled, the toxin enables lethal factor to enter the cell. This deadly triumvirate makes blood pressure plummet, causes hemorrhaging, and can lead to coma and death. These findings could lead to an antidote to the anthrax toxin and help clarify the mechanism by which it kills.

Acknowledgements

This study was supported by the grant of Ministry of Defence of the Czech Republic, project No. 6602129911

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