Anthrax Lethal Factor Cleaves Mouse Nlrp1b in Both
Toxin-Sensitive and Toxin-Resistant Macrophages
Kristina A. Hellmich, Jonathan L. Levinsohn, Rasem Fattah, Zachary L. Newman, Nolan Maier,
Inka Sastalla, Shihui Liu, Stephen H. Leppla, Mahtab Moayeri*
Microbial Pathogenesis Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland,
United States of America
Anthrax lethal factor (LF) is the protease component of anthrax lethal toxin (LT). LT induces pyroptosis in macrophages of
certain inbred mouse and rat strains, while macrophages from other inbred strains are resistant to the toxin. In rats, the
sensitivity of macrophages to toxin-induced cell death is determined by the presence of an LF cleavage sequence in the
inflammasome sensor Nlrp1. LF cleaves rat Nlrp1 of toxin-sensitive macrophages, activating caspase-1 and inducing cell
death. Toxin-resistant macrophages, however, express Nlrp1 proteins which do not harbor the LF cleavage site. We report
here that mouse Nlrp1b proteins are also cleaved by LF. In contrast to the situation in rats, sensitivity and resistance of Balb/
cJ and NOD/LtJ macrophages does not correlate to the susceptibility of their Nlrp1b proteins to cleavage by LF, as both
proteins are cleaved. Two LF cleavage sites, at residues 38 and 44, were identified in mouse Nlrp1b. Our results suggest that
the resistance of NOD/LtJ macrophages to LT, and the inability of the Nlrp1b protein expressed in these cells to be activated
by the toxin are likely due to polymorphisms other than those at the LF cleavage sites.
Citation: Hellmich KA, Levinsohn JL, Fattah R, Newman ZL, Maier N, et al. (2012) Anthrax Lethal Factor Cleaves Mouse Nlrp1b in Both Toxin-Sensitive and Toxin-
Resistant Macrophages. PLoS ONE 7(11): e49741. doi:10.1371/journal.pone.0049741
Editor: Nicholas J. Mantis, Wadsworth Center, New York State Dept. Health, United States of America
Received September 3, 2012; Accepted October 12, 2012; Published November 12, 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: This research was supported by the Intramural Research Program of the NIH, National Institute of Allergy and Infectious Diseases. 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@example.com
Anthrax lethal toxin (LT) consists of a receptor-binding protein,
protective antigen (PA), and a protease, lethal factor (LF). LT is a
major virulence factor, and its injection alone is sufficient to induce
the vascular collapse associated with anthrax disease in animal
models (for review see ). Rodent macrophages from certain
inbred strains are rapidly lysed by LT. Susceptibility to LT is
controlled by alleles encoding variants of the NOD-like receptor
(NLR) Nlrp1b in mice , and its ortholog in rats . Nlrp
proteins are intracellular pattern recognition receptors that detect
a variety of signals associated with pathogens and other dangers to
the cell . While a wide range of stimuli are recognized by
intracellular sensors such as Nlrp3 , the only known activator of
rodent Nlrp1 is LT. Treatment of macrophages and dendritic cells
from some inbred rodent strains such as Balb/cJ (mice) and
Fischer (rats) to LT results in Nlrp1/Nlrp1b-mediated activation of
caspase-1 and subsequent cleavage of IL-1b and IL-18, initiating
an immune response concurrent with rapid cell death (pyroptosis)
[2,5]. Macrophages from other rodent strains such as C57BL/6J
and NOD/LtJ (mice) and Lewis (rats) express Nlrp1 variants
which are not activated by LT and these cells are resistant to the
toxin [2,5]. Our laboratory recently demonstrated that LT
sensitivity in rats is determined by polymorphisms at residues
40-48 in the N-terminus of rat Nlrp1 . LF cleaves this site in rat
Nlrp1 of inbred strains harboring LT-sensitive macrophages, but
not the Nlrp1 of LT-resistant strains .
Mice harbor three Nlrp1 paralogs in their genomes, but the
existing data indicate that only Nlrp1b controls LT sensitivity .
Mouse Nlrp1b proteins are highly polymorphic , unlike the
situation in rats, where Nlrp1 is almost identical in all strains
except in the small region in the N-terminus noted above.
