Adherent-invasive Escherichia Coli LF82 Research Articles

Identification of autophagy receptors for the Crohn's disease-associated adherent-invasive Escherichia coli.

Crohn's disease (CD) is a chronic inflammatory bowel disease, of which the etiology involves genetic, environmental and microbial factors. Adherent-invasive Escherichia coli (AIEC) and polymorphisms in autophagy-related genes have been implicated in CD etiology. Autophagy is a key process for the maintenance of cellular homeostasis, which allows the degradation of damaged cytoplasmic components and pathogens via lysosome. We have shown that a functional autophagy is necessary for AIEC clearance. Here, we aimed at identifying the autophagy receptor(s) responsible to target AIEC to autophagy for degradation. The levels of autophagy receptors p62, NDP52, NBR1, TAX1BP1 and Optineurin were knocked down in human intestinal epithelial cells T84 using siRNAs. The NDP52 knock-out (KO) and p62 KO HeLa cells, as well as NDP52 KO HeLa cells expressing the wild-type NDP52 or the mutated NDP52Val248Ala protein were used. We showed that, among the tested autophagy receptors (p62, NDP52, NBR1, TAX1BP1 and Optineurin), diminished expression of p62 or NDP52 increased the number of the clinical AIEC LF82 strain inside epithelial cells. This was associated with increased pro-inflammatory cytokine production. Moreover, p62 or NDP52 directly colocalized with AIEC LF82 and LC3, an autophagy marker. As the NDP52Val248Ala polymorphism has been associated with increased CD susceptibility, we investigated its impact on AIEC control. However, in HeLa cell and under our experimental condition, no effect of this polymorphism neither on AIEC LF82 intracellular number nor on pro-inflammatory cytokine production was observed. Together, our results suggest that p62 and NDP52 act as autophagy receptors for AIEC recognition, controlling AIEC intracellular replication and inflammation.

VARIOUS ADHERENT-INVASIVE E. COLI (AIEC) AND NON-AIEC STRAINS ISOLATED FROM CROHN’S DISEASE PATIENTS GROW DIFFERENTLY WITH RUMINOCOCCUS GNAVUS PRE-DIGESTED MUCUS AND R. GNAVUS-RELEASED PRE-DIGESTED ILEAL AND COLONIC MUCUS MONOSACCHARIDES

Abstract BACKGROUND Mucus-digesting Ruminococcus gnavus (R. gnavus), adherent-invasive E. coli (AIEC), and hydrogen sulfide (H2S) are enriched in active Crohn’s disease (CD) patients compared to healthy subjects. Dual colonization of R. gnavus and E. coli LF82 in ex-germ-free mice enhanced distal colitis and H2S production in cecal contents. R. gnavus pre-digestion of mucus enhanced the ileal CD isolate AIEC LF82 growth and H2S production in vitro. H2S enhanced LF82 adhesion and invasion both in vitro and in vivo along with related LF82 adhesion gene expression and Caco-2 cell expression of CEACAM6, a receptor for AIEC ligand FimH. Interactions between other CD-derived AIEC/non-AIEC and R. gnavus are unknown. HYPOTHESIS Different AIEC and non-AIEC may grow and produce H2S differently using mucus digested by R. gnavus, which releases mucus-derived monosaccharides and sulfur substrates. METHODS We studied CD-derived clinical isolates of AIEC 541-15 (phylotype A), 541-1 (B1), LF82 and NRG857c (B2) and non-AIEC T75 (A) and lab strain K12 (A). M9 minimal media was supplemented with porcine gastric mucin (PGM), ileal (MIM) and colonic (MCoM) mucus isolated from germ-free mice. We measured growth kinetics and H2S production by these AIEC and non-AIEC following culture with sterile supernatants of each mucin/mucus media precultured with or without R. gnavus, monosaccharides obtained by R. gnavus digestion of mucus, and several sulfur substrates in the gut added to M9 minimal media. RESULTS AIEC, especially LF82 (B2), grew better with ileal mucus aerobically and, to a lesser extent, anaerobically than non-AIEC strains, with even greater AIEC growth with R. gnavus pre-digested mucus (Fig.1). AIEC produced more H2S with R. gnavus-precultured mucus than with mucus alone. R. gnavus digestion of PGM released mucus monosaccharides (Fig.2A). AIEC strains grew differently with each monosaccharide constituent of R. gnavus pre-digested PGM. AIEC grew better than non-AIEC with each monosaccharide in aerobic conditions, but AIEC (B2) grew better than others in all monosaccharides in anaerobic conditions (Fig.2B). Amongst mucus-relevant sulfur substrates, L-cysteine was the only substrate to enhance E. coli H2S production by all AIEC and non-AIEC strains. All mucus-relevant sulfur substrates supported R. gnavus survival for 24 hrs, while H2S sustained R. gnavus for 48 hrs in minimum media. CONCLUSIONS AIEC grew better and produced more H2S with R. gnavus pre-digested mucus suggesting that co-existence of AIEC with R. gnavus mutually support each other and may explain why they are often observed together in active CD patients. In addition, enhanced AIEC growth with ileal vs. colonic mucus may explain why AIEC are frequently isolated from ileal CD patients. AIEC phylotype growth differences with mucus substrates may explain their persistence in the ileal mucosa. Figure 1 Figure 2

