Anthrax Lethal Toxin Induced Lysosomal Membrane
Permeabilization and Cytosolic Cathepsin Release Is
Kathleen M. Averette1, Matthew R. Pratt2, Yanan Yang3, Sara Bassilian4, Julian P. Whitelegge4, Joseph A.
Loo3, Tom W. Muir2, Kenneth A. Bradley1*
1Department of Microbiology, Immunology & Molecular Genetics, University of California Los Angeles, Los Angeles, California, United States of America, 2Laboratory of
Synthetic Protein Chemistry, The Rockefeller University, New York, New York, United States of America, 3Department of Chemistry and Biochemistry, University of
California Los Angeles, Los Angeles, California, United States of America, 4The Pasarow Mass Spectrometry Laboratory, The NPI-Semel Institute, David Geffen School of
Medicine, University of California Los Angeles, Los Angeles, California, United States of America
NOD-like receptors (NLRs) are a group of cytoplasmic molecules that recognize microbial invasion or ‘danger signals’.
Activation of NLRs can induce rapid caspase-1 dependent cell death termed pyroptosis, or a caspase-1 independent cell
death termed pyronecrosis. Bacillus anthracis lethal toxin (LT), is recognized by a subset of alleles of the NLR protein Nlrp1b,
resulting in pyroptotic cell death of macrophages and dendritic cells. Here we show that LT induces lysosomal membrane
permeabilization (LMP). The presentation of LMP requires expression of an LT-responsive allele of Nlrp1b, and is blocked by
proteasome inhibitors and heat shock, both of which prevent LT-mediated pyroptosis. Further the lysosomal protease
cathepsin B is released into the cell cytosol and cathepsin inhibitors block LT-mediated cell death. These data reveal a role
for lysosomal membrane permeabilization in the cellular response to bacterial pathogens and demonstrate a shared
requirement for cytosolic relocalization of cathepsins in pyroptosis and pyronecrosis.
Citation: Averette KM, Pratt MR, Yang Y, Bassilian S, Whitelegge JP, et al. (2009) Anthrax Lethal Toxin Induced Lysosomal Membrane Permeabilization and
Cytosolic Cathepsin Release Is Nlrp1b/Nalp1b-Dependent. PLoS ONE 4(11): e7913. doi:10.1371/journal.pone.0007913
Editor: Adam J. Ratner, Columbia University, United States of America
Received September 23, 2009; Accepted October 18, 2009; Published November 18, 2009
Copyright: ? 2009 Averette et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was funded by National Institute of Health (NIH) awards AI077791 and AI057870 to K.A.B. K.M.A-M was supported by the NIH Microbial
Pathogenesis Training Grant T32 AI007323 and M.R.P. by an American Heart Association postdoctoral fellowship. The UCLA Mass Spectrometry and Proteomics
Technology Center was established and equipped with a grant from the W. M. Keck Foundation. The authors also acknowledge support to the UCLA flow
cytometry core funded through NIH grants CA16042 and AI28697. 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
The innate immune system is the first defense against invading
microorganisms, and functions to either clear or limit infection
until an adaptive response can be mounted. Innate immune cells
recognize pathogen-associated molecular patterns through pattern
recognition receptors such as toll-like receptors and nucleotide
oligomerization domain-like receptors (NLRs) [1,2]. NLRs identify
cytosolic danger signal(s) or foreign molecules and activate
protective cellular responses. Some NLRs bind directly, or
indirectly, to caspase-1 (GeneID: 12362) within large molecular
weight complexes called inflammasomes, and facilitate activation
of caspase-1 resulting in processing and release of the proin-
flammatory cytokines IL-1b (GeneID: 16176) and IL-18 (GeneID:
16173) . Multiple types of inflammasomes exist that vary by the
NLR that activates formation (e.g. NLRP3-inflammasome,
Nlrp1b-inflammasome, etc.). In macrophages and dendritic cells
(DCs), NLR-induced inflammasome activation can lead to
pyroptosis, a newly described necrosis-like programmed cell death
Pyroptosis is induced in response to numerous pathogens
including Shigella, Salmonella, Listeria, Legionella, Pseudomonas,
Mycobacterium, Yersinia, Burkholderia, and bacterial products
flagellin and B. anthracis lethal toxin (LT) [1,4–9]. B. anthracis LT is
produced during infection and typically functions to suppress
innate immunity [10–12]. The NLR family member Nlrp1b (also
known as Nalp1b; GeneID: 637515) recognizes the activity of B.
anthracis LT in the host cytosol, but is highly polymorphic in mice
with only a subset of alleles conferring a pyroptotic response to LT
. Macrophages that express an LT-sensitive allele of Nlrp1b
(LTS) undergo pyroptosis in the presence of this toxin, releasing
inflammatory cytokines that activate innate immunity [9,13]. It is
not understood how Nlrp1b controls recognition of LT or what
downstream events lead to cell death [1,7]. Here we used LT to
investigate the mechanism of cell death that occurs during
LT is secreted by B. anthracis as two proteinaceous subunits,
protective antigen (PA; GeneID: 2820165) and lethal factor (LF;
GeneID: 2820148) . The binding subunit, PA, attaches to host
cell receptors and oligomerizes to form a binding site for the
catalytic subunit, LF [15–18]. PA-LF complexes are endocytosed
and trafficked to acidic vesicles, where PA forms a membrane pore
and translocates LF into the cytosol . LF is a zinc-dependent
metalloproteinase that cleaves the N-terminus of mitogen activated
protein kinase kinases (MKKs) 1–4, 6, and 7 [19,20]. Cleavage of
MKKs by LT occurs at or near MKK-MAPK binding sites,
PLoS ONE | www.plosone.org1 November 2009 | Volume 4 | Issue 11 | e7913
disrupting downstream MAPK signaling [21,22]. Although
disruption of MAPK signaling alters numerous signaling pathways
and transcription, the activating danger signal(s) that induce
pyroptosis are unknown.
