Anthrax Lethal Toxin Disrupts Intestinal Barrier Function

and Causes Systemic Infections with Enteric Bacteria

Chen Sun
1
, Hui Fang
1
, Tao Xie
1
, Roger D. Auth
1
, Nayana Patel
2
, Patrick R. Murray
2
, Philip J. Snoy
{3
,
David M. Frucht
1
*
1Laboratory of Cell Biology, Division of Monoclonal Antibodies, Office of Biotechnology Products, Center for Drug Evaluation and Research, United States Food and Drug
Administration, Bethesda, Maryland, United States of America, 2Department of Laboratory Medicine, Warren Magnusen Clinical Center, National Institutes of Health,
Bethesda, Maryland, United States of America, 3Division of Veterinary Services, Center for Biologics Evaluation and Research, United States Food and Drug Administration,
Bethesda, Maryland, United States of America
Abstract
A variety of intestinal pathogens have virulence factors that target mitogen activated protein kinase (MAPK) signaling
pathways, including Bacillus anthracis. Anthrax lethal toxin (LT) has specific proteolytic activity against the upstream
regulators of MAPKs, the MAPK kinases (MKKs). Using a murine model of intoxication, we show that LT causes the dose-
dependent disruption of intestinal epithelial integrity, characterized by mucosal erosion, ulceration, and bleeding. This
pathology correlates with an LT-dependent blockade of intestinal crypt cell proliferation, accompanied by marked apoptosis
in the villus tips. C57BL/6J mice treated with intravenous LT nearly uniformly develop systemic infections with commensal
enteric organisms within 72 hours of administration. LT-dependent intestinal pathology depends upon its proteolytic
activity and is partially attenuated by co-administration of broad spectrum antibiotics, indicating that it is both a cause and
an effect of infection. These findings indicate that targeting of MAPK signaling pathways by anthrax LT compromises the
structural integrity of the mucosal layer, serving to undermine the effectiveness of the intestinal barrier. Combined with the
well-described immunosuppressive effects of LT, this disruption of the intestinal barrier provides a potential mechanism for
host invasion via the enteric route, a common portal of entry during the natural infection cycle of Bacillus anthracis.
Citation: Sun C, Fang H, Xie T, Auth RD, Patel N, et al. (2012) Anthrax Lethal Toxin Disrupts Intestinal Barrier Function and Causes Systemic Infections with Enteric
Bacteria. PLoS ONE 7(3): e33583. doi:10.1371/journal.pone.0033583
Editor: Stefan Bereswill, Charite-University Medicine Berlin, Germany
Received February 6, 2012; Accepted February 16, 2012; Published March 16, 2012
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for
any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Funding: The United States government provided the funding to support this work. The information presented in this manuscript reflects the work of the
authors and does not necessarily represent the policy of the United States Food and Drug Administration. The funders had no role in study design, data collection
and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: [email protected]
Current address: BD Diagnostics, Sparks, Maryland, United States of America
{ Deceased
Introduction
Bacillus anthracis (anthrax) infection most commonly occurs in
herbivores, which in the natural infection cycle consume infectious
spores that are present in contaminated soils [1]. Bacillus anthracis
carries an essential virulence plasmid, pX01, that encodes the
three components of anthrax toxin: edema factor (EF), lethal
factor (LF), and the host receptor-binding protective antigen (PA)
[2]. PA facilitates intracellular delivery of EF, an adenylate cyclase,
and/or LF, a metalloprotease with specific activity against MAPK
kinases (MKKs) [3]. The effects of the administration of the
combination of PA and LF, termed lethal toxin (LT), have been
the subject of intense research interest, because LT reproduces
many of the clinical features of anthrax infection in animal models
[4,5].
As anthrax is generally a gastrointestinal infection in the natural
setting [1], it would be predicted that this pathogen has evolved
mechanisms to facilitate infection via this route. In common with
the enteric pathogens, Shigella flexneri, Yersinia enterocolitica and
Salmonella enterica, Bacillus anthracis has virulence factors that target
components of the MAPK signaling pathways [6,7,8,9]. This
shared strategy of enteric pathogens would suggest that the
targeting of MKKs by LT might represent a mechanism to
promote infection via the gastrointestinal route of entry.