A Cranberry Concentrate Decreases Adhesion and Invasion of Escherichia coli (AIEC) LF82 In Vitro.

While many beneficial host–microbiota interactions have been described, imbalanced microbiota in the gut is speculated to contribute to the progression and recurrence of chronic inflammatory diseases such as Crohn’s disease (CD). This in vitro study evaluated the impact of a cranberry concentrate Type M (CTM) on adherent-invasive Escherichia coli (AIEC) LF82, a pathobiont associated with CD. Different stages of pathogenic infection were investigated: (i) colonization of the mucus layer, and (ii) adhesion to and (iii) invasion of the epithelial cells. Following 48 h of fecal batch incubation, 0.5 and 1 mM of CTM significantly altered AIEC LF82 levels in a simulated mucus layer, resulting in a decrease of 50.5% in the untreated blank, down to 43.0% and 11.4%, respectively. At 1 mM of CTM, the significant decrease in the levels of AIEC LF82 coincided with a stimulation of the metabolic activity of the background microbiota. The increased levels of health-associated acetate (+7.9 mM) and propionate levels (+3.5 mM) suggested selective utilization of CTM by host microorganisms. Furthermore, 1 mM of both fermented and unfermented CTM decreased the adhesion and invasion of human-derived epithelial Caco-2 cells by AIEC LF82. Altogether, this exploratory in vitro study demonstrates the prebiotic potential of CTM and supports its antipathogenic effects through direct and/or indirect modulation of the gut microbiome.

The Role of OmpR in Bile Tolerance and Pathogenesis of Adherent-Invasive Escherichia coli.

Gut microbiota dysbiosis toward adherent-invasive Escherichia coli (AIEC) plays an important role in Crohn’s disease (CD). The OmpR transcriptional regulator is required for the AIEC LF82 prototype strain to adhere and invade intestinal epithelial cells. In this study, we explored the role of OmpR in AIEC pathogenesis using a panel of eight Escherichia coli strains isolated from CD patients and identified as AIEC. The deletion of ompR together with the implementation of two cell-based assays revealed that the role of OmpR in adhesion in vitro was not conserved in AIEC clinical strains. Nevertheless, we showed that OmpR was required for robust gut colonization of transgenic mice expressing human CEACAM receptors, suggesting that OmpR is involved in alternative virulence mechanisms in AIEC strains. We found that deletion of ompR compromised the ability of AIEC strains to cope with the stress induced by bile salts, which may be key for AIEC pathogenesis. More specifically, we demonstrated that OmpR was involved in a tolerance mechanism toward sodium deoxycholate (DOC), one of bile salts main component. We showed that the misregulation of OmpF or the loss of outer membrane integrity are not the drivers of OmpR-mediated DOC tolerance, suggesting that OmpR regulates a specific mechanism enhancing AIEC survival in the presence of DOC. In conclusion, the newly discovered role of OmpR in AIEC bile tolerance suggests that OmpR inhibition would interfere with different aspects of AIEC virulence arsenal and could be an alternative strategy for CD-treatment.

The Crohn\u2019s disease-related bacterial strain LF82 assembles biofilm-like communities to protect itself from phagolysosomal attack

Patients with Crohn’s disease exhibit abnormal colonization of the intestine by adherent invasive E. coli (AIEC). They adhere to epithelial cells, colonize them and survive inside macrophages. It appeared recently that AIEC LF82 adaptation to phagolysosomal stress involves a long lag phase in which many LF82 cells become antibiotic tolerant. Later during infection, they proliferate in vacuoles and form colonies harboring dozens of LF82 bacteria. In the present work, we investigated the mechanism sustaining this phase of growth. We found that intracellular LF82 produced an extrabacterial matrix that acts as a biofilm and controls the formation of LF82 intracellular bacterial communities (IBCs) for several days post infection. We revealed the crucial role played by the pathogenicity island encoding the yersiniabactin iron capture system to form IBCs and for optimal LF82 survival. These results illustrate that AIECs use original strategies to establish their replicative niche within macrophages.

