Dichotomous metabolism of Enterococcus faecalis
induced by haematin starvation modulates colonic
Toby D. Allen,1,2Danny R. Moore,1,2Xingmin Wang,1,2Viviana Casu,1,2
Randal May,2Megan R. Lerner,3Courtney Houchen,2Daniel J. Brackett3,4
and Mark M. Huycke1,2
Mark M. Huycke
1Muchmore Laboratories for Infectious Disease Research, Department of Veterans Affairs Medical
Center, Oklahoma City, OK 73104, USA
2Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK
3Department of Surgery, University of Oklahoma Health Sciences Center, Oklahoma City, OK
4Research Service, Department of Veterans Affairs Medical Center, Oklahoma City, OK 73104, USA
Received 4 December 2007
Accepted 10 June 2008
Enterococcus faecalis is an intestinal commensal that cannot synthesize porphyrins and only
expresses a functional respiratory chain when provided with exogenous haematin. In the absence
of haematin, E. faecalis reverts to fermentative metabolism and produces extracellular superoxide
that can damage epithelial-cell DNA. The acute response of the colonic mucosa to haematin-
starved E. faecalis was identified by gene array. E. faecalis was inoculated into murine colons
using a surgical ligation model that preserved tissue architecture and homeostasis. The mucosa
was exposed to haematin-starved E. faecalis and compared with a control consisting of the same
strain grown with haematin. At 1 h post-inoculation, 6 mucosal genes were differentially regulated
and this increased to 42 genes at 6 h. At 6 h, a highly significant biological interaction network
was identified with functions that included nuclear factor-kB (NF-kB) signalling, apoptosis and
cell-cycle regulation. Colon biopsies showed no histological abnormalities by haematoxylin and
eosin staining. Immunohistochemical staining, however, detected NF-kB activation in tissue
macrophages using antibodies to the nuclear localization sequence for p65 and the F4/80 marker
for murine macrophages. Similarly, haematin-starved E. faecalis strongly activated NF-kB in
murine macrophages in vitro. Furthermore, primary and transformed colonic epithelial cells
activated the G2/M checkpoint in vitro following exposure to haematin-starved E. faecalis.
Modulation of this cell-cycle checkpoint was due to extracellular superoxide produced as a result
of the respiratory block in haematin-starved E. faecalis. These results demonstrate that the
uniquely dichotomous metabolism of E. faecalis can significantly modulate gene expression in the
colonic mucosa for pathways associated with inflammation, apoptosis and cell-cycle regulation.
For several decades, the colonic microbiota has been
postulated to play a role in the aetiology of sporadic
colorectal cancer (CRC) (Augenlicht et al., 2002; McGarr
et al., 2005; Rowland, 1995). This hypothesis is based on
the observation that intestinal cancers occur almost
exclusively in the colon where metabolically active bacteria
are in direct proximity to mucosal surfaces at densities of
1011c.f.u. (g faecal material)21. Several epidemiological
studies have attempted to identify associations between at-
risk populations for CRC and colonic bacteria (Benno et al.,
1986; Moore & Moore, 1995). This approach has been
hampered, however, by the enormous complexity of the
colonic microbiota and inadequate understanding of what
constitutes exposure risks for commensals (Eckburg et al.,
2005). Despite these limitations, a credible rationale
remains for studying the role of colonic commensals in
Abbreviations: CRC, colorectal cancer; DAPI, 49,6-diamidino-2-phenyl-
indole; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; HRP,
horseradish peroxidase; IL, interleukin; MnSOD, manganese superoxide
sequence; p.i. post-inoculation; qRT-PCR, quantitative real-time RT-PCR.
Journal of Medical Microbiology (2008), 57, 1193–1204
47798Printed in Great Britain1193
Perhaps the best evidence supporting a role for colonic
commensals in CRC derives from genetically engineered
mice that develop intestinal tumours when conventionally
colonized but have fewer tumours or no pathology when
housed in pathogen-free or germ-free environments
(Balish & Warner, 2002; Chu et al., 2004; Dove et al.,
1997; Engle et al., 2002; Kado et al., 2001; Kim et al., 2005;
Maggio-Price et al., 2006). The mechanisms by which
commensals trigger inflammation or initiate genomic
instability (a characteristic feature of sporadic CRC)
remain uncertain. Commensals can modulate the intestinal
mucosa through the metabolism of faecal steroids, by
producing short-chain fatty acids and by inducing host
genes (Augenlicht et al., 2002; Ba ¨ckhed et al., 2005;
Debruyne et al., 2001; McGarr et al., 2005). None of these
effects, however, is known to initiate or promote genomic
or epigenetic changes in epithelial cells as antecedents to
oncogenic transformation. Commensals that cause epithe-
lial-cell DNA damage, in contrast, are more likely to
initiate chromosomal instability than bacteria that modu-
late epithelial-cell metabolism but are otherwise not
Enterococcus faecalis is a Gram-positive minority constitu-
ent of the colonic microbiota that can directly damage
epithelial-cell DNA (Huycke et al., 2002), promote
chromosomal instability through a macrophage-induced
bystander effect (Wang & Huycke, 2007) and trigger colitis
and cancer in interleukin (IL)-10 knockout mice (Balish &
Warner, 2002; Kim et al., 2005). The bystander effect refers
to oxidatively stressed or irradiated cells that produce
chromosomal instability in neighbouring cells through the
production of diffusible mutagens (Lorimore & Wright,
2003). These effects are derived in part from the redox-
active phenotype of E. faecalis that occurs when this micro-
organism is grown without haematin. E. faecalis cannot
synthesize porphyrins and requires this nutrient to form
functional cytochrome bd, establish a proton gradient and
respire (Bryan-Jones & Whittenbury, 1969; Ritchey &
Seeley, 1974). In contrast, growth of E. faecalis in the
absence of haematin leads to a block in respiration and the
production of superoxide, hydrogen peroxide and hydroxyl
radical (Huycke et al., 2001, 2002). These reactive oxygen
species are known to damage DNA (Marnett, 2000),
although it remains to be determined whether this redox-
active phenotype promotes inflammation and colon cancer
in IL-10 knockout mice that are monoassociated with E.
