The roles of anthrax toxin in pathogenesis

Mahtab Moayeri and Stephen H Leppla

Anthrax lethal toxin is a multi-functional virulence factor that has
evolved to target multiple host functions to allow for optimal
establishment of Bacillus anthracis infection. The toxin appears
to play a role in all stages of infection, from germination to the
induction of vascular collapse leading to host death. Early in
infection, at sublethal doses, it acts to suppress immune cell and
cytokine responses, thereby promoting bacterial outgrowth.
Later in the disease, lethal levels of toxin induce the cytokineindependent shock-like death associated with anthrax. The
understanding of the molecular events induced by anthrax toxin
in different target cells at each stage of infection will aid in
deciphering the pathogenesis of this bacterium and developing
therapies.
Addresses
Building 30, Room 307, National Institute of Allergy and Infectious
Diseases, National Institutes of Health, Bethesda, MD 20892-4350, USA
Corresponding author: Stephen H Leppla
e-mail: sleppla@niaid.nih.gov

Current Opinion in Microbiology 2004, 7:1924

This review comes from a themed issue on
Hostmicrobe interactions: bacteria
Edited by Craig Roy and Philippe Sansonetti
1369-5274/$ see front matter
2003 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.mib.2003.12.001

Abbreviations
CMG capillary morphogenesis protein
EF
edema factor
ET
edema toxin
GR
glucocorticoid receptor
LF
lethal factor
LT
lethal toxin
MEK mitogen-activated protein kinase kinase
PA
protective antigen
TEM
tumor endothelial marker
TNF
tumor necrosis factor

Introduction
Bacillus anthracis, the causative agent of anthrax, produces
three polypeptides that comprise anthrax toxin. Protective antigen (PA) binds to cellular receptors, is cleaved by
cellular furin, oligomerizes, and transports lethal factor
(LF, a protease) and edema factor (EF, an adenyl cyclase)
into cells (reviewed in [1,2]; Figure 1). The lethal toxin
(LT, the combination of PA and LF) and edema toxin
(ET, the combination of PA and EF) are sufficient to
produce many of the symptoms of anthrax infection
[3,4,5], and isogenic mutant bacteria lacking single
toxin components are greatly attenuated [6,7]. Recent
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advances provide new clues as to the role these binary

toxins play in anthrax pathogenesis.
Recent work on anthrax toxin has provided a detailed
understanding of the steps involved in toxin entry into
target cells (Figure 1), but will not be the primary focus
of this review. Crystal structures have been obtained for
all three polypeptides [810]. Two related proteins,
tumor endothelial marker 8 (TEM8) and capillary morphogenesis protein 2 (CMG2), have been shown to
function as cellular receptors, although it is not clear
which is relevant in vivo [11,12]. Toxin internalization
occurs via membrane lipid rafts [13] and does not
depend on the intracellular region of TEM8 [14]. A
detailed understanding of amino acid residues involved
in PA binding to receptor, PA oligomerization steps, and
structural/functional aspects of binding to LF and EF
have emerged through numerous studies [1523]. Studies on the interaction of LF with mitogen activated
protein kinase-kinases (MEKs) and MEK substrate
peptides [2426] have begun to explain the highly
restricted specificity of LF and to provide a basis for
design of protease inhibitors. There have also been
notable advances in understanding the contribution to
virulence of the anti-phagocytic poly-glinked-D glutamic acid capsule [27,28], and in regulation of virulence
gene expression [2932]; topics which will not be discussed here.
In this review, we focus on the role anthrax plays at
various stages of establishing anthrax in the host.
Spores, germination and toxin

Inhalational anthrax occurs when inhaled spores are

phagocytosed by alveolar macrophages, carried to local
lymph nodes, and germinate there to produce vegetative
bacteria [3335]. For successful infection, intracellular
bacteria must resist killing by the macrophage. In vitro
studies have described alternative outcomes to the battle
between the macrophages and bacteria. One report
shows that spores germinate and grow to high numbers
within the macrophage, eventually escaping by lysing the
cell [36]. Others have found that germinated spores
survive in macrophages, but cannot multiply within
them. LT and ET are required for this survival and
apparently contribute to the eventual death of the cell
[37]. Welkos and co-workers [38] have also suggested
that increased germination in macrophages could be
associated with increased bacterial killing by the macrophage. Differences in the macrophages and Bacillus
anthracis strains used in these studies might account
for the contrasting results.
Current Opinion in Microbiology 2004, 7:1924

