MAJOR ARTICLE

Staphylococcus aureus Activation of Caspase

1/Calpain Signaling Mediates Invasion Through
Human Keratinocytes
Grace Soong,1 Jarin Chun,2,a Dane Parker,1 and Alice Prince1,2
1

Department of Pediatrics and 2Department of Pharmacology, College of Physicians amd Surgeons, Columbia University, New York, New York

(See the editorial commentary by Otto, on pages 14835.)

The USA300 strains of Staphylococcus aureus are the major cause of skin and soft tissue infection in the
United States. Invasive USA300 infection has been attributed to several virulence factors, including protein A
and the -hemolysin (Hla), which cause pathology by activating host signaling cascades. Here we show that S.
aureus exploits the proinammatory bias of human keratinocytes to activate pyroptosis, a caspase 1dependent form of inammatory cell death, which was required for staphylococci to penetrate across a keratinocyte
barrier. Keratinocyte necrosis was mediated by calpains, Ca2+-dependent intracellular proteases whose
endogenous inhibitor, calpastatin, is targeted by Hla-induced caspase 1. Neither Panton-Valentine leukocidin
nor protein A expression was essential, but inhibition of either calpain or caspase 1 activity was sufcient to
prevent staphylococcal invasion across the keratinocytes. These studies suggest that pharmacological interruption of specic keratinocyte signaling cascades as well as targeting the Hla might prevent invasive skin
infection by staphylococci.

The morbidity and mortality associated with the epidemic USA300 strains of methicillin-resistant Staphylococcus aureus (MRSA) in the United States has been
well documented [1]. In addition to the invasive infections that are associated with signicant morbidity
and mortality, there is also substantial economic cost
associated with the skin and soft tissue infections due
to these strains [2]. Exactly which of the many
USA300 virulence factors cause this excessive morbidity is widely debated but of great interest in vaccine
development [3]. Participation of the -hemolysin

Received and accepted 5 December 2011; electronically published 28 March

2012.
Presented in part: Annual Meeting of the Infectious Diseases Society of
America, October 2011, Boston, Massachusetts. Abstract 31592.
a
Present afliation: Memorial Sloan-Kettering Cancer Center, New York,
New York.
Correspondence: Alice Prince, MD, Department of Pediatrics, Columbia University, 650 W 168th St, BB416, New York, NY 10032 (asp7@columbia.edu).
The Journal of Infectious Diseases 2012;205:15719
The Author 2012. Published by Oxford University Press on behalf of the Infectious
Diseases Society of America. All rights reserved. For Permissions, please e-mail:
journals.permissions@oup.com.
DOI: 10.1093/infdis/jis244

(Hla) in the pathogenesis of skin infection is well

documented [46], but it remains unclear exactly how
these nonmotile bacteria invade through the barrier
posed by the multiple layers of proliferating and cornied keratinocytes that comprise normal human skin.
Staphylococcus aureus has been shown to invade into
human keratinocytes and cause both necrotic and
apoptotic forms of cell death, a process attributed to
several adhesins and virulence factors [7]. Murine
models of skin infection are problematic because staphylococci have difculty obtaining iron from murine
hemoglobin [8] and mouse models require large intradermal inoculations of bacteria, obviating the relevance of the physical and immunological barrier
properties of normal human skin [4].
Human keratinocytes are dynamic cells involved in
a highly ordered set of developmental activities;
initially proliferation, then maturation, cornication,
and shedding [9]. As active participants in innate
immune signaling, they express Toll-like receptors
(TLRs) either constitutively or by induction [10],
NODs, and caspases, components of the NLRP3 inammasome [11, 12]. In response to pathogens,
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keratinocytes rapidly produce antimicrobial peptides as well as

chemokines and cytokines to recruit and activate phagocytes.
Given these defenses, normal human skin is remarkably resistant to bacterial infection, even that associated with USA300
MRSA strains. Staphylococcus aureus strains have evolved with
their human hosts and are especially adept at exploiting the
immune responses that they evoke. Much of the pathology
they induce is not necessarily due to the direct toxicity of their
virulence factors but instead is due to the immune response
elicited. Recent studies have demonstrated that S. aureus
protein A, by activating epithelial RhoA/MLC and calpain signaling, mediates staphylococcal invasion through the paracellular junctions of the airway epithelium [13]. We postulated
that staphylococcal exploitation of keratinocyte signaling
could be responsible for penetration of these organisms
through the barrier posed by human keratinocytes and characterized the signaling pathways that are involved.

