Bacillus anthracis

Bacillus anthracis is the etiologic agent of anthrax â€" a common disease of livestock and, occasionally, of humans â€" and the only obligate pathogen within the genus Bacillus. B. anthracis is a Gram-positive, endospore-forming, rod-shaped bacterium, with a width of 1.0â€"1.2 µm and a length of 3â€"5 µm. It can be grown in an ordinary nutrient medium under aerobic or anaerobic conditions.

It is one of few bacteria known to synthesize a protein capsule (poly-D-gamma-glutamic acid). Like Bordetella pertussis, it forms a calmodulin-dependent adenylate cyclase exotoxin known as (edema factor), along with lethal factor. It bears close genotypical and phenotypical resemblance to Bacillus cereus and Bacillus thuringiensis. All three species share cellular dimensions and morphology. All form oval spores located centrally in an unswollen sporangium. B. anthracis spores, in particular, are highly resilient, surviving extremes of temperature, low-nutrient environments, and harsh chemical treatment over decades or centuries.

The spore is a dehydrated cell with thick walls and additional layers that form inside the cell membrane. It can remain inactive for many years, but if it comes into a favorable environment, it begins to grow again. It is sometimes called an endospore because it initially develops inside the rod-shaped form. Features such as the location within the rod, the size and shape of the endospore, and whether or not it causes the wall of the rod to bulge out are characteristic of particular species of Bacillus. Depending upon the species, the endospores are round, oval, or occasionally cylindrical. They are highly refractile and contain dipicolinic acid. Electron micrograph sections show they have a thin outer spore coat, a thick spore cortex, and an inner spore membrane surrounding the spore contents. The spores resist heat, drying, and many disinfectants (including 95% ethanol). Because of these attributes, B. anthracis spores are extraordinarily well-suited to use (in powdered and aerosol form) as biological weapons. Such weaponization has been accomplished in the past by at least five state bioweapons programs â€" those of the United Kingdom, Japan, the United States, Russia, and Iraq â€" and has been attempted by several others.

Historical background

French physician Casimir Davaine (1812-1882) demonstrated the symptoms of anthrax were invariably accompanied by the microbe B. anthracis. German physician Aloys Pollender (1799â€"1879) is also credited for this discovery. B. anthracis was the first bacterium conclusively demonstrated to cause disease, by Robert Koch in 1876. The species name anthracis is from the Greek anthrax (ἄνθραξ), meaning "coal" and referring to the most common form of the disease, cutaneous anthrax, in which large, black skin lesions are formed.

Genome structure

B. anthracis has a single chromosome which is a circular, 5,227,293-bp DNA molecule. It also has two circular, extrachromosomal, double-stranded DNA plasmids, pXO1 and pXO2. Both the pXO1 and pXO2 plasmids are required for full virulence and represent two distinct plasmid families.

pXO1 plasmid

The pXO1 plasmid (182 kb) contains the genes that encode for the anthrax toxin components: pag (protective antigen, PA), lef (lethal factor, LF), and cya (edema factor, EF). These factors are contained within a 44.8-kb pathogenicity island (PAI). The lethal toxin is a combination of PA with LF and the edema toxin is a combination of PA with EF. The PAI also contains genes which encode a transcriptional activator AtxA and the repressor PagR, both of which regulate the expression of the anthrax toxin genes.

pXO2 plasmid

pXO2 encodes a five-gene operon (capBCADE) which synthesizes a poly-γ-D-glutamic acid (polyglutamate) capsule. This capsule allows B. anthracis to evade the host immune system by protecting itself from phagocytosis. Expression of the capsule operon is activated by the transcriptional regulators AcpA and AcpB, located in the pXO2 pathogenicity island (35 kb). Interestingly, AcpA and AcpB expression are under the control of AtxA from pXO1.

Strains

The 89 known strains of B. anthracis include:

• Sterne strain (34F2; aka the "Weybridge strain"), used by Max Sterne in his 1930s vaccines
• Vollum strain, formerly weaponized by the US, UK, and Iraq; isolated from cow in Oxfordshire, UK, in 1935
  • Vollum M-36, virulent British research strain; passed through macaques 36 times
  • Vollum 1B, weaponized by the US and UK in the 1940s-60s
  • Vollum-14578, UK biotesting contaminated Gruinard Island, Scotland, in 1940s
  • V770-NP1-R, the avirulent, nonencapsulated strain used in the BioThrax vaccine
• Anthrax 836, highly virulent strain weaponized by the USSR; discovered in Kirov in 1953
• Ames strain, isolated from a cow in Texas in 1981; famously used in AMERITHRAX letter attacks (2001)
  • Ames Ancestor
  • Ames Florida
• H9401, isolated from human patient in Korea; used in investigational anthrax vaccines

Evolution

Whole genome sequencing has made reconstruction of the B. anthracis phylogeny extremely accurate. A contributing factor to the reconstruction is B. anthracis being monomorphic, meaning it has low genetic diversity, including the absence of any measurable lateral DNA transfer since its derivation as a species. The lack of diversity is due to a short evolutionary history that has precluded mutational saturation in single nucleotide polymorphisms.

A short evolutionary time does not necessarily mean a short chronological time. When DNA is replicated, mistakes occur which become genetic mutations. The buildup of these mutations over time leads to the evolution of a species. During the B. anthracis lifecycle, it spends a significant amount of time in the soil spore reservoir stage, a stage in which DNA replication does not occur. These prolonged periods of dormancy have greatly reduced the evolutionary rate of the organism.