Exosomes transfer miRNAs from cell-to-cell to inhibit autophagy during infection with Crohn’s disease-associated adherent-invasive E. coli

ABSTRACT Adherent-invasive E. coli (AIEC), which abnormally colonize the intestinal mucosa of Crohn’s disease (CD) patients, are able to adhere to and invade intestinal epithelial cells (IECs), survive and replicate within macrophages and induce a pro-inflammatory response. AIEC infection of IECs induces secretion of exosomes that increase AIEC replication in exosome-receiving IECs and macrophages. Here, we investigated the mechanism underlying the increased AIEC replication in cells receiving exosomes from AIEC-infected cells. Exosomes released by uninfected human intestinal epithelial T84 cells (Exo-uninfected) or by T84 cells infected with the clinical AIEC LF82 strain (Exo-LF82), the nonpathogenic E. coli K12 strain (Exo-K12) or the commensal E. coli HS strain (Exo-HS) were purified and used to stimulate T84 cells. Stimulation of T84 cells with Exo-LF82 inhibited autophagy compared with Exo-uninfected, Exo-K12 and Exo-HS. qRT-PCR analysis revealed increased levels of miR-30c and miR-130a in Exo-LF82 compared to Exo-uninfected, Exo-K12 and Exo-HS. These miRNAs were transferred via exosomes to recipient cells, in which they targeted and inhibited ATG5 and ATG16L1 expression and thereby autophagy response, thus favoring AIEC intracellular replication. Inhibition of these miRNAs in exosome-donor cells infected with AIEC LF82 abolished the increase in miR-30c and miR-130a levels in the released Exo-LF82 and in Exo-LF82-receiving cells, thus suppressing the inhibitory effect of Exo-LF82 on ATG5 and ATG16L1 expression and on autophagy-mediated AIEC clearance in Exo-LF82-receiving cells. Our study shows that upon AIEC infection, IECs secrete exosomes that can transfer specific miRNAs to recipient IECs, inhibiting autophagy-mediated clearance of intracellular AIEC.

Enhanced O-linked Glcnacylation in Crohn's disease promotes intestinal inflammation.

BackgroundTreatment of Crohn's disease (CD) remains to be a challenge due to limited insights for its pathogenesis. We aimed to determine the role of O-Linked β-N-acetylglucosamine (O-GlcNAc) in the development of CD and evaluate therapeutic effects of O-GlcNAc inhibitors on CD.MethodsO-GlcNAc in intestinal epithelial tissues of CD, adherent-invasive Escherichia coli (AIEC) LF82-infected cells and mice was determined by immunoblot and immunohistochemistry. AIEC LF82 and dextran sulfate sodium were administrated into C57BL/6 mice for estabolishing inflammatory bowel disease model and for therapeutic study.FindingsO-GlcNAc was increased in intestinal epithelial tissues of CD patients and AIEC LF82-infected mice. Infection of AIEC LF82 up-regulated the level of UDP-GlcNAc and increased O-GlcNAc in human colon epithelial HCT116 and HT-29 cells. We identified that IKKβ and NF-κB were O-Glycosylated in AIEC LF82-treated cells. Mutations of IKKβ (S733A) and p65 (T352A) abrogated the O-GlcNAc in IKKβ and NF-κB and inhibited AIEC LF82-induced activation of NF-κB. Application of 6-diazO-5-oxO-L-norleucine, an agent that blocks the production of UDP-GlcNAc and inhibits O-GlcNAc, inactivated NF-κB in AIEC LF82-infected cells, enhanced the formation of autophagy, promoted the removal of cell-associated AIEC LF82, alleviated intestinal epithelial inflammation, and improved the survival of the colitis mice.InterpretationIntestinal inflammation in CD is associated with increased O-GlcNAc modification, which is required for NF-κB activation and suppression of autophagy. Targeting O-GlcNAc could be an effective therapy for inflammatory bowel disease.FundingNational Natural Science Foundation of China (Nos. 81573087 and 81772924) and International Cooperation Foundation of Jilin Province (20190701006GH).

