A Microbial Biorealm page on the genus Bacillus anthracis
Higher order taxa
Bacteria (domain); Firmicutes (phylum); Bacilli (class); Bacillales (order); Bacillaceae (family); Bacillus (genus); Bacillus cereus group (Wheeler, D.).
Description and significance
Bacillus anthracis is a Gram-positive, rod-shaped bacterium, 1 - 1.2µm in width and 3 - 5µm in length. It lives in soils worldwide at mesophilic temperatures [#http://microbewiki.kenyon.edu/index.php/Bacillus_anthracis#cite_note-9]. It can be grown in aerobic or anaerobic conditons (facultative anaerobe) in a medium with essential nutrients, including carbon and nitrogen sources (7). In 1877, this organism was the first to be shown to cause disease by Dr. Robert Koch and verified by Dr. Louis Pasteur. The organism was isolated from sick animals and grown in the laboratory to study endospore formation. It is similar to Bacillus cereus, Bacillus subtilis, and Bacillus thuringiensis in cellular size, morphology, and spore formation (7).
Bacillus anthracis is an important organism to study genome sequence because it's used as a biological weapon (1). Genome sequencing can also be useful for the development of vaccines. The interactions between the host's immune system cells and the spores are an important area of research that will give us a better understanding of the anthrax disease. Development of better spore detectors will also be helpful (6).
Other names for this organisms include Bacteridium anthracis and Bacillus cereus var. anthracis. Common names include "anthrax" and "anthrax bacterium" (16).
The single chromosome found in the Bacillus anthracis genome is a circular, 5,227,293 bp DNA molecule (8). The main virulent factors are encoded on two plasmids, pXO1 (189 kb, anthrax toxin) and pXO2 (96 kb, capsule genes). The plasmids are circular, extrachromosomal, double-stranded DNA molecules (11). The toxin is a complex of three plasmid-encoded proteins. Two of the proteins are directly toxic, including LF (lethal factor) and EF (edema factor). High LF levels destroy white blood cells and release the bacterium, while EF increases cyclic AMP levels. The host is more susceptible to infection with EF. Energy and water balance is impaired by the increase in cyclic AMP, resulting in the accumulation of fluid in cells. The other plasmid-encoded protein, named PA (protective antigen), ushers the two toxic proteins in cells. PA forms a multimeric ring, which inserts into the cell membranes of the host. PA is not toxic alone, but if it is inactivated, the two toxic proteins would not be harmful. This is because PA allows the toxic components to pass through the membrane via a special toxin delivery system (7). Capsule production depends on the pX02 plasmid. Capsule formation is important because it allows the organism to resist phagocytosis.
Cell structure and metabolism
The vegetative Bacillus anthracis cells are Gram-positive, therefore they contain an extensive peptidoglycan layer, lipoteichoic acids, and crystalline cell surface proteins (S-layer proteins). Bacillus anthracis differs from other Gram-positive bacteria in that it does not contain teichoic acids and the S-layer proteins are not glycosylated. Cell wall polysaccharides function in anchoring the protective S-layer to the cell wall. The cell wall polysaccharides are composed of galactose (Gal), N-acetylglucosamine (Glc-NAc), and N-acetylmannose (ManNAc)in a 3:2:1 ratio (2).
The capsule (slime layer) is a polymer of amino acids (D-glutamate), unlike most other bacteria which have polysaccharide capsules (2). The cells excrete the capsule for protection and virulence. The capsule and the S-layer are compatible, but they can both be formed independently (without the presence of the other). A characteristic mucoid or "smooth" colony variant is correlated with capsule production ability. Virulent strains all form the capsule, and "rough" colony capsules are avirulent. Growth in atmospheric CO2 cause the antiphagocytic capsule and anthrax toxin proteins to be synthesized. The nontoxic capsule has an important role in infection establishment, while the end disease phases are mediated by the toxin (14).
The genome of Bacillus anthracis contains one flagellin gene, however four essential proteins contain point mutations and frameshifts. Therefore, the flagella are nonfunctional and the organism lacks motility. In addition to the pXO1 and pXO2 plasmids, this is what distinguishes Bacillus anthracis from other Bacillus cereus group members (10).
When vegetative cells are deprived of certain nutrients, endospores are formed. Oxygen is necessary for spore formation. Initially, the septum forms asymmetrically in the nutrient deprived cells that produce large (mother cell) and small (forespore) genome containing compartments. The forespore is engulfed by the mother cell and surrounded with three layers (cortex, coat, and exosporium), which are simultaneously formed. The thickest and innermost layer is the cortex made of peptidoglycan. The coat, consisting of a large number of different proteins, tightly covers the cortex. The exosporium is a loose-fitting structure that encloses the spore and serves as a source of surface antigens, which are involved in detection and interaction with the soil environment (13). It is composed of an external hair-like nap and a paracrystalline basal layer. The hair-like nap has filaments that are mostly formed by a single collagen-like glycoprotein (called BclA), and the basal layer consists of a dozen different proteins. One of the proteins, BxpB (also called ExsF), is required for the attachment of the hair-like nap to the basal layer. Suppressing spore germination is another one of its roles. Large molecules that are a potential harm are excluded by the exosporium, which also serves as a semipermeable barrier (1).
