Enterococcus faecalis Infection Activates Phosphatidylinositol 3-Kinase

Signaling To Block Apoptotic Cell Death in Macrophages

Jun Zou, Nathan Shankar
Department of Pharmaceutical Sciences, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA

Apoptosis is an intrinsic immune defense mechanism in the host response to microbial infection. Not surprisingly, many patho-
gens have evolved various strategies to manipulate this important pathway to benefit their own survival and dissemination in the
host during infection. To our knowledge, no attempts have been made to explore the host cell survival signals modulated by the
bacterium Enterococcus faecalis. Here, we show for the first time that during early stages of infection, internalized enterococci
can prevent host cell (RAW264.7 cells, primary macrophages, and mouse embryonic fibroblasts [MEFs]) apoptosis induced by a
wide spectrum of proapoptotic stimuli. Activation of caspase 3 and cleavage of the caspase 3 substrate poly(ADP-ribose) poly-
merase were inhibited in E. faecalis-infected cells, indicating that E. faecalis protects macrophages from apoptosis by inhibiting
caspase 3 activation. This antiapoptotic activity in E. faecalis-infected cells was dependent on the activation of the phosphatidyl-
inositol 3-kinase (PI3K)/Akt signaling pathway, which resulted in the increased expression of the antiapoptotic factor Bcl-2 and
decreased expression of the proapoptotic factor Bax. Further analysis revealed that active E. faecalis physiology was important
for inhibition of host cell apoptosis, and this feature seemed to be a strain-independent trait among E. faecalis isolates. Employ-
ing a mouse peritonitis model, we also determined that cells collected from the peritoneal lavage fluid of E. faecalis-infected mice
showed reduced levels of apoptosis compared to cells from uninfected mice. These results show early modulation of apoptosis
during infection and have important implications for enterococcal pathogenesis.

E nterococci are ubiquitous in the gastrointestinal tracts of most

complex metazoans, where they occur as part of the commen-
sal flora at approximately 104 to 107 organisms per gram of feces.
means by which apoptosis is triggered: the extrinsic and intrinsic
pathways. Stimulation of the transmembrane death receptors
with their cognate ligands can activate the extrinsic pathway (12,
Despite being a commensal, Enterococcus faecalis is endowed with 13). Upon stimulation, these receptors transmit external apop-
traits that make it an opportunistic pathogen, especially in immu- totic signals and result in the activation of caspase 3 within the cell.
nocompromised hosts (1, 2). Enterococci have emerged as a lead- In contrast, the intrinsic pathway is initiated by signaling factors
ing cause of antibiotic-resistant infections and now rank among released from the mitochondria (14). Various intrinsic stimuli
the most common nosocomial pathogens infecting the blood- activate Bcl-2 homology 3 (BH3)-only proteins, which overcome
stream, surgical sites, and urinary tract (3, 4). While substantial the inhibitory effects of antiapoptotic Bcl-2 proteins (15, 16). The
progress has been made in recent years in understanding the ge- activated BH3-only proteins promote the oligomerization of pro-
netic makeup of these organisms, much remains to be learned apoptotic proteins, such as Bax and Bak, in the mitochondrial
regarding how they interact with host cells during enterococcal outer membrane, which results in the release of cytochrome c into
infection. the cytoplasm. Cytochrome c induces the formation of the apop-
It has been previously reported that E. faecalis can translocate tosome, a multimeric protein complex that serves as a scaffold for
through the intact epithelial cell monolayer in vitro (5) and dis- caspase activation and proteolytically activates procaspase 9. The
seminate from the intestinal lumen into the bloodstream, liver, activated caspase 9 then cleaves and activates other caspases to
and spleen in an antibiotic-treated murine model of superinfec- induce apoptosis (17).
tion (6). Enterococci that gain access to the sterile abdominal cav- Numerous studies have shown that many pathogens can block
ity, either from the endogenous flora or as a result of surgical or delay host cell death to promote their intracellular survival (18,
intervention, encounter tissue and peritoneal macrophages in- 19). Chlamydia infection activates phosphatidylinositol 3-kinase
volved in the first line of host defense. For these commensals to (PI3K), which leads to Akt activation and subsequent phosphor-
cause systemic infection, they must, at least in part, escape the ylation of the proapoptotic protein Bad (20). Helicobacter pylori
bactericidal activity of macrophages to enter draining lymph
nodes or the bloodstream. Certain strains of E. faecalis are known
to survive within macrophages for extended periods (7–9). The Received 31 July 2014 Returned for modification 25 August 2014
resulting failure of macrophages to kill intracellular E. faecalis Accepted 16 September 2014
likely promotes the systemic spread of infection (6). Therefore, Published ahead of print 29 September 2014
elucidation of E. faecalis survival mechanisms in macrophages, Editor: G. S. Deepe, Jr.
and the effect on host signaling pathways, may provide new ways Address correspondence to Nathan Shankar, [email protected].
to prevent enterococcal infection. Supplemental material for this article may be found at http://dx.doi.org/10.1128
Apoptosis is a highly conserved pathway designed to maintain /IAI.02426-14.
tissue homeostasis by elimination of aged and damaged cells and Copyright © 2014, American Society for Microbiology. All Rights Reserved.
also serves as an important defense mechanism to control bacte- doi:10.1128/IAI.02426-14
ria, viruses, and parasites during infection (10, 11). There are two