Previous studies addressing the effects of LT on the intestine
generated conflicting results. Nearly a decade ago, fecal blood was
reported in the intestines of occasional, LT-treated mice, however
there was no microscopic evidence of intestinal pathology [10]. A
recent study by the same group reported multifocal intestinal
ulcerations in the setting of immunocompromised MyD88-
deficient mice treated with LT, but not in heterozygous animals,
which were reported to have minimal incidence of intestinal
ulceration [11]. In contrast, our findings demonstrated marked
ulceration and hemorrhage in wild-type C57BL/6J mice treated
with LT [12]. This apparent contradiction presented a compelling
basis for additional investigation to clarify whether LT mediates
pathogenic effects in the intestines of immunocompetant hosts, a
question potentially relevant to pathogenesis in the natural anthrax
infection cycle.
Using a series of experiments involving histological and
microbiological assessments, we have extensively characterized
the effects of LT on intestinal tissues. At a high dose of intravenous
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Figure 1. Anthrax LT causes intestinal damage in C57BL/6J and BALB/c mice. (A) C57BL/6J and BALB/c mice were injected
intravenously with anthrax LT or the PBS vehicle alone. Intestines obtained from moribund (LT-treated) or control (PBS-treated) animals were
photographed and assessed for gross pathological changes. Arrowheads indicate areas of hemorrhage. Areas of edema are marked by the letter E.
(B and C) C57BL/6J and BALB/c mice were injected intravenously with anthrax LT (n =34 and n =14, respectively) or the PBS vehicle alone (n=15 and
n =10, respectively). LT-treated mice were sacrificed when they became moribund; PBS-treated mice were sacrificed simultaneously as controls.
Shown are representative H&E sections from the small intestines of LT- and PBS-treated C57BL/6J mice (B, n=34), and BALB/c mice (C, n =14). All
mice within each strain displayed similar histological findings. Arrows indicate mucosal erosions/ulcerations, and arrowheads identify areas of
hemorrhage. Aperio ScanScope-acquired images are shown at 56. (D) Shown are adjacent sections from a typical ulcer in a moribund LT-treated
C57BL/6J mouse stained with H&E (left panels) or Brown & Brenn (right panels). High magnification reveals the submucosal penetration of bacteria.
Aperio ScanScope-acquired images are shown at 26or 206.
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Figure 2. Pathological effects of LT progress in a time-dependent manner following exposure and require the enzymatic activity of
LF. (A) C57BL/6J mice were euthanized at the indicated time points following intravenous LT treatment (2496 h, 20 animals/cohort), along with
control animals that received the PBS vehicle. Representative H&E-stained sections from the small intestines of these animals are shown; all mice in
each cohort showed similar histological findings. Aperio ScanScope-acquired images are shown at 56. Arrowheads indicate hemorrhage, asterisks
indicate villous blunting, and arrows indicate mucosal erosion/ulceration. (B) C57BL/6J mice were injected intravenously with LT (LT 14), LT
comprising wild-type PA and a protease-deficient LF mutant (mLT 12), or the PBS vehicle (PBS 12). Protein levels of MKK3 and MKK4 in intestinal
tissue samples were analyzed by western blotting 24 h post administration is shown. b-actin protein levels were assessed to demonstrate loading.
(C) C57BL/6J mice were injected intravenously with LT (n =5) or mLT (n=5). Mice that received LT alone showed signs of intoxication 4896 hours
post-treatment, whereas those that received mLT showed no signs of intoxication. When moribund, LT-treated mice were euthanized, simultaneously
with mLT-treated control mice. Shown are representative H&E sections from LT-treated (left panel) and mLT-treated mice (right panel); all mice in
each cohort showed similar histological findings. Arrows indicate areas of mucosal erosion/ulceration.
