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Future Microbiology

Review

Pathogenesis of adherentinvasive
Escherichia coli
Emma J Smith1, Aoife P Thompson1, Adam ODriscoll1 & David J Clarke*1
Department of Microbiology & Alimentary Pharmabiotic Centre, University College Cork, Cork, Ireland
*Author for correspondence: Tel.: +353 21 4903624 n david.clarke@ucc.ie
1

The etiology of Crohns disease (CD) is complex and involves both host susceptibility
factors (i.e., the presence of particular genetic alleles) and environmental factors,
including bacteria. In this regard, adherentinvasive Escherichia coli (AIEC), have
recently emerged as an exciting potential etiological agent of CD. AIEC are
distinguished from commensal strains of E. coli through their ability to adhere to
and invade epithelial cells and replicate in macrophages. Recent molecular
analyses have identified genes required for both invasion of epithelial cells and
replication in the macrophage. However, these genetic studies, in combination
with recent genome sequencing projects, have revealed that the pathogenesis
of this group of bacteria cannot be explained by the presence of AIEC-specific
genes. In this article, we review the role of AIEC as a pathobiont in the pathology
of CD. We also describe the emerging link between AIEC and autophagy, and
we propose a model for AIEC pathogenesis.

Idiopathic inf lammatory bowel disorders

(IBDs) are a complex of intestinal disorders
typified by chronic relapsing inflammation of
the GI tract. The major IBDs, Crohns disease
(CD) and ulcerative colitis, have a combined
prevalence of approximately 150200 cases
per 100,000 individuals in the western world,
with 35,000 new cases of CD being reported
annually [1]. Despite the high incidence of CD,
the etiology of the disease still remains elusive.
However, it has become widely accepted that
CD is due to an interaction between certain host
susceptibility factors and the environment [2,3].
Therefore, CD is thought to be the consequence
of an abnormal inflammatory response to the
presence of commensal enteric bacteria in
genetically susceptible individuals [46].
The role of host genetics in the development of
CD has been established for some time. Indeed
genome-wide association studies have identified
a number of alleles that increase the risk of
developing CD [7,8]. Perhaps the most significant
of the risk alleles are associated with NOD2
(CARD15), encoding an important protein in
the host innate immune response to bacteria
[9,10]. NOD2 has a C-terminal leucine-rich
repeat domain, which has been shown to interact
with a derivative of peptidoglycan (muramyl
dipeptide), thus sensing the presence of bacteria
in the cytosol of the host cell [11]. Several of the
most common CD-associated alleles are in
the leucine-rich repeat domain of NOD2 and
it is therefore suspected that these alleles will
interfere with the ability of NOD2 to respond
to its ligand [11,12]. Other common risk alleles
are associated with ATG16L1 (a nonsynonomous
10.2217/FMB.13.94 2013 Future Medicine Ltd

single nucleotide polymorphism resulting in a

single amino acid change that alters the activity
of the mature protein) and IRGM (a single
nucleotide polymorphism in the promoter
region of the IRGM gene that is thought to affect
tissue-specific expression of this gene) [8]. Both
ATG16L1 and IRGM encode proteins involved
in autophagy. The potential link between CD
and defects in autophagy will be discussed later
in this review.
In addition to the role of host susceptibility,
there is significant evidence to suggest that
microbes contribute to CD. First, as described
above, CD is strongly associated with genetic
mutations in the innate immune response of the
host [1315]. Second, one of the main pathological
features of CD is the presence of aphthous ulcers
of the mucosa, a disease manifestation commonly
associated with several infectious diseases
involving Salmonella spp., Shigella spp., Yersinia
enterocolitica and Mycobacterium tuberculosis [16].
Third, antibiotic treatment, which decreases
the luminal bacterial concentration, results
in a marked clinical improvement in some
CD patients, consistent with the involvement
of luminal bacteria in the pathology of CD
[4,17,18]. Finally, in animal models of IBD the
components of the microbial flora are essential
for the development of colitis, as gnotobiotic
mice exhibit none of the symptoms of IBD [19,20].
Mycobacterium avium subspecies
paratuberculosis

Keywords
autophagy n hostmicrobe
interactions n inflammation
n inflammatory bowel disease
n intestinal barrier
n intracellular replication
n pathobiont
n

During the 1980s and 1990s, much attention

was placed on Mycobacterium avium subspecies
paratuberculosis (MAP) as a potential cause of
Future Microbiol. (2013) 8(10), 12891300

part of

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Smith, Thompson, ODriscoll & Clarke