Nearest neighbors

B. anthracis belongs to the B. cereus group consisting of the strains: B. cereus, B. anthracis, B. thuringiensis, B. weihenstephanensis, B. mycoides, and B. pseudomycoides. The first three strains are pathogenic or opportunistic to insects or mammals, while the last three are not considered pathogenic. The strains of this group are genetically and phenotypically heterogeneous overall, but some of the strains are more closely related and phylogenetically intermixed at the chromosome level. The B. cereus group generally exhibits complex genomes and most carry varying numbers of plasmids.

B. cereus is a soil-dwelling bacterium which can colonize the gut of invertebrates as a symbiont and is a frequent cause of food poisoning It produces an emetic toxin, enterotoxins, and other virulence factors. The enterotoxins and virulence factors are encoded on the chromosome, while the emetic toxin is encoded on a 270-kb plasmid, pCER270.

B. thuringiensis is an insect pathogen and is characterized by production of parasporal crystals of insecticidal toxins Cry and Cyt. The genes encoding these proteins are commonly located on plasmids which can be lost from the organism, making it indistinguishable from B. cereus.

Pseudogene

PlcR is a global transcriptional regulator which controls most of the secreted virulence factors in B. cereus and B. thuringiensis. It is chromosomally encoded and is ubiquitous throughout the cell. In B. anthracis, however, the plcR gene contains a single base change at position 640, a nonsense mutation, which creates a dysfunctional protein. While 1% of the B. cereus group carries an inactivated plcR gene, none of them carries the specific mutation found only in B. anthracis.

The plcR gene is part of a two-gene operon with papR. The papR gene encodes a small protein which is secreted from the cell and the reimported as a processed heptapeptide forming a quorum-sensing system. The lack of PlcR in B. anthracis is a principle characteristic differentiating it from other members of the B. cereus group. While B. cereus and B. thuringiensis depend on the plcR gene for expression of their virulence factors, B. anthracis relies on the pXO1 and pXO2 plasmids for its virulence.

Clinical aspects

Pathogenesis

B. anthracis possesses an antiphagocytic capsule essential for full virulence. The organism also produces three plasmid-coded exotoxins: edema factor, a calmodulin-dependent adenylate cyclase, causes elevation of intracellular cAMP, and is responsible for the severe edema usually seen in B. anthracis infections; lethal toxin is responsible for tissue necrosis; protective antigen (so named because of its use in producing protective anthrax vaccines) mediates cell entry of edema factor and lethal toxin.

Manifestations in human disease

Three forms of human anthrax disease are recognized based on their portal of entry.

• Cutaneous, the most common form (95%), causes a localized, inflammatory, black, necrotic lesion (eschar).
• Pulmonary, the highly fatal form, is characterized by sudden, massive chest edema followed by cardiovascular shock.
• Gastrointestinal, a rare but also fatal (causes death to 25%) type, results from ingestion of spores.

Prevention and treatment

A number of anthrax vaccines have been developed for preventive use in livestock and humans. Infections with B. anthracis can be treated with β-lactam antibiotics such as penicillin, and others which are active against Gram-positive bacteria. Penicillin-resistant B. anthracis can be treated with fluoroquinolones such as ciprofloxacin or tetracycline antibiotics such as doxycycline.

Laboratory research

Components of tea, such as polyphenols, have the ability to inhibit the activity both of B. anthracis and its toxin considerably; spores, however, are not affected. The addition of milk to the tea completely inhibits its antibacterial activity against anthrax. Activity against the B. athracis in the laboratory does not prove that drinking tea affects the course of an infection, since it is unknown how these polyphenols are absorbed and distributed within the body.

Recent research

Advances in genotyping methods have led to improved genetic analysis for variation and relatedness. These methods include multiple-locus variable-number tandem repeat analysis (MLVA) and typing systems using canonical single-nucleotide polymorphisms. The Ames ancestor chromosome was sequenced in 2003 and contributes to the identification of genes involved in the virulence of B. anthracis. Recently, B. anthracis isolate H9401 was isolated from a Korean patient suffering from gastrointestinal anthrax. The goal of the Republic of Korea is to use this strain as a challenge strain to develop a recombinant vaccine against anthrax.

The H9401 strain isolated in the Republic of Korea was sequenced using 454 GS-FLX technology and analyzed using several bioinformatics tools to align, annotate, and compare H9401 to other B. anthracis strains. The sequencing coverage level suggests a molecular ratio of pXO1:pXO2:chromosome as 3:2:1 which is identical to the Ames Florida and Ames Ancestor strains. H9401 has 99.679% sequence homology with Ames Ancestor with an amino acid sequence homology of 99.870%. H9401 has a circular chromosome (5,218,947 bp with 5,480 predicted ORFs), the pXO1 plasmid (181,700 bp with 202 predicted ORFs), and the pXO2 plasmid (94,824 bp with 110 predicted ORFs). As compared to the Ames Ancestor chromosome above, the H9401 chromosome is about 8.5 kb smaller. Due to the high pathogenecity and sequence similarity to the Ames Ancestor, H9401 will be used as a reference for testing the efficacy of candidate anthrax vaccines by the Repbulic of Korea.

Host interactions

As with most other pathogenic bacteria, B. anthracis must acquire iron to grow and proliferate in its host environment. The most readily available iron sources for pathogenic bacteria are the heme groups used by the host in the transport of oxygen. To scavenge heme from host hemoglobin and myoglobin, B. anthracis uses two secretory siderophore proteins, IsdX1 and IsdX2. These proteins can separate heme from hemoglobin, allowing surface proteins of B. anthracis to transport it into the cell.

References

External links

• Bacillus anthracis genomes and related information at PATRIC, a Bioinformatics Resource Center funded by NIAID
• Hazards in Animal Research Database - Bacillus anthracis
• Pathema-Bacillus Resource