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LT, mice develop intestinal ulcerations and bleeding; these effects
depend upon the proteolytic activity of its LF component. LT-
induced intestinal pathology is distinguished by a blockade in
epithelial progenitor cell proliferation, accompanied by the
marked enhancement of apoptosis in the villus tips. We herein
report that this intestinal pathology is associated with a breakdown
in the host intestinal barrier, as nearly all wild-type C57BL/6J
mice and a substantial fraction of BALB/c mice treated with high-
dose LT develop systemic infections with enteric organisms within
72 h of exposure. This effect is at least as rapid as the development
of infectious complications reported following radiation or
chemotherapy [13,14,15,16]. These findings indicate that target-
ing of MKKs by anthrax LT results in severe compromise of the
intestinal barrier in immunonocompetant hosts, suggesting a
potential mechanism for bacterial entry via the enteric route.
Results
Anthrax LT induced intestinal pathology is not route- or
strain-dependent
We recently reported that wild-type C57BL/6J mice adminis-
tered intraperitoneal LT develop marked multi-focal ulcerations in
the small intestine [12]. To confirm that our findings were not
route- or strain-dependent, we administered intravenous LT to both
C57BL/6J and BALB/c mice. Pathological samples obtained from
moribund animals revealed evidence of gross intestinal bleeding in
both strains of mice; however, C57BL/6J mice exhibited more
intestinal edema following LT treatment (Figure 1A, left panel). In
contrast, BALB/c mice showed greater amounts of gross bleeding
than C57BL/6J mice (Figure 1A, right panel).
Despite some gross pathological differences, histological exam-
ination revealed similarities between the two strains. Mice that
became moribund and/or succumbed to LT within 48 h post-
administration showed little evidence of intestinal ulceration (not
shown), whereas animals that survived beyond this point exhibited
a distinct pattern of damage, characterized by multi-focal intestinal
erosions and ulcerations, with associated bleeding and abscess
formation (Figures 1BD). Preparations of adjacent sections
stained using Brown & Brenn revealed penetration of bacteria into
the submucosa (Figure 1D). Although LT-treated C57BL/6J and
BALB/c mice shared pathological features, sections from LT-
treated BALB/c mice generally showed more evidence of
hemorrhage (Figure 1C), corresponding with gross histological
findings (Figure 1A). Sections from LT-treated C57BL/6J mice,
in contrast, showed evidence of more severe and widespread ulcer
formation (Figure 1B).
Time-course progression of anthrax LT-induced intestinal
pathology
BALB/c mice succumb rapidly in response to LT, as they carry
a sensitive allele of Nalp1b that promotes caspase-1 activation and
inflammatory cytokine release in response to LT, whereas
C57BL/6J mice carry a resistant Nalp1b allele [17]. For this
reason, BALB/c mice are more likely than C57BL/6J mice to
succumb to LT within the first 48 h following toxin administra-
tion, prior to developing intestinal pathology. To better under-
stand how LT mediates pathological effects in the intestine, we
used C57BL/6J mice in the subsequent experiments to avoid early
animal deaths. In a time-course study (Figure 2A), LT-induced
pathological changes became evident in the intestine 48 h post LT
exposure. These changes included the onset of villous damage and
minor hemorrhage. By 72 h post LT exposure, focal areas showed
marked destruction of the normal villous structures, accompanied
by areas of ulceration. This pathology persisted in the few animals
that survived to 96 h post exposure. The pathological effects of LT
on the intestinal barrier were mediated by the proteolytic activity
of its LF component on MKKs, as demonstrated by mice
administered LT comprising wild-type PA and protease-deficient
LF (mLT). In contrast to mice receiving wild-type LT that showed
disrupted MKK signaling and intestinal ulceration, mice receiving
mLT showed no defect in MKK signaling pathways and no
pathological or histological evidence of intestinal damage
(Figures 2B and 2C).