Ca2+ Imaging

HaCaT cells were grown to 80% conuence in coverglass chamberslides and loaded for 1 hour at room temperature with 2 M
Fluo-3/AM in the presence of 0.02% pluronic acid in Minimum
Essential Medium Eagle. Cells were washed with PBS and incubated at 37C for 1 hour in RPMI 1640 medium. Immediately
after adding 108 colony-forming units (CFUs) of USA300,
Fluo-3/AM uorescence imaging was obtained and collected at
6-second intervals using a Zeiss LSM 510 META scanning confocal microscope and analyzed using the ImageJ program.
Apoptosis-Pyroptosis Assays

MRSA USA300 (LAC) wild-type (WT) and protein A (SpA)

mutant were grown as described elsewhere [14]. USA300 WT
and Hla mutant were provided by Juliane Bubeck-Wardenburg (University of Chicago), and USA300 WT and PantonValentine leukocidin (PVL) mutant were provided by Frank
DeLeo (National Institute of Allergy and Infectious Diseases).
The human keratinocyte HaCaT cell line was obtained from
Angela Christiano (Columbia University) and grown in
Roswell Park Memorial Institute (RPMI) 1640 medium with
10% fetal bovine serum.

HaCaT cells were grown to conuence in chamber slides, and

108 CFU/mL bacteria at 1:4 dilution was added to chambers.
For inhibitor studies, 20 M calpeptin (calpain inhibitor) was
incubated with cells for 30 minutes prior to bacterial stimulation.
Pan-caspase inhibitor, Z-VAD-FMK, and the specic caspase 1
inhibitor Z-WEHD-FMK were added at the same time as bacteria. After 2 hours of incubation at 37C, cells were washed 3
times with cold PBS, then incubated for 30 minutes on ice
with Molecular Probes YO-PRO-1 + PI assay according to the
manufacturers instructions. For uorescence microscopy, after
3 cold PBS washes, 1% paraformaldehyde in PBS was added to
wells, which were then imaged by confocal microscopy using a
Zeiss LSM 510 Meta Inverted microscope. All detector gain
and amplication settings were identical for each image. For
ow cytometry, after incubation with YO-PRO-1 + PI and PBS
washes, cells were incubated with 0.02% ethylene glycol tetraacetic acid in PBS to induce detachment. Cells were scraped and
transferred to tubes containing paraformaldehyde to a nal concentration of 1% for ow cytometry analysis by means of a BD
FACSCalibur using Cell Quest software (Becton Dickinson).

Organotypic Cultures and Microscopy

Statistics

Organotypic cultures of human keratinocytes in primary

culture were obtained from the Cell and Tissue Kinetics Core of
the Columbia University Department of Dermatology Skin
Disease Research Center. Human keratinocytes were grown at
an air-liquid interface supported by a dermal substitute matrix
as a 3-dimensional model system composed of the dermal and
the epidermal compartments. Following 24 hours of stimulation
with USA300 or phosphate-buffered saline (PBS), human organotypic skin equivalents were stained with hematoxylin-eosin.

Samples with normal distribution were analyzed by Student t

test. Differences between groups were considered signicant at
P < .05. Multiple comparisons were analyzed using 1-way
analysis of variance with an appropriate posttest. Statistical
analysis was performed using GraphPad Instat version 3.0.