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 E. faecalis Targets PI3K To Inhibit Apoptosis

inhibits gut epithelial cell apoptosis to dampen its self-renewal Western blotting. Following infection with E. faecalis strains as de-
and to enhance gastric colonization (21). Cells infected by Toxo- scribed above, the cells were washed with cold PBS and placed on ice. The
plasma gondii show reduced activation of apoptotic cascades, and cells in each well were harvested by scraping into lysis buffer consisting of
this effect is mainly dependent on the transcription factor NF-␬B, 50 mM Tris (pH 7.4), 1.0% NP-40, 150 mM sodium chloride, 1 mM
which regulates the host prosurvival machinery (22). The precise EDTA, 1 mM sodium fluoride, 1 mM sodium orthovanadate, and 1 mM
phenylmethylsulfonyl fluoride (PMSF) for 15 min. The lysate was centri-
molecular mechanisms of apoptosis inhibition vary depending on
fuged at 14,000 ⫻ g for 10 min, and the protein concentration of the
the infectious agent (11).
supernatant was determined using the Bio-Rad protein assay. Total pro-
In this study, we investigated the interaction of enterococci tein (25 ␮g) was subjected to polyacrylamide gel electrophoresis on either
with macrophages and found that E. faecalis-infected host cells 10 or 12% gels by using a Bio-Rad Mini Protean II apparatus and trans-
were profoundly resistant to apoptosis induced by either exoge- ferred electrophoretically onto a polyvinylidene difluoride (PVDF) mem-
nous or intrinsic apoptotic stimuli. The antiapoptotic activity cor- brane, which was then blocked with 5% skim milk. The PVDF membrane
related with the Enterococcus-induced blockade of caspase 3 acti- was incubated for 1 h with primary antibodies (dilution, 1:1,000) in 3%
vation. The molecular mechanism for E. faecalis-infected cells skim milk. The blots were further incubated with appropriate secondary
inhibiting apoptosis in vitro was PI3K-dependent activation of the antibodies conjugated to horseradish peroxidase (dilution, 1:3,000). Fol-
antiapoptotic factor Akt. Activation of Akt resulted in increased lowing three washes with Tris-buffered saline containing 0.1% Tween 20
expression of the antiapoptotic factor Bcl-2 and decreased expres- (TBST), the substrate was detected using a chemiluminescence detection
sion of the proapoptotic factor Bax. In agreement with these data, kit (Thermo Scientific, Rockford, IL). Densitometric calculations were
conducted on the images of blots with Image J software.
results from a mouse peritonitis model confirmed that peritoneal
Bacterial viability within RAW264.7 cells. Survival of E. faecalis
cells from E. faecalis-infected mice had reduced apoptosis scores within macrophages was assessed as described previously (27). Briefly,
compared to those from uninfected mice. To our knowledge, this RAW264.7 macrophages were seeded onto six-well plates at approxi-
is the first report of E. faecalis modulating apoptosis in infected mately 106 cells/well and incubated at 37°C under 5% CO2 for 24 h prior
macrophages. to infection. Triplicate wells of RAW264.7 cells were infected with E99 at
an MOI of 10 for 1 h at 37°C under 5% CO2. The cells were then washed
MATERIALS AND METHODS thrice with PBS and further incubated with DMEM plus 10% FBS con-
taining vancomycin (16 ␮g/ml) and gentamicin (150 ␮g/ml) to kill all
Bacterial strains and growth conditions. The E. faecalis strains used in
extracellular bacteria. At 3, 6, 12, 24, 48, and 72 h, the macrophages were
this study included E99, a clinical isolate from a urinary tract infection
washed twice with PBS and harvested in 1 ml of PBS. The viability and cell
(UTI), multilocus sequence type 4 (MLST4) (23); OG1RF, a clinical oral
count were assessed by trypan blue staining using a TC10 Automated Cell
isolate free of any plasmids and a strain used commonly in the laboratory
Counter (Bio-Rad, Hercules, CA). The macrophages were then lysed by
(24); MMH594, a clinical isolate that caused multiple infections in a hos-
adding 1/10 volume of a saponin cell lysis solution (saponin [40 mg/ml],
pital ward outbreak and the prototype for the pathogenicity island (1);
and V583, the first clinical isolate with vancomycin resistance identified in polypropylene glycol [P-2000; 8 ml/liter], and sodium polyanetholsulfon-