CD [21,22]. MAP is the causative agent of Johnes

disease, an intestinal disorder of ruminants; the
pathophysiology of which bears many of the
hallmarks of CD [23]. MAP was also found to be
present in pasteurized milk leading to the notion
that CD may be transmissible to susceptible
individuals through the consumption of
milk contaminated with MAP [24]. However,
therapies directed at MAP do not produce a
cure, and the evidence supporting a role for
MAP in the etiology of CD is far from being
conclusive [22,25]. On the one hand, there is
evidence that the prevalence of MAP DNA
in intestinal tissue samples (and the level of
antibodies against MAP antigens) is higher in
CD patients than in controls [2628]. At the same
time, the culturing of MAP from CD-affected
tissue has been inconsistent, with detection rates
ranging from 0 to 100% [4,29].
Escherichia coli

Over the last 1015 years, the microbe that has

attracted the most attention, with respect to
CD etiology, is Eschericia coli [30,31]. The colonic
mucosa and overlying mucus represents a unique
environmental niche, with bacteria adherent on
the surface of the mucus coat differing from those
underneath the mucus. Although the mucosa
in healthy individuals is relatively sterile, there
is a marked increase in the number of bacteria
found in the submucosal niche in CD patients
and >50% of these bacteria were shown to be
E. coli [32,33]. Several independent studies have
reported that the levels of mucosa-associated
E. coli are significantly increased in CD patients
compared with healthy controls. Laser capture
microdissection in conjunction with PCR
identified E. coli DNA within 12 (out of 15)
CD granulomas, compared with one (out of ten)
control granulomas [34]. Analysis of bacterial flora
associated with early and chronic ileal lesions of
CD patients showed that the ileal mucosa of up
to 36.7% of CD patients is abnormally colonized
by E. coli strains compared with only 510% of
control patients [3537]. E. coli counts from the
rectal mucosa were also observed to be higher in
active CD compared with inactive and healthy
controls [38]. Elevated levels of anti-E. coli
OmpC antibodies have been identified in 55%
of CD patients compared with less than 5% of
healthy controls [39,40]. Interestingly, reactivity to
OmpC is associated with more severe forms of
CD that are characterized by aggressive disease
progression and longer disease duration [39].
Interestingly, the majority of CD-associated
E. coli strains have been shown to adhere to and
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invade intestinal epithelial cells whilst also being

able to extensively replicate within macrophages.
Furthermore, it has been proposed that E. coli
exhibiting these phenotypes may form a new
pathotype referred to as adherentinvasive E. coli
(AIEC) [36,41].
Adherentinvasive E. coli

Based on the phenotypes mentioned previously,

many studies have isolated AIEC strains from
healthy individuals as well as CD patients.
Indeed, AIEC strains have also been isolated
from other mammals and have been implicated
in granulomatous colitis in Boxer dogs and
persistent mastitis in cows [42,43]. Phylogenetic
ana lysis of these strains has revealed that
AIEC appears to be a highly diverse group
with strains isolated from all four of the major
E. coli phylogroups (i.e., A, B1, B2 and D)
[44,45]. In terms of evolution, this phylogenetic
distribution strongly suggests that AIEC is not
a clonal group of strains and it is therefore likely
that the AIEC phenotype has independently
evolved several times. However, recent studies
examining the microbial diversity in biopsy
tissues from patients have demonstrated a
significantly higher prevalence of E. coli from
the B2 and D phylogroups in patients with CD
compared with controls [46,47]. Interestingly,
this may suggest that these phylogroups have a
fitness advantage in the CD gut. However, there
is also evidence to suggest that the prevalence
of the B2 phylogroup is generally increasing
in frequency in the human gut [48,49]. The B2
phylogroup is often considered to contain more
of what might be considered to be aggressive
commensals than the other phylogroups, as this
phylogroup contains a number of E. coli strains
that are associated with extraintestinal infections
(ExPEC); for example, urinary tract infections
and meningitis [50,51]. However, a survey of
ExPEC strains found that although there was
a significant degree of phylogenetic relatedness
between ExPEC and AIEC strains, the majority
of ExPEC strains did not act like AIEC strains,
suggesting that, although related, AIEC and
ExPEC also possess virulence-specific features
[45]. A comparison of the genome sequences of
AIEC strains LF82 and NRG685c reinforced
the phylogenetic closeness to ExPEC strains and
also identified some potential AIEC-specific
genes [52]. However, further comparisons with
a third, more divergent AIEC strain, HM605,
revealed that the similarity between LF82 and
NRG685c was probably indicative of their shared
serotype [53]. Moreover the addition of another
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Pathogenesis of adherentinvasive Escherichia coli

AIEC genome sequence, UM146, has confirmed

that there do not appear to be any genes that are
exclusively associated with this group of E. coli
[54]. Therefore, the AIEC phenotype is not due
to the presence of a specific virulence factor(s),
an observation that goes a significant way to
support the nonclonal phylogenetic distribution
of AIEC.
AIEC & the epithelial barrier