METHODS
Bacterial Strains and Cell Line

Bacterial Transmigration, Confocal Microscopy, and Dextran

Permeability

All analyses were performed as described elsewhere [13] with

the following inhibitors: calpeptin (20 M), cytochalasin D
(20 M), TAPI (50 M), GM6001 (20 M), or caspase 3
inhibitor I (10 M) from Calbiochem, EMD; Z-VAD-FMK
(10 M) or Z-WEHD-FMK (10 M) from G-Biosciences; and
Z-YVAD-FMK (10 M) from Enzo.
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RESULTS
USA300 Accumulates Within Keratinocytes

We examined the effects of S. aureus USA300, the epidemic

MRSA clone responsible for the majority of skin as well as
invasive infection in the United States [1], on human keratinocytes in primary organotypic culture (Figure 1A). Following a
24-hour incubation with USA300 S. aureus, human keratinocytes in primary culture remained intact; the basal layers of
keratinocytes showed vacuolization and the proliferating layers
were obviously thickened, an effect that has been attributed to
Hla-EGFR interactions [15]. Confocal images demonstrated

Figure 1. Interactions of Staphylococcus aureus strain USA300 and human keratinocytes. A, Light microscopic images of organotypic cultures of
human keratinocytes grown at an air-liquid interface supported by a dermal substitute matrix as a 3-dimensional model system composed of the dermal
and epidermal compartments under control conditions ( phosphate-buffered saline [PBS]) and following a 24-hour incubation with wild-type USA300. B,
z-section confocal images of HaCaT cells grown in a polarized fashion on transwells, following 4-hour and 24-hour incubation with USA300, imaged
after staining with phalloidin (red ) and antiS. aureus (green). C, x-y sections of infected HaCaTs demonstrating focal localization of organisms primarily
restricted to the apical surfaces of the cells. Staining is phalloidin (red ) and antiS. aureus (green). Left, enlarged rst apical section. Right, series of
sections from apical to basal; sequence, left to right, top to bottom.

USA300 on the surface of polarized HaCaT cells, a human

keratinocyte cell line, at 4 hours but no evidence of internalization or paracellular invasion at that time (Figure 1). At 24
hours staphylococci appeared to have accumulated within keratinocytes, in a focal distribution pattern, primarily at the
apical surface of the cell cultures (Figure 1B and 1C). Staphylococci were not observed within the paracellular spaces.
Participation of S. aureus Virulence Factors in the Activation
of Inammasome Signaling

These images of keratinocytes whose cytoplasm appeared to

be entirely replaced by S. aureus suggested the induction of
apoptosis/necrosis or pyroptosis, a caspase 1dependent form
of necrotic, proinammatory cell death [16]. Staphylococcal

activation of the NLRP3 inammasome has been well documented in immune cells [17] and mouse models of skin infection [11], although there are data to suggest the participation
of additional pathways in the induction of apoptosis and necrosis as well [7, 12]. Staphylococcus aureus activation of the
NLRP3 inammasome is thought to involve 2 stimuli, TLR2or NOD2-associated induction of prointerleukin 1 (IL-1)
production and Hla pore-dependent K+ efux inducing a sufcient cell stress to activate caspase 1 [18]. Caspase 1 activity
results in production of IL-1, a potent proinammatory cytokine that is critical for polymorphonuclear leukocyte (PMN)
recruitment and eventual clearance of staphylococcal skin infection [6]. Whether virulence factors such as protein A,
which activates TNFR1 signaling and thus is potentially linked
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Figure 2. Activation of keratinocyte inammasome signaling by Staphylococcus aureus USA300 strains. A, Constitutive production of prointerleukin
1 (IL-1) shown by immunoblot of cell lysates following exposure to wild-type (WT) USA300 and mutant strains. B, Immunodetection of caspase 1, 3,
and 7 production in keratinocytes 2 hours after exposure to USA300 strains. C, Quantication of caspase 1 activity in culture supernatants by colorimetric assay (G-Biosciences) following incubation with USA300 strains for 20 hours. *P < .01 compared with WT (1-way analysis of variance, Dunnett
posttest). Data are mean (SD). D, Secretion of IL-1 into culture supernatant following 2-hour exposure to USA300 strains and effect of the pan-caspase
inhibitor Z-VAD-FMK (Z-VAD) quantied by enzyme-linked immunosorbent assay. *P < .001 compared with WT; **P < .001 compared with untreated
(1-way analysis of variance, Tukey posttest); n = 6 for each. Data are mean (SD). Abbreviations: Hla, -hemolysin; PVL, Panton-Valentine leukocidin;
SpA, protein A.