the United States (25). All the E. faecalis strains were grown for 16 h in ate [9.6 mg/ml]) to release intracellular bacteria. The bacteria were quan-
Todd-Hewitt broth (THB) containing 1% glucose supplemented with the tified by serial dilution and plating. The number of viable bacteria at each
appropriate antibiotics (kanamycin [25 ␮g/ml] for E99, gentamicin [500 time point was expressed as CFU per 105 macrophages. Experiments were
␮g/ml] for MMH594 and V583, and rifampin [25 ␮g/ml] and fusidic acid performed in triplicate three independent times, and the means and stan-
[10 ␮g/ml] for OG1RF), and bacteria were counted by serial dilution and dard errors were determined for each time point.
plating. Before use, the bacteria were pelleted by centrifugation and Apoptosis assays. For analysis of apoptosis, RAW264.7 cells seeded on
washed with phosphate-buffered saline (PBS). glass coverslips in 24-well plates 24 h prior to infection were infected with
Cell culture and infection. RAW264.7 macrophages and mouse em- E99 at an MOI of 10 for 1 h at 37°C under 5% CO2. Subsequently the
bryonic fibroblasts (MEFs) were cultivated initially in Dulbecco’s modi- monolayers were washed three times with PBS and then incubated in fresh
fied Eagle medium (DMEM) plus 10% fetal bovine serum (FBS) to con- culture medium containing 10% FBS, vancomycin (16 ␮g/ml), and gen-
fluence in T-25 flasks. Bone marrow-derived macrophages (BMDMs) tamicin (150 ␮g/ml) for 12 h to kill all extracellular bacteria. The cells were
were isolated according to the method of a previous study (26). The cells then stimulated with 0.5 mg/ml actinomycin D for 8 h, cycloheximide
were seeded in 6-well culture dishes and then infected with E. faecalis for 1 (CHX) (10 ␮g/ml) plus tumor necrosis factor alpha (TNF-␣) (25 ng/ml)
h. For longer periods of incubation, the cells were washed thrice with PBS for 5 h, or 1 ␮M staurosporine for 3 h. Levels of apoptosis in infected
to remove unbound bacteria and further incubated with medium con- RAW264.7 cells were measured by the terminal deoxynucleotidyltrans-
taining vancomycin (16 ␮g/ml) and gentamicin (150 ␮g/ml) to kill all ferase-mediated dUTP-biotin nick end labeling (TUNEL) assay with the
extracellular bacteria. For some experiments, host cells were infected with DeadEnd Fluorometric TUNEL System (Promega, Madison, WI) ac-
E. faecalis for various periods and then treated with apoptosis inducers in cording to the manufacturer’s instructions. Cells from five random
medium containing gentamicin and vancomycin. To inhibit bacterial in- fields for each sample were counted using a 40⫻ objective lens. The
ternalization by RAW264.7 cells, the cells were pretreated with cytochala- percentage of apoptotic cells was calculated as follows: number of
sin D (2.5 ␮g/ml) for 1 h and then infected with E99 at a multiplicity of apoptotic cells/number of total cells counted ⫻ 100.
infection (MOI) of 10 for 1 h, followed by removal of cytochalasin D and Mouse peritonitis model. In vivo studies were conducted under an
extracellular bacteria. The uninfected cells were treated in a manner iden- animal protocol approved by the University of Oklahoma Health Sciences
tical to the experimental groups in all respects. Center Institutional Animal Care and Use Committee and adapted from
E. faecalis cell-free culture supernatants. E. faecalis E99 was inocu- previous reports (27, 28). Bacteria were cultivated overnight at 37°C in
lated into DMEM plus 10% fetal bovine serum in 6-well plates without THB containing 1% glucose supplemented with appropriate antibiotics.
RAW264.7 cells at the same inoculum used for infection experiments. The bacteria were collected by centrifugation at 6,000 ⫻ g for 10 min,
After 1 h, the supernatants were collected by centrifugation at 6,000 ⫻ g washed twice in PBS (pH 7.4), and resuspended in 5% hog gastric mucin
for 10 min and filtered twice using 0.22-␮m syringe filters before being (pH 7.0; Pfaltz and Bauer, Waterbury, CT) at a concentration of 1.0 ⫻ 109
used to replace the culture media of 6-well dishes that had confluent CFU/ml. Six- to 8-week-old female C57BL/6 mice (Harlan, Indianapolis,
RAW264.7 cells. After 12 h, the cells were stimulated with 1 ␮M stauro- IN) were administered 200 ␮l of the suspension (2.0 ⫻ 108 CFU) via
sporine (STS) for 5 h to induce apoptosis. intraperitoneal injection. Control mice were injected with 200 ␮l of 5%

