Dissemination of invasive Salmonella via
bacterial-induced extrusion of mucosal epithelia
Leigh A. Knodlera,1, Bruce A. Vallanceb, Jean Cellia, Seth Winfreea, Bryan Hansenc, Marinieve Monterob,
and Olivia Steele-Mortimera
a
Laboratory of Intracellular Parasites and cResearch Technologies Branch, Microscopy Unit, Rocky Mountain Laboratories, National Institute of Allergy and
Infectious Diseases, National Institutes of Health, Hamilton, MT 59840; and bDivision of Gastroenterology, Department of Paediatrics, University of British
Columbia, British Columbia Children’s Hospital, Vancouver, BC, Canada V6H 3V4
Edited by Pascale Cossart, Institut Pasteur, Paris, France, and approved August 24, 2010 (received for review May 5, 2010)
Salmonella enterica is an intracellular bacterial pathogen that resides
and proliferates within a membrane-bound vacuole in epithelial cells
of the gut and gallbladder. Although essential to disease, how Sal-
monella escapes from its intracellular niche and spreads to secondary
cells within the same host, or to a new host, is not known. Here, we
demonstrate that a subpopulation of Salmonella hyperreplicating in
the cytosol of epithelial cells serves as a reservoir for dissemination.
These bacteria are transcriptionally distinct from intravacuolar Sal-
monella. They are induced for the invasion-associated type III secre-
tion system and possess flagella; hence, they are primed for invasion.
Epithelial cells laden with these cytosolic bacteria are extruded out of
the monolayer, releasing invasion-primed and -competent Salmo-
nella into the lumen. This extrusion mechanism is morphologically
similar to the process of cell shedding required for turnover of the
intestinal epithelium. In contrast to the homeostatic mechanism,
however, bacterial-induced extrusion is accompanied by an inflam-
matory cell death characterized by caspase-1 activation and the apical
release of IL-18, an important cytokine regulator of gut inflammation.
Although epithelial extrusion is obviously beneficial to Salmonella
for completion of its life cycle, it also provides a mechanistic explana-
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tion for the mucosal inflammation that is triggered during Salmonella
infection of the gastrointestinal and biliary tracts.
caspase-1 | epithelial cells | flagella | IL-18 | type III secretion
I ntracellular pathogens reside either within a membrane-bound
vacuole or freely within the host cell cytosol. However, regardless
of their specific lifestyle, three distinct steps are common to their
infectious cycle: the ability to enter host cells, their establishment of
an intracellular niche, and their escape from the host cell (1). Entry
and intracellular survival are critical virulence stages for these
pathogens, but exit from the infected cell is essential for dissemi-
nation and transmission to other hosts. Although considerable
progress has been made in elucidating the first two facets of this
cycle, the mechanism by which intracellular pathogens escape from
host cells has been comparably neglected.
The Gram-negative bacterium Salmonella enterica causes a wide
range of food- and water-borne diseases ranging from self-limiting
gastroenteritis to systemic typhoid fever in both humans and ani-
mals. In enteric infections, Salmonella preferentially targets the
single layer of polarized columnar epithelial cells lining the surface
of the gastrointestinal tract (2–4), triggering an extensive in-
flammatory response. After invading epithelial cells from the apical
side, Salmonella resides and replicates within a membrane-bound
vacuole, known as the Salmonella-containing vacuole (SCV).
Symptomatic and asymptomatic infections are characterized by
the fecal shedding of bacteria (5, 6), suggesting that Salmonella
escapes from its intracellular niche back into the gut lumen as part
of its infectious cycle. Here, we report that Salmonella exits from
polarized epithelia by coopting a mechanism normally used by the
host to remove senescent cells from the mucosal epithelium.
Results and Discussion
WT Salmonella Hyperreplicates in the Cytosol of Epithelial Cells.
Human colonic epithelial cells (C2BBe1, a subclone of Caco-2)
www.pnas.org/cgi/doi/10.1073/pnas.1006098107
grown on filters were used as a model polarized monolayer to ex-
amine the infectious cycle of Salmonella. Confocal microscopy
analysis of infected monolayers revealed two distinct populations of
proliferating bacteria following the onset of replication ≥4 h post-
infection (p.i.) (Fig. 1A and Fig. S1A). Compared with an average
doubling time of ≥95 min for the total population (Fig. S1A), some
bacteria were replicating at a much faster rate, with a doubling time
of ∼20 min (Movie S1). There was a temporal increase in the in-
cidence of these “hyperreplicating” Salmonella (defined as >50
bacteria per cell) (Fig. 1 A and B). By 10 h p.i., 11 ± 4.2% of infected
cells contained hyperreplicating bacteria (Fig. 1A). A similar phe-
notype was previously described for a Salmonella sifA mutant,
which hyperreplicates in the host cell cytosol because of a defect in
maintaining vacuolar integrity (7). We therefore assessed whether
the hyperreplicating WT Salmonella we observed in polarized epi-
thelial cells are also free in the cytosol. Confocal microscopy in-
dicated that many of these bacteria were not in a lysosome-
associated membrane protein 1 (LAMP1)-positive compartment
(Fig. 1B and Fig. S1B), and thus not in a mature SCV (8). Selective
membrane permeabilization followed by immunostaining with
polyclonal anti-Salmonella LPS antibody revealed that at least one-
third of the hyperreplicating bacteria are cytosolic (Fig. 1C and Fig.