DOP49 Quantitative proteomics analysis of macrophages from Crohn’s disease patients and infected with adherent-invasive Escherichia coli

Abstract Background Crohn’s disease (CD) is a disabling inflammatory bowel disease. Currently, the available treatments only alleviate the symptoms. The anti-TNFα represent the most efficient therapeutic class, but its mechanisms are poorly known. Experimental data have highlighted the role of intestinal macrophages in the pathogenesis of CD. These macrophages present a defect in the control of CD-associated adherent-invasive E. coli (AIEC) replication, which is linked to altered autophagy. The main aim of this study was to compare the proteomic profile of macrophages from CD patients with and without anti-TNFα treatment, to those from ulcerative colitis (UC) patients or healthy subjects, both at the basal state and after AIEC infection. Methods Peripheral blood monocyte-derived macrophages (MDM) were obtained from 44 CD patients including 22 with and 22 without anti-TNFα treatment, 22 UC patients and 22 healthy subjects. MDM was infected or not with AIEC LF82 reference strain. The numbers of intracellular bacteria were determined at 1h and 6h post-infection, using gentamicin protection assay. The ‘bottom-up’ proteomic analysis of macrophages after 6h of AIEC LF82 infection or not, was assessed by a mass spectrometer, using the label-free quantification. Results AIEC survival was reduced within MDM from CD patients with anti-TNFα treatment compared with those from CD without TNFα treatment (p = 0.0235). The proteomic analysis identified 1359 proteins, of which, 71 were differentially expressed between MDM at the basal state and infected by AIEC LF82 whatever the origin, 30 proteins were specific in CD patients with anti-TNFα treatment and 16 in CD patients without anti-TNFα treatment (fold change >1.3). The bioinformatics analysis shows an impact of several pathways involved in cellular activation, immune process and lysosomal activity. Interestingly, CD macrophages expression levels of β3 integrin (ITGB3), glutathione peroxidase-1 (GPx-1) and peroxiredoxin 3 (PRDX3) are significantly correlated with AIEC survival at 6h post-infection and faecal calprotectin. The stratification of MDM from CD patients according to the level of faecal calprotectin highlighted that levels of calprotectin and α-L-Fucosidase 1 (FUCA-1) increased in a similar way, both at the basal state and after AIEC LF82 infection. Conclusion Our study highlighted that the anti-TNFα treatment limits bacteria replication and acts on macrophages function. For the first time, global proteomic analysis suggests that CD macrophages-AIEC interaction could be orchestrated by ITGB3, GPx-1, PRDX3 and FUCA-1. These proteins could represent potential therapeutic targets in CD patients that need to be further investigated.

The Crohn's disease-associated Escherichia coli strain LF82 relies on SOS and stringent responses to survive, multiply and tolerate antibiotics within macrophages.

Adherent Invasive Escherichia coli (AIEC) strains recovered from Crohn's disease lesions survive and multiply within macrophages. A reference strain for this pathovar, AIEC LF82, forms microcolonies within phagolysosomes, an environment that prevents commensal E. coli multiplication. Little is known about the LF82 intracellular growth status, and signals leading to macrophage intra-vacuolar multiplication. We used single-cell analysis, genetic dissection and mathematical models to monitor the growth status and cell cycle regulation of intracellular LF82. We found that within macrophages, bacteria may replicate or undergo non-growing phenotypic switches. This switch results from stringent response firing immediately after uptake by macrophages or at later stages, following genotoxic damage and SOS induction during intracellular replication. Importantly, non-growers resist treatment with various antibiotics. Thus, intracellular challenges induce AIEC LF82 phenotypic heterogeneity and non-growing bacteria that could provide a reservoir for antibiotic-tolerant bacteria responsible for relapsing infections.

Bovine Lactoferrin Pre-Treatment Induces Intracellular Killing of AIEC LF82 and Reduces Bacteria-Induced DNA Damage in Differentiated Human Enterocytes.

LF82, a prototype of adherent-invasive E. coli (AIEC), is able to adhere to, invade, survive and replicate into intestinal epithelial cells. LF82 is able to enhance either its adhesion and invasion by up-regulating carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM-6), the main cell surface molecule for bacterial adhesion, and its intracellular survival by inducing host DNA damage, thus blocking the cellular cycle. Lactoferrin (Lf) is a multifunctional cationic glycoprotein of natural immunity, exerting an anti-invasive activity against LF82 when added to Caco-2 cells at the moment of infection. Here, the infection of 12 h Lf pre-treated Caco-2 cells was carried out at a time of 0 or 3 or 10 h after Lf removal from culture medium. The effect of Lf pre-treatment on LF82 invasiveness, survival, cell DNA damage, CEACAM-6 expression, apoptosis induction, as well as on Lf subcellular localization, has been evaluated. Lf, even if removed from culture medium, reduced LF82 invasion and survival as well as bacteria-induced DNA damage in Caco-2 cells independently from induction of apoptosis, modulation of CEACAM-6 expression and Lf sub-cellular localization. At our knowledge, this is the first study showing that the sole Lf pre-treatment can activate protective intracellular pathways, reducing LF82 invasiveness, intracellular survival and cell–DNA damages.