Lysosomal membrane permeabilization (LMP), the loss of
proton gradients in acidic compartments and leakage of lysosomal
proteins into the cytosol, is associated with both apoptosis and
necrosis [23–28]. Severe LMP, characterized by rapid loss of
lysosomal membrane stability, is primarily associated with the final
stages of necrosis while mild LMP, or slow leakage of lysosomal
contents, alters cellular signaling and can induce caspase-
dependent apoptosis or caspase-independent apoptosis-like cell
death [24,27,29,30]. A role for LMP in LT-mediated pyroptosis
was recently described . We provide confirmatory evidence
that LMP occurs during LT-mediated pyroptosis and reveal that
LMP is dependent on the presence of an LT-responsive Nlrp1b.
Acidic compartments are compromised during LT-
A hallmark of LMP is the loss of lysosomal acidity. To
determine if lysosomal pH is affected by LT, we analyzed
macrophages for alterations in acridine orange (AO) staining
following toxin challenge. AO is a cell permeable, lysosomotropic
dye that is protonated and sequestered within acidic compart-
ments such as late endosomes and lysosomes. The fluorescence
emission of AO is concentration dependent, such that at high
concentrations (e.g. in lysosomes) it fluoresces red, while under
diffuse conditions (e.g. in the cytosol) it fluoresces green. LMP can
be recognized by a decrease in red AO fluorescence while
maintaining high green AO fluorescence. RAW 264.7 cells, a
murine macrophage-like cell line that expresses LTSalleles of
Nlrp1b, were pre-loaded with AO and treated with or without LT
for various incubation times then analyzed by flow cytometry. In
LT treated cells, there was a significant increase in a subpopu-
lation of cells that emit low red and high green (LR/HG)
fluorescence compared to control-treated cells (Figure 1A) and this
population increased over time following LT challenge. Of note, a
separate population of cells appeared that displayed both low red
fluorescence and low green fluorescence compared to the entire
population (Figure S1). The loss in fluorescence in both channels is
consistent with loss of cell membrane integrity resulting in the
combined loss of acidic compartments and cytoplasmic contents
containing AO. Indeed, the number of cells in this population was
proportional with duration of LT treatment and analysis of
forward versus side scatter profiles is consistent with non-viable
Next, we tested whether appearance of the AO LR/HG
subpopulation depends on Nlrp1b allelic variations. RAW 264.7
cells are derived from BALB/c mice which express LTSNlrp1b,
whereas IC-21 macrophage-like cells are derived from LT-
resistant (LTR) Nlrp1b expressing C57BL/6 mice and do not
undergo pyroptotic death in response to LT. IC-21 cells showed
no increase in LR/HG population in response to LT (Figure S2A).
To directly test whether Nlrp1b allelic differences were sufficient to
explain differential AO staining, we tested bone marrow derived
macrophages (BMDMs) derived from C57BL/6 mice expressing a
transgenic LT-responsive Nlrp1b allele from 129S1 mice (C57BL/
6Nlrp1b(129S1)mice; Tg+), or littermate controls (Tg2). C57BL/6
Tg- BMDMs showed no change in geometric mean fluorescence
when subjected to flow cytometry following AO staining and LT
treatment (Figure 1B).However,
BMDMs showed a time-dependent shift into LR/HG following
Figure 1. LT causes LMP in LTSmacrophages. (A) RAW 264.7
(RAW) cells pretreated with AO and subjected to LT (3 mg/mL LF and
1 mg/mL PA) for 60 or 75 minutes or media alone (NT). Cells were
analyzed by flow cytometry, live cells were gated based on forward and
side scatter and cells were analyzed for red (FL3) and green (FL1)
fluorescence. Cells are depicted here as a density plot. Upper left
quadrants represent cells with low red and high green fluorescence (LR/
HG). Numbers correspond to percent of cell population in LR/HG
quadrant. (B) C57BL/6Nlrp1b(129S1)BMDMs (B6 Tg+) or littermate controls
(B6) were pretreated with AO and subjected to either LT (1 mg/mL LF
and 1 mg/mL PA) for 85 or 95 minutes or media alone (NT). Cells were
analyzed as in (A). Density plot represent BMDMs from one of three
C57BL/6Nlrp1b(129S1)or C57BL/6 littermate controls and are representa-
tive of results obtained. (C) C57BL/6Nlrp1b(129S1)BMDMs were treated
with 1 mg/mL of LF, PA, LF and PA (LT), PA and LF-H719C (PA/mLF), or
10 ng/mL of lipopolysaccharide (LPS) for 90 min. Cells were collected
and analyzed for red and green fluorescence as in (A). BMDMs from
three C57BL/6Nlrp1b(129S1)were used for each condition and error bars
represent standard deviation.
LT Induces Nlrp1b-Mediated LMP
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LT-treatment (Figure 1B). Thus, in both BMDMs and immortal-
ized macrophage-like cell lines, LT causes relocalization of AO
that is dependent on expression of an LT-responsive Nlrp1b allele.
During intoxication, PA forms cation-selective, ion-conducting
channels in endosomal membranes that translocate LF in a
voltage-dependent manner . To determine if the LR/HG
population observed in response to LT was due to PA pore
formation rather than LMP, we performed AO staining of cells
treated with PA alone or PA in the presence of a catalytically
inactive lethal factor, LF-H719C, which binds but does not cleave
MKKs . We observed a pronounced increase in LR/HG only
in cells treated with the catalytically active LF and PA in both
C57BL/6Nlrp1b(129S1)BMDMs (Figure 1C) and RAW 264.7 cells
(data not shown). Therefore, alterations in AO staining in response
to LT are not explained by PA pore formation, but rather require
the catalytic activity of LF.