Intestinal epithelial cells act as a physical barrier

to prevent enteric bacteria from entering and
interacting with immune cells in the lamina
propia. However, these cells also act as innate
immune sensors for pathogens, as well as
commensals [55]. Pathogenic organisms, such as
Shigella, Salmonella and Listeria, penetrate the
mammalian intestine by either directly invading
through the epithelial layer or by entering
through the microfold (M) cells that constitute
approximately 5% of the epithelial cells in
the dome epithelium that overlies the Peyers
patches in the distal small intestine and the
lymphoid follicles in the colon [56]. M cells have
a unique role to play in mucosal immunity as
they are required to sample and deliver luminal
antigens from the GI tract to immune cells,
such as dendritic cells and lymphocytes [57,58].
There is some evidence that, following invasion,
AIEC can reduce epithelial barrier function by
displacing and redistributing ZO-1, a protein
required for the formation of apical tight juctions
[59,60]. This decrease in epithelial barrier integrity
would result in increased translocation of AIEC
across the epithelial barrier and may therefore
exacerbate AIEC pathogenesis.
Although AIEC are defined, at least in part, by
their ability to invade cultured epithelial cells it is
probable that, like other pathogens, AIEC utilize
M cells as a portal of entry into the epithelium
of CD patients. Indicative of this, the initial
lesions of CD, that is, aphthoid ulcers, generally
occur at the site of Peyers patches and colonic
lymphoid follicles [61]. Studies have shown that
GP2, a protein specifically located on the apical
plasma membranes of M cells, selectively binds
the type I fimbriae of Salmonella enterica serovar
Typhimurium and E. coli [62]. GP2 specifically
recognizes the FimH adhesion of type I fimbriae
and mice deficient for Nod2 express increased
amounts of GP2 [62]. Murine ligated intestinal
loop assays have shown that such binding allows
entry of bacteria into the Peyers patches, which
subsequently results in an antigen specific
mucosal immune response [63]. Moreover,
deficiency in either FimH or GP2 expression
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Review

leads to a significant decrease in transcytosis of

type I fimbriated bacteria through M cells [62].
In addition to type I fimbriae, the AIEC strain
LF82 also express long polar fimbriae (LPF) [64].
LPF of S. Typhimurium specifically adhere to M
cells of the murine follicle-associated epithelium
[65]. It has been shown that LPF-expressing
AIEC bacteria can adhere to M cells and
translocate into the Peyers patches, whereas
LFP-deficient AIEC cannot [64]. Moreover
AIEC LPF adherence to M cells and entry into
Peyers patches is independent of type I pili
adherence and transcytosis. In addition, 47%
of CD patients harbored E. coli expressing LPF
compared with 17% of control patients [64].
In addition to GP2, FimH of AIEC strains
has also been shown to recognize the host
glycoprotein CEACAM6, a surface glycoprotein
that is abnormally expressed in the ileal mucosa
of 35% of CD patients [66,67]. AIEC strains have
been shown to express FimH protein variants
with recently acquired amino acid substitutions;
these mutations confer a significantly higher
ability to adhere to CEACAM6-expressing
intestinal epithelial cells to AIEC [68].
Replacement of fimH in the AIEC strain LF82
with fimH from an E. coli K-12 strain decreased
the ability of LF82 to persist and to induce
severe colitis and gut inflammation in infected
transgenic mice expressing human CEACAM6
receptors [68]. Phylogenetic ana lysis of the
AIEC-associated fimH allele suggests that the
acquired point mutations are relatively recent
and thus may have been selected to allow AIEC
to colonize a new niche (i.e., the CD gut) in
a process called pathoadaptation. Moreover,
CEACAM6 expression has been shown to be
upregulated during infection with LF82 and
by the proinf lammatory cytokines TNF-
and IFN-, suggesting that AIEC may be
capable of promoting their own colonization
in CD patients [67,69]. Infection of CEACAM6expressing mice with AIEC also resulted in a
significant decrease in epithelial barrier function
and this was dependent on the presence of
type 1 fimbriae [70]. Finally, dose-dependent
degradation of type 1 fimbriae in the presence
of the protease meprin impairs the ability of
LF82 to bind to mannosylated host receptors
on epithelial T84 cells, leading to decreased
production of the proinflammatory cytokine
IL-8 [71]. Decreased levels of protective meprin
observed in CD patients are believed to promote
increased AIEC colonization [71]. The emerging
evidence for the importance of FimH in the
interaction of AIEC with the epithelial cells
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Smith, Thompson, ODriscoll & Clarke