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Zou and Shankar

FIG 1 Intracellular survival of E. faecalis strain E99 and effect on caspase 3 processing. (A) RAW264.7 cells were infected as described in Materials and Methods,
and the number of viable E. faecalis bacteria at each time point is expressed as CFU per 105 macrophages. Experiments were performed three times, and the means
and standard errors are reported for each time point. (B) Western blot of lysates at different time points from uninfected RAW264.7 cells (control) or infected
with E. faecalis E99, processed using antibody against cleaved caspase 3.

hog gastric mucin without bacteria. The control animals were treated in a h postinfection, and the activation of caspase 3 was detected at 24
manner identical to the experimental groups in all respects. Groups of 3 h, which is consistent with the TUNEL-staining assay (Fig. 1B). In
mice were euthanized 6 h or 12 h postinfection by isoflurane overdose, contrast, RAW264.7 cells infected with E. coli DH5␣ showed an
followed by cervical dislocation. Three milliliters of PBS was injected into increase in cleaved caspase 3 at 6 or 12 h postinfection (see Fig.
the peritoneal cavity of each mouse, and the peritoneal lavage fluid was
S1A in the supplemental material). These results showed that en-
collected from each group and mixed together. The cells in the lavage fluid
were subjected to Western blot analysis by using anti-cleaved caspase 3 terococcus-infected RAW264.7 macrophages are not apoptotic at
antibody according to the method described above. the early stage of infection and implied that a survival signal might
Statistical analyses. The statistical significance of results was deter- be triggered during the early stage of E. faecalis infection.
mined by using Student’s t test. Differences between experimental groups E. faecalis-infected cells are resistant to stimulus-induced
were considered significant when P values were ⱕ0.05. apoptosis. To evaluate how E. faecalis-infected cells respond to
apoptosis inducers, RAW264.7 cells with or without bacterial in-
RESULTS
fection were treated with the kinase inhibitor STS, a potent apop-
Resistance to killing is accompanied by antiapoptotic signals in tosis inducer. We found that STS could efficiently induce unin-
host macrophages early during E. faecalis infection. To charac- fected RAW264.7 cells to become apoptotic, as revealed by
terize the survival of E. faecalis in macrophages in vitro, we infected TUNEL staining (⬃30%). However, RAW264.7 cells infected
RAW264.7 cells with E. faecalis strain E99 at an MOI of 10. Exam- with E. faecalis significantly resisted STS-induced apoptosis
ination of the number of viable intracellular bacteria during 72 h
(⬃5%) (Fig. 2A and B). We also found similar antiapoptotic ac-
postinfection revealed that the number remained constant until
tivity in RAW264.7 cells infected with E. faecalis strains V583 and
about 12 h postinfection, followed by a significant decrease by 24
OR1GF (data not shown), suggesting that antiapoptotic activity
h, and declined gradually thereafter (Fig. 1A). Viable E. faecalis
may be a strain-independent trait in E. faecalis. Besides analysis of
bacteria were recovered up to 72 h postinfection, in contrast to
apoptosis induced by STS, we also evaluated the effect of E. faecalis
Escherichia coli DH5␣ (control), which could be eliminated from
infection on apoptosis initiated by TNF-␣ and actinomycin D.
macrophages to undetectable levels by 24 h (data not shown).
Taken together with our previous results (27) and those of others Enterococcus infection also significantly inhibited host cell apop-
(7), which showed that infected macrophages essentially remain tosis induced by these two stimuli, although the induction poten-
intact at least until 24 h after infection, these observations sug- cies of the different agents varied (Fig. 2A and B). Because the
gested that intracellular E. faecalis may be able to modulate host reagents use different pathways to induce apoptosis, these obser-
cell signaling and apoptosis early during infection to promote sur- vations suggested that E. faecalis infection may regulate a common
vival. step or block multiple steps leading to host cell apoptosis.
To examine host cell apoptosis during E. faecalis infection, we Activation of caspase 3 is inhibited by intracellular E. faeca-
studied two major hallmarks of apoptosis, namely, DNA fragmen- lis. Since caspase 3 is a critical executioner of apoptosis and cleav-
tation and caspase 3 cleavage in infected cells (29). TUNEL stain- age of caspase 3 often happens in the late stages of cellular apop-
ing revealed DNA fragmentation in the nuclei, and based on this tosis, we hypothesized that E. faecalis might inhibit host cell
staining pattern, we quantitated the apoptotic cells in culture, with apoptosis through inhibition of caspase 3 activation. As shown in
or without E. faecalis infection, at defined time points. At 6 and 12 Fig. 3A, when antibody to cleaved caspase 3 was used to detect
h postinfection, a level of ⬍1% apoptotic cells was detected in endogenous levels of activated caspase 3, the results revealed that
either the infected or uninfected cell populations from most cul- the processing of caspase 3 induced by STS or TNF-␣ in E. faecalis-
ture samples, with fewer apoptotic cells in infected samples than in infected RAW264.7 cells was significantly inhibited. In contrast, E.
the uninfected cell population, and the difference between the two coli-infected RAW264.7 cells did not significantly inhibit STS-in-
groups was not statistically significant (P ⬎ 0.05). By 24 h, there duced caspase 3 activation (see Fig. S1B in the supplemental ma-
were increased apoptotic events in infected cultures compared terial). We next evaluated the caspase enzymatic activity by mea-
with uninfected cultures (data not shown). Immunoblotting with suring the cleavage of the caspase 3 substrate poly(ADP-ribose)
an anti-cleaved caspase 3 antibody showed RAW264.7 cells in- polymerase (PARP). PARP p112 was reduced to the fragment p86
fected with E99 to have lower levels of cleaved caspase 3 at 6 or 12 in RAW264.7 cells treated with STS or TNF-␣, and this was

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 E. faecalis Targets PI3K To Inhibit Apoptosis

FIG 2 Effects of E. faecalis infection on host cell apoptosis induced by proapoptotic stimuli. RAW264.7 cells, alone or infected with E. faecalis E99 for 1 h, were
treated with 0.5 mg/ml actinomycin D for 8 h, with 10 ␮g/ml CHX and 25 ng/ml TNF-␣ for 5 h, or with 1 ␮M STS for 3 h. (A) Representative fluorescence
microscope images of TUNEL staining. (B) Average percentages of TUNEL-positive stained cells. Cells from five random fields were counted under a 40⫻
objective lens, and the percentage of apoptotic cells was calculated. The graph shows the results of three independent experiments and standard deviations (SD).
*, P ⬍ 0.05.

blocked in E. faecalis-infected cells. These observations suggested Fig. 4A, E. faecalis-induced antiapoptotic activity correlated with
that antiapoptotic factors of E. faecalis may function by preventing an increasing bacterial MOI in a dose-dependent manner. The
the activation of caspase 3. To test whether these observed effects inhibition of apoptosis, as a function of time, appeared as early as
were cell line dependent, we evaluated the effects of E. faecalis 2 h after infection and persisted for ⬃24 h (Fig. 4B), when apop-
infection on apoptosis progression in BMDMs and MEFs, again totic activity became evident. Since E. faecalis survival patterns in
by measuring cleavage of caspase 3. Similar to RAW264.7 cells, RAW264.7 cells showed that virtually all bacteria remained viable
inhibition of caspase 3 cleavage was noted during E. faecalis infec- at least up to 12 h postinfection, these observations suggested that
tion of BMDMs and MEFs (Fig. 3B and C). Finally, a murine active intracellular resistance to killing is required for antiapop-
peritonitis model was used to evaluate the status of caspase 3 dur- totic activity in RAW264.7 cells.
ing enterococcal infection. In the peritoneal cells of E99-infected Inhibition of host cell apoptosis is strain independent but
mice, a lower level of cleaved caspase 3 was observed than in mice requires active E. faecalis physiology. To investigate whether in-
infected with vehicle alone (Fig. 3D). hibition of apoptosis by E. faecalis E99 was unique to this partic-
E. faecalis-induced antiapoptotic activity is dose and time ular E. faecalis isolate, we evaluated strains V583, MMH594, and
dependent. To understand the mechanism of E. faecalis-induced OG1RF, with differing phenotypic characteristics (24). Our re-
antiapoptotic activity, we evaluated the kinetic relationship be- sults showed that there were no significant differences among the
tween the infectious dose and antiapoptotic activity. As shown in strains (Fig. 5A and B), indicating that the antiapoptotic activity