The mother cell lyses and the spore is released when spore formation is finished. Spores can live in the soil and other inhospitable environments for many years because, once spores have matured, they are resistant to physical and chemical damage. They are highly resistant to heat, cold, dessication, radiation, and disinfectants. Spores germinate and grow as vegetative cells when they find an aqueous environment with the proper nutrients. Small-molecule germinants, including inosine and L-alanine, are recognized by spore receptors and activate germination. The receptors are found within the membrane of the spore that is under the cortex. Spores that enter a host germinate and grow, producing a fatal toxin (1).
Defense mechanisms are necessary for bacteria to survive antimicrobial responses in the macrophage. Some of the antibacterial killing mechanisms include superoxide production by NADPH oxidase, hydrogen peroxide formation, generation of nitric oxide by nitric oxide synthase (NOS 2), defensin synthesis, and cationic protein activation. Superoxide dismutase (SOD) is the enzyme that regulates superoxide levels (9). Arginase, another protein, catalyzes the formation of L-ornithine and urea from L-arginine. Arginase regulates the production of nitric oxide by competing with NOS 2 for L-arginine. It is also involved in metabolite formation, including glutamic acid (9).
Bacillus anthracis lives in soils worldwide. Soil is the main habitat of aerobic, endospore-forming bacilli. Spore formers are ubiquitous. Therefore, when they are isolated from a certain environment, it does not necessarily imply that the specific environment is their habitat. Other organisms that live in aerobic soil include actinomycetes and filamentous fungi. Different members of the Bacillus species that live in the soil are classified as acidophiles, alkaliphiles, halophiles, thermophiles, psychrophiles, denitrifiers, nitrogen fixers, antibiotic producers, and pathogens. Some of the bacilli that live in the soil include Bacillus subtilis (antibiotic producer); Bacillus cereus, Bacillus thuringiensis, and Bacillus larvae (pathogens); Bacillus macerans and Bacillus polymyxa (nitrogen fixers); and Bacillus azotoformans, Bacillus laterosporus, Bacillus licheniformis, Bacillus pasteurii, and Bacillus stearothermophilus (denitrifiers). Many Bacillus species play an important role in degradation of biopolymers (such as starch and protein) and carbon and nitrogen cycles (14).
It is found that Bacillus anthracis organisms are capable of forming biofilms, which are resistant to a broad variety of antibiotics. Biofilms are the cause of many disease. Microorganisms in biofilms associate themselves with surfaces and they are covered by an extracellular matrix. Properties of biofilms are essential for survival and pathogenicity. Bacillus anthracis biofilms cause the anthrax disease. The organism causes disease mainly in ruminants in North America. Humans are rarely directly infected with Bacillus anthracis. Rather, infection in humans generally results from contact with an infected animal (5).
Bacillus anthracis causes the anthrax disease, which represents a complex interaction between the host and parasite. The particles of anthrax that are infectious are the Bacillus anthracis endospores. The organism penetrates into the blood stream and harms the host by producing toxins within the body. The slimy capsule layer that surrounds the organism allows it to resist phagocytosis by white cells (6).
The common disease forms are cutaneous, pulmonary, and gastrointestinal. The cutaneous form is caused by handling contaminated materials, and the pulmonary form is caused by inhalation. Skin abrasions allow spores to enter and cause local lesions by germinating there and developing gelatinous edema. Patients with a cutaneous anthrax disease mostly recover within 10 days, although a few progress to a life-threatening disease. Gastrointestinal anthrax is similar to cutaneous, but occurring on the intestinal mucosa. It is rare and has an extremely high mortality rate. The pulmonary form of the disease results in a higher mortality rate because the organism spreads through circulation. Macrophages in the lung's alveoli take up the spores and permit entry into the body. The infected macrophages lyse and bacteria are released into the blood stream, spreading though circulatory and lymphatic system. This results in septic shock, respiratory distress, and organ failure. Herbivorous animals become infected when they ingest spores from the soil. When humans contact infected animals (including flesh, bones, hides, hair and excrement), they become infected as well. Anthrax is almost never transmitted between people (14).
Until the 20th century, anthrax was a prevalent disease in humans and cattle. It is still an important pathogen in some countries today. Some scholars believe that the Egyptian plagues in the Bible may have been caused by anthrax. However, most people had not heard of anthrax until the recent 2001 scare in the United States. Robert Koch and Louis Pasteur developed a vaccine against anthrax, which was the first infectious disease they studied (Schaechter et al). The vaccines today are not fully effective. However, if the disease is diagnosed soon enough after infection, antibiotic treatment is effective (3). Methods to detect the organism quickly and new vaccines are under development. Because Bacillus anthracis lives in many soils, outbreaks are still reported. In fact, in the upper Midwest of the United States, many farms are under quarantine due to anthrax (Schaechter et al).