S2). These experiments demonstrate that WT Salmonella can rep-
licate in a vacuole and the cytosol in epithelial cells, but they pro-
liferate more efficiently in the cytosolic environment (7, 9, 10).
Cytosolic Salmonella Are Invasion-Primed. We hypothesized that the
two distinct intracellular environments, intravacuolar and cyto-
solic, would differentially influence the expression of bacterial
virulence genes. To assess this, we used a plasmid-derived tran-
scriptional fusion assay based on destabilized GFP(LVA). Pro-
moters were selected from well-characterized genes in each of the
three type III secretion systems (T3SSs): PfliC-gfp[LVA] (flagel-
lar T3SS), PprgH-gfp[LVA] (T3SS1), and PssaG-gfp[LVA]
(T3SS2) (11). The number of fluorescent bacteria was monitored
with time. Under the infection conditions used here, efficient
invasion requires both T3SS1 and flagellar-based motility (11–13)
(Fig. S1A). This single-cell assay confirmed that both T3SS1
(PprgH-gfp[LVA]) and flagella (PfliC-gfp[LVA]) were rapidly
down-regulated after bacterial internalization (11, 14, 15) (Fig.
S1C). Surprisingly, these virulence factors were not completely
inactivated (Fig. 2 and Fig. S1C). At 10 h p.i., ∼6% of infected
MICROBIOLOGY
cells contained fluorescent PprgH-GFP[LVA] bacteria (3.8 ±
1.2% of the total bacterial population) (Fig. 2 and S1C). Strik-
Author contributions: L.A.K., B.A.V., J.C., S.W., and O.S.-M. designed research; L.A.K., B.A.V.,
J.C., S.W., B.H., and M.M. performed research; L.A.K., B.A.V., J.C., and S.W. analyzed data;
and L.A.K. and O.S.-M. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Freely available online through the PNAS open access option.
1
To whom correspondence should be addressed. E-mail: lknodler@niaid.nih.gov.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1006098107/-/DCSupplemental.
PNAS | October 12, 2010 | vol. 107 | no. 41 | 17733–17738
 Fig. 1. Infected epithelial cells contain two distinct populations of replicating Salmonella. (A) Polarized C2BBe1 monolayers were infected with WT Sal-
monella constitutively expressing mCherry. Monolayers were fixed at the indicated times and immunostained for the tight junction marker ZO-1 and the late
endosomal/lysosomal marker LAMP1. The percentage of infected cells containing >50 bacteria per cell was scored by fluorescence microscopy (mean ± SD, n ≥
3 independent experiments). (B) Confocal image showing the two different populations of replicating Salmonella at 8 h p.i. Many hyperreplicating bacteria
are not in a LAMP1-positive vacuole. The overlay shows mCherry Salmonella in green, LAMP1 in red, and ZO-1 in blue. (Scale bar, 10 μm; Inset scale bar, 2 μm.)
(C) Confocal image showing that some hyperreplicating bacteria are accessible to anti-LPS antibody delivered to the cytosol. At 8 h p.i., the plasma membrane
of infected monolayers was selectively permeabilized with digitonin. Cells were then incubated with anti-Salmonella LPS and anti-GM130 antibodies to detect
cytosolic bacteria and the cytosolic face of the Golgi, respectively. Nuclei were stained with Hoechst 33342. The overlay shows mCherry Salmonella in red,
cytosolic LPS in green, and nuclei in blue. GM130 is shown in the gray scale. Asterisks in the overlay indicate digitonin-permeabilized cells. (Scale bar, 10 μm;
GM130 scale bar, 10 μm; Inset scale bar, 2 μm.)