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Zou and Shankar

FIG 3 Inhibition of caspase 3 activity in E. faecalis-infected cells. (A to C) RAW264.7 cells (A), BMDMs (B), and MEFs (C) were infected as described in Materials
and Methods. After treatment or not with apoptotic stimuli, the samples were lysed for Western blot analysis using antibodies against cleaved caspase 3 or PARP.
(D) C57BL/6 mice were injected intraperitoneally (i.p.) with 2.0 ⫻ 108 CFU of E. faecalis E99 suspended in 5% hog gastric mucin or with 5% hog gastric mucin
alone, and the peritoneal lavage fluid was collected at 6 or 12 h postinfection. After centrifugation, the pelleted cells were lysed and subjected to Western blot
analysis.

induced by E. faecalis is strain independent. To further investigate take was prevented. Furthermore, supernatants from bacterial
whether intact bacterial physiology was critical for the ability to conditioned medium failed to inhibit host cell apoptosis, suggest-
inhibit host cell apoptosis, we exposed RAW264.7 cells to either ing that no soluble factor secreted by E. faecalis was responsible for
live bacteria or bacteria that were killed by 4% paraformaldehyde. inhibiting host cell apoptosis (Fig. 5D).
E. faecalis-induced antiapoptotic activity was significantly re- E. faecalis inhibits apoptosis via the PI3K/Akt pathway. Sev-
duced for the killed bacteria, but not completely abolished (Fig. eral studies suggest that the PI3K/Akt signaling pathway is impor-
5C). We tested the importance of bacterial internalization for in- tant for host cell survival and protects apoptotic cell death induced
hibition of host cell apoptosis using cytochalasin D and found a by various stresses (19). Hence, we evaluated the effect of E. faeca-
significant reduction in antiapoptotic activity when bacterial up- lis infection on the PI3K/Akt signaling pathway in RAW264.7 cells

FIG 4 Effects of dose and duration of infection on host cell antiapoptotic activity. (A) RAW264.7 cells were infected as described in Materials and Methods with
E. faecalis E99 at an MOI of 0.5, 1, 10, or 20. Apoptosis was induced at 12 h postinfection with 1 ␮M staurosporine for 5 h. (B) RAW264.7 cells were infected with
E99 at an MOI of 10, and apoptosis was induced at 2, 12, and 24 h postinfection with 1 ␮M staurosporine for 5 h. Western blots of cell lysates at the indicated time
points were processed for detection of cleaved caspase 3 levels. Cleaved caspase 3 levels were normalized by actin densitometry, and fold activations relative to
untreated samples were determined. The graphs show the relative ratios of cleaved caspase 3, which are averages of three independent experiments and SD.

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 E. faecalis Targets PI3K To Inhibit Apoptosis

FIG 5 Multiple E. faecalis strains can induce host cell antiapoptotic response. (A and B) RAW264.7 cells were infected with E. faecalis strains OG1RF, V583,
MMH594, and E99 as described in Materials and Methods before treatment (Treat) with staurosporine (A) or TNF-␣ (B) and analysis of cleaved caspase 3 by
Western blotting. (C) The levels of cleaved caspase 3 in RAW264.7 cells were detected after different treatments, i.e., conditioned medium from E99 (Medium),
killed E99 (KE99), and cytochalasin D plus E99 (CytD&E99), and compared to cells treated with live E99 (E99) used as a control. The images are representative
of three independent experiments. (D) Cleaved caspase 3 levels were normalized by actin densitometry, and fold activations relative to untreated samples were
determined. Each data point in the graph represents the mean and SD of three independent experiments. *, P ⬍ 0.05; **, P ⬍ 0.01.

by using antibody specific to phosphorylated Akt (phospho-Akt) dent on PI3K activity, since the PI3K inhibitor LY294002 blocked
(Ser473). As shown in Fig. 6A, phosphorylated Akt was signifi- E. faecalis-induced Akt phosphorylation (data not shown).
cantly elevated in E. faecalis-infected cells at 6 and 12 h postinfec- We next investigated whether the upregulation of Akt phos-
tion. We further determined that the activation of Akt was depen- phorylation during E. faecalis infection was associated with inhi-

FIG 6 Blockade of PI3K activation reverses E. faecalis-induced antiapoptotic activity. (A) RAW264.7 cells were infected with E. faecalis E99 as described in
Materials and Methods and analyzed for pAkt levels at 6, 12, and 24 h by Western blotting. (B) RAW264.7 cells were pretreated with LY294002 (20 ␮M) for 30
min and then infected with enterococcus (MOI ⫽ 10). After 1 h of incubation, the cells were washed and replenished with fresh medium containing vancomycin
plus gentamicin and LY294002 (20 ␮M) for 12 h and then treated with STS to induce apoptosis. TUNEL staining was used to measure cell apoptosis. (C) Cell
lysates after different treatments were harvested for Western blotting. Cleaved caspase 3 levels were normalized by actin densitometry, and fold activations relative
to untreated samples were determined. Each data point in the graph represents the mean and SD of three independent experiments. *, P ⬍ 0.05; ns, not significant.