to apoptosis [19], or the PVL toxin might also contribute to

inammasome signaling is not established.
We postulated that constitutive expression of proIL-1 by
keratinocytes [20] as shown (Figure 2A) would prime them for
activation of the inammasome. Because caspase 1 is required
for proIL-1 processing, we similarly documented the production of caspases 1, 3, and 7, which were induced by WT
USA300, PVL mutant, or SpA mutant (which lack the
conserved surface protein that interacts with TNFR1 [19]) with
the exception of the Hla strain that induced signicantly less
caspase 1 (Figure 2B), which was quantied by a colorimetric
assay (G-Biosciences) (Figure 2C). In response to these S.
aureus strains, there was signicant production of IL-1 that
was signicantly decreased in cells treated with a pan-caspase
inhibitor (Figure 2D), indicating that the majority of IL-1
production in this setting is from inammasome activation.
USA300 Activation of Keratinocyte Calpain Activity

Caspase 1 also targets calpastatin, the endogenous inhibitor of

the calpains, intracellular proteases that participate in apoptosis and degradation of intracellular components [21]. To determine whether caspase expression in human keratinocytes was
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associated with the activation of calpains, we rst conrmed

the expression of calpains 1 and 2 and calpastatin in the
HaCaT cells (Figure 3A) as well as the generation of Ca2+ ux
(Figure 3B) required for calpain activity in response to
USA300 [22]. We predicted that WT USA300, but not the
Hla mutant, would activate the degradation of calpastatin,
through caspase 1 activation. This was conrmed in immunoblots showing calpastatin breakdown in keratinocytes stimulated with WT S. aureus that are capable of activating caspase
1, but not in response to the Hla mutant or in cells pretreated with calpeptin, a calpain inhibitor expected to interfere
with caspase binding (Figure 3C).
USA300 Activates Apoptosis and Pyroptosis in Human
Keratinocytes

Having demonstrated caspase 1 and inammasome activation

in HaCaT cells exposed to WT but not Hla S. aureus, we
next examined the biological consequences of this cellular
response to infection. We differentiated the induction of pyroptosis, or necrosis associated with caspase 1 activity, which
typically involves the rapid loss of the integrity of the plasma
membrane [16, 23] from apoptosis associated with caspases 3

Figure 3. Calpain expression in keratinocytes. A, Calpain expression in HaCaT cells was demonstrated by reverse-transcription polymerase chain
reaction using primers for calpain 1 (C1), calpain 2 (C2), calpastatin (Cst), and an actin control (A). B, Activation of Ca2+ uxes in Fluo-3 loaded HaCaTs
following the addition of Staphylococcus aureus strain USA300 or thapsigargin (Thaps) as a positive control. C, Immunodetection of calpastatin in
HaCaT lysates following 6-hour incubation with wild-type (WT) alone or pretreated with calpeptin (Calp) or -hemolysin (Hla) mutant as compared
with a medium control (M).

and 7, using differential uorescent labeling (Vybrant apoptosis assay kit, YO-PRO-1/propidium iodide, Molecular
Probes). After 2 hours of exposure to WT USA300 but not the
Hla mutant, both apoptotic and necrotic keratinocytes were
observed (Figure 4A). PVL and SpA mutants activated
apoptosis and necrosis at rates similar to the WT strain (data
not shown). In the presence of a pan-caspase or a caspase-1
specic inhibitor, the numbers of both apoptotic and necrotic/
pyroptotic cells decreased signicantly (Figure 4B).
An additional marker of cell necrosis is HMGB1, an
alarmin that is released from the cell nucleus and a potent activator of inammation [24]. Release of HMGB1 from the cells
exposed to WT USA300 but not the Hla mutant further indicated the induction of necrosis and activation of another
proinammatory signal (Figure 4C).
Calpain or Caspase 1 Inhibitors Rescue Keratinocytes From
Hla-Mediated Pyroptosis