ingly, these T3SS1-induced bacteria were almost exclusively
found in cells containing hyperreplicating bacteria (Fig. 2A) and
associated with flagella (Fig. 2D). As expected, T3SS2 was in-
duced intracellularly (16) (Fig. S1C); fluorescent PssaG-GFP
[LVA] bacteria were not detected until >2 h p.i., and 32 ± 6.6% of
the bacteria were GFP-positive by 10 h p.i. (Figs. S1C and S3). At
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8 h p.i., ∼60% of the T3SS1-induced bacteria were cytosolic and
23 ± 6% were in LAMP1-positive SCVs (Fig. 2 B, C, and E). By
contrast, the T3SS2-induced bacteria were intravacuolar (91 ±
6% LAMP1-positive) and typically found in cells containing 5–20
bacteria (Fig. 2E and Fig. S3). Using live cell imaging, the motility
of intracellular Salmonella was assessed at 8 h p.i. (Fig. 2F).

Fig. 2. Flagella and the invasion-associated T3SS1 are expressed by cytosolic bacteria late during infection. (A–D) Polarized C2BBe1 monolayers were infected
with WT Salmonella carrying a plasmid that expresses destabilized GFP [GFP(LVA)] under the control of the T3SS1-associated prgH promoter. At 8 h p.i.,
monolayers were fixed and immunostained for confocal microscopy. (A) T3SS1 is induced late during infection in “hyperreplicating” bacteria. PprgH-induced
bacteria (green in overlay), Salmonella LPS (red), and the tight junction marker ZO-1 (blue) are shown. (Scale bars, 10 μm.) (B) T3SS1-induced bacteria are not in
a mature LAMP1-positive SCV. PprgH-induced bacteria (green in overlay), LAMP1 (red), and ZO-1 (blue) are shown. (Scale bar, 10 μm; Inset scale bar, 2 μm.) (C)
T3SS1-induced bacteria are cytosolic. The plasma membrane of polarized cells was selectively permeabilized with digitonin before the cytosolic delivery of anti-
LPS and anti-GM130 (permeabilization control) antibodies. PprgH-induced bacteria (green in overlay), cytosolic bacteria (LPS, red), and Hoechst 33342 (blue) are
shown. Asterisks indicate cells permeabilized by digitonin. GM130 is shown in the gray scale. (Scale bar, 10 μm; GM130 scale bar, 10 μm; Inset scale bar, 2 μm.) (D)
T3SS1-induced bacteria are flagellated. PprgH-induced bacteria (green in overlay), FliC (red), and ZO-1 (blue) are shown. (Scale bar, 10 μm; Inset scale bar, 2 μm.)
(E) T3SS1-induced bacteria are cytosolic, whereas T3SS2-induced bacteria are vacuolar. Infected cells were fixed at 8 h p.i. and immunostained for LAMP1 or
selectively permeabilized with digitonin and incubated with anti-LPS antibodies to detect cytosolic bacteria. The number of T3SS1-induced (PprgH-GFP[LVA])
and T3SS2-induced (PssaG-GFP[LVA]) bacteria positive for LAMP1 or cytosolic LPS staining was scored by fluorescence microscopy (mean ± SD, n ≥ 3 independent
experiments.) (F) Some T3SS1-induced bacteria are motile. The motility of T3SS1-induced (PprgH-GFP[LVA]) and T3SS2-induced (PssaG-GFP[LVA]) bacteria was
assessed at 8–9 h p.i. by live cell imaging. (Inset) Average instantaneous velocities (μm/s) for motile PprgH-induced bacteria (n = 3 independent experiments).

17734 | www.pnas.org/cgi/doi/10.1073/pnas.1006098107 Knodler et al.

 Consistent with flagellin (FliC) expression (Fig. 2D), a subset of
T3SS1-induced bacteria was motile (Fig. 2F and Movie S2). This
population moved at speeds consistent with flagellar-based mo-
tility (4–15 μm/s) (17), whereas T3SS2-induced bacteria were
immobile (Fig. 2F and Movie S2). Hence, there are at least two
transcriptionally distinct intracellular populations of replicating
bacteria in epithelial cells: T3SS2-induced intravacuolar bacteria
and T3SS1-induced flagellated bacteria that are cytosolic.
Invasion-Primed Salmonella Are Released into the Lumen by
Extruding Cells. Coincident with the onset and kinetics of hyper-
replication, bacteria-laden cells extruding toward the apical side
were observed by EM (Fig. 3 A and B). Bacteria in these extruded
cells were not surrounded by a vacuolar membrane [Fig. 3B (ii)],
in agreement with the confocal microscopy data for hyper-
replicating bacteria (Figs. 1C and 2E). By contrast, SCV mem-
branes were readily apparent around replicating bacteria in cells
within the monolayer (Fig. 3C). To examine whether extrusion of
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Fig. 3. Apical extrusion of infected cells from mucosal epithelium in vitro and
in vivo. (A) SEM of the apical surface of an infected monolayer. Polarized
C2BBe1 cells were infected with WT Salmonella, and at 10 h p.i., samples were
fixed and processed for SEM. (i) Extruding cell is bacteria-laden. Microvilli on
the apical surface of the underlying monolayer are evident. (Scale bar, 2 μm.)