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Zou and Shankar

FIG 7 Internalized live E. faecalis bacteria can activate the PI3K/Akt pathway and inhibit apoptosis induced by STS. RAW264.7 cells were pretreated with
LY294002 (20 ␮M) for 30 min and then infected with live or killed E99 (KE99) at an MOI of 10. After 1 h of incubation, the cells were washed and replenished
with fresh medium containing vancomycin and gentamicin without LY294002. (A) The macrophages were further incubated for 6, 12, and 24 h and then
processed for Western blotting. (B and C) The macrophages were further incubated for 12 h, and then STS was used to induce host cell apoptosis, which was
measured by Western blotting with anti-cleaved caspase 3 antibody (B) and TUNEL staining (C). (D) The supernatants from live-E99-infected (⫹) or uninfected
(⫺) RAW264.7 cells treated as for panel A for 12 h were collected and, after centrifugation, separately added to freshly prepared RAW264.7 cells for 12 h before
induction of apoptosis by STS. *, P ⬍ 0.05; ns, not significant. The error bars indicate SD.

bition of host cell apoptosis. As shown in Fig. 6B, treatment of activation during bacterium-cell contact still led to apoptosis in-
RAW264.7 cells with the PI3K-specific inhibitor LY294002 signif- hibition in RAW264.7 cells containing internalized E. faecalis.
icantly reversed E. faecalis-induced apoptosis inhibition as mea- This inhibition, however, was lost when killed E. faecalis was used.
sured by TUNEL assays. When cleavage of caspase 3 was used as a It has been previously shown that cytokines secreted during infec-
marker to monitor host cell apoptosis, the inhibition of apoptosis tion can delay host cell apoptosis in an autocrine fashion (30). To
induced by STS was reversed in the presence of the PI3K inhibitor address this issue, RAW264.7 cells were cultured in supernatants
LY294002 in E. faecalis-infected RAW264.7 cells (Fig. 6C). collected from E. faecalis-infected and uninfected macrophages in
LY294002 had no effect on macrophage phagocytosis of E. faecalis culture. No difference in RAW264.7 cell apoptosis was observed
and did not directly affect bacterial growth or viability in culture between the two groups (Fig. 7D), implying that the cytokines
medium (data not shown). Therefore, these results indicate that induced by E. faecalis are not likely involved in the antiapoptotic
infection with E. faecalis activates the PI3K/Akt pathway to delay activity seen in these assays.
apoptosis caused by the pathogen. These observations were not E. faecalis infection regulates the abundance of Bax and
limited to the RAW264.7 cells, since LY294002 similarly reversed Bcl-2, which are dependent on the PI3K-Akt pathway. Since Akt
E. faecalis-induced antiapoptotic activity in MEFs (data not directly affects the apoptotic machinery by regulating the expres-
shown). sion of the proapoptotic protein Bax and the antiapoptotic pro-
Since the contact of bacteria with cells before internalization tein Bcl-2 (31), we wished to examine the expression of these
may induce the PI3K/Akt pathway, we investigated whether en- factors in RAW264.7 cells during E. faecalis infection. The level of
terococci could activate PI3K/Akt signaling after internalization. Bax decreased following infection, while Bcl-2 increased prior to
When the cells were coincubated with the PI3K inhibitor for 1 h 24 h postinfection (Fig. 8A, B, and C). Blocking PI3K activity using
during infection, the inhibitor completely inhibited Akt phos- LY294002 increased Bax and decreased Bcl-2 in RAW264.7 cells
phorylation during internalization of E. faecalis by RAW264.7 containing internalized E. faecalis (Fig. 8D, E, and F). Thus, these
cells (data not shown). After bacterial internalization and removal results suggest that Bax and Bcl-2 are downstream targets of the
of the inhibitor, PI3K/Akt activation was evident at ⬃12 h postin- PI3K/Akt pathway and that they may promote antiapoptotic
fection and subsided by 24 h (Fig. 7A). These results show that responses in macrophages following phagocytosis of viable E.
internalized E. faecalis induces Akt phosphorylation in RAW264.7 faecalis.
cells. To further confirm the correlation between antiapoptotic
activity and PI3K/Akt activation induced by internalized E. faeca- DISCUSSION
lis, we employed TUNEL staining and cleavage of caspase 3 to In this study, we demonstrate for the first time, that phagocytosis
quantify apoptosis. As shown in Fig. 7B and C, inhibition of PI3K of E. faecalis by macrophages induces a dose- and time-dependent

5138 iai.asm.org Infection and Immunity

 E. faecalis Targets PI3K To Inhibit Apoptosis

FIG 8 Role of PI3K in regulating antiapoptotic and proapoptotic proteins in E. faecalis-infected cells. (A, B, and C) Lysates from infected RAW264.7 cells were
prepared for Western blot analysis to detect the relative levels of Bax and Bcl-2 proteins using anti-Bax and anti-Bcl-2 antibodies, respectively. (D, E, and F)
Untreated RAW264.7 cells or cells pretreated for 30 min with the PI3K inhibitor LY294002 (20 ␮M) were infected with E. faecalis for 1 h, and then the cells were
washed and replenished with fresh medium containing vancomycin plus gentamicin and LY294002 (20 ␮M) for 12 h. The cells were harvested for Western blot
analysis using Bax- and Bcl-2-specific antibodies. The relative densities of Bax and Bcl-2 protein bands on Western blots were compared to that of actin in each
group, and fold activations relative to untreated samples were determined. Each data point in the graph represents the mean and SD of three independent
experiments. *, P ⬍ 0.05 compared with control; ns, not significant compared with control.