An uncharacterised protein mediates motility, biofilm formation, and host colonisation in adherent invasive Escherichia coli

Adherent Invasive Escherichia coli (AIEC) is a non-diarrhoeagenic intestinal E. coli patho type with a putativea etiological role in Crohn’s Disease (CD). AIEC pathogenes is ischaracterised by adhesion to, invasion of, and replication within intestinal epithelial cells and macrophages, and biofilm formation. Using a heterologous expression screen, we identified a gene in the AIEC LF82 genome encoding a protein which self-assembles into filaments in HeLa cells, which we hypothesised was a novel pilin or biofilm matrix component, designated bcmA (biofilm coupled to motility in AIEC). Using a crystal violet-based assay, we found LF82ΔbcmA have defective biofilm formation, which can be fully complemented by episomal bcmA expression. Microscopic analysis of LF82 biofilms demonstrated LF82ΔbcmA form patchy, sparse biofilms, and revealed an intracellular localisation for GFP-tagged bcmA, suggesting the protein is not a surface-exposed adhesin or biofilm matrix component. We therefore assessed the role of bcmA in flagellar-mediated motility and found that – despite displaying wild-type flagellar morphologies – LF82ΔbcmA have profound swimming and swarming defects. Work in a Caenorhabdit is elegans infection model suggests bcmA is not required for full virulence; however, preliminary evidence suggests LF82ΔbcmA have defective C. elegans gut colonisation. Taken together, our data demonstrates roles for bcmA in AIEC host colonisation via an undefined role in motility. bcmA is highly conserved among pathogenic gammaproteobacteria, including Salmonella Typhi, Shigella boydii and Klebsiella pneumoniae. This suggests bcmA may not only be significant for AIEC pathogenesis, but may also represent an important virulence determinant in several major gammaproteobacterial pathogens.

Flagellin-mediated activation of IL-33-ST2 signaling by a pathobiont promotes intestinal fibrosis.

Intestinal fibrosis is a severe complication in patients with Crohn’s disease (CD). Unfortunately, the trigger leading to the development of intestinal fibrosis in the context of CD remains elusive. Here, we show that colonization by a CD-associated pathobiont adherent-invasive Escherichia coli (AIEC) promotes the development of intestinal fibrosis. Exogenously inoculated AIEC strain LF82 and commensal E. coli HS were gradually eradicated from the intestine in healthy mice. In Salmonella- or dextran sodium sulfate-induced colitis models, AIEC exploited inflammation and stably colonize the gut. Consequently, persistent colonization by AIEC LF82 led to substantial fibrosis. In contrast, commensal E. coli HS was unable to derive a growth advantage from inflammation, thereby failing to colonize the inflamed intestine or promote intestinal fibrosis. AIEC colonization potentiated the expression of the IL-33 receptor ST2 in the intestinal epithelium, which is crucial for the development of intestinal fibrosis. The induction of ST2 by AIEC LF82 was mediated by flagellin, as the FliC mutant failed to induce ST2. These observations provide novel insights into pathobiont-driven intestinal fibrosis and can lead to the development of novel therapeutic approaches for the treatment of intestinal fibrosis in the context of CD that target AIEC and/or its downstream IL-33-ST2 signaling.

Preventive Effect of Spontaneous Physical Activity on the Gut-Adipose Tissue in a Mouse Model That Mimics Crohn's Disease Susceptibility.

Crohn’s disease is characterized by abnormal ileal colonization by adherent-invasive E. coli (AIEC) and expansion of mesenteric adipose tissue. This study assessed the preventive effect of spontaneous physical activity (PA) on the gut-adipose tissue in a mouse model that mimics Crohn’s disease susceptibility. Thirty-five CEABAC10 male mice performed spontaneous PA (wheel group; n = 24) or not (controls; n = 11) for 12 weeks. At week 12, mice were orally challenged with the AIEC LF82 strain for 6 days. Body composition, glycaemic control, intestinal permeability, gut microbiota composition, and fecal short-chain fatty acids were assessed in both groups. Animals were fed a high fat/high sugar diet throughout the study. After exposure to AIEC, mesenteric adipose tissue weight was lower in the wheel group. Tight junction proteins expression increased with spontaneous PA, whereas systemic lipopolysaccharides were negatively correlated with the covered distance. Bifidobacterium and Lactobacillus decreased in controls, whereas Oscillospira and Ruminococcus increased in the wheel group. Fecal propionate and butyrate were also higher in the wheel group. In conclusion, spontaneous physical activity promotes healthy gut microbiota composition changes and increases short-chain fatty acids in CEABAC10 mice fed a Western diet and exposed to AIEC to mimic Crohn’s disease.