(ii) Inset from i. (Scale bar, 0.5 μm.) (B) Transmission EM (TEM) of an extruded
cell. Cells were infected as for A, and samples were fixed and processed for
TEM. (i) Extruded cell is filled with bacteria and is adjacent to the apical surface
of the monolayer, distinguished by microvilli (MV). (Scale bar, 2 μm.) (ii) Inset
from i showing the absence of a vacuolar membrane around Salmonella in
extruded cells. N, epithelial cell nucleus. (Scale bar, 0.5 μm.) (C) TEM of an
infected C2BBe1 cell that was not extruding. Cells were infected as for A, and
samples were fixed and processed for TEM. Salmonella are clearly surrounded
by a vacuolar membrane (arrowheads). (Scale bar, 0.5 μm.) (D and E) EM of
infected gallbladders. Gallbladders from C57BL/6 mice were collected 6 d p.i.
and processed for TEM. Apical extrusion of Salmonella-infected epithelial cells
into the lumen (L) is evident. Some bacteria are also free in the lumen. (Scale
bars, 2 μm.) (E) Infected cell is undergoing extrusion into the luminal space (L),
with an obvious constriction site formed by the junctional complexes of
neighboring cells (arrowheads). Microvilli (MV) on the apical surface of the
mucosal epithelium are indicated.
Knodler et al.
infected epithelial cells occurs in vivo, we used a mouse model in
which Salmonella rapidly breaches the intestinal barrier and
spreads systemically to various tissues, including the mucosal
epithelium lining the gallbladder. In this murine infection model,
Salmonella causes inflammation similar to cholecystitis seen in
humans during acute typhoid fever (18). We observed both free
bacteria and bacteria-laden epithelial cells in the gallbladder lu-
men (Fig. 3 D and E). In agreement with our observations,
sloughing of enterocytes laden with Salmonella from villus tips has
also been reported in rabbit ileal loop studies (2). Therefore,
extrusion of Salmonella-infected epithelial cells is evident in both
enteric and systemic infections.
Salmonella-associated extrusion resembles cell extrusion in-
volved in the rapid turnover of polarized epithelial cells in the gut.
This process occurs when neighboring cells contract to push
a dying cell out of the monolayer and is characterized by re-
organization of adherens and tight junctions to maintain the in-
tegrity of the epithelial monolayer (19–22). Here, we observed
similar actin contractile rings and tight junction “rosettes” at the
base of extruding Salmonella-infected cells (Fig. S4 and Movie
S3), suggesting that the Salmonella-associated and homeostatic
extrusion events occur via similar cell biological processes.
The incidence of extrusion was significantly increased on in-
fection: 10 ± 2.7% of cells containing Salmonella showed evidence
of extrusion at 10 h p.i. compared with only 0.85 ± 0.89% of un-
infected cells (Fig. 4A). Furthermore, cells containing T3SS1-
induced Salmonella were more likely to be extruded than those
containing T3SS2-induced bacteria (20 ± 4.7% vs. 2.4 ± 0.9%;
Fig. 4 A and B). In infected gallbladders, T3SS1-induced bacteria
were found within epithelial cells lining the gallbladder at 4 d p.i.
and did not immunostain for FliC (Fig. S5). By 5 d p.i., flagellated
T3SS1-induced bacteria were predominantly found within cells
that had been sloughed into the lumen or were free in the lumen
(Fig. 4C and Fig. S5). Altogether, these data imply a strong cor-
relation between T3SS1 induction and Salmonella-induced ex-
trusion in vitro and in vivo. To address the invasion competence
of these bacteria, we developed a secondary infection assay
(Fig. S6A). Consistent with the onset of cell extrusion from the
polarized monolayer, significant numbers of bacteria could be
recovered from a naive population of epithelial cells from 6 h p.i.
and increasing thereafter. This demonstrates that Salmonella re-
leased from extruded cells are invasion-primed and -competent.