makes it an attractive therapeutic target to

block the interaction between AIEC and the
gut mucosa in the early stages of IBD.
Outer membrane vesicles (OMVs) have also
been shown to contribute to AIEC invasion.
Deletion of the yfgL gene, which encodes
the YfgL lipoprotein, in AIEC LF82 led to a
significant decrease in OMV release and a
marked reduction in the ability of LF82 yfgL bacteria to invade intestinal epithelial cells [72].
The deletion of yfgL in LF82 was also shown
to negatively affect the release of the outer
membrane proteins OmpA and OmpC [73]. It
has recently been demonstrated that an ompA
mutant in LF82 has a reduced ability to invade
intestinal epithelial cells in comparison to the
wild-type strain [74]. OmpA promotes fusion
of OMVs with the Gp96 receptor expressed on
the surface of epithelial cells, thus promoting
invasion. Gp96 is overexpressed at the apical
plasma membrane of ileal epithelial cells of
patients with CD [74]. This suggests that in CD
patients, AIEC may take advantage of Gp96
overexpression to allow delivery of virulence
factors that contribute to the invasion process
via OMVs into host cells.
Recent studies have implicated exogenous
polysaccharides which are commonly added
to processed foods as emulsifiers, stabilizers and
bulking agents as having a role in promoting
the invasion of epithelium cells by AIEC [75,76].
Maltodextrin, a polysaccharide derived from
starch hydrolysis, has been shown to induce
type I fimbriae expression in LF82, resulting
in a significant increase in bacterial adhesion to
human intestinal epithelial cells [75]. Moreover,
there is an increased prevalence of the malX
gene, essential for maltodextrin metabolism, in
mucosa-associated bacteria taken from the ileums
of 71% of CD patients compared with 18% of
controls [75]. The commonly used emulsifier
polysorbate-80 has also been implicated in
the enhanced translocation of the AIEC
strain HM605 across both M cells and Caco2
monolayers [76]. More recently, it has been shown
that a western diet (i.e., a diet that contains high
levels of fat and sugar) can induce a dysbiosis
in the gut microbiota of CEACAM6-expressing
mice that reduces epithelial barrier function and
selects for AIEC colonization [77]. These studies
demonstrate that dietary components may
influence bacterial adhesion and translocation
across intestinal epithelial cells in CD patients,
and suggest a mechanism by which western diets
rich in exogenous fats and polysaccharides may
contribute to disease susceptibility.
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AIEC replication within macrophages

Host survival is critically dependent on the

effective recognition and killing of pathogens by
professional immune cells such as neutrophils,
macrophages and dendritic cells. These cells can
directly kill microorganisms by phagocytosis,
coupled with the production of antimicrobial
compounds such as reactive oxygen species and
proteases. In the intestine, macrophages are
located underneath the intestinal epithelium,
with the lamina propria containing the largest
number of macrophages in the body [78].
AIEC strains isolated from CD patients are
capable of surviving and replicating to high
levels within J774A.1 murine macrophages and
human monocyte-derived macrophages [79,80].
AIEC strain LF82 can survive intracellularly
without inducing host cell death resulting in
the secretion of large amounts of TNF- [79].
This cytokine is produced after macrophage
stimulation, showing that macrophages are still
active in the presence of intracellular bacteria.
Therefore, continuous production of TNF- is
possibly due to the persistence of these bacteria
within the macrophage. The persistence of LF82
in macrophages is similar to that of Salmonella
[81], in that it does not require bacterial escape
into the cytoplasm. Rather, LF82 was shown to
induce the formation of a large, spacious vacuole
in J774A.1 macrophages, presumably achieved
by the fusion of initial phagosomes [79]. This
may be a key step in its ability to persist, as the
large vacuole may result in a dilution of toxic
compounds in the phagolysosome.
Upon phagocytosis of LF82 by macrophages,
plasma membrane proteins surrounding the
phagosome were rapidly replaced by early
endosome antigen 1, which allows the AIEC
phagosome to reach the late endosomal stage of
maturation, characterized by the acquisition of
the Rab7 GTPase [80]. The Rab7 GTPase was
not retained on the AIEC phagosome, suggesting
that the phagosome may have evolved into a
phagolysosome [80]. In addition, peripheral
transmembrane glycoproteins (Lamps) were
observed on the AIEC-containing phagosome,
and intraluminal concentrations of cathepsin D
(degradative protease) were increased [80]. This is
in contrast to Salmonella phagosomes, which do
not fuse with lysosomes, and thus do not contain
high levels of hydrolytic enzymes [82,83]. Upon
analysis of these LF82-containing phagosomes,
it was shown that cathepsin D was present in an
active proteolytic form and that the phagosome
was highly acidic. Moreover, when macrophages
were treated with pH-neutralizing agents,
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Pathogenesis of adherentinvasive Escherichia coli