Crohn's Disease-Associated Adherent-Invasive Escherichia coli Manipulate Host Autophagy by Impairing SUMOylation.

The intestinal mucosa of Crohn’s disease (CD) patients is abnormally colonized with adherent-invasive Escherichia coli (AIEC) that are able to adhere to and to invade intestinal epithelial cells (IECs), to survive in macrophages, and to induce a pro-inflammatory response. AIEC persist in the intestine, and induce inflammation in CEABAC10 transgenic mice expressing human CAECAM6, the receptor for AIEC. SUMOylation is a eukaryotic-reversible post-translational modification, in which SUMO, an ubiquitin-like polypeptide, is covalently linked to target proteins. Here, we investigated the role of SUMOylation in host responses to AIEC infection. We found that infection with the AIEC LF82 reference strain markedly decreased the levels of SUMO-conjugated proteins in human intestinal epithelial T84 cells. This was also observed in IECs from LF82-infected CEABAC10 transgenic mice. LF82-induced deSUMOylation in IECs was due in part to increased level of microRNA (miR)-18, which targets PIAS3 mRNA encoding a protein involved in SUMOylation. Over-expression of SUMOs in T84 cells induced autophagy, leading to a significant decrease in the number of intracellular LF82. Consistently, a decreased expression of UBC9, a protein necessary for SUMOylation, was accompanied with a decrease of LF82-induced autophagy, increasing bacterial intracellular proliferation and inflammation. Finally, the inhibition of miR-18 significantly decreased the number of intracellular LF82. In conclusion, our results suggest that AIEC inhibits the autophagy response to replicate intracellularly by manipulating host SUMOylation.

Phagocytosis and Epithelial Cell Invasion by Crohn's Disease-Associated Adherent-Invasive Escherichia coli Are Inhibited by the Anti-inflammatory Drug 6-Mercaptopurine.

Adherent-invasive Escherichia coli (AIEC) strains are overrepresented in the dysbiotic microbiota of Crohn’s disease (CD) patients, and contribute to the onset of the chronic inflammation typical of the disease. However, the effects of anti-inflammatory drugs used for CD treatment on AIEC virulence have not yet been investigated. In this report, we show that exposure of AIEC LF82 strain to amino-6-mercaptopurine (6-MP) riboside, one of the most widely used anti-inflammatory drugs in CD, impairs its ability to adhere to, and consequently to invade, human epithelial cells. Notably, phagocytosis of LF82 treated with 6-MP by human macrophages is also reduced, suggesting that 6-MP affects AIEC cell surface determinants involved both in interaction with epithelial cells and in uptake by macrophages. Since a main target of 6-MP in bacterial cells is the inhibition of the important signal molecule c-di-GMP, we also tested whether perturbations in cAMP, another major signaling pathway in E. coli, might have similar effects on interactions with human cells. To this aim, we grew LF82 in the presence of glucose, which leads to inhibition of cAMP synthesis. Growth in glucose-supplemented medium resulted in a reduction in AIEC adhesion to epithelial cells and uptake by macrophages. Consistent with these results, both 6-MP and glucose can affect expression of cell adhesion-related genes, such as the csg genes, encoding thin aggregative fimbriae (curli). In addition, glucose strongly inhibits expression of the fim operon, encoding type 1 pili, a known AIEC determinant for adhesion to human cells. To further investigate whether 6-MP can indeed inhibit c-di-GMP signaling in AIEC, we performed biofilm and motility assays and determination of extracellular polysaccharides. 6-MP clearly affected biofilm formation and cellulose production, but also, unexpectedly, reduced cell motility, itself an important virulence factor for AIEC. Our results provide strong evidence that 6-MP can affect AIEC-host cell interaction by acting on the bacterial cell, thus strengthening the hypothesis that mercaptopurines might promote CD remission also by affecting gut microbiota composition and/or physiology, and suggesting that novel drugs targeting bacterial virulence and signaling might be effective in preventing chronic inflammation in CD.