Sequencing Nlrp1b from 18 inbred mouse strains identified five
different Nlrp1b protein sequences, two associated with LT
sensitivity and three with resistance . The proteins encoded
by four of the five different Nlrp1b alleles have .88% homology to
one another. Especially intriguing is the high degree of homology
between proteins encoded by Nlrp1b allele 1 (expressed in Balb/cJ
and numerous other strains with LT-sensitive macrophages),
Nlrp1b allele 3 (expressed in NOD/LtJ and AKR/J mice, which
harbor LT-resistant macrophages) and Nlrp1b allele 5 (expressed in
CAST/EiJ mice, which harbor LT-sensitive macrophages). We
wished to determine whether the differential responses of these
highly homologous proteins were determined by LF cleavage of
Nlrp1b, as it is in rats . We report here that Nlrp1b expressed in
both Balb/cJ macrophages (LT-sensitive) as well as Nlrp1b from
NOD/LtJ macrophages (LT-resistant) are cleaved by LT within
the N-terminal region, at the same sites. These results suggest that
the resistance of NOD/LtJ macrophages to LT, and the inability
of the Nlrp1b protein expressed in these cells to be activated by the
toxin, is likely due to polymorphisms that render the protein
inactive in a manner independent of LT cleavage.
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Results and Discussion
Our earlier studies identified an LF cleavage site within the N-
terminus of rat Nlrp1. Toxin cleavage at this site leads to
macrophage pyroptosis . An alignment of rat Nlrp1 sequences
of Fischer (CDF, LT-sensitive) and Lewis (LEW, LT-resistant) rats,
as well as Nlrp1b sequences from four mouse strains is shown in
Figure 1A. The alignment shows that the LF cleavage site within
rat Nlrp1 lies in an inserted sequence that is absent in mouse
Nlrp1b proteins. Perplexingly, unlike the situation in rats, the
Balb/cJ (BALB, LT-sensitive) and NOD/LtJ (NOD, LT-resistant)
Nlrp1b sequences, present in mice having macrophages of
opposing sensitivities to LT, are identical over the first 55 amino
acids of the protein. Examination of the BALB (S) and NOD (R)
Nlrp1b sequences revealed two nearby potential LF cleavage sites
having characteristics like those of the established cleavage sites
identified in rat Nlrp1, mitogen activated protein kinase kinase 4
(MEK4), and other LF substrates [6–9] (Red box in Figure 1A and
Figure 1B). Nlrp1 proteins are expressed endogenously at low
levels that are difficult to detect via Western blotting. Therefore we
expressed full-length N-terminally hemagglutinin (HA) epitope-
tagged rat Nlrp1 (CDF-S) and mouse Nlrp1b (BALB-S and NOD-
R) proteins in HT1080 human fibroblasts by stable transfection.
Immunoprecipitation(IP) with anti-HA antibodies showed
expression of full-length HA-tagged Nlrp1 proteins, as well as
multiple C-terminally truncated variants (Figure 2). As previously
reported , LF cleavage of HA-tagged rat Nlrp1 (CDF) expressed
in fibroblasts produced a 6-kDA HA-antibody reactive cleavage
fragment (Figure 2A). In a new result, we found that LF cleaved
the HA-tagged BALB (S) Nlrp1b protein, producing a (slightly
(Figure 2A), suggesting that the BALB (S) Nlrp1b cleavage site is
slightly upstream of the rat Nlrp1 insertion sequence which
contains the cleavage site. This would also mean that this cleavage
site would lie within a region of identical sequence for both BALB
(S) and NOD (R) Nlrp1b.
We also found that LF cleaves the HA-NOD (R) Nlrp1b protein
(Figure 2B), as might be expected given its sequence identity to the
HA-BALB (S) protein. Careful assessment in repeated experiments
of the fragments generated by LF cleavage of these Nlrp1b
proteins showed that the BALB cleavage fragment was slightly
smaller than the NOD cleavage fragment, suggesting cleavage at
two unique sites within the N-terminal regions of these proteins
(Figure 2C). This finding suggested that one or more of the six
polymorphisms immediately downstream of the potential LF
cleavage sites (with the following amino acid changes: R56K,
R67K, L79P, C85Y, I93V, V101I) may influence the site at which
LF cleaves these proteins.
To identify the exact cleavage sites, the first 118 aa of BALB (S)
and NOD (R) Nlrp1b were expressed and purified as GST fusions
(designated BALB118 and NOD118) (Figure 1B, lines 3 and 4).
