Bacillus anthracis and Anthrax (page 3)
(This chapter has 5 pages)
© Kenneth Todar, PhD
Anthrax is primarily a disease of domesticated and wild animals,
herbivorous animals, such as cattle, sheep, horses, mules and goats.
become infected incidentally when brought into contact with diseased
which includes their flesh, bones, hides, hair and excrement.
The natural history of Bacillus anthracis is obscure.
the spores have been found naturally in soil samples from around the
the organisms cannot be regularly cultivated from soils where there is
an absence of endemic anthrax. In the United States there are
areas of infection in South Dakota, Nebraska, Arkansas, Texas,
Mississippi, California and small areas that exist in other states.
endemic areas, anthrax occurs irregularly, often with many years
In the United States, the incidence of naturally-acquired anthrax is
extremely rare (1-2 cases of cutaneous disease per year). Worldwide,
incidence is unknown, although B. anthracis is present in most
the world. Unreliable reporting makes it difficult to estimate the true
incidence of human anthrax worldwide. However, in fall 2001, 22 cases
anthrax (11 inhalation, 11 cutaneous) were identified in the United
following intentional contamination of the mail.
The most common form of the disease in humans is cutaneous
which is usually acquired via injured skin or mucous membranes. A minor
scratch or abrasion, usually on an exposed area of the face or neck or
arms, is inoculated by spores from the soil or a contaminated animal or
carcass. The spores germinate, vegetative cells multiply, and a
gelatinous edema develops at
the site. This develops into papule
12-36 hours after infection. The papule changes rapidly to a vesicle,
a pustule (malignant pustule),
and finally into a necrotic ulcer
infection may disseminate, giving rise to septicemia. Lymphatic
also occurs within seven days. In severe cases, where the blood stream
is eventually invaded, the disease is frequently fatal.
Another form of the disease, inhalation anthrax (woolsorters'
disease), results most commonly from inhalation of spore-containing
where animal hair or hides are being handled. The disease begins
with high fever and chest pain. It progresses rapidly to a systemic
pathology and is often fatal if treatment cannot stop the invasive
of the infection.
Gastrointestinal anthrax is analogous to cutaneous anthrax
occurs on the intestinal mucosa. As in cutaneous anthrax, the organisms
probably invade the mucosa through a preexisting lesion. The bacteria
from the mucosal lesion to the lymphatic system. Intestinal anthrax
from the ingestion of poorly cooked meat from infected animals.
anthrax is rare but may occur as explosive outbreaks associated with
of infected animals. Intestinal anthrax has an extremely high mortality
Meningitis due to B. anthracis is a very rare
that may result from a primary infection elsewhere.
Pathogenicity of Bacillus anthracis
Bacillus anthracis clearly owes its pathogenicity to two
determinants of virulence: the formation of a poly-D-glutamyl capsule,
which mediates the invasive stage of the infection, and the production
of the multicomponent anthrax toxin
which mediates the toxigenic stage.
Bacillus anthracis forms a single antigenic type of capsule
consisting of a poly-D-glutamate polypeptide. All virulent strains of B.
anthracis form this capsule. Production of capsular material is
with the formation of a characteristic mucoid or "smooth" colony type.
"Smooth" (S) to "rough" (R) colonial variants occur, which is
with ability to produce the capsule. R variants are relatively
Capsule production depends on a 60 megadalton plasmid, pX02;
transfer to nonencapsulated B. anthracis via transduction
the encapsulated phenotype.
Figure 8. Two microscopic
to demonstrate the presence of the poly-D-glutamyl capsule of Bacillus
anthracis. Left. India ink capsule outline 1000X. Right a
antibody is reacted specifically with the capsular material which
the capsule fluorescent - FA stain 1000X.
The poly-D-glutamyl capsule is itself nontoxic, but functions to
the organism against complement and the bactericidal components of
and against phagocytic engulfment and destruction. The capsule plays
its most important
role during the establishment of the infection, and a less significant
role in the terminal phases of the disease, which are mediated by the
The poly-D-glutamyl capsule is formed in vivo or in the laboratory
the bacterium is grown on serum plates in a 5% CO2
The capsular material can be detected by the McFadyean reaction which
staining with polychrome methylene blue. Blue rods in a background of
capsular material is a positive test (Figure 9). Neither B.
nor B. thuringiensis synthesizes this capsular polymer, so the
of capsular material can be used to distinguish B. anthracis
its closest relatives.