antiapoptotic response characterized by reduced DNA fragmen- such as Mycobacterium tuberculosis, Legionella pneumophila, and
tation and caspase 3 activation. Enterococci are able to survive for Brucella spp., use differing mechanisms to block apoptosis in or-
prolonged periods in cultured macrophages, unlike other Gram- der to use the host cell as a vehicle for in vivo dissemination (18).
positive cocci, such as Lactococcus lactis, but the exact mechanism Gram-positive pathogens known to modulate apoptosis in
by which this occurs remains unclear. Previous data from our host cells include Streptococcus, Staphylococcus, Bacillus, Listeria,
laboratory (27) and that of others (7) have shown that most strains and Clostridia species, although there appears to be limited con-
of E. faecalis are rapidly phagocytized by macrophages and appear servation of the mechanisms involved (35). Among those more
within phagolysosomes within the first 24 h postinfection. Subse- closely related to Enterococcus, group A streptococcus (GAS) in-
quently, based on electron microscopic examination of infected duces rapid, dose-dependent apoptosis in primary and cultured
cells at later time points, intracellular enterococci are found in the macrophages and neutrophils, and it has been shown that the
cytoplasm at 24 to 48 h and eventually lead to lysis and fragmen- pore-forming cytolysin streptolysin O is necessary and sufficient
tation of host macrophages by 48 to 72 h postinfection. Consistent for the apoptosis-inducing phenotype (36). Induction of host cell
with these previous observations, our data presented here further death by group B streptococcus (GBS) includes apoptotic mech-
show that during early infection of macrophages by E. faecalis, the anisms in several cell types, including macrophages, and was ini-
bacteria effectively resist killing, as their numbers remain constant tially attributed to ␤-hemolysin production, leading to caspase-
for at least up to 12 h, and during this time, the viable intracellular dependent apoptosis (37, 38). Similarly, dual mechanisms are
E. faecalis bacteria can interfere with host cell apoptotic signaling observed during apoptosis of Streptococcus pneumoniae-infected
pathways. These results also imply that at the early stage of infec- cell types mediated by the pore-forming exotoxin pneumolysin.
tion, E. faecalis has characteristics similar to those of some intra- In tissue macrophages and respiratory epithelial cells, pneumoly-
cellular bacteria that can communicate with host cells to benefit sin contributes to a caspase-dependent pathway of apoptosis (39,
bacterial survival and dissemination. 40). Interestingly, in macrophages, Staphylococcus aureus induces
Apoptosis is a highly regulated host process and a prominent strong caspase 3 activation concomitant with activation of the
feature of macrophage biology in the context of infectious disease antiapoptotic machinery (41). The ability of S. aureus to persist
and inflammation. Different pathogens potentially use various within host macrophages before escaping the intracellular niche
mechanisms to induce or inhibit apoptosis in macrophages and has been proposed as a mechanism for systemic dissemination,
thereby influence the disease pathology (32). For instance, mac- similar to the case for Listeria monocytogenes (42, 43). In contrast
rophage death caused by Shigella and Salmonella is linked to re- to the conversion to small-colony variants (SCVs), which allows
lease of proinflammatory cytokines, and the ensuing inflamma- survival for months or years (44), the limited survival within mac-
tion leads to the spread of bacteria within host tissues (33). rophages is considered a short-term solution.
Likewise, Yersinia spp. induce apoptosis by suppressing NF-␬B Enterococci are occupants of the gastrointestinal tract as part
activation and TNF-␣ synthesis, which are essential to pathogen of the commensal flora, and it is conceivable that they may use
clearance (34). Highly adapted intracellular bacterial pathogens, macrophages as vehicles for systemic dissemination. While our