To explore further the effect of E. faecalis on the colonic
mucosa, we developed an in vivo colonic ligation model to
examine host gene expression in wild-type mice. This
model preserves tissue architecture and homeostasis, and
permits an analysis of gene expression in stromal and
epithelial-cell compartments. We compared E. faecalis
supplied with haematin with E. faecalis grown without
haematin to determine the effect of altered bacterial
metabolism on colonic mucosal gene expression. We
believe this comparison reflects the naturally occurring
potential dichotomous metabolism of E. faecalis in the
intestinal environment. We found the induction and
suppression of a small subset of genes involved in cell-
cycle regulation, apoptosis and nuclear factor-kB (NF-kB)
signalling by haematin-starved E. faecalis. These findings
indicate that the colonic mucosa rapidly responds to
alternate metabolisms of E. faecalis and suggest novel
mechanisms by which this commensal may promote
inflammatory or potentially transforming events.
Bacteria, growth conditions and superoxide assay. E. faecalis
strain OG1RF is a human oral isolate that produces extracellular
superoxide (~20 mmol min21per 109c.f.u. in vitro), promotes
chromosomal instability, and produces colonic inflammation and
cancer when monoassociated with IL-10 knockout mice (Balish &
Warner, 2002; Huycke & Moore, 2002; Kim et al., 2005; Wang &
Huycke, 2007). E. faecalis was grown overnight in closed tubes using
brain heart infusion broth (BD Diagnostics) with or without 10 mM
haematin (Sigma), and washed in sterile PBS before use in
experiments. E. faecalis cannot synthesize porphyrins and is unable
to form functional cytochrome bd unless supplied with exogenous
haematin (Huycke, 2002). Cytochrome bd is one of two terminal
quinol oxidases expressed by E. faecalis that, when active, allows
oxidative phosphorylation, promotes growth and suppresses extra-
cellular superoxide (Huycke et al., 2001). Growth with haematin
attenuates superoxide production by .10-fold, an effect that persists
for .6 h in vitro. For all experiments, haematin-replete growth of E.
faecalis was confirmed by measuring the attenuation of superoxide
using a ferricytochrome c assay as described previously (Huycke et al.,
1996). Unless otherwise specified, all chemicals were of analytical or
molecular biology grade from Sigma.
Murine colonic ligation model. To assess the short-term effects of
haematin-starved E. faecalis on the intact colonic mucosa, we
developed an intestinal ligation model. The technique is analogous
to the ileal loop model used to investigate diarrhoeal toxins.
Conventionally housed and fed 25–28 g adult male BALB/c mice
(Jackson Laboratory) were anaesthetized using 1–2% isoflurane in a
carrier gas composed of 95% O2and 5% CO2. Through a 5 mm
midline abdominal incision, the proximal colon was identified at its
juncture with the caecum. Two ligatures 0.5 cm apart were placed
around the colon and a 2 mm incision was made into the colon. A
gavage needle was inserted into the colon and the contents were
completely flushed through the rectum using sterile PBS. The rectum
was closed with a purse-string ligature and the colon backfilled with
1.0 ml PBS alone or PBS containing enterococci at a concentration of
16108c.f.u. ml21. Immediately preceding instillation, D-glucose was
added to the PBS or the bacterial inoculum to a final concentration of
5 mM. This sugar initiates extracellular superoxide production by E.
faecalis, but not for bacteria grown with haematin (Huycke et al.,
2001). Following inoculation, both colonic ligatures were tied to
prevent backflow of enterococci and peritoneal contamination from
proximal intestinal contents. Care was taken to preserve blood flow to
the colon. The surgical area was washed with sterile PBS, the colon
was gently returned to the peritoneal cavity, the abdominal incision
was closed and mice were allowed to recover.
At 1 or 6 h post-inoculation (p.i.), mice were anaesthetized, the
abdomens reopened and the colons surgically removed. The surgical
manipulations were well tolerated, with only one mouse not surviving
to the end of the protocol. Contents were cultured for enterococci as
described previously (Huycke et al., 1992). Colon biopsies of 5 mm
were obtained for histopathology and immunohistochemistry.
T. D. Allen and others
1194 Journal of Medical Microbiology 57
Biopsies were examined using haematoxylin and eosin staining, and a
modified Brown and Brenn stain. The remaining colon segments were
opened longitudinally and the mucosal surfaces scraped with sterile
razors for RNA extraction. Biopsies were fixed in formalin and
scrapings were snap frozen in liquid nitrogen. Mice (n520) were
exposed to E. faecalis or PBS (n56) with independent experiments
analysed by group (1 and 6 h p.i.) for mice exposed to haematin-
starved E. faecalis (n55 per group), E. faecalis grown with haematin
(n55 per group) or PBS (n53 per group). The animal protocol was
approved by the Animal Studies Subcommittee of the Veterans Affairs
Research and Development Committee.