Mice harboring pathobiont-free microbiota do not develop intestinal inflammation that normally results from an innate immune deficiency.

BackgroundInability to maintain a stable and beneficial microbiota is associated with chronic gut inflammation, which classically manifests as colitis but may more commonly exist as low-grade inflammation that promotes metabolic syndrome. Alterations in microbiota, and associated inflammation, can originate from dysfunction in host proteins that manage the microbiota, such as the flagellin receptor TLR5. That the complete absence of a microbiota (i.e. germfree conditions) eliminates all evidence of inflammation in TLR5-deficient mice demonstrates that this model of gut inflammation is microbiota-dependent. We hypothesize that such microbiota dependency reflects an inability to manage pathobionts, such as Adherent-Invasive E. coli (AIEC). Herein, we examined the extent to which microbiota mismanagement and associated inflammation in TLR5-deficient mice would manifest in a limited and pathobiont-free microbiota. For this purpose, WT and TLR5-deficient mice were generated and maintained with the 8-member consortium of bacteria referred to as “Altered Schaedler Flora” (ASF). Such ASF animals were subsequently inoculated with AIEC reference strain LF82. Feces were assayed for bacterial loads, fecal lipopolysaccharide and flagellin loads, fecal inflammatory marker lipocalin-2 and microbiota composition.ResultsRelative to similarly maintained WT mice, mice lacking TLR5 (T5KO) did not display low-grade intestinal inflammation nor metabolic syndrome under ASF conditions. Concomitantly, the ASF microbial community was similar between WT and T5KO mice, while inoculation with AIEC strain LF82 resulted in alteration of the ASF community in T5KO mice compared to WT control animals. AIEC LF82 inoculation in ASF T5KO mice resulted in microbiota components having elevated levels of bioactive lipopolysaccharide and flagellin, a modest level of low-grade inflammation and increased adiposity.ConclusionsIn a limited-complexity pathobiont-free microbiota, loss of the flagellin receptor TLR5 does not impact microbiota composition nor its ability to promote inflammation. Addition of AIEC to this ecosystem perturbs microbiota composition, increases levels of lipopolysaccharide and flagellin, but only modestly promotes gut inflammation and adiposity, suggesting that the phenotypes previously associated with loss of this innate immune receptor require disruption of complex microbiota.

Adaptation of adherent-invasive E. coli to gut environment: Impact on flagellum expression and bacterial colonization ability

ABSTRACT The pathogenesis of Crohn's disease (CD) is multifactorial and involves genetic susceptibility, environmental triggers and intestinal microbiota. Adherent-invasive Escherichia coli (AIEC) are flagellated bacteria more prevalent in CD patients than in healthy subjects and promote chronic intestinal inflammation. We aim at deciphering the role of flagella and flagellin modulation by intestinal conditions. AIEC flagellum expression is required for optimal adhesion to and invasion of intestinal epithelial cells. Interestingly, differential flagellin regulation was observed between commensal E. coli (HS) and AIEC (LF82) strains: flagellum expression by AIEC bacteria, in contrast to that of commensal E. coli, is enhanced under intestinal conditions (the presence of bile acids and mucins). Flagella are involved in the ability of the AIEC LF82 strain to cross a mucus layer in vitro and in vivo, conferring a selective advantage in penetrating the mucus layer and reaching the epithelial surface. In a CEABAC10 mouse model, a non-motile mutant (LF82-ΔfliC) exhibits reduced colonization that is restored by a dextran sodium sulfate treatment that alters mucus layer integrity. Moreover, a mutant that continuously secretes flagellin (LF82-ΔflgM) triggers a stronger inflammatory response than the wild-type strain, and the mutant's ability to colonize the CEABAC10 mouse model is decreased. Overexpression of flagellin in bacteria in contact with epithelial cells can be detrimental to their virulence by inducing acute inflammation that enhances AIEC clearance. AIEC pathobionts must finely modulate flagellum expression during the infection process, taking advantage of their specific virulence gene regulation to improve their adaptability and flexibility within the gut environment.