We postulated that targeting the effectors of cell necrosis, blocking either caspase 1 or the calpains, would prevent keratinocyte
cell death. Treatment of the keratinocytes with calpeptin signicantly decreased the numbers of both apoptotic and necrotic/
pyroptotic cells, consistent with the predicted role of caspase 1
in activating calpains that contribute to cell autolysis. By uorescence-activated cell sorting analysis, a population of smaller,
condensed keratinocytes was readily apparent in the HaCaTs
exposed to WT but not Hla staphylococci (Figure 5A). Pretreatment of the keratinocytes with either calpeptin or a caspase
1 inhibitor, but not a caspase 3 inhibitor, signicantly decreased

this fraction of necrotic, and in this case specically pyroptotic,

cells (Figure 5B). Despite the generation of pyroptotic cells, the
tissue culture wells appear to remain fully populated with intact
monolayers of keratinocytes as viewed with transmission
microscopy; the cells exposed to WT staphylococci for 6 hours
had condensed nuclei, rounded cytoplasm, and scattered permeability to trypan blue and were distinct from the larger cells
with well-dened borders observed in the Hla or mediumexposed controls (Figure 5C).
Pyroptotic Keratinocytes Facilitate S. aureus Penetration
Through Keratinocytes

Pyroptotic keratinocytes could provide a focus for S. aureus to

accumulate within the dead and dying cells, eventually reaching sufcient numbers to breach the keratinocyte barrier. This
hypothesis was tested by comparing the numbers of WT
versus Hla mutants able to penetrate through HaCaTs grown
on Transwells (Corning-Costar) (Figure 6A). There was signicantly greater transmigration of the WT USA300 strain
than the Hla mutant (P < .0001), whereas the SpA strain
invaded as well as the WT control. The PVL strain was also
less capable of transmigration but not as decient as the Hla
strain. To assess the integrity of the keratinocyte barrier, we
monitored translocation of 3000 MW uorescent dextran
from the apical to the basal chamber of the transwells
(Figure 6B). Only the Hla strain was associated with signicantly less dextran permeability, suggesting that Hla but not
PVL was critical in the disruption of the integrity of the keratinocyte barrier.
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Figure 4. Staphylococcus aureus strain USA300 induces pyroptosis in keratinocytes. A, Fluorescence imaging was used to detect apoptotic keratinocytes permeable to YO-PRO-1 (green) that binds DNA of cells with semipermeable cytoplasmic membranes and pyroptotic, fully permeable necrotic
cells (PI) (red) following a 2-hour exposure to USA300: i, YO-PRO-1(+)apoptotic (green); ii, PI(+)pyroptotic (red); iii, transmission brighteld image;
iv, merged. B, Flow cytometric quantication of apoptotic (YO-PRO-1+) (green) vs necrotic/pyroptotic (PI+) (red) cells as identied in (A), from cultures
treated with wild-type (WT) USA300 under control conditions or with pan-caspase inhibitor Z-VAD-FMK (ZV), a caspase 1specic inhibitor, Z-WEHDFMK (ZW), or calpeptin (Cpep), as compared with the effects of the -hemolysin (Hla) mutant strain. *P < .01 compared with WT (1-way analysis of
variance, Dunnett posttest). Data are mean (SD), representative of 2 individual experiments. C, Immunodetection of HMGB1 in keratinocyte supernatants
following 6-hour incubation with WT or Hla USA300. Abbreviation: MFI, mean uorescence activity.

Caspase 1 or Calpain Inhibitors Prevent USA300 Penetration

Through Keratinocytes

The predicted mechanism of staphylococcal penetration

through the keratinocytes, via caspase 1mediated cleavage of
calpastatin, activation of calpains, and pyroptosis, was further
documented. USA300 transmigration across HaCaTs treated
with calpeptin, a calpain inhibitor, or a caspase 1 inhibitor
was signicantly decreased whereas treatment of the keratinocytes with a caspase 3 inhibitor did not alter rates of transmigration (Figure 6C). Cytochalasin D treatment of the HaCaTs,
which prevents the endocytosis of S. aureus or its components
[25], the metalloproteinase inhibitor TAPI, and a general protease inhibitor, GM6001, did not inhibit transmigration (data
not shown). Thus, inhibition of caspase 1 or calpain activity
protected the keratinocytes from pyroptosis and staphylococcal penetration.
DISCUSSION
These experiments further characterize the signaling cascades
exploited by S. aureus to cause skin infection. Our data
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suggest that it is the proinammatory bias of human keratinocytes and the expression of specic staphylococcal
toxins, particularly Hla, that result in focal skin infection.
Human keratinocytes, even in the absence of recruited phagocytes, are remarkably resistant to S. aureus penetration. Keratinocytes have several unique features that contribute to their
ability to withstand invasion by S. aureus and other common
pathogens. Despite the high prevalence of S. aureus nasal colonization, even with the potentially virulent USA300 strains,
remarkably few individuals actually become infected. Production of several types of antimicrobial peptides is likely to
substantially inhibit supercial colonization by these organisms [26]. However, once staphylococci are sensed by the keratinocytes, they are poised to undergo pyroptosis to eradicate
infected cells. In contrast to macrophages, which require
the internalization of particulate peptidoglycan to activate
inammasome signaling [17], keratinocytes constitutively
express IL-1 and thus are primed for activation by
surface contact with staphylococci. While the keratinocytes are
undergoing degradation, the organisms appear to proliferate
intracellularly, having activated the destruction of the