Gene expression, network response and transcriptional reg-
ulatory element analyses. Total RNA was isolated from colonic
scrapings for mice exposed to haematin-replete or haematin-starved
E. faecalis at 1 and 6 h p.i. using an Atlas pure total RNA labelling
system (BD Biosciences Clontech). Probes were synthesized by reverse
transcription using [a-33P]dATP. As the quantities of mucosal
scrapings from individual colons were small, each sample was used
in its entirety for probe synthesis. Extracted and labelled cDNA
probes were hybridized overnight to 5000 cDNA murine arrays (BD
Biosciences Clontech). Separate arrays (n55 per group for a total of
20 arrays) were used for each probe prepared from colon scrapings.
After high-stringency washes, membranes were quantified (Storm 820
PhosphorImager; Amersham Biosciences) and expression of indi-
vidual genes was determined as absorbance readings minus
background (ArrayVision software; Imaging Research). Significantly
GeneSpring software version 6.2 (Silicon Genetics). After background
subtraction, raw signals were normalized per spot and by array, using
an intensity-dependent Lowess protocol. Signal intensities were
normalized to the 50th percentile, and comparisons between array
results at the 1 and 6 h time points were made using Student’s t-test
with P,0.005 considered significant. Fold changes were calculated
using the GeneSpring fold change filter option. The Benjamini–
Hochberg method was used to correct for multiple testing and to
minimize false discovery rates.
Biologically relevant response networks for significantly modulated
genes were constructed using Ingenuity Pathways Analysis (Ingenuity
Systems; www.ingenuity.com). The Ingenuity Pathways Knowledge
Base is the largest curated database on mammalian biology in the
published literature. Findings on genes in human, mouse and rat
studies from peer-reviewed publications are encoded into an ontology
by content and modelling experts. Manual extraction and curation
identifies specific interactions that result in fewer false positives than
automated methods. Networks are algorithmically generated based on
their connectivity, and pathways of highly interconnected genes are
identified by statistical likelihood testing (Calvano et al., 2005).
In silico analysis of differentially expressed genes was performed for
transcription factor-binding sites using the web-based Promoter
Analysis and Interaction Network Tool software (Vadigepalli et al.,
2003). Comparisons were carried out using all genes on the murine
array as the reference library.
Immunohistochemistry. Immunohistochemical analysis of the p65
component of NF-kB was performed on serial sections of paraffin-
embedded murine colon tissue. Antigen retrieval of deparaffinized
sections was performed using a decloaking chamber (Biocare
Medical) with citrate buffer or 0.1% pronase (Dako) and processed
using the Sequenza staining method (Thermo Scientific). Endogenous
peroxidase activity was quenched using peroxidase-blocking reagent
(Dako) followed by a blocking step with buffer containing 1% BSA
(Jackson ImmunoResearch), 1% normal horse serum (Jackson
ImmunoResearch), coldwater fish gelatin and Tween 20. Sections
were stained using nuclear localization sequence (NLS)-specific
anti-p65 antibody (diluted 1:300; Rockland Immunochemicals) or
anti-F4/80 mAb (diluted 1:150; AbD Serotec). The former antibody
recognizes the NLS on p65 that is masked by IkB. The F4/80 antigen is
a surface marker expressed by mature murine macrophages (Austyn
& Gordon, 1981). After primary incubation with NLS-specific anti-
p65 antibody, sections were incubated in horseradish peroxidase
(HRP)-labelled EnVision + (Dako). Sections stained with anti-F4/80
antibody were developed using anti-rat secondary antibody (diluted
1:1000; Jackson ImmunoResearch), followed by incubation with
ready-to-use streptavidin–HRP solution (Dako). Following incuba-
tion, sections were developed with 3,3-diaminobenzidine substrate or
Bajoran Purple (Biocare Medical) and counterstained with haema-
toxylin (Biocare Medical). The distribution of positive cells per field
(magnification 100 6 ) between groups was assessed in a randomized
and blind fashion, and compared using ridit analysis, with P,0.05
considered significant (Fleiss, 1981). This method assumes that
discrete measures represent intervals in an underlying continuous
distribution without any assumptions about the distribution. Ridits
range from 0 to 1 and the ridit for the control (or comparator)
distribution is 0.50. A mean ridit is .0.50 when more than half of the
time randomly selected measures from the experimental distribution
have a value greater than randomly selected measures from the
Colon sections were processed for netrin-1 immunohistochemistry
using UltraVision LP detection system HRP polymer and AEC
chromogen (LabVision). Sections were blocked with H2O2for 10 min
to inhibit endogenous peroxidase activity, followed by washes in Tris-
buffered saline with Tween 20 at pH 8.0. Following antigen retrieval,
Ultra V block (Dako) was applied for 5 min followed by a 1:20
dilution of rabbit anti-netrin-1 (Ab-1) primary antibody or control
peptide following the manufacturer’s instructions (Calbiochem).
Sections were counterstained with Immuno* master haematoxylin
(American Master*Tech Scientific).