A measured decrease in acidic compartments could be due to
LMP or from loss of acidic vesicles through exocytosis, macro-
autophagy or disintegration of lysosomal membranes. To
determine if LT-treated cells show signs of lysosomal loss or
fusion, we stained and visualized acidic vesicles with Lysotracker
Red, a fluorescent probe that associates with the membranes of
acidic compartments, but unlike AO, continues to stain de-
acidified lysosomes. When added prior to a cytotoxic stimulus,
Lysotracker Red will continue to stain lysosomal membranes that
undergo LMP, but will disperse throughout the cell if the
lysosomal membrane disintegrates or fuses with the plasma
membrane [33,34]. Likewise, fusion of lysosomal membranes with
other compartments would result in a decrease in fluorescence
intensity or an increase in acidic vesicle size [35,36]. In LT treated
cells, Lysotracker Red-stained lysosomes were clearly visible
following LT-treatment up to the time of death (Figure 2).
Interestingly, Lysotracker Red continued to stain lysosomal
membranes up to 30 min following cell death, as determined by
membrane permeability to trypan blue (data not shown). This
observation is consistent with LMP, in which lysosomes lose
acidity and release lysosomal contents but appear structurally
normal [24,37]. Furthermore, no significant increase in lysosome
size was observed following LT treatment, indicating that
macroautophagy does not occur under the assay conditions
employed here (Figure 2) [38,39]. Therefore, the increase in the
AO LR/HG population following LT treatment is not due to
major lysosomal exocytosis or lysosomal fusion with non-acidic
vesicles, but is consistent with LMP.
Cathepsin B is active in the cytosol and inhibition of
cathepsins blocks LT-mediated pyroptosis
Cathepsins are a subtype of lysosomal acid hydrolases that
participate in protein turnover, antigen processing, pro-hormone
activation and, when released into the cytosol, cell death .
Cathepsin B (ctsB) activity was reported to be required for LMP-
mediated apoptosis and necrosis in response to multiple insults
including TNF-a, the chemotherapeutic pyrimethamine, the
antibiotics nigericin and staurosporine and the Mycobacterium
tuberculosis vaccine Bacillus Calmette-Guerin [41–46]. We tested
whether cathepsin proteolytic activity is required for LT-induced
cytotoxicity. Using CA074Me and z-FA-FMK, two compounds
that inhibit cathepsins including ctsB and ctsL, we observed that
both C57BL/6Nlrp1b(129S1)BMDMs and RAW 264.7 cells were
protected from LT (Figure 3A and 3B). In addition to their
cytosolic roles in cell death, cathepsins function within the
lysosomal lumen and extracellularly. To differentiate between
effects of CA074Me on intracellular versus extracellular cathepsin
activity, we utilized CA074, a ctsB inhibitor that is not cell
membrane permeable. Neither C57BL/6Nlrp1b(129S1)BMDMs, nor
RAW 264.7 cells, were protected from LT by pretreatment with
CA074 (Figure 3A and 3B). Therefore, the protection afforded by
CA074Me and z-FA-FMK is from inhibition of intracellular
To ensure that cathepsin inhibitors did not negatively impact
LT entry or activity, LF activity was analyzed by probing for
MEK2 cleavage following LT challenge. No detectable change in
LF activity was observed in the presence of z-FA-FMK (Figure 3C).
Although a slight delay in MEK1 cleavage was detected in cells
pretreated with 100 mM CA074Me (data not shown), no defect in
LF activity was apparent at 50 mM (Figure 3C), a concentration
that still provided maximal protection from LT (Figure 3B).
Therefore, inhibition of intracellular cathepsin activity blocks LT-
mediated cell death.
To directly test if ctsB is active in the cytosol during LT-
mediated cell death, we utilized a novel ctsB-specific molecular
probe, Ak-EVD-AMK . This probe specifically reacts with
Figure 2. Lysosome ultrastructure appears unaltered during Nlrp1b-mediated pyroptosis. (A) RAW 264.7 cells were pre-stained with
Lysotracker Red DND-99 followed by LT or untreated (NT) for 75 min and imaged on glass slides at 406magnification. Black arrows correspond to
condensed nuclear DNA observed in pyroptotic cells.
LT Induces Nlrp1b-Mediated LMP
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Figure 3. CtsB is active in the cytosol and inhibition of cathepsin activity blocks LT-mediated pyroptosis. (A) C57BL/6Nlrp1b(129S1)BMDMs
were pre-treated withvaryingconcentrationsof z-FA-FMK, CA074Me, or CA074for 4 hours, followedby addition of 400 ng/mL of PA and300 ng/mL of
LF (LT), or no treatment (NT), for an additional 3.5 hours. Cytotoxicity was measured using ATP-lite. BMDMs from three C57BL/6Nlrp1b(129S1)were used
with each condition tested in quadruplicate. Error bars represent standard deviation. (B) RAW 264.7 cells were pre-treated with varying concentrations
of z-FA-FMK, CA074Me, or CA074for 4 hours, followed by addition of 400 ng/mL of PAand300 ng/mLof LF (LT), or notreatment (NT), for anadditional
3.5 hours.Cytotoxicity wasmeasuredusingATP-lite. Thedatapresented herearerepresentativeof threeor moreindependentexperiments.Eachpoint
represents the mean of triplicate or quadruplicate samples from a single experiment, with error bars representing standard error. (C) RAW 264.7 cells
were pre-treated with 100 mM z-FA-FMK or 50 mM CA074Me for 4 hours followed by LT (400 ng/mL PA and 300 ng/mL LF; +) or no toxin (2) for
2.5 hours. Cell lysates were subjected to western blot analysis and probed with an antibody that recognizes the N-terminus of MEK2. (D) Specificity of
the ctsB probe Ak-EVD-AMK was determined by treating RAW 264.7 or NIH 3T3 cells with or without high concentration (5 or 10 mM) of probe.
Cycloaddition assays were performed on Ak-EVD-AMK labeled cellular lysates with azido-rhodamine, and Ak-EVD-AMK labeled proteins were analyzed
by in-gel fluorescence. The pro-form of ctsB is 43 kDa, whereas active ctsB is seen as either 31 kDa or 25 kDa. (E) In-gel fluorescence showing
electrophoretically separated proteins from RAW 264.7 cells treated with 400 ng/mL of PA and 300 ng/mL of LF and/or, low dose (625 nM) Ak-EVD-
AMK. Underthese conditions,cytosolic ctsBis preferentially labeled. Cycloadditionwas preformed as in (D). Thegel was then stained withcoomassie to
indicate equal sample loading.