Figure 5. Calpain and caspase 1 inhibitors prevent keratinocyte pyroptosis. A, Fluorescence-activated cell sorting analysis of HaCaTs incubated for 2
hours with medium alone, wild-type (WT) Staphylococcus aureus strain USA300, or -hemolysin (Hla) mutant USA300 demonstrating the generation
of a population of smaller, condensed cells (circled). B, Quantication of the numbers of condensed cells following exposure to WT USA300 in the
presence of calpeptin (Cpep), a caspase 1 inhibitor (C1inh), or a caspase 3 inhibitor (C3inh) as compared with cells exposed to the Hla mutant strain,
expressed as a percentage of the total cells. *P < .01 compared with WT (1-way analysis of variance, Dunnett posttest). Data are mean (SD), representative of 2 individual experiments. C, Transmission light microscopy of representative trypan bluestained wells of HaCaTs after 6 hours of exposure to
WT or Hla staphylococci and medium alone as a control. Blue staining due to membrane permeability indicated apoptosis/necrosis in WT, less in
Hla, and not at all in medium-treated cells.

keratinocyte and evaded the toxicity of the keratinocyte

products.
The ability of keratinocytes to proliferate in response to
S. aureus is also likely to prevent invasive infection. As we
observed, by 24 hours organotypic cultures of human keratinocytes appeared to be substantially thicker when exposed to
these bacteria. As S. aureus Hla stimulates epithelial proliferation via EGFR [15], keratinocyte infection does not result in
widespread loss of barrier function but instead results in focal
areas of infection and induction of more keratinocytes to
replace the necrotic cells. This was evident in the transmission
microscopy of the HaCaT cultures, which despite the induction of pyroptosis remained remarkably intact for at least 24
hours following infection. This is in contrast to what occurs at
more vulnerable mucosal sites, such as the airway, that do not
have such proliferative capacity and would be rapidly destroyed by a similar exposure [13].
It is interesting that different components of staphylococci
appear to mediate invasion through specic tissues. Hla

production is clearly critical for skin infection, whereas

protein A, which was entirely dispensable in the activation of
pyroptosis of keratinocytes, was essential to enable staphylococci to penetrate across an airway epithelial barrier [13]. Hla
also contributes to barrier dysfunction in A549 cells through
its interaction with ADAM-10 and cleavage of E-cadherin
[27]. This interaction may also contribute to penetration
through the skin, accounting for the transmigration that is not
inhibited in the presence of calpain or caspase 1 inhibitors. It
is also noteworthy that neither SpA nor Hla is unique to the
USA300 strains of staphylococci.
The PVL strain was less effective in transmigration across
the keratinocytes, but the same mutant was as effective as the
parental strain in stimulating pyroptosis. It is possible that
PVL pore formation acts in concert with Hla to enhance keratinocyte pyroptosis but is not a critical factor.
The induction of keratinocyte signaling and specically
calpain activity was critical for staphylococcal invasion. The
participation of calpains in mediating inammation in
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response to bacterial infection has become increasingly recognized. We have previously found that calpains participate in
PMN recruitment across airway epithelial barriers [22]. In
those studies, the stimulus for calpain activity was the Ca2+
ux induced through TLR2 signaling, a factor that is relevant
to staphylococcal skin infection. Calpains are also central to
the pathology that is caused by staphylococcal infection of the
skin. Although our in vitro system was restricted to the keratinocytes themselves, in vivo, PMNs would be recruited and activated through inammasome-mediated signaling: by IL-1
and by calpain-mediated cleavage of paracellular junctional
proteins to facilitate their recruitment [22]. As a consequence
of inammasome activation in the skin, focal accumulation of
PMNs results in the pustule or abscess formation characteristic of S. aureus skin infection. Recognition that the caspase
1/calpain cascade in keratinocytes is a major cause of the pathology induced by S. aureus could provide new targets for
topical antistaphylococcal therapy, as well as provide new
impetus for drugs that inhibit S. aureus Hla activity.
Notes
Acknowledgments. We thank Taylor Cohen for help with statistical
analyses.
Financial support. This work was supported by the National Institutes of Health (grant number RO1 HL79395 to A. P.) and the Columbia
University Department of Dermatology Pilot and Feasibility Grant
Program (grant number P30AR44535).
Potential conicts of interest. All authors: No reported conicts.
All authors have submitted the ICMJE Form for Disclosure of Potential
Conicts of Interest. Conicts that the editors consider relevant to the
content of the manuscript have been disclosed.