such as chloroquine and ammonium chloride,

intracellular replication of the AIEC strain,
LF82, was inhibited [80]. We have shown similar
results with another strain of AIEC [ODriscoll A,
Unpublished Data] indicating that the acidic pH may
switch on expression of virulence genes that allow
AIEC to persist in this niche [80].
Previous studies have identified the HtrA
stress protein as having an essential role in
the ability of LF82 to survive and replicate in
acidic pH conditions. An LF82 htrA mutant
had a reduced growth rate in comparison with
the wild-type strain when cultured in a growth
medium that simulated the low nutrient, low
pH microenvironment of the phagosome [84].
Moreover, LF82 htrA gene expression was
upregulated 38-fold in intramacrophagic bacteria.
Interestingly, upregulation of the LF82 htrA gene
was not observed in a nonpathogenic E. coli K-12
strain after phagocytosis, indicating that the LF82
genetic background is essential for the upregulation
of htrA in macrophages [84]. Similarly, htrA has
also been shown to be important for intracellular
replication of Salmonella, Legionella pneumophila
and Brucella abortus in vitro [8588].
The Dsb protein family are responsible for
the formation of intramolecular disulfide bonds,
which are essential for the correct functioning
of proteins normally found in the periplasm or
on the surface of Gram-negative bacteria. The
Dsb oxidative pathway involves DsbA, which
is responsible for the formation of disulfide
bonds (through the specific reduction of the
thiol group associated with the Cys amino
acids) in newly synthesized proteins [89]. Other
proteins of the Dsb family are: DsbB, which is
responsible for the reoxidation of DsbA; DsbC,
which has proof-reading functions; and DsbD,
which oxidizes DsbC [89]. The role of dsbA in
virulence has already been reported for many
pathogens. It is involved in the biogenesis of the
toxins from Vibrio cholera and enterotoxigenic
E. coli, and also in the biogenesis of bacterial cell
surface appendages such as flagella and fimbriae
in enteropathogenic and uropathogenic E. coli
[9094]. An AIEC LF82 dsbA mutant was unable
to replicate in macrophages [95]. Transcription of
the dsbA gene was upregulated when the strain
was grown in a medium that partly mimicked
conditions expected to be encountered in the
phagolysosome, suggesting that DsbA may be
required to allow these bacteria to survive the
harsh conditions it encounters in the macrophage
[95]. DsbA also plays an important role in the
pathogenicity of the Gram-negative pathogen
Shigella flexneri. S. flexneri is the causative
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Review

agent of shigellosis, which is characterized by

acute inflammation of the colon and mediated
through interaction of the pathogen with
intestinal macrophages. Similarly to the LF82
dsbA mutant, the S. flexneri dsbA mutant also
grew poorly within macrophages in comparison
with the parent strain [96].
Studies have also identified the RNA binding
protein Hfq as having an important role in LF82
survival and replication within macrophages
[97]. Hfq is a protein that binds to small
regulatory RNA molecules thus facilitating the
interaction between the regulatory RNA and
its target, usually mRNA [98]. Deletion of hfq
in LF82 resulted in reduced host epithelial cell
invasion as well as reduced intracellular survival
and replication in macrophages [97]. Moreover,
deletion of hfq increased the sensitivity of LF82
to stress conditions encountered within the
phagolysosome, such as low pH, reactive oxygen
species and reactive nitrogen species [97]. This
suggests that sRNA molecules may play an
important role in the regulation of the AIEC
phenotype.
As previously mentioned, genome-wide studies
suggest that AIEC genetically resembles ExPEC,
a group of E. coli associated with infection outside
of the gut (including neonatal meningitis E. coli
and uropathogenic E. coli). However, the only
member of the ExPEC group that has been shown
to be capable of surviving and replicating within
macrophages is the neonatal meningitis E. coli
strain, E. coli K1. OmpA expression is necessary
for efficient binding to, and internalization of, K1
by macrophages [99]. OmpA mutants may enter
the macrophage, albeit at lower levels, via OmpAindependent mechanisms; however, ompA
mutants fail to replicate within the macrophage
[99]. As previously mentioned, LF82 ompA mutant
bacteria have a reduced ability to invade intestinal
epithelial cells; however, the involvement of
OmpA in the intramacrophagic replication of
AIEC has not yet been explored [100].
In summary, many genes that are required
for replication in the macrophage have been
identified. However, none of these genes are
AIEC-specific, and they all encode proteins
that are found to be conserved throughout
the Enterobacteriaceae. Therefore, the factor(s)
that differentiate AIEC from other strains of
E. coli and allow this pathotype to persist in the
macrophage remain unidentified.
Autophagy & AIEC

Macrophages can resolve bacterial infections using either phagocytosis or autophagy.