LT Induces Nlrp1b-Mediated LMP
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active but not the pro-form of ctsB by covalently modifying the
active site cysteine of this protease. Ak-EVD-AMK reacts with
both cytosolic and lysosomal ctsB at high concentrations (5-
10 mM) (Figure 3D), but is specific for cytosolic ctsB at lower
concentrations (625 nM). Ak-EVD-AMK contains an alkyne
functional group that allows for subsequent covalent modification
of labeled proteins with azide-containing imaging probes via a
copper catalyzed cycloaddition reaction. RAW 264.7 cells were
treated with LT, labeled with low dose Ak-EVD-AMK to detect
cytosolic ctsB, lysed and the alkyne-modified proteins were
detected by cycloaddition of azido-rhodamine followed by SDS-
PAGE and direct in-gel fluorescence detection (Figure 3E). A
strong increase in Ak-EVD-AMK labeling of ctsB was observed at
low probe concentration in LT treated cells compared with no-
toxin controls. Thus, LT induces ctsB release into the cytosol,
consistent with LMP. Taken together, our data indicate that ctsB is
active in the cytosol during LT-mediated pyroptosis and that
cathepsins are required to induce cell death.
LT-induced LMP is a late event in pyroptosis
Several recent studies have begun to elucidate the pyroptotic
response to LT. Events that occur following intoxication include
(in order of occurrence) cleavage of MEKs and/or an unidentified
target(s), proteasome cleavage of unknown target(s), mitochondrial
dysfunction, potassium efflux, caspase-1 inflammasome activation,
plasma membrane permeability and cellular lysis with release of
IL-1b and IL-18 [9,13,48–52]. To determine the stage at which
LMP contributes to pyroptosis, we performed epistasis experi-
ments using chemical inhibitors and conditions that prevent LT-
induced pyroptosis. The observation that LMP is not detected in
LT-treated wildtype C57BL/6 BMDMs nor IC-21 cells (Figure 1B
and Figure S2A) suggests that LMP is downstream of Nlrp1b
activity. We found that heat shock or the presence of proteasome
inhibitors, two conditions that inhibit LT-induced caspase-1
activation and pyroptosis [48,49,51,53,54], prevented a shift to
LR/HG in C57BL/6Nlrp1b(129S1)BMDMs (Figure 4) or RAW
264.7 cells (Figure S2). Interestingly, the presence of 150 mM
exogenous potassium chloride, conditions that protect macro-
phages from LT lysis (data not shown), did not prevent AO
relocalization (Figure 4B). Of note, potassium chloride protection
from LT-mediated cellular lysis is also downstream of disruption of
mitochondrial membrane potential . Thus, our data supports
potassium efflux as a late event in pyroptosis.
Cellular stress proteins are altered during LT-mediated
LT-induced pyroptosis appears to be independent of gross
transcriptional changes [55-57], and is likely governed by changes
in the proteome. We investigated the affect of LT on the
macrophage proteome using two-dimensional difference gel
electrophoresis (2D-DIGE). MEK1 was cleaved by 20 min,
MEK2 by 40 min and cellular lysis occurred between 75 and
90 min post-LT challenge under the conditions employed here
(data not shown). We found generalized proteolysis following
70 min of LT intoxication (data not shown), consistent with LMP
or extrinsic apoptosis . Since we detected LF cytosolic activity
by 20 min, we chose to analyze the proteomic changes following
LT challenge at 30 and 40 min post-LT to identify early events in
the pyroptotic death pathway. LF-specific events were further
elucidated by comparison with macrophages treated with the
binding component (PA) alone.
2D-DIGE identified several proteins whose abundance increased
or decreased following LT challenge. The identities of proteins
whose abundance changed most significantly were determined by
excising protein spots, followed by trypsin digestion and mass
spectrometric analysis (Table 1). Proteome changes were validated
by western blot analysis, which confirmed that microtubule-
associated protein, RP/EB family, member 1 (Mapre1; GeneID:
13589), eukaryotic translation elongation factor 2 (EF-2; GeneID:
13629)(Figure 5A) and heat shockprotein 70 kDa(Hsp70;GeneID:
15511) (Figure 5B) increase following LT challenge. Interestingly,
we see fluctuations in protein abundance followed by loss of both
Hsp70 and EF-2 at later intoxication time points (Figure 5A and
5B). Bcl-2-associated athenogene 1 (Bag-1; GeneID: 12017) is an
anti-apoptoticgene that associateswith Mapre1, Hsp70andnuclear
Figure 4. Heat shock, proteasome and ctsB/L inhibition, but
not potassium chloride, prevents LT-induced LMP. (A) C57BL/
6Nlrp1b(129S1)BMDMs were heat shocked at 42uC (HS) for 15 min prior to
addition of LT (1 mg/mL of PA and 500 ng/mL of LF) for 90 min.
Proteasome inhibition was accomplished with co-incubation of cells
with 10 mM MG-132 and either LT (1 mg/mL of PA and 500 ng/mL of LF)
or PA only for 90 min. Cells were collected for flow cytometry and % LR/
HG was determined as in Figure 1A. Experiments were preformed using
BMDMs from three C57BL/6Nlrp1b(129S1)and samples were collected in
triplicate. Error bars represent standard deviation. (B) In a separate
experiment C57BL/6Nlrp1b(129S1)BMDMs were pretreated with LT
(400 ng/mL PA and 300 ng/mL LF) or without toxin (NT) for 2.5 hours.
Cells were also either pre-incubated with 50 mM CA074Me for 4 hours
or co-treated with 150 mM potassium chloride (KCl) followed by LT.
Cells were collected for flow cytometry and % LR/HG was determined as
in Figure 1A. Experiments were preformed using BMDMs from three
C57BL/6Nlrp1b(129S1)and samples were collected in triplicate. Error bars
represent standard deviation.