References

Figure 6. Staphylococcus aureus invasion through keratinocytes is

decreased by calpain and caspase 1 inhibition. A, Quantication
(in colony-forming units [CFUs]) of organisms recovered from the basal
compartment of transwells 24 hours after application of a 108-CFU inoculum in the upper chamber. Approximately 90% of the added inoculum of
wild-type (WT) strain was retrieved in the basal compartment following
24-hour incubation. Mutant strains were compared with their matching
WT strain (P values are shown; Student t test). Data are mean (SD),
representative of at least 3 individual experiments. B, Recovery of uorescent 3000 MW dextran from the basal compartment of transwells incubated with the staphylococcal mutant and WT paired strains indicated.
All were compared with medium alonetreated HaCaT cells (P value
shown is for mutant compared with WT; Student t test). Data are mean
(SD), representative of at least 2 individual experiments. C, Effects of
caspase 1 inhibitor (C1 inh), caspase 3 inhibitor (C3 inh), and calpeptin
treatment on S. aureus transmigration, showing quantication of CFUs of
WT USA300 recovered from the basal compartment of transwells treated
with the inhibitors. *P values shown are compared with WT (1-way
analysis of variance, Dunnett posttest). Data are mean (SD), representative of at least 2 individual experiments. Abbreviations: Hla, -hemolysin;
PVL, Panton-Valentine leukocidin; SpA, protein A.

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1. Klevens RM, Morrison MA, Nadle J, et al. Invasive methicillin-resistant Staphylococcus aureus infections in the United States. JAMA
2007; 298:176371.
2. Talan DA, Krishnadasan A, Gorwitz RJ, et al. Comparison of Staphylococcus aureus from skin and soft-tissue infections in US emergency
department patients, 2004 and 2008. Clin Infect Dis 2011; 53:1449.
3. Otto M. A MRSA-terious enemy among us: end of the PVL controversy? Nat Med 2011; 17:16970.
4. Hruz P, Zinkernagel AS, Jenikova G, et al. NOD2 contributes to
cutaneous defense against Staphylococcus aureus through alpha-toxindependent innate immune activation. Proc Natl Acad Sci U S A
2009; 106:128738.
5. Kennedy AD, Bubeck Wardenburg J, Gardner DJ, et al. Targeting of
alpha-hemolysin by active or passive immunization decreases severity
of USA300 skin infection in a mouse model. J Infect Dis 2010;
202:10508.
6. Miller LS, Pietras EM, Uricchio LH, et al. Inammasome-mediated
production of IL-1beta is required for neutrophil recruitment against
Staphylococcus aureus in vivo. J Immunol 2007; 179:693342.
7. Mempel M, Schnopp C, Hojka M, et al. Invasion of human keratinocytes by Staphylococcus aureus and intracellular bacterial persistence
represent haemolysin-independent virulence mechanisms that are
followed by features of necrotic and apoptotic keratinocyte cell death.
Br J Dermatol 2002; 146:94351.
8. Pishchany G, McCoy AL, Torres VJ, et al. Specicity for human
hemoglobin enhances Staphylococcus aureus infection. Cell Host
Microbe 2010; 8:54450.