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Zou and Shankar

observations here show that E. faecalis resists apoptosis of macro- cells may provide time for E. faecalis to adapt to the intracellular
phages early during infection, we (27) and others (7) have shown environment before emerging from this niche to disseminate
in previous studies that viable E. faecalis bacteria can be recovered and cause systemic infection (48). Experiments to determine
at significantly later time points, beyond when apoptosis is evident whether E. faecalis-infected epithelial cells respond to proapo-
(24 h). Electron micrographs of E. faecalis-infected macrophages ptotic stimuli in the same manner as macrophages are under
further suggested that some bacteria at these later time points way in our laboratory.
appear capable of cytoplasmic existence and continued survival, An important finding of this study is that E. faecalis in
eventually leading to macrophage disintegration and lysis (7). RAW264.7 cells activates the PI3K/Akt pathway, although the
Thus, it appears that E. faecalis may employ two distinct strategies mechanism by which this occurs is not fully understood. As shown
during macrophage infection, similar to those described for S. by previous studies, cytokines induced during infection can stim-
aureus (41). One is to delay cell death during early infection to ulate PI3K/Akt activation (30). Enterococcal infection can induce
allow intracellular adaptation and dissemination, while the other the robust production of proinflammatory cytokines and chemo-
is to induce later cell death to promote bacterial egress from in- kines from macrophages (9, 53). However, culture medium from
fected macrophages. While the molecular mechanisms involved E. faecalis-infected macrophages did not inhibit host cell apopto-
in these plausible scenarios require further investigation, a ques- sis induced by proapoptotic stimuli. While we cannot completely
tion raised by the viable intracellular E. faecalis bacteria regards rule out a role for cytokines in E. faecalis-induced antiapoptotic
their genetic makeup. Do these bacteria coordinate the expression activity, it does not appear to be a significant mechanism for our
of genes specifically required for intracellular survival, likely genes observations. A more plausible explanation could be that E. faeca-
that are on the E. faecalis core genome, or do they represent genetic lis produces one or more novel antiapoptotic factors in the host
variants of the parent strain that are better adapted to survive cell to manipulate the PI3K/Akt pathway directly. This strategy is
inside the host cell? Although the transcriptional changes occur- widely used by many intracellular pathogens to manipulate host
ring in the E. faecalis genome in response to the intramacrophage cell kinase signaling, and a number of viral and bacterial antiapo-
environment has not been reported, SCVs of E. faecalis isolated ptotic factors have been identified (54, 55). Interestingly, a novel
from clinical specimens have been described (45). The SCV phe- mode of modulation of apoptosis and release of intracellular bac-
notype has been characterized as cells of different sizes with aber- teria from infected epithelial cells has recently been described for
rant shapes forming smaller colonies than the clonally related nor- S. aureus, which is primarily an extracellular pathogen (56). This
mal strain on culture media, growing with an extended lag phase strategy involves the ESAT-6-like secretion system (Ess), which is
with delayed entry into stationary phase, failing to grow on simple similar to the Esx-1 secretion system described for M. tuberculosis
media without the addition of blood, and showing changes in and is apparently conserved in the genomes of many Gram-posi-
metabolic pathways. Along these lines, internalization of Salmo- tive bacteria, including Bacillus subtilis, Bacillus anthracis, and L.
nella enterica serovar Typhimurium by macrophages has been re- monocytogenes (57, 58). The proteins EsxA and EsxB, substrates of
ported to induce the formation of nonreplicating persisters (46). the S. aureus Ess, belong to the WXG100 motif superfamily (58)
Multiple toxin-antitoxin (TA) modules contributed to the forma- and were shown to be important during intracellular S. aureus
tion of intracellular persisters, and the macrophage environment infection (56). EsxA interfered with S. aureus-induced apoptosis
was found to induce phenotypic heterogeneity, leading to either in human epithelial cells in vitro and required unimpeded secre-
bacterial replication or the formation of nonreplicating persisters tion of the protein. Overexpression of EsxA in transfected cells
that could provide a reservoir for relapsing infection. The exact increased protection from apoptotic cell death. Further, it was
nature of E. faecalis isolates that survive the antimicrobial activi- shown that EsxA acted in concert with EsxB to mediate release of
ties of macrophages remains to be investigated. S. aureus from host cells. While orthologous Esx proteins in M.
It is also important to consider apoptosis in the context of tuberculosis have been implicated in several aspects of pathogene-
nonphagocytic cells, such as epithelial cells, because our studies sis, including apoptosis and host cell lysis (59), the functions of
show that enterococcus infection can induce antiapoptotic activ- other orthologs, such as those in L. monocytogenes, remain unclear
ity in both professional phagocytes and nonphagocytic cells. (60). Enterococcal genomes also appear to encode EsxA-like pro-
Apoptosis is a normal occurrence for epithelial cells in order to teins, but their contribution to enterococcal physiology and
maintain a critical balance of cells in the crypt and villus during pathogenesis remains to be studied (61, 62). Although their pre-
self-renewal of the intestinal lining, but it may also function as a cise role(s) during infection remains to be determined, the studies
mechanism to eliminate infected cells so as to prevent pathogen described above raise the prospect that the Esx proteins in Gram-
proliferation and access to deeper tissues (47, 48). The same strat- positive bacteria may represent conserved factors that control host
egy is evident in superficial epithelial cells lining the urinary blad- cell apoptosis and cell lysis. In any case, the results obtained with
der. Uropathogens, such as Proteus mirabilis and certain strains of the Esx proteins of S. aureus demonstrate that bacterial proteins
E. coli, effectively invade the urothelium, form intracellular reser- are active in host cells and can regulate apoptosis in infected cells.
voirs that evade antibiotics and the immune response, and allow Consistent with this notion, we found that nonviable E. faecalis
recurring infections (49). The host response to such infections is bacteria after internalization could not protect cells from apopto-
sloughing of cells into the urine (50). E. faecalis accounts for a sis, suggesting that a newly synthesized enterococcal factor(s)
significant proportion of chronic bladder infections worldwide might be responsible for the inhibition of apoptosis. While our
and is also a leading cause of catheter-associated UTIs (51). Recent data show that the activation of the PI3K/Akt pathway modulates
studies have shown that E. faecalis bacteria are harbored within expression of the key apoptosis effectors Bax and Bcl-2, the fact
urothelial cells shed from the bladders of patients with chronic that enterococcal infection inhibited apoptosis induced by a wide
UTIs (52). The ability to persist in epithelial cells lining the intes- range of stimuli makes it likely that other apoptosis effectors are
tine or the bladder and to resist apoptosis and shedding of these affected, as well.

5140 iai.asm.org Infection and Immunity

 E. faecalis Targets PI3K To Inhibit Apoptosis

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ACKNOWLEDGMENTS .doi.org/10.1371/journal.ppat.0020045.
This project was funded, in part, by a President’s Associates Presidential 21. Mimuro H, Suzuki T, Nagai S, Rieder G, Suzuki M, Nagai T, Fujita Y,
Nagamatsu K, Ishijima N, Koyasu S, Haas R, Sasakawa C. 2007.
Professorship awarded by the University of Oklahoma to N.S.
Helicobacter pylori dampens gut epithelial self-renewal by inhibiting apop-
We gratefully acknowledge the assistance of Mark Huycke and Xing- tosis, a bacterial strategy to enhance colonization of the stomach. Cell Host
min Wang for critical reading of the manuscript. Microbe 2:250 –263. http://dx.doi.org/10.1016/j.chom.2007.09.005.
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