LT Induces Nlrp1b-Mediated LMP
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hormone receptors . We did not, however, observe changes in
Bag-1 protein levels following LT challenge (Figure 5A). Lamin A
(GeneID: 16905) appears to be processed during LT-treatment
since we found Lamin A to decrease in one excised spot while
increasing in another. We also found alterations in levels of a-
enolase, a known substrate of caspase-1 that colocalizes with Nlrp1b
inflammasomes [13,59]. Interestingly, cathepsin 7 precursor (also
known as CTS1 and cts7; GeneID: 56092) is homologous to ctsL
but is understudied and primarily associated with embryonic
development. Cathepsin 7 is expressed in RAW 264.7 cells and
may be proteolytically activated or change subcellular localization
in response to LT. Of the proteins found to change following LT
treatment, the vast majority are activated by or involved with the
cellular stress or heat shock response (Table 1). In addition to
inflammasome formation, it appears that LT activation of Nlrp1b
causes a stress response that results in numerous changes in the
proteome followed by generalized proteolysis.
Finally, we also found that Bid (GeneID: 12122), a potential
mediator of LMP-mediated cell death [24,60,61], is processed to
its active form, tBid, in the presence of LT (Figure 5B).
Interestingly, Bid can be cleaved by cathepsins B, H, L, S, K
[30,62], and potentially caspase-1 . Therefore, Bid may
amplify an LMP positive feedback loop (Figure 6A) and is a
potential mediator of LT-induced pyroptosis. Our data supports
pyroptosis as a type of programmed cell death mediated by
Nlrp1b, cathepsins and potentially Bid.
Pyroptosis is a pro-inflammatory PCD that occurs in response to
cellular recognition of danger signals [1–3,64]. Although requiring
caspase-1, pyroptosis maintains characteristics of necrosis [65,66],
a caspase-independent cell death. Here we show that LMP occurs
during LT-mediated pyroptosis, which leads to the release of ctsB
into the cytosol and resultant cell death. A causal role for cytosolic
cathepsin activity in pyroptosis is indicated by the ability of
CA074Me and z-FA-FMK, but not CA074, to block LT-mediated
cytolysis. Lysosomal membranes can be observed up to and after
the time of plasma membrane permeability, suggesting that LMP,
rather than lysosomal exocytosis, disintegration or fusion, occurs
during LT-mediated pyroptosis.
Previously, pyroptosis was differentiated from pyronecrosis by
the requirement for caspase-1 and ctsB, respectively . Although
casp-12/2macrophages show reduced sensitivity to LT, they are
partially susceptible to LT-mediated cytolysis by an unknown
process . In the absence of caspase-1 activity, other mediators of
LMP and pyroptosis, such as cathepsins, may be sufficient to
induce cell death. Since ctsB can induce cell death similar to
Figure 5. Proteomic changes in LT-treated cells. Western blots
showing electrophoretically separated proteins from RAW 264.7 cells
treated with LT for various time points, or untreated (NT). Arrows
indicate protein isoforms detected using protein-specific primary
antibodies and fluorescently labeled secondary antibodies. (A) Identical
cellular lysates were subjected to SDS-PAGE, transferred to PVDF and
probed with different primary and secondary antibodies. b-tubulin was
used as an equal loading control in each experiment. This blot
represents one of three independent experiments showing similar
results. (B) Identical cellular lysates were subjected to SDS-PAGE,
transferred to PVDF and probed with different primary and secondary
antibodies. Anti-Bid antibody recognizes both full-length Bid (FL-Bid)
and the truncated form (tBid). The membrane was exposed to film for
1 sec (low exposure) or 45 sec (high exposure) to detect both FL-Bid
and tBid. tBid protein surfaces at 50 min following LT treatment at high
toxin concentrations. This blot represents one of three independent
experiments showing similar results.
Table 1. Proteomic changes that occur in RAW 264.7 cells
treated with LT.
Accession Protein IDProtein nameStress (*)I/D
NP_062412 Cathepsin 7 precursorI
NP_001002011 Lamin A* I/D
reductase core protein 1
NP_542364 Nuclear aco2D
AAA40075Ribosomal protein S4D
NP_001002011 Lamin A*D
NP_034860 annexin A1*D
Proteins whose abundance was altered at both 30 and 40 min post-LT
challenge in 2D-DIGE were identified through LC-MS/MS. Stars represent
proteins whose abundance change is recorded in the literature to occur
following heat shock or stress. Proteins that increased (I) following exposure to
LT, versus PA only, and those that decreased (D) relative to PA only are
indicated. Fold increase or decrease varied from 0.5 to 3.0.
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pyroptosis in the absence of caspase-1 , the role of cathepsins in
pyroptosis may be substantial.
Inflammasome complexes are typically comprised of caspase-1,
caspase-11, a NOD/NLR family member and a caspase adaptor
protein, ASC. Unlike other NLRPs, Nlrp1b lacks a pyrin domain,
the ASC-binding domain, but does encode a caspase recruitment
domain (CARD) that may directly bind caspase-1 . In support
of an ASC-independent role in Nlrp1b-mediated cell lysis, RAW
264.7 cells, which lack ASC expression , respond to LT
similarly to ASC-expressing J774A.1 macrophage cell line and
C57BL/6Nlrp1b(129S1)BMDMs (Figure 1) [9,68,69]. Further, size
exclusion chromatography of J774A.1 cells treated with LT
revealed a shift in localization of caspase-1 from a low-molecular
weight fraction containing ASC to a high molecular weight
fraction containing Nlrp1b, but not ASC . Interestingly, ASC
is required for IPAF/NLRC4-mediated caspase-1 activation and
IL-1b production, but is not required for IPAF-mediated
pyroptosis [70–72]. Finally, ASC is not required for human
NALP1 inflammasome activation . Therefore, ASC is not
likely required for LT-mediated cell lysis, but may enhance IL-1b
and IL-18 release in response to this toxin. The role, or lack
thereof, for ASC in Nlrp1b-mediated pyroptosis is currently being
pursued in our laboratory.