Both BALB118 and NOD118 were cleaved by LF (Figure 3A and
Figure 1. Nlrp1 protein alignments and constructs. (A). Alignment of amino acid sequences from the N-terminus of mouse Nlrp1b and rat
Nlrp1 proteins. Sequences shown are those of 4 mouse and 2 rat strains, including strains having macrophages that are either sensitive (S) or resistant
(R) to LT. The previously identified LT cleavage site after residue 44 in rat CDF Nlrp1 is indicated by an arrow. The red box indicates the region of
mouse sequence shown in (B). (B) Nlrp1b constructs used in this study with focus on N-terminal regions containing putative LF cleavage sites. The
top two constructs represent the full-length HA-tagged Nlrp1b proteins from the LT-sensitive Balb/cJ (BALB) and the LT-resistant NOD/LtJ (NOD)
macrophages, which were expressed in HT1080 cells. Full length NOD Nlrp1b is shorter (1172 aa) than BALB Nlrp1b due to a region downstream of
the leucine rich repeat domain that is missing in this protein. The next four constructs represent proteins where aa 3-118 of Nlrp1b were expressed
and purified from E. coli as N-terminal GST-tagged proteins. These proteins also contain a C-terminal His6 tag (not represented in figure). In the
sequence alignments, residues identical to those in the construct listed above are indicated by quotation marks (‘‘). Putative LF cleavage sites based
on previously described motifs are drawn as vertical dotted lines below filled arrows. The MEK4 cleavage site is also aligned with both putative Nlrp1b
cleavage sites. The last two sequences are those of constructs having two key lysine residues substituted with alanine.
Anthrax Toxin Cleaves Mouse Nlrp1b
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3B). Interestingly, in repeated experiments we noted that NOD118
appeared to be more efficiently cleaved by LF than BALB118
(data not shown). This result corresponds to a similar phenomenon
seen in cell lysates from HT1080 cells expressing full-length HA-
Nlrp1b proteins, where NOD (R) Nlrp1b was cleaved more
efficiently than BALB (S) Nlrp1b (data not shown). Furthermore,
in analyses of the canonical cleavage pathway, where HA-tagged
Nlrp1b proteins were immunoprecipitated from cells after delivery
of LF to the cytosol by PA, NOD Nlrp1b protein appeared to be
more efficiently cleaved (Figure S1).
Mass spectrometry analyses of BALB118 and NOD118
following cleavage by LF yielded masses of 30,486 and 31,257,
indicating that LF cleaves between the two lysine-leucine bonds
(after K38 and K44) (Figure 1B, arrows). Both cleavage fragments
were found following LF treatment of both proteins, albeit with
differing prevalence in multiple independent cleavage runs (data
not shown). Thus, it appears probable that the two fragments of 5-
6 kDa observed following cleavage of the full length Nlrp1b
proteins in cell lysates result from cleavage at the K38 and K44
cleavage sites, and that BALB (allele 1) Nlrp1b is preferentially
cleaved at the first site while NOD (allele 3) Nlrp1b is
preferentially cleaved at the second site. Substitution of the lysines
at both sites with alanines (Figure 1B, lines 5, 6) abrogated LF-
mediated cleavage of the BALB118 and NOD118 proteins
Since the data presented above suggests that Nlrp1b of NOD
macrophages would be cleaved by LF delivered to the cytosol, the
question arises as to why these cells do not undergo pyroptosis.
One possible defect in downstream events could be a failure to
activate caspase-1. However, we found that nigericin activated the
Figure 2. Cleavage of full length rat and mouse Nlrp1b proteins by LF. (A) IP (anti-HA pulldown) followed by anti-HA Western blotting of
lysates from HT1080 cells expressing HA-tagged mouse Nlrp1b (BALB) or rat Nlrp1(CDF) proteins following treatment with LF (1 mg/ml) for 15 min or
2 h. Cleavage of CDF Nlrp1 leads to appearance of a 6-kDa HA-reactive band and cleavage of BALB Nlrp1b leads to a slightly smaller fragment. (B) IP
(anti-HA pulldown) followed by anti-HA Western blotting of lysates from HT1080 cells expressing HA-tagged Nlrp1b proteins or control vector
following treatment with LF (1 mg/ml, 30 min). Anti-HA cross-reactive bands not marked as HA-Nlrp1 also appear in vector-transfected controls. (C)
Comparison of size of cleavage fragments generated after cleavage of BALB and NOD HA-tagged Nlrp1b (using conditions same as 2B), indicating the
smaller size of the fragment generated following cleavage of the BALB protein (Western representative of five similar experiments).
Anthrax Toxin Cleaves Mouse Nlrp1b
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Nlrp3 inflammasome to elicit efficient activation of caspase-1 and
subsequent IL-1b cleavage in both NOD and BALB bone
marrow-derived macrophages (Figure 4), indicating that the
caspase-1 activation pathway was fully functional. NOD macro-
phages also had no defect in flagellin-mediated Nlrc4 inflamma-
some activation (data not shown). As expected, LT activated
caspase-1 in BALB (S) but not in NOD (R) macrophages (Figure 4).