20 Hostmicrobe interactions: bacteria

Figure 1

PA

LF or EF

PA20

PA63

TEM8
or CMG2

Furin

H+

ATP
cAMP

EF

LF

LF
MEKs

Unknown substrate
Current Opinion in Microbiology

Anthrax toxin in cells. Protective antigen (PA) binds anthrax toxin receptors TEM8 or CMG2 and is cleaved by furin. Cleaved PA oligomerizes and
provides binding sites for EF or LF. The complex is endocytosed and LF and EF are translocated to the cytosol after acidification of an intracellular
compartment. EF catalyzes the conversion of ATP to cAMP. LF cleaves members of the MEK family and other potential targets.

The anthrax toxins and antibodies to them can alter the

balance in the struggle between bacteria and the macrophage. Although anthrax toxin has commonly been associated with the final stages of disease, toxin components
are expressed at the spore stage [39] and by newly
germinated spores within macrophages [34,37], suggesting that ET and LT may play a role in very early stages of
anthrax. Early LT expression immediately after germination may promote survival of emergent vegetative cells
and enable release of bacteria into blood by lysing macrophages [37,40]. However, using ET knockout B. anthracis
mutants, a requirement for ET function has also been
shown for survival of germinated bacteria [37]. The recent
recognition that LF lysis of macrophages is restricted to a
few mouse strains and is not seen in other species (see
below) indicates that the roles LT and ET play inside the
macrophage are likely to be more subtle than direct lysis.
Additionally, it should be noted that studies on the role of
toxin in germination are also contradictory. While GuidiRontani et al. [37] have shown that B. anthracis strains
lacking any of the three toxin components are efficiently
killed after germination in macrophages, other studies
using toxin mutant strains indicate no toxin requirement
Current Opinion in Microbiology 2004, 7:1924

for germination, bacterial multiplication within, and lysis

of the macrophage [36]. The role of toxin in germination
and macrophage survival needs to be clarified in different
hosts.
Anthrax toxin is produced by vegetative bacteria, so it is
surprising that PA is also found on the dormant spore
surface by immunogold staining [39]. The presence of PA
on spores appears to be functionally relevant, because
antibody to PA enhances spore phagocytosis and spore
killing by macrophages in vitro [38,39,41]. Opsonic clearance of spores by antibodies to PA might in part explain
the lack of detectable bacteremia in PA-vaccinated
animals following experimental challenge with spores
[39,42]. Furthermore, this potential action of antibodies
to toxin should be considered in vaccine design, where
the current emphasis is on inducing antibodies that
neutralize the toxin.
Lethal toxin and macrophages

LT is a protease that cleaves members of the MEK family

[43,44] and causes rapid lysis of macrophages from some
inbred mouse strains [45,46]. LT-mediated lethality in
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The roles of anthrax toxin in pathogenesis Moayeri and Leppla 21