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Phagocytosis involves the detection of bacteria

by cell membrane receptors; in the case of E. coli,
Toll-like receptor (TLR) 4 and TLR5 [101,102].
Autophagy is a cytosolic process that involves
recognition of bacteria by NOD proteins such
as NOD2 [103]. Following recognition, a double
membrane is formed around the bacteria resulting
in the autophagosome. The autophagosome then
fuses with lysosomes leading to the degradation
of the contents of the autophagolysosome [15,103].
Genome-wide association studies have uncovered
autophagy genes that are linked to CD, including
NOD2, ATG16L1 and IGRM [7,8]. NOD2
activates autophagy following the binding of MDP
(a derivative of peptidoglycan), which is present
in the host cell cytosol. ATG16L1 is required
for the development of the autophagosome and
IGRM is involved in the regulation of autophagic
bacterial clearance [15,104,105]. Dendritic cells from
individuals expressing CD-associated NOD2 or
ATG16L1 variants show impaired autophagy
induction and reduced AIEC clearance [15,106].
Moreover, inhibition of autophagy (due to the
presence of the atg5-/- allele) in mouse embryonic
fibroblasts selectively increased the ability of
AIEC to replicate in these cells compared
with other pathotypes of E. coli [14]. A recent
study showed that macrophages infected with
AIEC strain LF82 rapidly activate autophagy
[106]. This study also showed that siRNAmediated knockdown of ATG16L1, NOD2 or
IRGM expression (expected to mimic what is
occurring in the CD patient) led to increased
intramacrophagic replication of AIEC [106].
Therefore, in macrophages, autophagosomes are
capable of killing AIEC and defects in autophagy,
associated with alleles linked to CD, permit
AIEC persistence and replication. ATG16L1,
a specific protein marker for autophagosomes,
was closely associated with actin rearrangements
induced during bacterial engulfment, suggesting
that autophagic proteins were recruited to the
LF82 entry site [106]. Interestingly, TLR4 has
also been shown to activate autophagy through
the recruitment of NOD2 to the site of bacterial
entry, a process dependent on the autophagy
cargo protein p62 [107]. Therefore, there appears
to be significant regulatory crosstalk between
autophagy and phagocytosis. Indeed it appears
that, upon phagocytosis by macrophages,
AIEC can enter either a phagosome or an
autophagosome, and this will determine the fate
of the bacteria. Interestingly AIEC infecting
neutrophil-like PLB-985 cells blocked autophagy
at the autolysosomal step, thus allowing
intracellular survival of the bacteria. On the
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other hand, stimulation of autophagy in the

same cell line by nutrient starvation or rapamycin
treatment reduced intracellular AIEC survival
[108]. Furthermore, the autophagolysosomes
in which AIEC resided in the PLB-985 cells
appeared to be nonacidic; signifying that LF82
is capable of either delaying or preventing the
full maturation of autolysosomes in neutrophils
[108]. Proinflammatory cytokine IFN-, a type II
interferon produced by T cells and NK cells, is
involved in promoting intracellular microbial
killing [109]. After binding with IFN- receptors,
IFN- typically activates Jak2STAT1 signaling.
The JAKSTAT cascade provides a direct
mechanism to translate an extracellular signal into
a transcriptional response, such as cell apoptosis
[109]. Recent studies have shown that, when
human epithelial cells are stimulated with IFN-
following AIEC infection, IFN--mediated
STAT1 phosphorylation was prevented [110].
This suggests that suppression of epithelial cell
STAT1 signal transduction by AIEC strains
isolated from patients with CD may represent a
novel mechanism by which AIEC overrides the
hosts immune response. Taken together, these
studies suggest that the process of autophagy
plays an important role in the clearance of AIEC,
and the defects in autophagy associated with CD
may contribute to the pathology of this disease by
reducing the clearance of this group of bacteria.
Proposed model for the role of AIEC
in CD

AIEC have been isolated in increased numbers

from the guts of CD patients; however, they are
also present in the guts of healthy individuals
where they do not cause disease [3]. Therefore
AIEC can be considered to be pathobionts, that
is, commensal organisms that can take advantage
of a certain environment to cause disease. As
discussed, CD patients harbor an underlying
genetic immunodeficiency in autophagy, and thus
it is possible that these patients will be unable to
clear AIEC that gain access to the lamina propria,
resulting in chronic inflammation [7,8]. Therefore,
AIEC could be directly involved in initiating CD.
However, this appears unlikely as CD patients do
not appear to have an increased susceptibility to
infection by other invasive intestinal pathogens
such as Salmonella and Shigella [3].
An alternative and, in our opinion, a more
likely theory, is that inflammation in the CD
gut licenses AIEC pathogenicity (see FIGURE 1)
[3]. Therefore, AIEC proliferation would be
secondary to, and not the primary cause of,
inflammation. It is generally accepted that CD
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Pathogenesis of adherentinvasive Escherichia coli