A possible reason for these findings is that NOD macrophages may
be deficient in protein’s that are important to inflammasome
assembly in an Nlrp1b-dependent manner, which does not impact
Nlrp3-mediated caspase-1 activation.
While this manuscript was in preparation, Frew et al. reported
that C-terminal autoproteolysis of Nlrp1b within the FIIND
(function-to-find) domain is required for inflammasome activation
and that this proteolytic processing was absent in allele 3 (NOD)
Nlrp1b due to a single polymorphism, V988D . The site for C-
terminal autoproteolysis in Nlrp1 has been identified .
Differentially C-terminally truncated variants of NOD (R) Nlrp1b,
and BALB (S) Nlrp1b were also present in our expression system
(Figure 2). Frew et al. were able to eliminate proteolytic processing
of BALB (allele 1) Nlrp1b by introducing the V988D substitution
(from NOD, allele 3), and this mutation prevented the protein
from being activated by LT . Intriguingly, the authors were
unable to restore LF responsiveness to the LT-nonresponsive
NOD (R) Nlrp1b protein even after restoration of its autoproteo-
lytic processing. These results, in combination with the findings
reported here, suggest that the resistance of NOD (R) Nlrp1b to
LF is not due to absence of a required LF cleavage event, or simply
due to a deficiency in C-terminal autoproteolysis. It is possible, but
unlikely, that preferential LF cleavage of NOD (R) Nlrp1b at
residue K44, instead of K38, likely due to the presence of
downstream polymorphisms altering folding in the N-terminus of
this protein, is the reason for the defect in activation of this protein.
It seems more likely that polymorphisms in other domains of this
protein render it nonresponsive to LT. The truncated domain
downstream of the leucine rich repeats in NOD Nlrp1b may result
in altered conformation and folding of this protein in a manner
that interferes with its unfolding to allow dimerization or caspase-1
recruitment. Thus, even when autoproteolysis at the C-terminus is
restored and LT cleaves the N-terminus efficiently, the protein
may be unable to act as an inflammasome platform. The
deciphering of the mechanism for resistance to LT requires
further experimentation. We propose, however, that cleavage of
the N-terminus of both mouse and rat Nlrp1 proteins by LF may
be required for activation of the inflammasome by LT, although it
may be insufficient in the absence of other processing events.
Materials and Methods
This study was carried out in strict accordance with the
recommendations in the Guide for the Care and Use of
Laboratory Animals of the National Institutes of Health. All bone
marrow harvests were performed in accordance to protocols
approved by the NIAID Animal Care and Use Committee.
PA, LF, and LF E687C purification from avirulent Bacillus
anthracis strains has been described . Concentrations of LT
correspond to the concentration of each toxin component (i.e.,
1 mg/ml LT has 1 mg/ml PA and 1 mg/ml LF). GST-fusion
proteins of BALB118 and NOD118 (described below) were
expressed from pGEX-KG vectors in Escherichia coli BL21(DE3)
Figure 3. Cleavage of mouse BALB118 and NOD118 Nlrp1
fusion proteins by LF. (A, B) In vitro cleavage of N-terminally 6His-
GST-tagged aa 3-118 of Nlrp1b proteins. Purified proteins (0.53 mg/ml
or 0.94 mg/ml, in A and B, respectively) were treated with the indicated
molar ratios of LF, or with a 1:10 molar ratio of the mutant LF E687C
(LFm), for 4 h prior to SDS gel electrophoresis and Coomasie staining.
F1 and F2 refer to two fragments generated following LF treatment. (C)
GST-tagged or double alanine mutant variants (0.44-0.66 mg/ml) were
treated with 33 mg/ml LF or LFm for 4 h prior to SDS gel electrophoresis
and Coomassie staining.
Figure 4. Caspase-1 activation in bone marrow-derived mouse
macrophages. LPS-primed (1 mg/ml, 2 h) bone marrow-derived
macrophages from Balb/cJ or NOD/LtJ mice were treated with LT
(1 mg/ml) for 60 or 80 min, or with nigericin at indicated doses for
20 min. Cell lysates were analyzed by Western blotting for IL-1b, and
the same samples were probed with caspase-1 p10 antibody to detect
Anthrax Toxin Cleaves Mouse Nlrp1b
PLOS ONE | www.plosone.org4November 2012 | Volume 7 | Issue 11 | e49741
and purified in a two-step process on glutathione-Sepharose and Download full-text
nickel chelate columns using standard purification protocols. High
affinity anti-HA (cat# 11867423001, Roche Diagnostics, India-
napolis, IN), anti-IL-1b (cat# AF-401-NA, R&D Systems,
Minneapolis, MN) and various IR-dye conjugated secondary
antibodies (Licor Biosciences, Lincoln, NE and Rockland Immu-
nochemicals, Gilbertsville, PA) were purchased. Nigericin was
purchased from Calbiochem (San Diego, CA).