animal models was previously thought to be due to this

rapid macrophage lysis and/or induction of pro-inflammatory cytokines leading to shock [47], but has recently been
shown to be independent of macrophage sensitivity and
cytokine response [5]. LT does not induce classic
cytokine-mediated shock or inflammatory-mediated
pathology in mice or rats [5]. The resistance of macrophages from humans and other anthrax-susceptible species to lysis by LT also indicates that macrophages do not
play a direct role in LT-induced lethality. Rats, and
especially the Fischer rat, are unique in their extreme
susceptibility to LT, but have macrophages that are not
lysed by LT. Doses of LT that kill mice in 4896 h will kill
Fischer rats in less than one hour [5,48], yet death is
accompanied by the same accumulation of pleural (and to a
lesser extent, peritoneal) fluid commonly observed in LTtreated mice [5] and in human cases of anthrax [4952].
Despite the LT resistance of most species macrophages,
recent studies show that in vitro activation of LT-resistant
mouse macrophages with lipopolysaccharide (LPS) or
treatment with tumor necrosis factor (TNF) renders them
sensitive to killing by LT [53,54]. LT treatment alone
in mice does not activate macrophages (Moayeri and
Leppla, unpublished data) or sensitize them [5], but
components of the B. anthracis cell wall or capsule may
activate and sensitize macrophages in the course of a
normal infection. It is not clear if sensitized macrophages
undergo apoptosis [53], necrosis [54], or both, or if the
molecular mechanism of lysis is the same as in the
inherently LT-sensitive macrophages. Sensitization
requires inhibition of p38 activation and NfkB function
[53], resulting in removal of pathway responses that
inhibit cell death. Involvement of mTor (mammalian
target of rapamycin) kinase in the sensitization pathway
has also been proposed [54].
The dominant genetic locus determining mouse macrophage sensitivity to LT maps to a region on mouse
chromosome 11 that encodes the kinesin Kif1C [55].
The function of this kinesin is unknown and its link to
macrophage lysis remains to be elucidated. LT-mediated
cleavage of the only known LT substrates, the MEKs,
does not appear to be sufficient to cause macrophage
death, because cleavage also occurs in resistant [24,56]
and pharmacologically protected [57] macrophages. However, Kif1C and the cargo it moves along microtubules may
play a role in protection from the consequences of MEK
cleavage in resistant cells and be the deciding factor in LTmediated cell death. How the function or dysfunction of
kinesin relates to TNF- and LPS-mediated sensitization of
resistant macrophages remains to be elucidated.
Toxin and the host

Sensitization of resistant mouse macrophages to LT by

LPS, Gram-positive cell wall components and TNF
[53,54], and sensitization of resistant human macrowww.sciencedirect.com

phages by IFN-g, B. anthracis spores [58] and TNF [54]

suggest that LT-mediated macrophage lysis may play a
role at some stage of anthrax infection. Macrophage lysis
is clearly not required for LT lethality [5]. The
observed inverse correlation between the sensitivity of
an animal to LT and its susceptibility to spore infection
classically associated with anthrax [45,59] also calls into
question the relevance of macrophage lysis by LT in
infection. However, macrophage sensitization and killing
at a later stage in infection could inhibit protective cytokine responses and interfere with the immune response to
B. anthracis. LT also blocks cytokine responses through
shutdown of the MEK pathways [60,61], and recent
studies show that LT impairs host B and T cell immune
responses by its action on dendritic cells [62]. B. anthracis
cell wall components or other materials shed after germination and bacterial spread may induce IL-b, TNF and
IL-6 [58], leading to macrophage activation and sensitization at a later disease stage. Paradoxically, however, LT
would also be cleaving MEKs and thereby shutting down
the cytokine responses required for macrophage activation
and sensitization. It will be important to see if B. anthracis
components can in fact activate macrophages to LT
sensitivity in vivo and if this macrophage sensitivity can
be linked to exacerbation of LT function.
Variability in the extent of lethal toxin susceptibility
between different inbred mouse strains harboring LTsensitive and resistant macrophages suggests that macrophage sensitivity alone is not sufficient in defining a
strains response to toxin (Moayeri and Leppla, unpublished data). Testing of many different inbred mouse
strains and macrophage-deficient mice indicates that
there is not a clear correlation between macrophage
sensitivity and mouse susceptibility to LT (Moayeri
and Leppla, unpublished data). Thus, other genetic elements appear to play a role in determining mouse susceptibility to LT. Virtually all cultured cells tested have
receptors for toxin and therefore many cell types other
than macrophages could be damaged in a toxin-treated or
infected animal. MEK cleavage is expected to have
serious consequences in other cell types, given the central
role the MEK kinase pathways play in cellular physiology.
Endothelial cells are prime candidates as targets for LT
action, as alteration of their barrier function is clearly seen
in the LT-mediated vascular collapse. However, other
cell types are also affected [5]. Future studies in characterizing the role of LT in anthrax will benefit from
analysis of many different cell types and tissues.
Another fascinating recent discovery is the inhibition of
glucocorticoid receptor (GR) function by very low doses
of LT [63]. The GR response is primarily associated with
anti-inflammatory functions. At the simplest level, LTmediated loss of GR function constitutes the inhibition of
another crucial mediator of the immune response. GR
inhibition may play a role in multiple stages of anthrax
Current Opinion in Microbiology 2004, 7:1924