Pre-existing AIEC

Gut lumen

M cell

Review

AIEC proliferation

CEACAM6
GP2

TNF-
INF-

Macrophage
e

Autophagy

TNF-
IL-1

Lysosome

T cell
Lymphoid tissue

DC
Initiation
membrane

Autolysosome

Figure 1. Model for adherentinvasive Escherichia coli pathogenesis. (A) AIEC may be present as normal members of the gut
microbiota in humans. (B) Environmental insults, such as antibiotic therapy, induce a dysbiosis that selects for the proliferation of AIEC
within the gut lumen. The resulting inflammatory response further contributes to the microbial dysbiosis in the gut and induces increased
expression of CEACAM6, a receptor for AIEC on the surface of epithelial cells. (C) AIEC can invade epithelial cells and may also be taken
up through the Peyers patches via binding of long polar fimbriae (and type 1 fimbriae) to GP2 expressed on the surface of the M cells
overlying the patches. (D) The AIEC are translocated to the macrophages (underlying the Peyers patches or patrolling the lamina propria)
where the bacteria can replicate within the phagosomes. The presence of AIEC results in the rapid induction of autophagy, killing the
bacteria and resolving the infection. However, in some Crohns disease patients, the presence of particular susceptibility alleles decreases
the autophagy response and AIEC can therefore persist and replicate in their niche within the macrophages resulting in the
hypersecretion of proinflammatory cytokines.
AIEC: Adherentinvasive Escherichia coli; DC: Dendritic cell; M cell: Microfold cell.
Adapted with permission from [3] .

involves a hypersensitivity of the gut to the enteric

microbiota, resulting in continuous antigenic
stimulation and activation of the mucosal immune
system, leading to intestinal damage [6,111].
Indeed, strong evidence for this stems from the
observation that gnotobiotic mice only develop
colitis when the microbiota has been reintroduced
[19,20]. Moreover, a dysbiosis has been observed
in CD patients, characterized by an abundance
of aggressive species, such as Proteobacteria,
relative to protective species, such as Firmicutes
[111]. These changes in the microbiota (possibly
associated with elements of a modern lifestyle
[e.g., diet and antibiotics] and amplified by the
future science group

genetic defects present in some CD patients)

could stimulate a hyperimmunological response
resulting in inflammation that could, in turn, favor
the proliferation and invasion of AIEC (e.g., by
upregulating receptors such as CEACAM6 and
Gp96 or by inducing ulcerations that facilitate
AIEC translocation to the lamina propria). This
would exacerbate the inflammatory process and
result in a positive feedback loop of invasion and
inflammation [2,77].
Future perspective

AIEC are a potential new therapeutic target for

the treatment of CD, at least in some patients.
www.futuremedicine.com

1295

Review

Smith, Thompson, ODriscoll & Clarke

Therefore, a detailed understanding of what

differentiates AIEC from other, potentially
beneficial, strains of E. coli is vital before
the targeting of AIEC in the treatment of
CD may be possible. Although many genes
required for the pathogenesis of AIEC have
been identified, all of these genes are found
throughout the E. coli genus, including nonAIEC strains, suggesting that there might not
be any AIEC-specific genes that can explain
the characteristics of these bacteria. Therefore,
the pathogenicity-associated genes already
identified in AIEC may encode proteins that
have a slightly different role or activity in the
AIEC background compared with other E. coli
backgrounds, as has been reported for FimH.
Another possibility is that the regulation of
these genes may be different and these changes
may offer AIEC a fitness advantage, as has been
reported for the intramacrophagic regulation of
htrA in AIEC [82].
What are the selection pressures that have
driven these subtle changes in gene function
and/or regulation and, thus, the evolution
of AIEC? It is easy to see how incremental
improvements in the ability of E. coli to attach
to epithelial cells could offer these bacteria a
significant fitness advantage in the human gut,
and this might be exacerbated in the context of
a CD gut where particular ligands (e.g., GP2
and CEACAM6) might be overexpressed.
However, the selection pressure driving the
evolution of the ability of AIEC to replicate in
a macrophage may not be so obvious, as E. coli
are generally considered to be present in the gut
lumen. The coincidental evolution hypothesis
offers one possible explanation, suggesting that
the evolution of bacterial virulence factors is a
consequence of their adaptation to other environmental niches. In support of this hypothesis, it has been shown that some pathogens
(e.g., Legionella, Listeria and Salmonella) are
capable of replicating in macrophages, and are
also able to survive predation by environmental
protozoa such as ameba [112,113]. Indeed there is
some evidence that a number of E. coli strains,
particularly those from the B2 phylogroup, are
resistant to grazing by protozoa [114]. Given that
the majority of AIEC strains also belong to the
B2 phylogroup, this leads to the suggestion that
there may be a correlation between the resistance of E. coli to environmental predation and
replication in the macrophage. Therefore, we
might need to understand the life of E. coli outside of the mammalian host in order to fully
understand the environmental forces that may
1296