Cell lines. Chromosomally stable HCT116 colonic epithelial
cells (AmericanTypeCultureCollection) were grown in 5% CO2at
37 uC using McCoy’s 5A medium modified by
and 25 mM HEPES (Invitrogen) and supplemented with 10%
fetal bovineserum (FBS).RAW264.7
(American Type Culture Collection) were grown under the same
conditions using Dulbecco’s modified Eagle’s medium modified with
4.5 g glucose l21and L-glutamine (Invitrogen) and supplemented
with 10% FBS. For experiments involving co-incubation with E.
faecalis, bacteria were diluted to 16109c.f.u. ml21in fresh medium
without FBS. YAMC cells are a non-transformed intestinal epithelial
cell line derived from healthy tissue and were a gift from the Ludwig
Institute for Cancer Research (Whitehead et al., 1993). These cells
were grown in 5% CO2at 33 uC using RPMI 1620 (Invitrogen)
supplemented with 5% FBS, 5 U recombinant murine gamma
interferon (PeproTech) ml21and ITS Premix (BD) according to
the manufacturer’s instructions. After treatment, cells were washed
with PBS and complete medium was added containing gentamicin
(10 mg ml21) and penicillin (100 U ml21) to kill any remaining
extracellular bacteria. For H2O2-treated cells, catalase (1200 U ml21)
was also included in the complete medium to eliminate residual
Immunofluorescent assay. To visualize NF-kB activation, E.
faecalis-treated RAW264.7 cells and HCT116 cells grown on
chambered slides were fixed with paraformaldehyde and incubated
with polyclonalanti-p65 IgG
Biotechnology). A fluorescein isothiocyanate (FITC)-conjugated IgG
(diluted 1:200; Santa Cruz Biotechnology) was used as the secondary
antibody and nuclei were counterstained with 49,6-diamidino-2-
phenylindole (DAPI) prior to laser-scanning confocal microscopy
(LSM-510 META; Zeiss).
E. faecalis modulates mucosal gene expression
NF-kB–luciferase reporter assay. To verify NF-kB activation,
RAW264.7 cells were transfected with the pNFkB-Luc reporter vector
(Clontech) using Lipofectin reagent (Invitrogen). Transfected cells
were treated with E. faecalis for 1 h and further incubated for 24 h.
LPS treatment (10 mg ml21) served as a control. Cell lysates were
prepared using reporter lysis buffer according to the manufacturer’s
instructions (Promega). Luciferase activity was measured using a
luciferase assay system (Promega) and a TD-20/20 luminometer
(Turner Designs). Values were normalized to protein concentration.
Cell-cycle and apoptosis assays. The ability of E. faecalis to
activate colonic epithelial-cell checkpoints or initiate apoptosis was
assessed by flow cytometry. Following a 1 h treatment with E. faecalis,
HCT116 or YAMC cells were incubated in fresh medium containing
FBS, gentamicin and penicillin. Cells were fixed overnight with 70%
ethanol and stained with propidium iodide (0.02 mg ml21) contain-
ing 0.1% Triton X-100 and 0.2 mg RNase A ml21. Stained cells were
analysed using a FACSCalibur flow cytometer (BD Immunocytometry
Systems). Apoptotic cells were stained using the Annexin V FITC
apoptosis detection kit according to the manufacturer’s instructions
(Calbiochem, EMD Biosciences) and quantified by flow cytometry.
Data were analysed using CellQuest Pro software. Statistical analyses
were performed using ModFit version 2.2 software (Verity Software
House). For each sample, .10000 events were collected and groups
were compared using Student’s t-test with P,0.05 considered
Quantitative real-time RT-PCR (qRT-PCR) and gene silencing.
Total mRNA was isolated from E. faecalis-treated cells using a
NucleoSpin RNA II kit (BD Biosciences) and 2 mg was reverse-
transcribed with an iScript cDNA synthesis kit according to the
manufacturer’s instructions (Bio-Rad Laboratories). qRT-PCR was
performed using an Mx3005P Q-PCR System following the manu-
facturer’s instructions (Stratagene). Primers used to assess expression
of genes identified in the array experiment (Table 1) were purchased
from Integrated DNA Technologies. The gene for netrin-1 was
silenced by RNA interference using siGENOME SMARTpool reagent
specific for human NTN1 or with an siCONTROL non-targeting
siRNA pool (Dharmacon). Transient transfections were performed
using DharmaFECT 4 transfection reagent (Dharmacon) according to
the manufacturer’s protocol and gene silencing was confirmed by
qRT-PCR. Groups were compared using Student’s t-test with P,0.05
Western blotting. Total protein was extracted from cells and equal
amounts were analysed by SDS-PAGE before transfer to PVDF
membranes (Amersham Biosciences). Assays for netrin-1 were
performed using goat polyclonal anti-human netrin-1 antibody and
alkaline phosphatase-conjugated rabbit anti-goat IgG as the secondary
antibody (Santa Cruz Biotechnology) and antibody binding was
detected using an ECF Western blotting detection system (Amersham
Biosciences) according to the manufacturer’s instructions.
RESULTS AND DISCUSSION
E. faecalis activates NF-kB in colonic
To assess the short-term effects of haematin-starved E.
faecalis on colonic gene expression, we developed an in vivo
ligation model that preserved mucosal architecture and
homeostasis. The mean concentrations of E. faecalis
recovered from luminal contents at 1 and 6 h p.i. were
approximately 10-fold lower than the initial inocula but
were not significantly different for mice administered
haematin-starved E. faecalis compared with haematin-
replete E. faecalis. No histological abnormalities were noted
for colon biopsies at either time point for any group. In
addition, epithelial or submucosal cocci were not visible by
a tissue Gram stain, indicating that an acute mucosal
infection had not occurred (data not shown).