Anthrax LT induces systemic infections with enteric
bacteria in wild-type mice
The first indication that LT-treated mice might be developing
systemic bacterial infections was the observation that mice treated
with LT developed severe hypoglycemia with serum glucose levels
dropping precipitously prior to death (Figure S1). One potential
explanation for the hypoglycemia in LT-treated animals was that
it was secondary to the effects of systemic bacteremia [18,19]. To
investigate this possibility, we performed abdominal wash and
blood cultures on samples obtained from moribund LT-treated
C57LB/6J and BALB/c mice. Ninety-four percent of C57BL/6J
animals (32/34) had developed systemic bacterial infections, with
29/34 animals becoming bacteremic (Table 1). Systemic bacterial
infections were also observed in 5/14 LT-treated BALB/c mice
(Table S1). BALB/c mice are very sensitive to LT [17], and half
of the mice (7/14) in our experiment died within 48 h of LT
exposure. None of these animals developed systemic bacterial
infection. However, a majority of the mice (5/7) that survived
longer than 48 h post LT exposure (5266 hours) developed
systemic bacterial infections. In addition, three moribund animals
in each strain were culture positive for abdominal cavity infections
in the absence of bacteremia (6 animals total), whereas only one
Table 1. Bacterial Culture Results in LT-treated Mice at
Autopsy (C57BL/6J).
Bacterial Culture Results (n=34)
N Abdominal cavity Blood
2 2 2
3 + 2
1 2 +
28 + +
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Table 2. Bacterial Blood Culture Results in LT-treated Mice*.
Species N
Enterococcus faecalis 22
Enterobacter cloacae 15
Escherichia coli 12
Staphylococcus species 8
Klebsiella oxytoca 6
Acinetobacter 6
*Moribund, LT-treated C57BL/6J mice; n =34 total; some mice were co-infected
with 23 bacterial species.
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C57BL/6J mouse was bacteremic in the absence of abdominal
cavity infection (Table 1 and Table S1), suggesting that the
abdominal cavity was the primary site of infection prior to
hematological dissemination. LT-induced infection required the
proteolytic activity of its LF component; mice administered LT
with a protease-deficient LF component neither showed intestinal
MKK cleavage in vivo (Figure 2B) nor developed bacterial
infections (Table S2).
Next, we used an automated bacterial identification system,
supplemented with conventional biochemical tests, to identify the
bacterial species causing the underlying infections. These studies
revealed that the infections were caused by enteric flora, with
Enterococcus faecalis, Enterobacter cloacae, and Escherichia coli accounting
for over 70% of the isolates (Table 2). We then investigated the
development of systemic infections in time-course experiments
following exposure to LT (Table 3). Peritoneal and blood culture
results were negative in PBS-treated controls and in mice treated
with LT for 24 h prior to sampling. At 48 h post exposure, 4/15
animals exhibited bacterial growth in cultures of peritoneal
washes, whereas only 1/15 animals had a positive blood culture.
Animals euthanized 72 h and 96 h post exposure had nearly
uniformly positive blood and peritoneal wash cultures. These
findings closely correlated with the time-course of the intestinal
pathology observed in LT-treated mice and were consistent with
conclusion that peritoneal infection generally precedes hemato-
logical dissemination.
The pathological effects of anthrax LT on the intestine
are dose-dependent
To resolve potential inconsistencies in the literature, we
explored the contribution of toxin dose to the development of
intestinal pathology. We routinely used a dose of 200 mg PA and
80 mg LF for experiments with C57BL/6J mice, representing a 2.7
molar ratio of PA/LF that slightly exceeds the 7:3 molar ratio of
the fully occupied toxin complex. Our dosing regimen differed
with that used by the other group that reported minimal LT-
induced pathology in the intestines of wild-type or heterozygous
MyD88
+/2
mice (100 mg PA and 100 mg LF) [10,11]. We
hypothesized that our regimen could represent a higher effective
dose than the 1:1 weight-based ratio used by the other group.
Therefore, we investigated a lower dose to determine whether we
could explain the findings of these other reports. These
experiments revealed that mice that received a reduced dose of
LT (100 mg PA and 40 mg LF) still developed lethal intoxication,
but deaths in these animals were delayed relative to mice receiving
the higher dose (.1 wk vs. #4 d post-treatment, data not shown).
In addition, there was little evidence of intestinal pathology in mice
receiving the reduced LT dose (Figure 3A). Moreover, most mice
in the lower dose group were blood culture negative, whereas all of
the animals receiving the full dose developed systemic infections
with relatively high levels of circulating bacteria (Figure 3B and
Table S3). Thus, LT-induced pathology and the development of
systemic bacterial infections is dose-dependent, an observation that
could explain apparent discrepancies in the literature.