There are conflicting data on exactly where in the cell death
pathway LMP plays a role. On one hand, we find that LMP, like
mitochondrial outer membrane permeability (MOMP), occurs
after involvement of Nlrp1b and the proteasome, indicating that
this is a late event. Furthermore, heat shock prevents LMP and
protects macrophages from lysis even when applied late in the
intoxication process . In contrast, we observed that potassium
chloride, which blocks inflammasome activation in other systems
, did not block LMP, suggesting that LMP occurs prior to or
independent of inflammasome activation. Interestingly, Newman
et al. found that the potassium channel inhibitor quinidine did not
prevent LT-induced LMP , however data is not shown.
Although preventing cellular lysis, 150 mM KCl supplemented
media does not inhibit LT-induced MOMP . Since
mitochondrial membrane disruption induces LMP , we
hypothesize that LT-induced MOMP is sufficient to induce
LMP in an Nlrp1b-independent manner. It would be interesting
to determine if quinidine prevents LT-induced MOMP, thus
We propose a model whereby LMP participates in a positive
feedback loop that requires Nlrp1b to amplify an LT-mediated
danger signal (Figure 6). Precedence for such a positive feedback
loop exists. For example, cathepsins released into the cytosol
during LMP cleave the pro-apoptotic protein Bid that induces
MOMP and further LMP [25,26,75]. Cathepsins also cleave and
activate caspase-1 , and both cathepsins and caspase-1 cleave
Bid [30,60,62,63], further augmenting a positive feedback
amplification loop. In this model, LMP could be up- or
downstream of inflammasome activation. Indeed, LMP can itself
act as a danger signal, inducing inflammasome activation and cell
death through changes in calcium concentration, cytosolic cathep-
sin activity, oxidative stress, or induction of MOMP [77–79]. It is
possible that initial LMP occurs upstream of Nlrp1b but LMP is not
detected using our AO relocalization assay. In this case, both LTS
and LTRcells would initiate a cell death pathway that involves
LMP, but that only cells containing an LTSNlrp1b amplify and
propagate the signal. This would coincide with the observation that
Figure 6. Model of LF internalization and activation of LMP and MOMP. LF binds oligomerized PA pre-pore and is internalized into
endosomes where acidic pH triggers PA to form a pore in the endosomal membrane and translocate LF into the host cytosol. Following translocation,
LF cleaves MKKs and induces Nlrp1-dependent pyroptosis. LF directly, or indirectly, causes LMP, resulting in release of cathepsins into the cytosol.
Cathepsins or LMP-mediated signaling may directly activate the inflammasome. Alternatively, LMP may occur downstream of inflammasome
activation, potentially through caspase-1 mediated cleavage of Bid. Activation of caspase-1 or cytosolic release of cathepsins can result in cleavage of
Bid and a positive feedback amplification of LMP, inflammasome activation and mitochondrial outer membrane permeabilization (MOMP).
LT Induces Nlrp1b-Mediated LMP
PLoS ONE | www.plosone.org7 November 2009 | Volume 4 | Issue 11 | e7913
cathepsin inhibitors prevent detectable LMP. Therefore, a feedback
loop induced first by minor leakage of cathepsins into the cytosol
could be amplified by increased inflammasome/caspase-1 activa-
tion, Bid cleavage, MOMP and/or activation of unknown signaling
Our finding that LMP is involved in LT-mediated cell death
may explain the activity of various inhibitors reported to protect
cells from LT. For example, calpain inhibitors and secretory
phospholipase A2 (sPLA2) inhibitors, both of which protect
against LMP [80–82], also protect cells from LT-induced
pyroptosis [49,83]. In addition, antioxidants such as N-acetyl-
cysteine are potent inhibitors of LMP and prevent LT-induced
release of IL-1b, a downstream product of inflammasome
activation [17,84,85]. Finally, heat shock provides strong
protection against LT-mediated pyroptosis through an unknown
mechanism , and Hsp70 protects from both LMP [86–88]
and MOMP [88–93] and is capable of reducing cellular damage
associated with these death pathways. Heat shock leads to
increased levels of Hsp70 and therefore, Hsp70 may play a
substantial role in mediating heat shock-induced resistance to LT.
Of note, all proteomic studies report a change in Hsp70, with one
group reporting a decrease in Hsp70 , while other groups
report an increase in Hsp70 following LT treatment [95–97].
Whether Hsp70 overexpression is sufficient to protect against LT-
induced LMP and pyroptosis is currently being explored. We also
report multiple stress response proteins, specifically those involved
in heat shock response, are altered during LT-induced pyroptosis.
A heat shock-type response may be a reaction to LT- or
pyroptosis-associated cell damage, though this response fails to
protect cells from major LMP and cytolysis induced by the toxin
dose used in in vitro studies.
Inflammasome activation and pyroptosis has broad biological
significance. While caspase-1 activation protects the host from
various pathogens during infection, excessive caspase-1 activation
contributes to various inflammatory disorders and septic shock
[3,7,8]. The control of caspase-1 activation and pyroptosis is an
attractive target for protecting cells from both microbial and
autoinflammatory attack. Our data support a role for lysosomal
cathepsins in NLR-mediated pyroptosis. CtsB is recently impli-
cated in activation of NLRP3-inflammasomes by crystalline
structures [77,78] and in caspase-1 cleavage . Here we show
that lysosomal damage and release of cathepsins are central to
pyroptosis initiated by LT.