Future Microbiol. (2013) 8(10)

drive the evolution of important pathogenic

abilities.
In order to confidently establish a role for
AIEC in the etiology of CD, it is imperative
that the molecular work described in this review
is complemented with the appropriate animal
studies needed to link AIEC with inflammation
in the host. Several studies have already reported
increased murine gut inflammation in the
presence of AIEC (as indicated by cytokine
production and/or gut histology) [115]. However,
there is no universally accepted animal model
for CD and these published studies have been
carried out in a variety of backgrounds, where
inflammation was induced either by treating
the animal with dextran sodium sulphate [115]
or using Tlr5 knock-out mice [116]. Therefore, in
order to facilitate comparisons between studies,
it would be useful if researchers interested in
AIEC pathogenicity adapted a single model that
best reflected our current understanding of the
role of this bacterium in CD.
Finally, a significant weakness in our current
understanding of AIEC is that nearly all molecular
studies to date have been undertaken using only
one strain, LF82. This strain was isolated from the
ileum of a CD patient and it is entirely possible
that the pathogenesis of this strain will be different
from AIEC strains isolated from other niches;
for example, the colon. Therefore, if common
mechanisms that underpin the characteristics of
AIEC are to be found, it is essential that future
studies are undertaken with other strains of
AIEC. The identification of mechanisms related
to pathogenesis that are common to all AIEC is a
prerequisite for the development of new anti-AIEC
treatments for CD patients.
Acknowledgements

The authors would like to thank everyone who has

contributed to the ongoing research into adherent
invasive Escherichia coli in the Clarke laboratory.
Financial & competing interests disclosure

Research in the Clarke laboratory is funded by the Irish

Health Research Board and through the Alimentary
Pharmabiotic Centre. The Alimentary Pharmabiotic
Centre is a research center supported by Science
Foundation Ireland (grant no. 07/CE/B1368). The
authors have no other relevant affiliations or financial
involvement with any organization or entity with a
financial interest in or financial conflict with the
subject matter or materials discussed in the manuscript
apart from those disclosed.
No writing assistance was utilized in the production
of this manuscript.
future science group

Pathogenesis of adherentinvasive Escherichia coli

Review

Executive summary
Background
nMicrobes have long been considered an important etiological agent in Crohns disease (CD).
nAdherentinvasive Escherichia coli (AIEC) have been shown to be present in the submucosa of a significant number of CD patients.
nAIEC are distinguished from other types of E. coli though their ability to invade epithelial cells and replicate in macrophages.
AIEC & the epithelial barrier
nType 1 fimbriae bind to a variety of targets present on the surface of epithelial cells and these appendages are required for invasion.
nThe AIEC FimH adhesin appears to have evolved recently so that it is more able to adhere to targets present in the CD gut, a process
called pathoadaptation.
nOther surface proteins (e.g., long polar fimbriae and flagella) are also implicated in adhesion to, and invasion of, epithelial cells.
AIEC & the macrophage
nAIEC occupy a stressful niche in the macrophage.
nThe factor(s) that distinguish AIEC from other strains of E. coli (i.e., facilitate replication in this niche) have not yet been identified.
AIEC & autophagy
nA strong link between CD and defects in autophagy in humans is emerging.
nDefects in autophagy have been shown to selectively increase the replication of AIEC within epithelial cells and macrophages, thus
linking known host genetic risk factors with a potential environmental trigger of CD.
Conclusion
nAIEC are found in the guts of healthy individuals where the bacteria do not cause any disease. Therefore AIEC should be considered as
pathobionts rather than pathogens.
nAlthough there is evidence to support a role for AIEC in driving inflammation in cultured cells, the complex etiology of CD makes the
appropriate animal studies difficult, that is, relevent AIEC-associated phenotypes may only be seen in certain animal genetic
backgrounds.
nWhether AIEC are a viable therapeutic target for the treatment of CD is still not clear. However, these bacteria do present themselves as
an exciting model for studying the evolution of bacteriahost interactions (i.e., commensal to pathogen).

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