Extruding Cells Undergo Inflammatory Cell Death. During gut ho-
meostasis, epithelial cells are shed into the lumen as a result of
anoikis, a form of apoptosis characterized by activated caspase-2,
-3, and -9 but not caspase-1 or -8 (23–26). Because the Salmonella-
laden extruding cells also showed structural features typical of cell
death both in vivo and in vitro (Fig. 3 B, D, and E), we next assayed
for caspase activity. Epithelial monolayers were incubated with
cell-permeable fluorescent probes that bind irreversibly to acti-
vated caspases. Uninfected extruding cells were positive for active
caspase-3/-7 (74 ± 8.1%) but not for active caspase-1 (21 ± 9.4%),
as previously described (27) (Fig. 5B). By contrast, the majority of
infected extruding cells were positive for both active caspase-1
MICROBIOLOGY
(83 ± 4.5%) and caspase-3/-7 (85 ± 7.3%) (Fig. 5 A and B). Ho-
meostatic extrusion of epithelial cells is not prevented by general
caspase inhibition (21). Addition of a caspase-1 inhibitor did not
block, but significantly decreased, extrusion of infected cells
(Fig. 4A), suggesting that the signal for homeostatic and bacterial-
induced extrusion precedes caspase activation.
Caspase-1–dependent programmed cell death, also known as
pyroptosis, is characterized by pore formation in the plasma
membrane, followed by cell swelling and lysis, and the proteolytic
processing of proinflammatory cytokines, leading to the secretion
of mature active IL-1β and IL-18 (28). To monitor plasma
membrane integrity, infected cells were incubated with SYTOX
Orange nucleic acid dye, which can only enter cells with a com-
PNAS | October 12, 2010 | vol. 107 | no. 41 | 17735
 Fig. 4. Extruding epithelial cells contain invasion-primed Salmonella. (A) Quantification of extrusion. Polarized C2BBe1 monolayers were infected with
Salmonella constitutively expressing mCherry or carrying destabilized GFP reporters for T3SS1 (PprgH-GFP[LVA]) or T3SS2 (PssaG-GFP[LVA]). At 10 h p.i.,
monolayers were fixed and immunostained for ZO-1. DNA was stained with Hoechst 33342. Where indicated (+YVAD), 100 μM Ac-YVAD-CMK was added
before, and maintained throughout, the infection. Infected (mCherry bacteria) cells, or cells containing PprgH-positive or PssaG-positive bacteria, showing
signs of extrusion were scored by fluorescence microscopy (mean ± SD, n ≥ 4 independent experiments). Asterisks indicate significantly different from
infected cells (P < 0.05, ANOVA with Dunnett’s post hoc test). (B) Confocal image showing an extruding cell containing T3SS1-induced bacteria. Polarized
C2BBe1 monolayers infected with WT Salmonella carrying PprgH-GFP[LVA] were fixed and immunostained at 10 h p.i. DNA was stained with Hoechst 33342.
PprgH-induced bacteria (green in overlay), apical plasma membrane marker, villin (red), DNA (cyan), and tight junction marker ZO-3 (gray) are shown. The x–z
section is indicated by a dashed line. (Scale bar, 10 μm.) (C) Fluorescence microscopy image showing that epithelial cells containing T3SS1-induced, flagellated
bacteria are extruded into the gallbladder lumen (L). C57BL/6 mice were infected with Salmonella carrying a plasmid that expresses destabilized GFP under
the control of the T3SS1-associated invF promoter (PinvF-GFP[LVA]). Mice were killed 5–6 d p.i., and gallbladders processed for immunostaining. DNA was
stained with DAPI. PinvF-induced bacteria (green in overlay), FliC (red), and DNA (cyan) are shown. The arrowhead indicates an extruded cell containing
numerous T3SS1-induced, flagellated bacteria. (Scale bar, 10 μm; Inset scale bar, 2 μm.)

promised plasma membrane, and Hoechst 33342, a cell-perme-
able nuclear stain. Salmonella-infected extruding cells were pos-
itive for both dyes, indicating plasma membrane rupture, whereas
neighboring cells within the monolayer stained only with Hoechst
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extrusion, releasing the invasion-primed Salmonella into the lu-
men of the gastrointestinal and biliary tracts. Escape into the lu-
men allows Salmonella to infect secondary cells rapidly, and may
also contribute to host-to-host transmission. Thus, by subverting
a host-dependent cell turnover event, Salmonella completes its

33342 (Fig. 5C). We next quantified the apical and basolateral

release of cytokines from infected C2BBe1 cells. Although IL-1β
release was below the limits of detection (0.8 pg/mL), we observed
a steady temporal increase in the release of IL-18, which was re-
stricted to the apical side of monolayers (Fig. 5D) and dependent
on caspase-1 and -3 (Fig. 5E). Collectively, these data highlight
a clear difference in the activated cell death program between
Salmonella-induced and homeostatic extrusion.