Cell Culture and Transfections
Cell culture and transfection methods have been previously
described . For stable transfections, Nlrp1b-expressing cell lines
were derived by selection with hygromycin B (500 mg/ml;
Invitrogen) for 15 days. Western blot with anti-HA antibody was
performed to identify expression levels. Bone marrow-derived
macrophages (BMDMs) were generated from marrow obtained
from Balb/cJ or Nod/LtJ mice (Jackson Laboratories, Bar Harbor,
ME) as previously described .
cDNA sequences for BALB and NOD mNlrp1b were synthe-
sized by GeneArt Life Technologies (Grand Island, NY) and were
cloned into the pIREShyg3 vector using Nhe1 and Xma1 sites.
BALB118 and NOD118 sequences were synthesized by GeneArt
Life Technologies and cloned along with added C-terminal His6
tags into the pGEX-KG vector using BamHI and EcoRI sites.
Mutagenesis was performed using the QuikChange system
(Agilent Technologies, La Jolla, CA) and sequencing was
performed by Macrogen (Rockville, MD).
To assess Nlrp1 cleavage in cell lysates, cells were grown to
confluence and lysed in sucrose buffer (250 mM sucrose, 10 mM
HEPES, 0.05 M EDTA, 0.2% Nonidet-P40) containing ZnCl2
(1 mM) and NaCl (5 mM), followed by LF treatment at 37uC for
varying times. Alternatively, cells were first treated with LT at
1 mg/ml for 5 h (canonical cleavage), followed by lysis in sucrose
buffer containing 5 ng/ml LF inhibitor PT-168541-1 (gift of Alan
Johnson, Panthera Biopharma). Cleavage reactions were analyzed
by Western blot (WB) or immunoprecipitatoin (IP) followed by
WB. For in vitro cleavage assays with purified proteins, BALB118
and NOD118 were incubated for varying times at 37uC with
purified LF at varying concentrations in the presence of ZnCl2
(1 mM) and NaCl (5 mM). Samples were separated on an 8-25%
SDS-PAGE gel using the PhastSystem (GE Life Sciences, Piscat-
away, NJ) and visualized by Coomassie staining.
Western Blots and Immunoprecipitation
WB were performed using either anti-HA (1:1000), anti-
caspase-1 (1:200), or anti-IL-1b (1:2,500) and proteins were
detected using the Odyssey Infrared Imaging System (Licor
Biosciences). For IP, anti-HA antibody (Roche Diagnostics) was
added to cell lysates (5-15 mg/ml) and samples were continuously
mixed by rotation at 4uC for 1 h, followed by Protein A/G agarose
(Santa Cruz Biotechnology) addition and continued overnight 4uC
incubation with rotation. Beads were centrifuged at 4,000 rpm for
2 min and washed with 10 mM HEPES three times prior to
elution of proteins using SDS loading buffer (10% SDS, 0.6 M
DTT, 30% glycerol, 0.012% bromophenol blue, at 90uC, 5 min).
The molecular masses of the BALB118 and NOD118 proteins
and their cleavage products were determined by liquid chroma-
tography-electrospray mass spectrometry using an HP/Agilent
1100 MSD instrument (Hewlett Packard, Palo Alto, CA) at the
NIDDK core facility, Bethesda, MD.
Nlrp1b proteins by LT. HT1080 cells expressing HA-
tagged mouse Nlrp1b (BALB or NOD) proteins were first
treated with LF+ +PA (1 mg/ml, each) for 3 h. IP (anti-HA
pulldown) was then performed on lysates followed by anti-HA
Canonical cleavage of full length mouse
We thank Devorah Crown for bone marrow isolation and D. Eric
Anderson for assistance with mass spectrometry.
Conceived and designed the experiments: KAH JLL RF ZLN SHL MM.
Performed the experiments: KAH JLL RF NM MM. Analyzed the data:
KAH JLL RF ZLN NM SHL MM. Contributed reagents/materials/
analysis tools: RF ZLN IS SL SHL. Wrote the paper: KAH SHL MM.
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