Broad spectrum antibiotic therapy attenuates pathology
in LT-treated mice
Therapy with gentamicin and amoxicillin was used to assess the
role of bacterial infection in mediating the effects of LT, as this
antibiotic combination provided broad coverage against the
enteric organisms cultured from the abdominal cavities and blood
of LT-treated mice. Whereas all the LT-treated control animals
developed bacteremia, only 1/15 animals receiving antibiotics
developed a positive abdominal wash culture, and none developed
bacteremia (Table S4). The co-administration of broad spectrum
antibiotics did not prevent LT-induced structural damage to the
villi. Antibiotic treatment did, however, attenuate villous blunting
and reduce the size and frequency of the associated intestinal
erosions and ulcerations (Figure 4A). This suggested a role for
invading bacteria in enhancing some, but not all, of the features of
intestinal damage. Moreover, the administration of antibiotics
reversed the hypoglycemia observed in moribund, LT-treated
mice (Figure 4B). This finding was consistent with our hypothesis
that LT-induced hypoglycemia was secondary to systemic
bacterial infection.
Anthrax LT has anti-proliferative and pro-apoptotic
effects on the intestinal epithelium
As LT is known to have anti-proliferative effects on a broad
spectrum of cell types [4], we hypothesized that it could have this
effect on intestinal epithelial cells as well. Intestinal sections from
mice treated with LT or PBS control for 48 h were stained with
anti-Ki67, a marker of cellular proliferation. Ki67 staining was
observed in the crypts of the villi in sections from PBS-treated
control animals (Figure 5, upper left panel), the location of
intestinal epithelial progenitor cells. These cells are known to
divide every 2 to 5 days, replacing cells that slough off the tips of
the villi in a continual process [20]. LT treatment reduced cellular
proliferation in the villous crypts up to 48 h following exposure
(Figure 5, upper middle panel), and this was associated with a
breakdown of the normal villous structure. Administration of a
protease-deficient mLT had no effect on baseline proliferation in
the intestinal crypts (Figure 5, upper right panel). Staining with
anti-cleaved (activated) caspase-3 revealed marked apoptosis in the
villous tips of LT-treated animals (Figure 5, lower middle panel),
compared to the scant staining observed in samples from PBS-
treated (Figure 5, lower left panel) or mLT-treated animals
(Figure 5, lower right panel). Taken together, these data establish
that the specific proteolytic activity of LF on the MKKs is required
for the anti-proliferative and pro-apoptotic effects of LT on the
intestinal epithelium. As the blockade in intestinal cell proliferation
and the evidence of enhanced caspase-3-mediated apoptosis
Table 3. Bacterial Culture Results at Autopsy (C57BL/6J Mice).
Time course of LT treatment
PBS (n=15*) 24 h (n=15) 48 h (n=15) 72 h (n=15) 96 h (n=15)
Culture positive in
abdominal cavity
0 0 4 10 14
Bacteremia 0 0 1 9 14
*PBS-treated control mice were sacrificed at varying time points following administration: 24 h (n =3), 48 h (n =3), 72 h (n =3), 96 h (n =6).
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correlate temporally with the observed intestinal damage, the
findings support a role for these mechanisms in mediating the
pathological effects of LT on the intestine.
Discussion
Recent research efforts have focused on inhalation as a route of
anthrax infection, which is the preferred route for intentional
exposure of humans when using anthrax as a weapon [21,22],
rather than the gastrointestinal route of infection that characterizes
the natural epidemiology of Bacillus anthracis infection [23].
Presumably, Bacillus anthracis has evolved strategies to overcome
host defenses at the most common initial site of host penetration,
the intestinal tract. Our studies have extensively characterized the
timing and pattern of LT-induced intestinal pathology, which is
marked by villous blunting, mucosal erosions, and ulceration.
Figure 3. Dose-dependent effects of anthrax LT on the intestinal barrier. (A) C57BL/6J mice were injected intravenously with PBS (control,
n =5), 100 mg PA/40 mg LF (n =10), or 200 mg PA/80 mg LF (n=10). LT-treated animals were euthanized when they became moribund, each with a
simultaneously euthanized PBS-treated control. Samples from the small intestines of these animals were analyzed by H&E staining. Representative
sections from a control animal and animals from each of the two LT-dose cohorts are shown; all animals in each cohort showed similar histological
findings. Aperio ScanScope-acquired images are shown at 56. Arrows indicate mucosal ulcerations and asterisks identify the area of villous blunting.