As haematin-starved E. faecalis induces COX-2 expression
in macrophages in vitro (Wang & Huycke, 2007) and COX-
2 is regulated via NF-kB (Karin & Greten, 2005), we
initially determined whether E. faecalis activated this
redox-sensitive signalling pathway in the colon. NF-kB
was detected using an NLS-specific anti-p65 antibody that
detects p65 only after it dissociates from IkB. p65 is a
member of the canonical NF-kB pathway and is activated
by many stimuli including redox stress and exposure to
bacteria (Karin & Greten, 2005). We found significantly
increased numbers of cells with p65 nuclear staining in
colons exposed to E. faecalis at 6 h compared with PBS
controls (ridit50.57, P50.008). Although an increase in
NF-kB activation was found for colons exposed to
haematin-starved compared with haematin-replete E.
faecalis, this difference was not statistically significant
(ridit50.52, P50.25). To identify mucosal cells with NF-
kB activation, we stained serial colon sections with the
NLS-specific anti-p65 antibody and an anti-F4/80 mAb.
Nearly all cells positive for p65 also stained positive for
F4/80, indicating that this redox-sensitive signalling
pathway had been activated in tissue macrophages
(Fig. 1a, b). In contrast, no staining for the NLS of p65
was noted in epithelial cells.
To determine whether E. faecalis activated NF-kB in vitro,
we exposed macrophage and epithelial cell lines to
haematin-starved bacteria. Strong nuclear staining, along
Table 1. Primer pairs for qRT-PCR
Target geneForward primer (5§A3§)Reverse primer (5§A3§)
C3ar1 (human, murine)
Cyr61 (human, murine)
Akap8l (human, murine)
ActB (human, murine)
T. D. Allen and others
1196 Journal of Medical Microbiology 57
with cytoplasmic staining, was noted in macrophages by
laser-scanning confocal microscopy (Fig. 1c). NF-kB
activation was noted as early as 3 h p.i. and persisted for
.48 h. In comparison, HCT116 cells exposed to E. faecalis
did not lead to nuclear localization of p65 (data not shown).
To verify NF-kB activation in macrophages, we transfected
RAW264.7 cells with the pNFkB-Luc reporter plasmid.
Compared with thecontrol, there wasa .25-foldincreasein
NF-kB activation at 24 h following exposure to haematin-
starved E. faecalis (Fig. 1d). Furthermore, manganese
superoxide dismutase (MnSOD) significantly reduced NF-
kB activation (P50.005), indicating that extracellular
superoxide from haematin-starved bacteria contributed to
this effect. The addition of catalase did not further decrease
NF-kB activation by haematin-starved E. faecalis, although
H2O2alone activated NF-kB in these cells.
NF-kB regulates genes involved in cellular proliferation,
immunity andapoptosis(Karin& Greten,2005).
Activation of NF-kB requires the phosphorylation of IkB
by IkB kinase. This results in IkB degradation and
release of NF-kB homo- and heterodimers to translocate
into the nucleus. NF-kB promotes tumorigenesis by inhibi-
ting apoptosis, dysregulating tumour-specific immune
responses and producing reactive oxygen species that can
damage genomic DNA. Our findings indicate that
haematin-starved E. faecalis activates NF-kB, in part, by
producing extracellular superoxide. Although reactive
oxygen species (including superoxide) can activate NF-
kB, this effect is unpredictable and typically cell-dependent
(Gloire et al., 2006). Many studies use H2O2as an oxidative
stress, although superoxide should also be considered as it
may lead to dissimilar effects. For example, superoxide is
required for IL-1-dependent NF-kB activation in chon-
drocytes (Mendes et al., 2003), enhances LPS-dependent
NF-kB activation in macrophages (Khadaroo et al., 2003)
and initiates NF-kB activation in neutrophils (Mitra &
Abraham, 2006). In this study, MnSOD significantly
Fig. 1. Haematin-starved E. faecalis activates NF-kB in macrophages. Serial sections of colon exposed to E. faecalis for 6 h
were stained with NLS-specific anti-p65 antibody (a) and anti-F4/80 mAb (b). NF-kB activity was localized to F4/80-positive
tissue macrophages (indicated by arrows, magnification 40?). (c) RAW264.7 cells treated with E. faecalis (1 h, 1?109c.f.u.
ml”1) exhibited cytoplasmic and nuclear localization of p65 (stained green with FITC; magnification 63?) by laser-scanning
confocal microscopy at 24 h post-treatment, whereas untreated cells primarily exhibited cytoplasmic staining (nuclei stained
blue with DAPI). (d) pNFkB-Luc-transfected macrophages showed increased NF-kB induction following treatment with E.
faecalis; MnSOD significantly decreased E. faecalis-dependent NF-kB induction, whilst catalase caused no further reduction
(see Methods for details). Data are means±SEM for at least six experiments. *, P50.005; **, P50.01; ***, P,0.0002 compared
with E. faecalis.
E. faecalis modulates mucosal gene expression
decreased E. faecalis-dependent induction of NF-kB in
RAW264.7 cells, confirming that this anionic radical can
potentiate NF-kB activation beyond that seen with H2O2
Colonic mucosal gene response to haematin-
starved E. faecalis
To determine how broadly the haematin-starved physi-
ology of E. faecalis modulated gene expression in the
colonic mucosa, we compared mRNA from mice for
.5000 genes following exposure to haematin-starved E.
faecalis with mRNA following exposure to haematin-
replete E. faecalis. At 1 h p.i., six colonic mucosal genes
were differentially regulated (Sod2, Sod3, Agtrl1, Vav1,
Car4 and Nme6; P,0.01 for each). At 6 h, 25 genes were
significantly downregulated and 17 genes upregulated
(Table 2). Of the differentially regulated genes at the 1 h
time point, only Sod3 and Car4 were still differentially
regulated at 6 h. For the 42 colonic mucosal genes
differentially expressed by haematin-starved E. faecalis,
nine (21%) were related to inflammatory or stress
responses and ten (24%) involved pathways for cell-cycle
control, signalling and apoptosis. In addition, several were
expressed by immune effector cells, suggesting that acute
colonic mucosal responses to haematin-starved E. faecalis
involve innate and/or adaptive immunity.