During the first stage of inhaled anthrax illness, the symptoms are similar to influenza, including fever, coughing, sore throat, fatigue, sweating, vomiting, diarrhea, headache, nausea, chest pain, and shortness of breath. Symptoms are much more extreme in the second stage, which can result in death in 2-48 hours. The incidence (1-2 cases of cutaneous disease per year) of naturally acquired anthrax is rare in the United States. In fall 2001, intentional contamination of mail resulted in 22 cases of anthrax, of which 11 were inhalation and 11 cutaneous (3).
Research has suggested anthrax protective antigen (PA) can induce cellular lysis in human cells. PA-induced lysis is mediated by human cell receptor TEM8; this receptor is found in epithelial cells of the lungs, skin, and intestines as well as endothelial cells . PA is believed to first dissociate from this receptor and then insert into the membrane for toxicity . Consistent with this idea, TEM8 possesses a low biochemical affinity for PA; low affinity allows reversible binding . PA-induced lysis is believed to result from hyperporation of the cell. As pores compromise osmotic integrity, the cells lysed. No evidence of apoptosis was found during the experiments .
Other than TEM8, only one other human receptor associated with anthrax toxin has been identified: CMG2 . The affinity of CMG2 for PA is almost 1,000 times greater than that of TEM8 . Since it is thought that PA must first dissociate from its receptor before it can insert into the membrane, the high affinity of PA for CMG2 and vice versa prevents dissociation under normal homeostatic conditions. Therefore, according to the model of PA-induced cell lysis, it is unlikely CMG2 would mediate this event .
Application to Biotechnology
The toxin of Bacillus anthracis is used in biological warfare and bioterrorism (5). During the 20th century anthrax was used as a weapon in many countries. It has also been directed toward farm animals for warfare. The significance of anthrax as a terror weapon was realized in 2001. Although small outbreaks can result in a strong response, some people argue that anthrax is not an ideal biological weapon because the organism is not particularly pathogenic. To infect people, a large number of spores are needed. The most effective form of anthrax is a very fine powder. Therefore, to make anthrax a weapon, the preparation needs to be ground into a fine powder. Anticaking agents are necessary as well to prevent clumping of the spores. Bacillus anthracis can be grown easily, but it is important to have special containment facilities and to be careful when working with them. They can be engineered to be resistant to antibiotics even though they are usually sensitive to antibiotics including penicillin and ciprofloxacin (Schaechter).
Very recent research was conducted on the effects of anthrax toxin on cardiac function. The study found that anthrax toxin had direct effects on the cardiovascular system. Hypotensive shock usually occurs in anthrax infection cases. Varying doses of lethal toxin (LeTx) and edema toxin (EdTx) were administered to rats. The onset time, degree of hypotension and mortality was determined. Lethal toxin and edema toxin were both found to induce hypotension, which results in a shock followed by death. In rats that were treated with lethal toxin, the propagation velocity doubled and the left ventricular systolic and diastolic areas increased by 20 percent. This did not occur in rats treated with edema toxin. However, the rats treated with only edema toxin had an increase in heart rate. Therefore, it was concluded that edema toxin causes a reduction in preload, while lethal toxin results in a reduction of systolic function in the left ventricle (15).
One current research study characterizes the microbiology of a bacterium that caused anthrax-like disease and death in four chimpanzees and a gorilla in Côte d'Ivoire and Cameroon. The motility, gamma phage resistance, and penicillin G resistance (in Cameroon isolates) of the atypical isolates differ from that of the typical Bacillus anthracis strains. Toxin and capsule plasmids were present in these isolates, which were similar in size to the pXO1 and pXO2 plasmids in typical Bacillus anthracis. The sequence of the atypical strains were found to resemble that of the typical Bacillus anthracis strain and virulent Bacillus cereus and Bacillus thuringiensis strains (which are uncommon). The study led to the proposal that the atypical isolates share a common ancestor with the classic Bacillus anthracis. Another possibility is that the Bacillus anthracis strain transferred its plasmids to a Bacillus cereus group strain, resulting in the atypical strain (4).
Another experiment analyzed the role of the capsule in infection and suggests a possible treatment for anthrax infection. The poly-D-glutamic acid capsule of Bacillus anthracis, which prevents phagocytosis by the host cells, can be degraded by the use of CapD (a polyglutamic acid depolymerase). The capsule plasmid of Bacillus anthracis encodes CapD. When treated with CapD, the bacterium can be phagocytosed due to the degradation of the capsule. This allows the organism to be killed by the host neutrophils. The extent of capsule degradation and the degree of phagocytosis are dependent (12).
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Edited by Grace Ucar, student of Rachel Larsen, Kit Pogliano, and David Holzhauer