(B) C57BL/6J mice were injected intravenously with 200 mg PA/80 mg LF (n=8) or 100 mg PA/40 mg LF (n=8) as shown. Cardiac blood samples were
collected when the mice became moribund. Bacterial count data are shown (mean 6 SD; * p,0.001, Students t-test).
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These structural defects, when combined with the well-described
immunosuppressive effects of LT [24,25] lead to systemic infections
with enteric bacteria that would otherwise exist in a commensal
relationship with their mammalian hosts. LT-induced intestinal
damage and infectious sequelae require the proteolytic activity of its
LF component, highlighting the essential role of MKK-dependent
signaling in maintaining the integrity of the intestinal barrier. It
should be noted that mice treated with antibiotics in combination
with LT had a similar mortality curve to those receiving LT alone
(data not shown), indicating that LT-induced infection is not
required for the toxin to cause mortality as a stand-alone agent;
other toxin-mediated effects are sufficient to mediate lethality.
Nevertheless, these results are relevant in the setting of infection,
exposing a potential toxin-mediated mechanism that could serve to
promote bacterial entry via the intestinal route.
In addition, progress has been made toward identifying the
mechanisms that underlie the enterotoxic effects of LT. In this
regard, the LT-induced proliferative block observed in intestinal
epithelial progenitor cells would be predicted to disrupt the normal
cycle of displacement/replacement of terminally differentiated
enterocytes, thereby accounting for the observed accumulation of
apoptotic cells at the villous tips, accompanied by signs of villous
blunting and ulceration. Subsequent expansion of the intestinal
ulcers depends partially on bacterial activity, as antibiotics
attenuate ulcer progression. That LT-induced immunosuppression
contributes to bacterial invasion and dissemination appears a likely
hypothesis and is consistent with findings of intestinal pathology in
immunosuppressed MyD88
2/2
mice, but not MyD88
+/2
mice,
treated with a lower dose of LT [11]. In contrast to radiation- and
chemotherapy-induced immunosuppression that is maximal when
neutrophil counts drop days following exposure [13,14,15], the
immunosuppressive effect of anthrax LT on MKK signaling
occurs in hours [26,27,28], correlating with the relatively rapid
development of the intestinal syndrome in LT-exposed mice.
Nevertheless, other potential pathogenic mechanisms should be
considered as well. LT alters the cellular localization and
trafficking of key junction associated proteins, such as the
cadherins and ZO1, resulting in dysregulation of the junctional
complexes that establish functional cellular barriers [29,30].
Studies addressing a potential role for these pathways in mediating
the effects of LT on the intestine are warranted. Moreover, the
development of a rodent model for gastrointestinal anthrax
involving toxigenic Bacillus anthracis would advance progress in
this field as well; currently, this experimental tool is not available to
the scientific community [31].
Our experimental findings further define the critical host targets
of LT, as well as clarify inconsistencies in the literature. First, we
observed that the enterotoxic effect of LT is dose-dependent.
Administration of a decreased dose of LT results in mortality, but
without concomitant intestinal pathology, indicating that differ-
ences in the dose and/or potency of LT likely accounted for
previous reports of minimal LT-induced intestinal pathology in
wild-type hosts. Second, although LT drives the breakdown of the
intestinal barrier and resultant enteric bacterial infections, the
effects of LT on other host targets account for its lethality when
administered parenterally as a stand-alone agent. More impor-
tantly, these results firmly establish MKKs as critical regulators of
the interface between the host and potential pathogens in the
intestinal tract. Proteolysis and inactivation of MKKs by anthrax
LT induces intestinal ulceration and the subsequent development
of disseminated infection with enteric organisms. The rapidity (48
72 h) and severity of this effect matches or exceeds that associated
with radiation or chemotherapy injury [13,14,15,16]. These
findings not only have direct ramifications for the pathogenic
mechanisms of Bacillus anthracis, but they suggest intriguing
implications for enteric pathogens known to target MKK/MAPK
pathways via specific virulence factors [6,7,8,9].