P009 Autophagy-related host factors are implicated in the incapability of macrophages from Crohn’s disease patients to mediate adherent-invasive E. coli clearance

Several experimental data have highlighted the potential role of intestinal macrophages in the pathogenesis of Crohn’s disease (CD). These macrophages present a defect in the control of CD-associated adherent-invasive E. coli (AIEC) replication, which could be linked to altered autophagy. We investigated the impact of several CD-associated singer nucleotide polymorphisms (SNPs) including those implicated in autophagy on the ability of macrophages from CD patients to mediate AIEC bacterial clearance. Peripheral blood monocyte-derived macrophages (MDM) were obtained from 95 CD patients, 30 ulcerative colitis (UC) patients and 15 healthy subjects genotyped for CD-associated SNPs-related to autophagy, especially IRGM (rs10065172) and ULK1 (rs12303764) and, infected with AIEC LF82 reference strain. Functional assay were performed on MDM after AIEC infection and/or after a short-term silencing of ULK1 gene. The numbers of intracellular bacteria were determined using gentamicin protection assay. IRGM, ULK1 and p62 protein expression was determined by western blot. AIEC survival was increased within MDM from CD patients compared with those from UC patients or healthy subjects (p = 0.0019). MDM from CD patients failed to mediate AIEC bacterial clearance especially in patients harbouring the CD-associated IRGM SNP (p = 0.045). In contrast, AIEC survival was decreased in MDM from patients with the CD-associated ULK1 SNP (p = 0.046). Expression of ULK1, but not IRGM and p62, was increased in MDM from CD patients infected by AIEC bacteria compared with MDM from UC patients or healthy subjects (p = 0.0056) and are significantly correlated with the AIEC survival (p = 0.0369). In the same way, the down-regulation of ULK1 within MDM limits the AIEC survival (p = 0.0018) within MDM and could regulate the autophagic flux suggested by an increase of the expression of p62 (p = 0.0043). We confirmed that MDM from CD patients failed to mediate AIEC bacterial clearance compared with those of UC or healthy subjects. Our results highlight a role of CD-associated SNPs, related to autophagy, IRGM and ULK1, on the ability of macrophages from CD patients to mediate AIEC bacterial clearance. At the protein level, our data suggested a potential role of the protein ULK1, the cornerstone of autophagy initiation, to control AIEC bacteria in patients with CD. This protein could be a potential target to alter the interaction between macrophages and AIEC in patients with CD.

The effects of mucosal media on some pathogenic traits of Crohn's disease-associated Escherichia coli LF82.

Adherent-invasive Escherichia coli (AIEC) pathovar has been identified in intestinal mucosa of patients with Crohn's disease. Our aim was to compare the impact of sterile mucosal media (Muc-M) originated from different parts of the intestine on some pathogenic traits of AIEC LF82 strain. Muc-M composed of certain rates of cell culture medium or M63 minimal medium and mucosal contents obtained from different part of intestine were designed for cell-infection experiments and biofilm-formation assays. The results showed that Muc-M reduced usually pathogenic properties of AIEC LF82. However, LF82 adhesion, invasion and specific biofilm formations were markedly higher in Muc-MCR than those in Muc-MIR . In this context, the findings of present study could help the endeavors related to determining molecular targets for AIEC bacteria.

Adherent-Invasive E. coli enhances colonic hypersensitivity and P2X receptors expression during post-infectious period

ABSTRACTIrritable Bowel Syndrome (IBS) and Inflammatory Bowel Disease (IBD) are related gastrointestinal disorders characterized by abdominal pain associated with colonic hypersensitivity (CHS). Studies in humans have reported an abnormal colonization of Adherent-Invasive E. coli (AIEC) in the ileum of Crohn's disease (CD) patients associated with overexpression of the bacterial colonizing receptor CEACAM6. The aim of the present study was to investigate whether AIEC reference strain LF82 could induce intestinal impairment during infectious and/or post-infectious periods and subsequently the development of CHS. Transgenic mice overexpressing human CEACAM6 protein (TG) and their wild-type littermates were gavaged by CD-associated AIEC bacteria (reference strain LF82) or PBS for 3 d. Colonic hypersensitivity was assessed by colorectal distension (CRD) test during infectious (D4) and post-infectious periods (D21). Several markers of intestinal inflammation were monitored and the colonic expression of purinergic P2X receptors was quantified. At D4, an increased visceromotor response (VMR) to the CRD test was observed in TG mice infected with CD-associated AIEC LF82 in comparison with non-infected TG mice and persisted in a subgroup of infected animals at D21 after bacteria clearance. Increased VMR was associated with low-grade intestinal inflammation, increased intestinal permeability and expression of P2X 3, 4 and 7. This study shows that certain susceptible hosts infected with CD-associated AIEC bacteria can develop persistent CHS associated with low-grade inflammation and increased P2X receptors expression. Thus, CD-associated AIEC infection in CEACAM6 transgenic mice could be used as a novel post-infectious mouse model mimicking quiescent IBD with IBS-like symptoms such as visceral pain.