We have demonstrated that extruding Salmonella-infected cells
undergo inflammatory cell death. A complex but still largely un-
explained pathological feature of salmonelloses is an overwhelming
inflammatory response. We propose that bacterial-induced ex-
trusion provides one mechanistic explanation for the pathogenesis
of mucosal inflammation during Salmonella infections of the in-
testine and gallbladder. In support of our data, activation of IL-18 in
porcine intestinal mucosa has been reported for S. enterica serovar
Choleraesuis infections (29). Interestingly, in inflammatory bowel
diseases, chronic inflammation is also associated with elevated
IL-18 levels (30–32). Therefore, caspase-1–dependent IL-18 pro-
duction by intestinal epithelial cells might prove to be a mediator
of mucosal inflammation associated with both autoimmune dis-
orders (33, 34) and bacterial infections.
Conceptually, an increased turnover of mucosal epithelium
provides the host with an ideal defense mechanism against in-
fection. Indeed, it has been implicated as a protective mechanism
in the gut against both bacteria and parasites (35, 36). However, we
believe that Salmonella takes advantage of this process as a unique
means of bacterial egress. Critical to this is the finding that
a population of Salmonella is cytosolic and expresses the virulence
genes required for invasion. We hypothesize that a vacuole mat-
uration defect leads to the cytosolic release of a small, but signif-
icant, fraction of bacteria. The nutrient-rich cytosol supports
a high bacterial replication rate and reprograms virulence gene
expression toward invasion. The cytosolic load of bacteria is
sensed by the host cell, leading to inflammatory cell death and
infectious cycle (Fig. S6B). Given the prevalence of mucosal-
dwelling pathogens, other pathogens may also use this host cell
process as an exit strategy.
Materials and Methods
Bacterial Strains and Plasmids. WT S. enterica serovar Typhimurium
(S. Typhimurium) SL1344 (37) and ΔSPI2::kan (38), ΔSPI1::kan (15), and flgB::
Tn10 mutants (11) have been described previously. For constitutive expression of
GFP or mCherry, WT S. Typhimurium was electroporated with pFPV25.1 (39) or
pFPV-mCherry (40), respectively. S. Typhimurium carrying the destabilized GFP
(GFP[LVA]) (41) reporter plasmids pMPMA3ΔPlac-PprgH-gfp[LVA], pMPMA3Δ-
Plac-PinvF-gfp[LVA], pMPMA3ΔPlac-PfliC-gfp[LVA], or pMPMA3ΔPlac-PssaG-
gfp[LVA] (11) were used to analyze intracellular virulence gene expression.
Infection of Cultured Epithelial Cells with Salmonella. Cell lines were obtained
from the American Type Culture Collection and used at a passage number ≤13
for all experiments. C2BBe1 human colorectal adenocarcinoma cells (CRL-
2012), a clone of Caco-2, were maintained in DMEM (Mediatech) containing
10 μg/mL human transferrin (Sigma) and 10% (vol/vol) heat-inactivated FCS
(Gibco). HeLa human cervical adenocarcinoma cells were maintained in
Eagle’s modified Eagle medium (Mediatech) containing 10% (vol/vol) heat-
inactivated FCS. Polarized monolayers were established by seeding 2 × 105
C2BBe1 cells in basal seeding medium containing MITO+ serum extender
(Becton Dickinson) on BIOCOAT fibrillar collagen 24-well inserts with a 1-μm
membrane pore size (Becton Dickinson). After 24 h, the seeding medium was
replaced with enterocyte differentiation medium containing MITO+ serum
extender. Cells were incubated in differentiation medium for a total of 3 d,
replaced with fresh medium each day, until the transepithelial electrical re-
sistance was ≥250 Ω.cm2, as measured using a Millicell Electrical Resistance
System (Millipore). The medium was changed to DMEM containing 10% (vol/
vol) heat-inactivated FCS (growth medium, GM) before infection.