22 Hostmicrobe interactions: bacteria

infection, from promoting establishment of bacteremia to

exacerbating later molecular events induced by LT in
various tissues. GR response differences between rat
strains and among various species may define their relative abilities to resist the initial establishment of a B.
anthracis infection or may alter their susceptibility to the
effects of LT. The Fischer rat, for example, is known to
be a hypersecretor of glucocorticoids in response to infection. At a later stage of infection, the control by glucocorticoids of the host response to hypoxia could be critical,
because hypoxia was a prominent effect seen in LTtreated mice [5]. The link between GR shutdown and
LT pathology is an active area of study.

Conclusions
In summary, anthrax toxin plays a key role in virulence,
with its expression aiding bacterial survival at several
stages of the pathogenic process. Early in infection, both
LT and ET expression are likely to be required at the
spore germination stage to blunt the bactericidal activity
of phagocytes, promoting survival and release from
macrophages. During the initial stages after release from
macrophages, expression of sublethal levels of LT disarms many branches of the immune system through MEK
cleavage, allowing for better survival of bacteria by preventing cytokine responses [58,60,61], dendritic cell
responses, and B and T cell immunity [62]. Production
of ET also incapacitates phagocytes and cytokine pathways through cAMP induction [64,65]. The shutdown of
innate immune responses allows bacteria to evade host
defences so that the infection can progress to an extensive
bacteremia. As bacteremia increases, capsule and bacterial cell wall products shed by dying bacteria may sensitize
macrophages to LT and result in destruction of these cells
by the higher levels of toxin secreted into the blood at
later stages. Thus in these late stages, and after the
macrophage has served its carrier purpose for efficient
spore germination, LT disarms the immune system at a
new level by destruction of macrophages. More importantly, at these stages, LT also induces another set of
lethal molecular events, presumably in currently
unknown target cells, the ultimate result of which is
vascular leakage, systemic hypoxia and shock-like death
[5]. The unique nature of the shock-like death induced
by LT in mice is a starting point for understanding these
molecular events.
It is important to keep in mind that the stage-specific
roles discussed here for sublethal and lethal doses of LT
may only be applicable to the mouse and its macrophages,
which are the experimental models most easily and
extensively studied. The many differences seen in susceptibility to spore infection and the course of disease
among species could provide important clues for understanding how this bacterium functions in humans. A
recent study of the pathology of anthrax in cynomolgus
monkeys again confirms widespread hemorrhages, vascuCurrent Opinion in Microbiology 2004, 7:1924

litis and edema, similar to that found in human infections,

but absent in LT-treated mice [5,66,67,68,69]. This
contrast in pathology emphasizes the importance of
studying the whole organism and its entire arsenal, such
as ET, capsule and other virulence factors, in numerous
animal models. Alternatively, the value of a reductionist
approach that looks at the effects of factors individually
cannot be discounted. For example, the fascinating 40min LT-mediated death of the Fischer rat with vascular
leakage manifestations similar to those seen after 72 h or
longer in mice and other species may provide the simplest
model for understanding LT function. In the Fischer rat,
sensitization of macrophages to LT lysis, induction or
shutdown of cytokines, and induction of any major organ
pathology are irrelevant. Rapid occurrence of a series of
events in the rat, with very similar final consequence
(pleural fluid accumulation and vascular collapse) to other
species indicates that LT-mediated lethality may, in the
end, be due to a simple molecular event induced directly
by this toxin on a specific cell type and that the Fischer rat
may provide a model in which the initiating events can be
deciphered.
Future studies on B. anthracis and its virulence factors will
likely need to rely more and more on in vivo models to
achieve a full understanding of pathogenesis. This is not a
new idea in this field, as discovery and characterization of
anthrax toxin by H Smith in the early 1950s was the result
of in vivo studies, and he has often reminded researchers
of the value of studying pathogenic bacteria and their
virulence factors in vivo [70,71].

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