Materials and Methods
Cell Culture and Reagents
RAW 264.7 cells and J774A.1 were cultured in DMEM
(Cellgro, Mediatech, Inc cat#10-017-CV) and IC-21 cells were
cultured in RPMI. Cell lines were obtained from ATCC. Femur
exudates from C57Bl/6 or C57BL/6Nlrp1b(129S1)transgenic animals
were cultured for 7 days in DMEM both supplemented with 10%
fetal bovine serum (Atlanta, cat#S11550), 1% penicillin/strepto-
mycin/glutamine (Gibco), 2% 14–22 conditioned media and
incubated in a 5% CO2humidified incubator at 37uC. BMDMs
from three C57BL/6Nlrp1b(129S1)or C57BL/6 littermate controls
were used as replicates for each experiment. Intoxication medium
consisted of DMEM containing 25 mM Hepes (Cellgro, Media-
tech, Inc. cat#15-018-CV), supplemented with 10% FBS and 1%
PSG. PA, LF and LF-H719C were produced and purified as
previously described . CA074 (N-1475) and CA074Me (N-
1660) were purchased from Bachem (Torrance, CA). LPS was
from Escherichia coli. Z-FA-FMK (cat# 342000) was purchased
Acridine orange relocation assay
Cultured cells (56105per well) were seeded in 6-well plates in
DMEM the night before intoxication. The next morning, media
was replaced with intoxication media containing 5 mg/ml acridine
orange (Calbiochem, cat# 113000) for 15 min under otherwise
standard culture conditions. Wells were then rinsed twice with
intoxication media and 1 mg/mL PA and 1 mg/mL LF (unless
otherwise stated) was added to LT-treated cells. Cells were
detached by scraping with a rubber policeman, collected by
pelleting at 5,0006g and washed three times with PBS (1 mL).
PBS with 1% formaldehyde (300 mL) was used to fix cells and
samples were subjected to flow cytometric assessment of red (FL3
channel) and green (FL1 channel) AO fluorescence using a Becton
Dickinson FACSCalibur Analytic Flow cytometer. Analysis was
performed using FLOWJO flow cytometry analysis software (Tree
Star, Inc., Ashland, Oregon).
Lysotracker staining and imaging
RAW 264.7 cells (16105per well) were seeded on 12 mm poly-
D-lysine coverslips (BD Biosciences cat# 354086) within 12-well
plates the day before experiment. DMEM containing 50 nM
Lysotracker Red DND-99 (Invitrogen-Molecular Probes) was
added to cells for 90 min under normal growth conditions. Cells
were then washed twice with PBS, followed by addition of
intoxication media alone or containing 1 mg/mL of LF and 1 mg/
mL of PA. Coverslips were removed from dish and live cells were
imaged in PBS containing 2 mM MgCl2using an inverted Nikon
Eclipse TE300 fluorescence microscope.
For 384-well plate format, cells were seeded at 26103cells
per well in white-bottom plates. Toxin and/or inhibitors were
added to a total of 60 mL of total media and incubated for the
time indicated. Experiments were halted with addition of 20 mL
of ATP-lite (PerkinElmer, Waltham, Massachusetts) and lumi-
nescence was measured using Victor 3V (PerkinElmer) plate
reader. Luminescence data is obtained in relative light units
(RLU). Background luminescence was subtracted from all
Cytosolic ctsB activity assay
Cultured cells (66106) were seeded in 10 cm dishes and treated
with 400 ng/mL PA and 300 ng/mL of LF. After 90 minutes, Ak-
EVD-AMK (625 nM for cytosolic probing; 5 or 10 mM for total
cellular ctsB probing) or DMSO was added to intoxication media
for an additional 30 min. The cells were then collected by gentle
scraping, pelleted by centrifugation at 2,0006g, and washed twice
with PBS (1 mL). Cell pellets were resuspended in 100 mL of ice-
cold NP-40/TEA lysis buffer (1% NP-40, 50 mM triethanolamine
(TEA), 150 mM NaCl, pH 7.4, with Complete Mini protease
inhibitor cocktail (Roche Biosciences, Indianapolis, IN)) for
30 min and then centrifuged at 4uC for 10 min at 13,2006 g.
Post-nuclear supernatants were collected and protein concentra-
tion was determined using Bio-Rad Protein Assay (cat# 500-
0001). NP-40/TEA lysis buffer was added to lysates to equate
experimental sample volumes and cycloaddition reactions were
performed as follows: Azido-rhodamine tag (100 mM, 5 mM stock
in DMSO) was added, followed by 1 mM TCEP (50 mM stock in
H2O) and 100 mM triazole ligand (1.7 mM stock in DMSO:t-
butanol 1:4). The samples were gently vortexed and 1 mM
CuSO4 (50 mM stock in H2O) was added. Samples were vortexed
again and allowed to react at RT for 1 h. Reactions were
terminated by addition of ice-cold acetone (1 mL), incubated at
LT Induces Nlrp1b-Mediated LMP
PLoS ONE | www.plosone.org8November 2009 | Volume 4 | Issue 11 | e7913
220uC for 20 min and centrifuged at 13,2006g 4uC for 30 min
to precipitate proteins. The supernatants were carefully decanted
and the resulting pellets dried at RT for 5 min to remove excess
acetone. The protein pellet was subsequently resuspended in 2X
SDS-protein reducing buffer and boiled for 10 min. Proteins were
then separated by SDS-PAGE and analyzed by in-gel fluorescent
scanning using a Typhoon scanner (GE Healthcare; excitation at
532 nM, emission at 580 nM).
Cells were lysed in 1% Triton X-100 buffer (150 mM NaCl;
50 mM Tris-HCl, pH 8; 0.1% SDS; 1% Triton X-100; 5 mM
MgCl2) with Complete Mini protease inhibitor cocktail (Roche
Biosciences), incubated on ice for 30 min, spun down at
13,2006 g for 10 min unless otherwise noted. Post-nuclear
supernatant protein concentration was determined using Bio-
Rad Protein Assay. Proteins were denatured by addition of 6X
reducing SDS-protein loading buffer and boiled for 10 min.
Samples were vortexed, spun down at 13,2006g for 10 minutes
and separated by SDS-PAGE (10% gel for MEK2 and ctsB; 15%
gel for Bid/tBid) followed by transfer to PVDF. Membranes
were blocked with 5% nonfat dried milk in TBST (50 mM Tris,
150 mM NaCl, 0.5% Tween 20, pH 7.6) for 1 hr at RT, then
incubated with primary antibody in 5% nonfat dried milk in
TBST for 1 h at RT or overnight at 4uC. Membranes were
washed 3x with TBST for 10 min and either developed or
probed with secondary antibody (goat anti-rabbit 1:10,000,
Sigma Aldrich) followed by washing 3x with TBST for 20 min.