To identify potential biological interaction networks for
these differentially regulated genes, we subjected genes at
the 6 h time point to Ingenuity Pathways Analysis. Only
one highly significant mucosal response network was
identified (Fig. 2; P,0.0001). Functions within this
network included cell-cycle regulation, inositol phosphate
metabolism, NF-kB signalling (RelA or p65), ERK/MAPK
signalling, chemokines, T-cell receptors, integrins and
fibroblast growth factor. Exploration of potential regula-
tory responses within the network was performed using in
silico transcriptional regulatory element analysis. One
hundred and eleven transcriptional regulatory elements
were associated with the forty-two differentially regulated
genes. When we compared the frequency of these elements
with elements for all genes on the murine array, there were
only five significantly over-represented elements: three for
NF-kB (including the sequence for p65 binding), HEN1
and GATA-1. In addition, 66 (59%) of the 111
transcriptional regulatory elements were significantly
under-represented. Overall, these findings indicate that
haematin-starved E. faecalis acutely activates NF-kB
signalling in the colonic mucosa.
These in silico analyses identified a single response network
with seven upregulated mucosal genes. p65 was the major
transcription factor in this network, and in biopsies NF-kB
activity localized to tissue macrophages. The mechanism by
which E. faecalis contacts tissue macrophages was not
investigated, but may involve translocation of enterococci
(Kraehenbuhl & Neutra, 2000). These specialized epithelial
M cells inthe colon
cells facilitate uptake of luminal bacteria and coordinate
their interaction with innate and adaptive immune effector
cells. Enterococci readily translocate across the intact
intestinal epithelium and, in murine models, are often
recovered from the liver, spleen and mesenteric lymph
nodes (Wells et al., 1990). This phenomenon may derive, in
part, from ineffective killing of E. faecalis by macrophages
(Gentry-Weeks et al., 1999). In the colonic ligation model,
the concentration of luminal bacteria at 6 h was 10-fold
lower than the original inoculum. Epithelial translocation
is one possible explanation for a decrease in colony counts.
Several genes within the mucosal response network were
associated with NF-kB signalling including C3ar1, Cyr61
and Akap8l. C3AR1 (complement component 3a receptor
1) induces NF-kB activation when coupled to Ga16(Yang
et al., 2001). Similarly, Cyr61 (cysteine-rich protein 61) is
associated with NF-kB signalling, inflammation and
angiogenesis (Klein et al., 2002), as well as anti-apoptotic
effects when overexpressed in breast cancer (Lin et al.,
2004). Although CYR61 has been implicated in the
progression of breast cancer (Xie et al., 2001), it can also
act as a tumour suppressor (Chien et al., 2004). Finally,
AKAP8L (nuclear protein kinase A anchoring protein) can
bind NF-kB, although the significance of this interaction is
unclear (Bouwmeester et al., 2004). Several other genes in
the mucosal response network have been implicated in
cancer biology. For example, MCM2 (minichromosome
maintenance 2 protein) binds to the nuclear scaffold
created by AKAP8L and promotes apoptosis in cancer cells
(Feng et al., 2003). In contrast, TIMP2 (inhibitor of matrix
metalloproteinase 2) inhibits apoptosis and allows tumour
growth (Egeblad & Werb, 2002), although its overexpres-
sion is inhibitory (Gomez et al., 1997). The net long-term
effect of haematin-starved E. faecalis on the colonic mucosa
cannot be discerned from this study, although the
complexity of the early response is apparent.
E. faecalis blocks G2/M transition in intestinal
As the mucosal response network indicated differential
expression of genes involved in apoptosis and because
reactive oxygen species from E. faecalis can damage colonic
epithelial-cell DNA to initiate programmed cell death
(Huycke et al., 2002), we investigated the effect of
superoxide on cell-cycle checkpoints and apoptosis. Both
HCT116 and YAMC cells exposed to haematin-starved E.