Materials and Methods
Animal strains
Experiments involved mice that were treated in accordance with
an animal protocol (#WO2006-58), which was approved by the
CBER Institutional Animal Care and Use Committee. Experi-
Figure 4. Administration of antibiotics partially attenuates
intestinal pathology in LT-treated mice. C57BL/6J mice were
administered either LT alone or LT with concomitant amoxicillin and
gentamicin. Mice were euthanized when they became moribund, which
occurred at the same time frame for both cohorts (data not shown). (A)
Samples from the small intestine were obtained and analyzed by H&E
staining, as shown in representative sections from 2/15 animals from
each cohort. All mice in each cohort showed similar histological
findings. Arrowheads indicate hemorrhage, asterisks indicate villous
blunting, and arrows indicate mucosal erosions/ulcerations. Aperio
ScanScope-acquired images are shown at 56. (B) Blood samples
obtained by cardiac puncture were assessed for glucose concentrations.
(n =15 for each cohort, * p,0.0001, Students t test).
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ments involved female BALB/c and C57BL/6J mice that were 8
12 weeks of age, which were purchased from The Jackson
Laboratory. Mice were allowed at least one week to acclimatize to
the animal facilities prior to experimentation.
Western blotting
Murine protein extracts were prepared using T-PER Tissue
Protein Extraction Reagent (Thermo Scientific, Rockford, IL),
following the manufacturers suggested protocol. Protein samples
were electrophoretically separated on 412% NuPage Bis-Tris
gradient gels (Invitrogen, Carlsbad, CA) and transferred to 0.45-
mm nitrocellulose membranes. The membranes were incubated
with the following primary antibodies at the indicated concentra-
tions: anti-mitogen activated protein kinase (MKK)-3, 200 ng/ml
(Santa Cruz Biotechnology, Santa Cruz, CA; sc-959); anti-MKK-
4, 100 ng/ml (Santa Cruz Biotechnology, Santa Cruz, CA; sc-964)
or anti-b-actin 200 ng/ml (Sigma, St. Louis, MO; A2228). After
incubation with the appropriate species-specific HRP-conjugated
secondary antibodies, the membranes were then incubated with
SuperSignal West Pico chemiluminescent substrate (Thermo
Scientific, Rockford, IL) and exposed to autoradiograph film.
In vivo intoxication experiments
Lyophilized recombinant PA, LF, and mutant LF (E687C) were
purchased from List Biological Laboratories, Inc. (Campbell, CA)
and reconstituted in sterile water (stock concentration: 1 mg/mL
in 5 mM HEPES, 50 mM NaCl, pH 7.5). Anthrax LT was
administered with a fixed ratio of PA/LF of 2.5:1 by weight (molar
ratio: 2.7:1). Unless otherwise indicated, mice were injected
intravenously with 200 mg PA/80 mg LF (C57BL/6J) or 100 mg
PA/40 mg LF (BALB/c), administered in a total volume of 0.3 mL
of PBS. As a negative control, selected mice received the same
volume of PBS alone. Mice were sacrificed when moribund or as
otherwise indicated. Blood samples were collected aseptically
either by cardiac puncture (Figure 4B) or from the tail vein
(Figure S1). Blood glucose levels were determined using an
automatic glucose analyzer (Ascensia Elite XL, Bayer). Central
blood glucose concentrations in cardiac puncture samples were
consistently higher than concentrations detected in tail vein
samples.
Some experiments involved the concomitant administration of
broad-spectrum antibiotics with anthrax LT. Both antibiotics were
administered subcutaneously beginning the day of LT treatment
(amoxicillin: 100 mg/kg every 8 hours, and gentamicin 16 mg/kg
one time per day, Sigma, St. Louis, MO). Antibiotic therapy was
maintained until the animal succumbed to toxemia or until the
termination of the experiment.
Gross pathological assessments were performed on dissected
murine gastrointestinal tracts, which were photographed against a
white background using a Nikon D40 camera.