Preparation of invasive Salmonella and infection of HeLa cells at a multi-
plicity of infection (MOI) of ∼50 were as previously described (42). Polarized
monolayers were infected apically at an MOI of ∼50–100 for 10 min and then
washed three times apically and twice basolaterally in HBSS (Mediatech). Cells
were incubated in antibiotic-free GM until 30 min p.i. Thereafter, GM con-
taining 50 μg/mL gentamicin (Sigma) was added for 1 h to kill any remaining

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Fig. 5. Salmonella-infected extruding epithelial cells undergo inflammatory
cell death. (A) Confocal image of an infected extruding cell positive for active
caspase-1. Polarized C2BBe1 monolayers were infected with WT Salmonella
constitutively expressing mCherry (red in overlay). At 9 h p.i., live cells were
incubated with the active caspase-1 probe FAM-YVAD-FMK (green). Nuclei
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were stained with Hoechst 33342 (cyan). (Upper) 3D-rendered view in the x–z
plane. (Scale bar, 10 μm.) (B) C2BBe1 cells were infected as in A and incubated
with FAM-YVAD-FMK (active caspase-1) or FAM-DEVD-FMK (active caspase-3
and -7) at 9 h p.i. Uninfected or infected extruding cells positive for active cas-
pases were scored by fluorescence microscopy (mean ± SD, n ≥ 3 independent
experiments). (C) Confocal image of an extruding cell labeled by a membrane
impermeant nucleic acid dye. Polarized monolayers were infected with WT
Salmonella constitutively expressing GFP (green in overlay). At 10 h p.i., live cells
were incubated with two nucleic acid dyes, the membrane impermeant SYTOX
Orange (red in overlay) and the membrane permeant Hoechst 33342 (cyan). The
extruding cell contains numerous Salmonella and is positive for both nucleic acid
stains, indicating a compromised plasma membrane. The x–z section is indicated
by a dashed line. (Scale bar, 10 μm.) (D) Apical release of IL-18 from infected
epithelial cells. Polarized monolayers were mock-infected (○) or infected with
Salmonella (●). Apical supernatants were collected and assayed for IL-18 con-
centration by sandwich ELISA (mean ± SD, n ≥ 3 independent experiments). (E)
Caspase-1 and caspase-3 activation are required for IL-18 release. Polarized
monolayers were mock-infected or infected with WT Salmonella. Where in-
dicated, monolayers were pretreated and incubated for the entire infection
with the general caspase inhibitor Z-VAD-FMK (50 μM), the caspase inhibitor
negative control Z-FA-FMK (50 μM), the caspase-1 inhibitor Ac-YVAD-CMK (100
μM), or the caspase-3 inhibitor, Ac-DEVD-CMK (50 μM). At 10 h p.i., apical
supernatants were collected and IL-18 was assayed by ELISA (mean ± SD, n ≥ 3
independent experiments). Asterisks indicate data significantly different from
WT Salmonella infection (P < 0.05, ANOVA with Dunnett’s post hoc test).
extracellular bacteria, followed by GM containing 10 μg/mL gentamicin for the
remainder of the experiment to restrict the extracellular growth of bacteria.
Enumeration of Intracellular and Extracellular Bacteria. For quantification of
viable intracellular bacteria, polarized monolayers were washed apically and
basolaterally three times with PBS and then lysed in 1 mL of 1% (vol/vol) Triton
X-100/0.1% (wt/vol) SDS (TX-100/SDS). Serial dilutions were plated on LB agar
plates. Bacterial doubling time was calculated as described previously (43).
To measure the invasion competence of apically released bacteria, we
developed a secondary infection protocol. Polarized C2BBe1 monolayers
were infected and treated with 50 μg/mL gentamicin to kill extracellular
bacteria as described above. At 1.5 h p.i., transwells were inverted onto
a monolayer of HeLa cells seeded in six-well plates (2 × 105 cells per well and
Knodler et al.
two transwells per well). Coincubation of C2BBe1 and HeLa cells continued
in GM containing 10 μg/mL gentamicin. Polarized monolayers were solubi-
lized in TX-100/SDS as described above. For quantification of the secondary
infection, the HeLa cells were washed extensively in HBSS, solubilized in 1 mL
of TX-100/SDS, and plated on LB agar plates.
Immunofluorescence Staining. Infected monolayers were washed twice, api-
cally and basolaterally, with PBS and then fixed apically and basolaterally with
3.5% (wt/vol) paraformaldehyde (PFA) for 20 min at room temperature (RT).
Monolayers were then washed apically and basolaterally with PBS, followed
by incubation in 75 mM ammonium chloride/20 mM glycine in PBS for 10 min
at RT to quench free aldehyde groups. After washing in PBS, monolayers
were permeabilized in 10% (vol/vol) normal goat serum/0.1% (wt/vol) sa-
ponin in PBS (SS-PBS) for 20 min at RT. Primary and secondary antibodies
were diluted in SS-PBS and applied apically and basolaterally in a humid box
for 1 h at RT. Filters were excised from transwell supports and placed cell side
up on a drop of Prolong Gold antifade reagent (Invitrogen) on a glass slide.
Another drop of mounting media was applied to the filter, and a coverslip
was placed directly on top. Samples were cured overnight at RT.