faecalis for 1 h developed marked arrest at the G2/M
transition by 48 h (Fig. 3a, b, c, d). This effect was partially
reversed with MnSOD and completely abolished when
MnSOD and catalase were both added to bacteria-treated
cells. The effect of catalase suggested that H2O2, which
spontaneously arises from the disproportionation of
superoxide, also contributed to activation of the G2/M
checkpoint. Treatment of HCT116 cells with H2O2alone is
known to cause this arrest (Chang et al., 2003), but our
findings showed a greater proportion of arrested cells
T. D. Allen and others
1198Journal of Medical Microbiology 57
Table 2. Colonic mucosal genes with significantly altered expression following a 6 h exposure to haematin-starved E. faecalis
compared with E. faecalis grown with haematin
Gene symbolGene productFold change
Inflammation and stress response
Complement component 3a receptor 1
Tumour necrosis factor (ligand) superfamily, member 10
X-ray repair complementing defective repair in Chinese
hamster cells 6
Chemokine (CXC) ligand 2
Tumour necrosis factor-induced protein 6
Superoxide dismutase 3, extracellular
Coagulation factor II (thrombin) receptor-like 1
Heat-shock protein 4
Cell-cycle regulation, apoptosis and cell signalling
Minichromosome maintenance deficient 2 mitotin
Gap junction membrane channel protein a10
Cysteine-rich protein 61
Tissue inhibitor of metalloproteinase 2
Cell division cycle 2-like 1
Protein tyrosine phosphatase 4a1
Mitogen-activated protein kinase 6
Transcriptional regulation and DNA binding
Calcium ion binding and transport
Structural and miscellaneous
T-cell acute lymphocytic leukaemia 2
Nuclear receptor superfamily 2, group C, member 1
Aryl hydrocarbon receptor nuclear translocator 2
Germ cell-less homologue (Drosophila)
Mesenchyme homeobox 2
Sine oculis-related homeobox 6 homologue (Drosophila)
DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 3, X-linked
Guanine nucleotide-binding protein, aq polypeptide
ADP-ribosylation factor-like 4
G protein-coupled receptor 34
Neuronal pentraxin 1
Calcium channel, voltage dependent, b3 subunit
YME1-like 1 (S. cerevisiae)
Carbonic anhydrase 4
Proteasome (prosome, macropain) subunit, b type 2
A-kinase (PRKA) anchor protein 8-like
Protein phosphatase 3, catalytic subunit, c isoform
Neuropathy target esterase
Guanosine diphosphatase dissociation inhibitor 1
*Genes that are part of the mucosal response network induced by haematin starvation (see Fig. 2).
DGenes also differentially regulated at the 1 h time point.
E. faecalis modulates mucosal gene expression
following exposure to haematin-starved E. faecalis than
To determine whether G2/M checkpoint activation by
haematin-starved E. faecalis was associated with an increase
in apoptosis, we examined HCT116 cells for early apoptotic
cells and noted no increase at 24 and 48 h compared with
no-treatment controls (Fig. 3e). In contrast, HCT116 cells
exposed to H2O2showed significantly increased apoptosis
at 24 h. These findings did not involve NF-kB, as nuclear
localization of p65 was not found by laser-scanning
confocal microscopy at 6, 24, 48 or 72 h (data not shown).
In contrast to transformed HCT116 cells, the primary non-
transformed YAMC cells showed significantly increased
apoptosis at 48 h following exposure to haematin-starved
E. faecalis or H2O2treatment (Fig. 3f).
We demonstrated previously that E. faecalis can damage
colonic epithelial-cell DNA (Huycke et al., 2002). In the
current study, we found that extracellular superoxide from
E. faecalis activates the G2/M checkpoint in HCT116 and
YAMC cells. This effect was partially reversed by MnSOD
and completely eliminated when both MnSOD and catalase
were used, suggesting that extracellular superoxide and
H2O2each contributed to cell-cycle modulation. Arrest at
the G2/M transition can be triggered by DNA double-
strand breaks to activate pathways that allow mitosis to
proceed after DNA repair or, alternatively, to initiate
apoptosis (Nougayrede et al., 2006; Taieb et al., 2006).
Differing outcomes following exposure to haematin-
starved E. faecalis were apparent for HCT116 and YAMC
cells, and demonstrated how cellular responses to DNA
damage vary by cell type. Finally, we noted in a prior study
that a minority of cells exposed to haematin-starved
E. faecalis failed to repair DNA damage or to initiate
instability (Wang & Huycke, 2007).
The DNA-damaging effects of commensals on the colonic
mucosa may not be limited to E. faecalis. Pathogenic and
commensal strains of E. coli express hybrid peptide–
polyketide and cytolethal distending toxins that produce
DNA double-strand breaks, arrest at the G2/M transition
and cell death (Nougayrede et al., 2006; Taieb et al., 2006).
Other examples include commensal bacteria that utilize
sulfate as an oxidant (in the assimilatory pathway) or
terminal electron acceptor (in the dissimilatory pathway)
to dispose of hydrogen-reducing equivalents (Gibson et al.,
1988). The net result of this metabolism is hydrogen sulfide
that, like superoxide, can be genotoxic to epithelial cells
(Attene-Ramos et al., 2006). DNA double-strand breaks
created by reactive oxygen species, hydrogen sulfide or
other clastogens should activate DNA damage repair
responses and activate the G2/M checkpoint (Su, 2006).
Ongoing DNA damage could lead to the accumulation of
Fig. 2. Colonic mucosal response network. Genes significantly upregulated by haematin-starved E. faecalis at 6 h p.i. are
highlighted in bold and the network was identified by Ingenuity Pathways Analysis. Direct interaction refers to gene products
with direct effects on targets; indirect interaction refers to gene product effects on targets through intermediate effectors.
T. D. Allen and others
1200 Journal of Medical Microbiology 57
Investigations are underway in our laboratory to explore
important to oncogenictransformation.
Regulation of netrin-1 by extracellular superoxide
from E. faecalis
To investigate further the gene response network in vitro,
we screened HCT116, YAMC and RAW264.7 cells by qRT-
PCR for differential regulation of selected upregulated
genes in the network known to be transcriptionally
regulated by NF-kB or involved in apoptosis (Table 1).