Figure 5. Anthrax LT blocks intestinal epithelial cell proliferation and promotes apoptosis. C57BL/6J mice were euthanized 48 h
following the administration of LT (n =5), protease-deficient mLT, or PBS (n=5, controls). (A) Sections from the small intestines of these animals were
evaluated by immunohistochemistry for the Ki67. Representative sections from damage-free areas of PBS-treated, LT-treated, and mLT-treated
animals are shown in the upper panels for comparison. All animals in each cohort showed similar histological findings. Aperio ScanScope-acquired
images are shown at 56. (B) Sections from the small intestines of these animals were evaluated by immunohistochemistry for activated caspase-3.
Representative sections from damage-free areas of PBS-treated, LT-treated, and mLT-treated animals are shown in the lower panels for comparison;
all animals in each cohort showed similar histological findings. Aperio ScanScope-acquired images are shown at 56.
doi:10.1371/journal.pone.0033583.g005
Anthrax Lethal Toxin Causes Enteric Infections
PLoS ONE | www.plosone.org 8 March 2012 | Volume 7 | Issue 3 | e33583
Histological assessments
To evaluate the intestinal damage caused by anthrax LT or
protease-deficient mutant LT, intestinal samples were collected
from each mouse and fixed in 10% neutral buffered formalin
(Sigma, St. Louis, MO). Paraffin sectioning, hematoxylin and
eosin staining (H&E), and Brown and Brenn (B&B) staining were
performed by Histoserv, Inc (Gaithersburg, MD). In addition,
Histoserv performed immunohistochemistry staining using the
following antibodies: anti-Ki67 (Abcam, Cambridge, MA, Ab
16667, dilution: 1:100) and anti-cleaved caspase-3 (Cell Signaling
Technology, Danvers, MA; #9664, dilution: 1:400). H&E-, B&B-
and immunohistochemistry-stained slides were scanned using an
Aperio ScanScope (ScanScope, Aperio, CA). The images were
acquired at 206magnification, and relevant areas were converted
into a TIF format at various magnifications for the generation of
figures. Stained tissue sections were analyzed by a board-certified
veterinary pathologist in a blinded fashion.
Bacterial isolation and identification
Bacterial isolates were cultured from aseptically-obtained blood
(cardiac puncture) or peritoneal wash samples using blood agar
plates (Remel, Lenexa, KS; RO1202) and/or MacConkey plates
(Remel, Lenexa, KS; RO1552). Peritoneal cavity washes were
performed using 3 mL of PBS; 100 mL of the post-wash samples
was cultured on blood agar plates. Bacterial identifications were
performed by the NIH Department of Laboratory Medicine, using
the Siemens Micro Scan Auto SCAN4 system (Deerfield, IL),
supplemented with conventional biochemical tests [32,33]. To
quantify bacteria in blood or peritoneal wash specimens, samples
were serially diluted and cultured on blood agar plates prior to
overnight culture and enumeration.
Supporting Information
Figure S1 LT causes a drop in serum glucose concen-
trations in vivo. C57BL/6J mice were injected intravenously
with LT (n =15) or PBS (n =15). Tail vein blood samples were
assessed for glucose concentration at varying time points following
administration as shown. (* p,0.001, ** p,0.0001, Students t-
test).
(TIF)
Table S1 Bacterial Culture Results at Autopsy (BALB/
c).
(DOC)
Table S2 Bacterial Culture Results at Autopsy.
(DOC)
Table S3 Bacterial Culture Results at Autopsy (C57BL/
6J Mice).
(DOC)
Table S4 Effect of Antibiotics on LT-induced Bacter-
emia in C57BL/6J Mice.
(DOC)
Acknowledgments
The authors thank Drs. Weiming Ouyang, Wen Jin Wu, Francisco
Borrego, and Kathryn King for thoughtful review of the manuscript. The
untimely death of one of our co-authors, Dr. Philip Snoy, brought special
meaning to this work. The information presented in this manuscript reflects
the work of the authors and does not necessarily represent the policy of the
U.S. Food and Drug Administration.
Author Contributions
Conceived and designed the experiments: CS HF TX PM DF. Performed
the experiments: CS HF TX RA NP PS. Analyzed the data: CS HF TX
RA NP PM PS DF. Contributed reagents/materials/analysis tools: PM PS
DF. Wrote the paper: CS TX DF.
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