9. Houben E, De Paepe K, Rogiers V. A keratinocytes course of life. Skin

Pharmacol Physiol 2007; 20:12232.
10. Kollisch G, Kalali BN, Voelcker V, et al. Various members of the Tolllike receptor family contribute to the innate immune response of
human epidermal keratinocytes. Immunology 2005; 114:53141.
11. Munoz-Planillo R, Franchi L, Miller LS, Nunez G. A critical role
for hemolysins and bacterial lipoproteins in Staphylococcus
aureus-induced activation of the Nlrp3 inammasome. J Immunol
2009; 183:39428.
12. Craven RR, Gao X, Allen IC, et al. Staphylococcus aureus alphahemolysin activates the NLRP3-inammasome in human and mouse
monocytic cells. PLoS One 2009; 4:e7446.
13. Soong G, Martin FJ, Chun J, Cohen TS, Ahn DS, Prince A. Staphylococcus aureus protein A mediates invasion across airway epithelial
cells through activation of RhoA GTPase signaling and proteolytic
activity. J Biol Chem 2011; 10.1074/jbc.M1111.295386.
14. Martin FJ, Gomez MI, Wetzel DM, et al. Staphylococcus aureus activates type I IFN signaling in mice and humans through the Xr repeated sequences of protein A. J Clin Invest 2009; 119:19319.
15. Haugwitz U, Bobkiewicz W, Han SR, et al. Pore-forming Staphylococcus aureus alpha-toxin triggers epidermal growth factor receptordependent proliferation. Cell Microbiol 2006; 8:1591600.
16. Miao EA, Leaf IA, Treuting PM, et al. Caspase-1-induced pyroptosis
is an innate immune effector mechanism against intracellular bacteria.
Nat Immunol 2010; 11:113642.
17. Shimada T, Park BG, Wolf AJ, et al. Staphylococcus aureus evades
lysozyme-based peptidoglycan digestion that links phagocytosis, inammasome activation, and IL-1beta secretion. Cell Host Microbe
2010; 7:3849.

18. Walev I, Martin E, Jonas D, et al. Staphylococcal alpha-toxin kills

human keratinocytes by permeabilizing the plasma membrane for
monovalent ions. Infect Immun 1993; 61:49729.
19. Gomez MI, Lee A, Reddy B, et al. Staphylococcus aureus protein A
induces airway epithelial inammatory responses by activating
TNFR1. Nat Med 2004; 10:8428.
20. Feldmeyer L, Keller M, Niklaus G, Hohl D, Werner S, Beer HD. The
inammasome mediates UVB-induced activation and secretion of interleukin-1beta by keratinocytes. Curr Biol 2007; 17:11405.
21. Kashio Y, Nakamura K, Abedin MJ, et al. Galectin-9 induces apoptosis through the calcium-calpain-caspase-1 pathway. J Immunol 2003;
170:36316.
22. Chun J, Prince A. TLR2-induced calpain cleavage of epithelial junctional proteins facilitates leukocyte transmigration. Cell Host Microbe
2009; 5:4758.
23. Bergsbaken T, Fink SL, Cookson BT. Pyroptosis: host cell death and
inammation. Nat Rev Microbiol 2009; 7:99109.
24. Pisetsky DS, Erlandsson-Harris H, Andersson U. High-mobility group
box protein 1 (HMGB1): an alarmin mediating the pathogenesis of
rheumatic disease. Arthritis Res Ther 2008; 10:209.
25. Edwards AM, Potter U, Meenan NA, Potts JR, Massey RC. Staphylococcus aureus keratinocyte invasion is dependent upon multiple highafnity bronectin-binding repeats within FnBPA. PLoS One 2011; 6:
e18899.
26. Otto M. Staphylococcus colonization of the skin and antimicrobial
peptides. Expert Rev Dermatol 2010; 5:18395.
27. Inoshima I, Inoshima N, Wilke GA, et al. A Staphylococcus aureus
pore-forming toxin subverts the activity of ADAM10 to cause lethal
infection in mice. Nat Med 2011; 17:13104.

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