Figure 9. McFadyean's reaction
showing short chains of Bacillus anthracis cells lying
amorphous, disintegrated capsular material. White blood cells can also
The toxigenic properties of Bacillus anthracis were not
until 1954. Prior to that time, because of the tremendous number of
bacilli observed in the blood of animals dying of the disease (109
bacteria/ml), it was assumed that death was due to blockage of the
popularly known as the "log-jam" theory. But experimentally it was
that only about 3 x 106 cells/ml are necessary to cause
of the animal. Furthermore, the cell-free plasma of animals dying of
infection contained a toxin which causes symptoms of anthrax when
into normal guinea pigs. These observations left little doubt that a
exotoxin plays a major role in the pathogenesis of anthrax.
One component of the anthrax toxin has a lethal mode of the
that is not entirely understood at this time. Death is apparently due
depletion, secondary shock, increased vascular permeability,
failure and cardiac failure. Death from anthrax in humans or animals
occurs suddenly and unexpectedly. The level of the lethal toxin in the
circulation increases rapidly quite late in the disease, and it closely
parallels the concentration of organisms in the blood.
Production of the anthrax toxin is mediated by a
pX01, of 110 megadaltons. The toxin consists of three distinct
components. Each component of the toxin is a thermolabile protein with
a mw of approximately 80kDa.
Factor I is the edema factor (EF) which is necessary for the
edema producing activity of the toxin. EF is known to be an inherent
adenylate cyclase, similar to the Bordetella pertussis
Factor II is the protective antigen (PA), because it induces
protective antitoxic antibodies in guinea pigs. PA is the binding
domain of the anthrax toxin which has two active (A) domains, EF
and LF (below).
Factor III is known as the lethal factor (LF) because it is
for the lethal effects of the anthrax toxin. Apart from their
each of the three factors exhibits no significant biological activity
an animal. However, combinations of two or three of the toxin
yield the following results in experimental animals.
PA+LF combine to produce lethal activity
EF+PA produce edema
EF+LF is inactive
PA+LF+EF produces edema and necrosis and is lethal
These experiments suggest that the anthrax toxin has the familiar
enzymatic-binding structure of bacterial exotoxins with PA acting as
B fragment and either EF or LF acting as the active A fragment.
EF+PA has been shown to elevate cyclic AMP to extraordinary levels
susceptible cells. Changes in intracellular cAMP are known to affect
in membrane permeability and may account for edema. In macrophages and
neutrophils an additional effect is the depletion of ATP reserves which
are needed for the engulfment process. Hence, one effect of the toxin
be to impair the activity of regional phagocytes during the infectious
The effects of EF and LF on neutrophils have been studied in some
Phagocytosis by opsonized or heat-killed Bacillus anthracis
is not inhibited by either EF or LF, but a combination of EF + LF
engulfment of the bacteria and the oxidative burst in the pmns. The two
toxin components also increased levels of cAMP in the neutrophils.
studies suggest that the two active components of the toxin, EF + LF,
increase host susceptibility to infection by suppressing neutrophil
and impairing host resistance.
LF+PA have combined lethal activity as stated above. The lethal
is a Zn++ dependent protease that induces cytokine
in macrophages and lymphocytes, and its mechanism of action is slowly
understood. The crystal structure of lethal factor is known to to
be a member of the
protein kinase (MAPKK) family of enzymes that disrupts cellular
Furthermore, the identity of the human receptor for anthrax PA, named anthrax
toxin receptor, has been demonstrated to be a type I membrane
that binds directly to PA.
In summary, the virulence of Bacillus anthracis is
to three bacterial components; 1. Capsular material composed of
poly-D-glutamate polypeptide; 2. EF component of exotoxin; 3. LF
component of exotoxin. Both the capsule and the anthrax toxin may play
a role in the early
stages of infection, through their direct effects on phagocytes.
anthrax bacilli multiply at the site of the lesion. Phagocytes migrate
to the area but the encapsulated organisms can resist phagocytic
or if engulfed, can resist killing and digestion. A short range effect
of the toxin is its further impairment of phagocytic activity and its
effect on leukocytes, including phagocytes, at the site. After the
and their toxin enter the circulation, the systemic pathology, which
be lethal, will result.
Bacillus anthracis coordinates the expression of its
factors in response to a specific environmental signal. Anthrax toxin
and the antiphagocytic capsule are produced in response to growth in
atmospheric CO2. This CO2 signal is thought to be
of physiological significance for a pathogen which invades mammalian