future microbiology aiec review

10.2217/FMB.13.94 © 2013 Future Medicine Ltd ISSN 1746-0913Future Microbiol. (2013) 8(10), 1289–1300

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Idiopathic inf lammatory bowel disorders (IBDs) are a complex of intestinal disorders typified by chronic relapsing inflammation of the GI tract. The major IBDs, Crohn’s disease (CD) and ulcerative colitis, have a combined prevalence of approximately 150–200 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 [4–6].

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

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 [13–15]. 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

During the 1980s and 1990s, much attention was placed on Mycobacterium avium subspecies paratuberculosis (MAP) as a potential cause of

Pathogenesis of adherent–invasive Escherichia coli

Emma J Smith1, Aoife P Thompson1, Adam O’Driscoll1 & David J Clarke*1

1Department of Microbiology & Alimentary Pharmabiotic Centre, University College Cork, Cork, Ireland *Author for correspondence: Tel.: +353 21 4903624 n [email protected]

The etiology of Crohn’s 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, adherent–invasive 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.

Keywords

n autophagy n host–microbe interactions n inflammation n inflammatory bowel disease n intestinal barrier n intracellular replication n pathobiont

ReviewFor reprint orders, please contact: [email protected]

Future Microbiol. (2013) 8(10)1290 future science group

Review Smith, Thompson, O’Driscoll & Clarke

CD [21,22]. MAP is the causative agent of Johne’s 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 [26–28]. 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 coliOver the last 10–15 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 5–10% of control patients [35–37]. 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

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 adherent–invasive E. coli (AIEC) [36,41].

Adherent–invasive E. coliBased 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 adherent–invasive Escherichia coli Review

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 barrierIntestinal 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 Peyer’s 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 Peyer’s 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 Peyer’s patches, which subsequently results in an antigen specific mucosal immune response [63]. Moreover, deficiency in either FimH or GP2 expression

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 Peyer’s patches, whereas LFP-deficient AIEC cannot [64]. Moreover AIEC LPF adherence to M cells and entry into Peyer’s 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-α and IFN-γ, suggesting that AIEC may be capable of promoting their own colonization in CD patients [67,69]. Infection of CEACAM6-expressing 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



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.

AIEC replication within macrophagesHost 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-α [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-α 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 ana lysis 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,



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 [O’Driscoll 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 [85–88].

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 [90–94]. 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

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 OmpA-independent 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 & AIECMacrophages can resolve bacterial infect-ions using either phagocytosis or autophagy.



Phago cytosis 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 siRNA-mediated 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

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-γ, a type II interferon produced by T cells and NK cells, is involved in promoting intracellular microbial killing [109]. After binding with IFN-γ receptors, IFN-γ typically activates Jak2–STAT1 signaling. The JAK–STAT 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-γ following AIEC infection, IFN-γ-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



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

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 perspectiveAIEC are a potential new therapeutic target for the treatment of CD, at least in some patients.

T cell

M cellGP2

Gut lumen Pre-existing AIEC AIEC proliferation

CEACAM6

TNF-αTNF-α IL-1βINF-γ

DCLymphoid tissue

LysosomeAutophagy

AutolysosomeInitiationmembrane

Macrophagee

Figure 1. Model for adherent–invasive 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 Peyer’s 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 Peyer’s 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 Crohn’s 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: Adherent–invasive Escherichia coli; DC: Dendritic cell; M cell: Microfold cell. Adapted with permission from [3].

Rebecca Dias



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 non-AIEC 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 envi-ronmental niches. In support of this hypoth-esis, 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 resist-ance of E. coli to environmental predation and replication in the macrophage. Therefore, we might need to understand the life of E. coli out-side of the mammalian host in order to fully understand the environmental forces that may

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 inf lammation 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.

AcknowledgementsThe 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 disclosureResearch 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.



Executive summary

Background�n Microbes have long been considered an important etiological agent in Crohn’s disease (CD).�n Adherent–invasive Escherichia coli (AIEC) have been shown to be present in the submucosa of a significant number of CD patients.�n AIEC are distinguished from other types of E. coli though their ability to invade epithelial cells and replicate in macrophages.

AIEC & the epithelial barrier�n Type 1 fimbriae bind to a variety of targets present on the surface of epithelial cells and these appendages are required for invasion.�n The 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.�n Other surface proteins (e.g., long polar fimbriae and flagella) are also implicated in adhesion to, and invasion of, epithelial cells.

AIEC & the macrophage�n AIEC occupy a stressful niche in the macrophage.�n The 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�n A strong link between CD and defects in autophagy in humans is emerging.�n Defects 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�n AIEC 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.�n Although 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.

�n Whether 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 bacteria–host interactions (i.e., commensal to pathogen).

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