Live cell staining was carried out by incubation for 10 min at 37 °C with 2 μg/
mL Hoechst 33342 (Invitrogen) to stain nucleic acids or 0.5 μM SYTOX Orange
(Invitrogen) to stain nucleic acids in cells with a compromised plasma mem-
brane. For determination of activated caspase-1 and caspase-3/-7, live cells
were incubated with FAM-YVAD-FMK or FAM-DEVD-FMK, respectively, in
GM for 1 h according to the manufacturer’s instruction (Immunochemistry
Technologies) before fixation.
To determine whether intracellular bacteria were vacuolar or cytosolic,
polarized C2BBe1 cells were infected as described, and at 8 h p.i., the apical
plasma membrane was selectively permeabilized using digitonin to allow
access of antibody to the cytosol. Briefly, transwells were washed three times
in KHM buffer [110 mM potassium acetate, 20 mM Hepes, 2 mM MgCl2 (pH
7.3)] and incubated apically with 150 μg/mL digitonin (Sigma) in KHM buffer
for 90 s at RT. Transwells were then immediately washed with KHM buffer.
Rabbit polyclonal anti-Salmonella LPS antibody and mouse anti-human
GM130 monoclonal antibody were added for 15 min at 37 °C to label cy-
tosolic bacteria and the cytosolic face of the Golgi, respectively. Monolayers
were then fixed and quenched as described above, followed by nonselective
permeabilization in 0.1% (wt/vol) saponin and 10% (vol/vol) horse serum in
PBS for 15 min at RT. Anti-Salmonella LPS antibody (1:200; Difco) and anti-
GM130 monoclonal antibody (1:50; BD Transduction Laboratories) were
detected using Alexa Fluor- and Cy5-conjugated secondary antibodies as
described above. Nucleic acids were subsequently stained with Hoechst
33342 (2 μg/mL) for 10 min at RT. Intracellular bacteria were then scored for
LPS staining. Extruding cells were excluded from analysis because they have
a compromised plasma membrane and delivery of antibodies could occur in
a digitonin-independent manner. For each experiment, one transwell was
used as a permeabilization control to ensure that the apical plasma mem-
brane (but not the endomembranes) was permeabilized. For this, the digi-
tonin-treated cells were incubated with two LAMP1 antibodies: rabbit
polyclonal antibody directed against the cytoplasmic tail of LAMP1 (1:250;
Novus Biologicals) and a mouse monoclonal antibody directed against the
luminal portion of LAMP1 (1:1,000; clone H4A3, Developmental Studies
Hybridoma Bank) (Fig. S2). We excluded any experiments in which cells
stained with both anti-LAMP1 antibodies.
Immunostaining of mouse gallbladder tissues was performed using pre-
viously described procedures (44). Gallbladders were fixed in 4% (wt/vol) PFA
for 1 h at RT, washed in PBS, embedded in optimal cutting template com-
pound (Sakura Finetek), and then frozen with isopentane and liquid N2 and
stored at −70 °C. Serial sections were cut at a thickness of 4 μm for immu-
nohistochemical staining with Alexa Fluor 488-conjugated rabbit anti-GFP
MICROBIOLOGY
antibody (1:500; Invitrogen) and mouse monoclonal antibody anti-FliC
(1:100; BioLegend). Nucleic acids were stained with DAPI.
Animal Infections. WT S. Typhimurium (Fig. 3 D and E) or S. Typhimurium
carrying pMPMA3ΔPlac-PinvF-gfp[LVA] (Fig. 4C) were grown overnight at
37 °C with shaking in LB or LB containing 50 μg/mL carbenicillin, respectively.
Cultures were diluted in PBS to ∼5–8 × 103 cfu/mL. Female C57BL/6 mice
(Charles River) were infected with 100 μL of the diluted culture by tail vein
injection. Additionally, for infections with plasmid-bearing Salmonella, mice
were treated with carbenicillin (100 mg/kg) by i.p. injection daily throughout
the infection to maintain the plasmid. The protocols used were in direct
accordance with guidelines drafted by the University of British Columbia’s
Animal Care Committee and the Canadian Council on the Use of Laboratory
Animals. Gallbladders were collected 4–6 d p.i. and processed for EM or
immunohistochemistry as described above.
PNAS | October 12, 2010 | vol. 107 | no. 41 | 17737
 Information on reagents, quantification of cytokine release, fluorescence
microscopy, determination of bacterial velocities, and EM is provided in SI
Materials and Methods.
ACKNOWLEDGMENTS. We thank Caixia Ma and Tina Huang for their expert
technical assistance; Anita Mora for graphics assistance; the Genomics Core
Facility at Rocky Mountain Laboratories for DNA sequence analysis; and Rey
Carabeo, Ed Miao, Staffan Svärd, and members of the Steele–Mortimer lab-
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