Of these, only NTN1 in HCT116 cells was significantly
upregulated by haematin-starved E. faecalis (Fig. 4). The
inability to detect similar changes in gene expression in
vitro using these cells compared with the in vivo model
probably represents inherent differences between trans-
formed cells and complex multicellular tissues in living
animals (Waddell et al., 2007). Indeed, the rationale for the
in vivo model was to avoid oversimplification of host–
commensal interactions found in homogeneous in vitro
Netrin-1 is an extracellular ligand secreted by intestinal
epithelial cells that binds basolateral epithelial receptors
such as DCC (Arakawa, 2004). When left unbound, these
Change in G1 phase (%)
24 h48 h
3 4 1 2 3 4
Early apoptotic cells (%)
H2O2 (200 mM)
24 h 48 h
3 4 1 2 3 4
Change in G2/M phase (%)
24 h 48 h
3 41 2 3 4
24 h 48 h
3 41 2 3 4
Change in S phase (%)
24 h48 h
3 41 2 3 4
24 h 48 h
3 4 1 2 3 4
Early apoptotic cells (%)
H2O2 (200 mM)
Fig. 3. Haematin-starved E. faecalis alters the epithelial cell cycle and fails to induce apoptosis in colonic epithelial cells. (a, b)
Changes in the HCT116 (a) and YAMC (b) cell cycle at 24 and 48 h following a 1 h exposure to E. faecalis, and (c, d)
representative histograms from HCT116 (c) and YAMC (d) cells demonstrating a pattern of arrest at the G2/M transition.
Treatments: 1, H2O2(200 mM); 2, E. faecalis (1?109c.f.u. ml”1); 3, as in treatment 2 plus MnSOD (1200 U ml”1); 4, as in
treatment 3 plus catalase (1200 U ml”1). Data are the means±SD of at least three experiments. (e, d) The percentage of early
apoptotic cells in HCT116 (e) and YAMC (f) cells at 24 h (white bars) and 48 h (black bars) following 1 h exposure to E.
faecalis or H2O2(200 mM) compared with untreated cells. Data are the means±SD of at least five experiments. *, P50.03; **,
P,0.002; ***, P,0.0001 compared with the control at each time point.
E. faecalis modulates mucosal gene expression
receptors initiate signalling pathways that lead to apopto-
sis. The role of netrin-1 in tumorigenesis was established
mice where overexpression led to
intestinal hyperplasia and high-grade tumours (Mazelin
et al., 2004). Addition of MnSOD reduced E. faecalis-
induced NTN1 expression by 2.5-fold (P50.001), indic-
ating that extracellular superoxide was partially responsible
Although we detected abundant expression of netrin-1 in
the murine colonic mucosa by immunohistochemistry, no
significant differences in immunoreactivity were found for
any group of mice. The lack of differential staining may
have been due to insufficient time for tissue protein
concentrations to change (i.e. only 6 h). To determine
whether increased netrin-1 expression in HCT116 cells
explained the lack of increased apoptosis in HCT116 cells
following exposure to E. faecalis (Fig. 3e), we used short
interfering RNA to knock down NTN1 expression. Gene
silencing led to an 83–90% reduction in NTN1 mRNA for
cells exposed to haematin-starved E. faecalis compared with
untransfected controls or cells transfected with scrambled
short interfering RNA. The proportion of cells undergoing
apoptosis in NTN1-silenced cells, however, proved no
different than for untransfected controls (data not shown).
This result could have been due to a lack of functional
receptors for netrin-1 on HCT116 cells or to defects in the
secretion of netrin-1. When Western blots were performed,
netrin-1 was not detected (data not shown), implying that
other mechanisms are responsible for the inhibition of
apoptosis in these cells following exposure to haematin-
starved E. faecalis.
Several factors merit consideration when interpreting the
results of this study. Because cDNA from mucosal
scrapings were used, by necessity, in their entirety to
synthesize probes, we could not check changes in gene
expression for arrays by qRT-PCR. To overcome this
limitation, we performed ten arrays per time point to
increase the number of independent replicates. This
resulted in a robust dataset for the identification of genes
with differing expression. Furthermore, we defined altered
gene expression using conservative rules in order to
identify only those genes that were highly likely to have
undergone induction or repression by haematin-starved E.
faecalis. Additional genes may have been identified using
less rigorous cut-offs. This, in turn, could have expanded
the gene response network or identified other networks,
but would not have changed the primary conclusion of this
study, which is that the metabolic activity of intestinal
commensals can acutely alter colonic mucosal gene
expression. The short-term nature of our colonic ligation
model only permitted an examination of early-response
genes. Longer-term studies will require intestinal coloniza-
tion or a gnotobiotic design. Finally, although superoxide
from haematin-starved E. faecalis induced NTN1, knock-
down of this gene had no affect on apoptosis, as secretion
of this ligand appeared defective in these cells.
In summary, we found that the uniquely dichotomous
metabolism of E. faecalis, a colonic commensal with a
rudimentary respiratory chain that requires exogenous
haematin for oxidative phosphorylation, can significantly
modulate gene expression in the colonic mucosa. In vivo, in
silico and in vitro analyses identified genes and signalling
pathways that are associated with inflammation, apoptosis
and cell-cycle regulation. Overall, these results suggest
mechanisms by which E. faecalis might enhance epithelial-
cell susceptibility to DNA damage through the activation of
tissue macrophages and by modulating apoptosis in
This work was supported by a grant from the Office of Research and
Development, Medical Research Service, Department of Veterans
Affairs Medical Center and Frances Duffy Endowment. Special thanks
to Richardo Saban and Robert Hurst for helpful advice, Stanley
Lightfoot for assistance with histopathology, Wei-Qun Ding for the
plasmid construct pNFkB-Luc, Jim Henthorn at the University of
Oklahoma Health Sciences Center Cytometry Laboratory and Ben
Fowler at the Oklahoma Medical Research Foundation Core Imaging
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