Anthrax lethal toxin and Salmonella elicit the
common cell death pathway of caspase-1-dependent
pyroptosis via distinct mechanisms
Susan L. Fink*, Tessa Bergsbaken†, and Brad T. Cookson†‡§
*Molecular and Cellular Biology Program, Departments of†Microbiology and‡Laboratory Medicine, University of Washington, Seattle, WA 98195
Edited by Charles A. Dinarello, University of Colorado Health Sciences Center, Denver, CO, and approved January 30, 2008 (received for review
August 5, 2007)
inflammatory cytokines. In Salmonella-infected macrophages,
caspase-1 also mediates a pathway of proinflammatory pro-
grammed cell death termed ‘‘pyroptosis.’’ We demonstrate active
caspase-1 diffusely distributed in the cytoplasm and localized in
discrete foci within macrophages responding to either Salmonella
infection or intoxication by Bacillus anthracis lethal toxin (LT). Both
stimuli triggered caspase-1-dependent lysis in macrophages and
dendritic cells. Activation of caspase-1 by LT required binding,
uptake, and endosome acidification to mediate translocation of
lethal factor (LF) into the host cell cytosol. Catalytically active LF
cleaved cytosolic substrates and activated caspase-1 by a mecha-
nism involving proteasome activity and potassium efflux. LT acti-
vation of caspase-1 is known to require the inflammasome adapter
Nalp1. In contrast, Salmonella infection activated caspase-1
through an independent pathway requiring the inflammasome
adapter Ipaf. These distinct mechanisms of caspase-1 activation
converged on a common pathway of caspase-1-dependent cell
death featuring DNA cleavage, cytokine activation, and, ulti-
mately, cell lysis resulting from the formation of membrane pores
between 1.1 and 2.4 nm in diameter and pathological ion fluxes
that can be blocked by glycine. These findings demonstrate that
distinct activation pathways elicit the conserved cell death effector
mechanism of caspase-1-mediated pyroptosis and support the
notion that this pathway of proinflammatory programmed cell
death is broadly relevant to cell death and inflammation invoked
by diverse stimuli.
single-cell analysis ? apoptosis ? inflammasome ? inflammation ?
programmed cell death
tory cytokines. Caspase-1 has emerged as a critical determinant
of both pathological inflammation and resistance to infectious
diseases. Notably, caspase-1-deficient mice are protected from
endotoxic shock (1, 2), ischemic injury (3, 4), and inflammatory
salmonellosis (6, 7). Salmonella enterica serovar Typhimurium is
a pathogen that invades host macrophages and stimulates
caspase-1-dependent cell death (8). Salmonella-infected macro-
phages produce activated IL-1? and IL-18 and undergo rapid
lysis with the release of inflammatory intracellular contents, and
thus the term ‘‘pyroptosis’’ is used to describe this form of
proinflammatory cell death (9).
Activation of caspase-1 occurs via induced proximity in in-
flammasomes or pyroptosomes, which are protein complexes
analogous to the apoptosis-inducing apoptosome (10, 11). In-
flammasomes contain NOD-like receptor (NLR) family pro-
teins, which are cytosolic pattern-recognition receptors stimu-
lated by infectious agents and endogenous danger signals.
Salmonella-induced activation of caspase-1 requires the host
NLR protein Ipaf, as well as the bacterial type III secretion
system (T3SS) and flagellin (10). The NLR protein Nalp3
he caspase-1 protease causes cell death and cleaves the
precursors of IL-1? and IL-18, producing mature inflamma-
activates caspase-1 in response to extracellular ATP binding to
cell surface P2X7receptors (12), and Nalp1 is required for the
activation of caspase-1 and macrophage death in response to
anthrax lethal toxin (LT), a critical virulence factor of Bacillus
anthracis (13). LT entry into host cells has been elegantly
characterized, yet the mechanism(s) of cytotoxicity are incom-
pletely defined. Further, it is unclear how inflammasomes are
regulated or how different stimuli activate caspase-1. Events
downstream of caspase-1 activation, other than IL-1? and IL-18
activation, have only recently been described for Salmonella
infection (14). Here we demonstrate that LT intoxication and
which converge to cause host cell death using an apparently
conserved program of caspase-1-mediated pyroptosis. Together
with the observations that caspase-1 is involved in a wide variety
of pathological conditions, our data support the idea that the
proinflammatory programmed cell death pathway of pyroptosis
is of broad biological relevance to cell death and inflammation
triggered by diverse stimuli.
LT and Salmonella Stimulate Caspase-1-Dependent Death of Macro-
phages and Dendritic Cells (DCs). We examined the activation of
caspase-1 by staining macrophages with a fluorescent peptide
(FAM-YVAD-FMK) that binds specifically and irreversibly to
active caspase-1. Whereas mock-infected macrophages have no
detectable active caspase-1 (Fig. 1A), those infected with Sal-
monella contain active caspase-1 in large bright foci and diffusely
throughout the cell [Fig. 1B and supporting information (SI) Fig.
9]. The localization of active caspase-1 in LT-treated macro-
phages was strikingly similar, with discrete brightly staining foci
and diffuse active caspase-1 (Fig. 1C). LT has been suggested to
stimulate macrophage apoptosis (15). However, a highly sensi-
tive assay did not detect activity of the central apoptotic effector,
caspase-3, in LT-treated macrophages (SI Fig. 10 and SI Mate-
rials and Methods).
LT and Salmonella are both cytotoxic for macrophages (8, 16),
and we confirmed cell lysis by measuring the release of cytosolic
lactate dehydrogenase (LDH). LT-intoxicated and Salmonella-
infected macrophages underwent caspase-1-dependent lysis that
was prevented by the specific caspase-1 inhibitor YVAD (17),
but not the negative control inhibitor zFA (Fig. 1D and SI Fig.
11). Caspase-1-independent release of LDH by cells treated with
H2O2was not affected by YVAD or zFA (Fig. 1D). DCs also are
Author contributions: S.L.F., T.B., and B.T.C. designed research; S.L.F. and T.B. performed
research; S.L.F., T.B., and B.T.C. analyzed data; and S.L.F. and B.T.C. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
§To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2008 by The National Academy of Sciences of the USA
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