Membranes were developed using ECL reagents (ImmunoStar,
BioRad) and autoradiography film (HyBlot, Danville Scientific).
To ascertain levels of endogenous proteins, SDS-PAGE gels
were stained with coomasie and photographed or membranes
were probed with anti-tubulin antibody (T5168 from Sigma
Aldrich) followed by goat anti-mouse IgG (cat# 28173 from
AnaSpec, Inc. San Jose, CA). Anti-Bid antibody (# 2003), anti-
Hsp70 antibody (#4872), anti-eEF2 (#2332) were purchased
from Cell Signaling Technology; anti-Bag-1 (C16) antibody
(DB004) from Delta Biolabs, Gilroy, CA, anti-MAPRE1 (EB1,
H-70: sc-15347) antibody and MEK-2 (N-20): sc-524 from Santa
Two dimensional difference gel electrophoresis (2D-DIGE)
was performed at Applied Biomics, Hayward, CA. Cells were
seeded the previous day at 107cells per 10 cm plate. Cells were
treated with either PA (1 mg/mL) or PA and LF (1 mg/mL each)
for 30 or 40 min. Timing of sample collection was carefully
calibrated by western blot analysis of MEK1 and MEK2
20 min. Following LT-challenge, cells were collected by gentle
scraping, washed 3x with PBS, pelleted at 2,0006 g for 5 min
and pellets were flash-frozen in ethanol-dry ice bath and shipped
to shipped to Applied Biomics. Proteins were extracted and
labeled with either Cy3 or Cy5. Isoelectric focusing in the first
dimension was carried out at pH 3–10, and size-based
separation in the second dimension was performed using a 9–
12% linear gradient SDS-PAGE. Proteomic changes were
detected using DeCyder software and spots were cut out at
Applied Biomics and mailed to UCLA. Spots were digested with
trypsin and subjected to LC-MS/MS (UCLA) using either a
QqTOF instrument or nanospray. Peptide sequencing was
accomplished with nanoflow high performance liquid chroma-
tography system (LC Packings, Sunnyvale, CA, USA) with a
nanoelectrospray (nano-ESI) interface (Protana, Odense, Den-
mark) and an Applied Biosystems/Sciex QSTAR XL (QqTOF)
mass spectrometer (Foster City, CA). The samples were first
loaded ontoa LC Packings
(150 mm63 mm; particle size 5 mm) and washed for two minutes
with the loading solvent, 0.1% formic acid. The samples were
then injected into a LC Packings PepMap C18 column
(75 mm6150 mm; particle size 5 mm) for nano-LC separation
at a flow rate of 220 nL/min. For each LC-MS/MS run,
typically 6 mL sample solution was loaded to the precolumn first
and washed with the loading solvent of 0.1% FA. The eluents
used for the LC were (A) 0.1% formic acid and (B) 95% ACN/
5% H2O/0.1% FA. The following gradient was used: 6% B to
24% B in 18 min, 24% B to 36% B in 6 min, 36% B to 80% B in
2 min and stayed at 80% B for 8 min. The column was finally re-
equilibrated with 6% B for 16 min before the next run.
A New Objective (Woburn, MA) PicoTip tip (i.d. 8 mm) was
used for spraying with the voltage set at 1750 V. Peptide product
ion spectra were automatically recorded during the LC-MS runs
by the information-dependent analysis (IDA) on the mass
spectrometer. Argon was employed as the collision gas. Collision
energies for maximum fragmentation were automatically calcu-
lated using empirical parameters based on the charge and mass-to-
charge ratio of the peptide. Protein identifications were deter-
mined using Mascot database search algorithm (Matrix Science).
low green subpopulation. C57BL/6Nlrp1b(129S1)BMDMs (B6 Tg+)
or littermate controls (B6) were analyzed for changes in AO
fluorescence as in Figure 1B, except cells were not gated via
forward and side scatter for normal cell morphology. Density plot
represents BMDMs from one of three C57BL/6Nlrp1b(129S1)or
C57BL/6 littermate controls that were tested with similar results
Found at: doi:10.1371/journal.pone.0007913.s001 (1.19 MB TIF)
AO relocalization in ungated cells contain a low red,
phenotype as BMDMs in AO relocalization. (A) RAW 264.7 cells
pretreated with AO for 20 minutes were treated with LT (500 ng/
mL LF and 1 mg/mL PA) or left untreated (NT) for 3 to 4 hours.
Cells were collected for flow cytometry and analyzed as in
Figure 1A. Results represent duplicate samples from two
independent experiments. Error bars represent standard error.
(B) RAW 264.7 were heat shocked at 42uC (RAW + HS) or left
untreated (RAW) for 15 min prior to addition of AO, followed by
LT (3 mg/mL LF and 1 mg/mL of PA) for 75 and 90 min. Cells
were analyzed for changes in AO fluorescence as in Figure 1A.
Density plot represents one of three independent experiments with
Found at: doi:10.1371/journal.pone.0007913.s002 (1.32 MB TIF)
RAW 264.7 and IC-21 cells display a similar
We would like to thank Jill Terra (UCLA), Jeremy Mogridge (University of
Toronto), Benhur Lee (UCLA), Robert Damoiseaux (MSSR, UCLA) and
Howard C. Hang (Rockefeller) for reagents and technical assistance and
Eric Boyden and Bill Dietrich for C57BL/6 Nlrp1b129S1transgenic mice.
Conceived and designed the experiments: KA MRP JPW JL TWM KAB.
Performed the experiments: KA MRP YY. Analyzed the data: KA MRP
YY SB KAB. Contributed reagents/materials/analysis tools: MRP JPW JL
TWM KAB. Wrote the paper: KA KAB.
LT Induces Nlrp1b-Mediated LMP
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