Adherent-invasive Escherichia Coli Strains Research Articles

Outer Membrane Vesicles Derived from Adherent-Invasive Escherichia coli Induce Inflammatory Response and Alter the Gene Expression of Junction-Associated Proteins in Human Intestinal Epithelial Cells.

Adherent-invasive Escherichia coli (AIEC) pathobionts, which are characterized by their ability to adhere to and invade intestinal epithelial cells, are associated with the etiopathogenesis of inflammatory bowel diseases (IBDs). Outer membrane vesicles (OMVs) released by AIEC strains can facilitate the interaction of these bacteria with host cells through delivering bacterial effectors. The aim of this study was to determine the ability of OMVs derived from AIEC strain LF82 to induce the host immune response, leading to production of proinflammatory cytokines and also altering the gene expression of junction-associated proteins in the human epithelial colorectal adenocarcinoma Caco-2 cell line. OMVs were extracted from AIEC strain LF82, and the cell viability of Caco-2 cells treated with these vesicles was assessed by MTT assay. The morphology and size distribution of vesicles were analyzed using transmission electron microscopy and dynamic light scattering, respectively. Gene expression of occludin, ZO-1, claudin-2, E-cadherin, TLR-2, and TLR-4 in response to OMVs was assessed in Caco-2 cells by RT-qPCR. Moreover, the secretion of IL-8 and TNF-α into the supernatant of Caco-2 cells upon treatment with OMVs was measured using ELISA. Our results demonstrated that OMVs upregulated the gene expression level of TLRs and also altered the gene expression level of junction-associated proteins. OMVs derived from AIEC may play a major role in the promotion of intestinal inflammation and epithelial barrier dysfunction. However, further investigations are needed to elucidate the putative role of OMVs in the pathogenesis of AIEC and IBD.

The Efficacy of Selected Probiotic Strains and Their Combination to Inhibit the Interaction of Adherent-Invasive Escherichia coli (AIEC) with a Co-Culture of Caco-2:HT29-MTX Cells.

The gastrointestinal tract's microbiota plays a crucial role in human health, with dysbiosis linked to the development of diseases such as inflammatory bowel disease (IBD). Whilst the pathogenic mechanisms underlying IBD remain poorly characterised, adherent-invasive Escherichia coli (AIEC) has been implicated as a microbiological factor in disease pathogenesis. These strains show an enhanced ability to diffusely adhere to and invade intestinal epithelial cells, along with the ability to survive and replicate within macrophages. Probiotics, such as Lactobacillus strains, have been identified as potential treatment options due to their abilities to compete with pathogens for binding sites and regulate the host immune response. In this study, we used four well-characterised Lactobacillus strains and their combination to test their ability to inhibit the adhesion, invasion, and translocation of a well-characterized AIEC strain, F44A-1, in a co-culture of Caco-2 and HT29-MTX cell lines representing the gut epithelium. The results demonstrated that the pre-inoculation of the probiotic candidates 90 min prior to the introduction of the AIEC was more effective in inhibiting AIEC interaction than the co-inoculation of the strains. While the individual probiotic strains greatly reduced AIEC colonisation and invasion of the co-cultured cells, their combination was only more effective in reducing the translocation of the AIEC. These results suggest that probiotics are more effective when used prophylactically against pathogens and that the combination of strains may enhance their efficacy against AIEC translocation once used as a prophylactic measure.

A CONSORTIA OF CLINICAL E. COLI STRAINS WITH DISTINCT IN-VITRO ADHERENT/INVASIVE PROPERTIES ESTABLISH THEIR OWN CO-COLONIZATION NICHE AND SHAPE THE INTESTINAL MICROBIOTA IN INFLAMMATION-SUSCEPTIBLE MICE

Abstract Mucosal colonization with adherent-invasive Escherichia coli (AIEC) are believed to perpetuate intestinal inflammation and associated inflammatory bowel disease (IBD). However, it remains unclear if the 24-year-old AIEC in-vitro definition fully predicts mucosal colonization in-vivo. To fill this gap, germ-free inflammation-susceptible interleukin-10-deficient (Il10-/-) and inflammation-resistant WT mice were colonized with a consortia of AIEC and non-AIEC strains, then given a murine fecal transplant to provide niche competition. E. coli strains isolated from human intestinal tissue were each marked with a unique molecular barcode that permits identification and quantification by barcode-targeted sequencing. Our data reveal that specific AIEC and non-AIEC strains reproducibly colonize the intestinal mucosa of WT and Il10-/- mice. These E. coli expand in Il10-/- mice during inflammation and induce compositional dysbiosis to the microbiome in an inflammation-dependent manner based on histological analysis and 16S sequencing. Because select AIEC and non-AIEC strains colonize the mucosa, this suggests the in vitro AIEC definition may not fully predict in vivo colonization potential. Further comparison of seven E. coli genomes pinpointed unique genomic features contained only in highly colonizing strains (two AIEC and two non-AIEC). Those colonization-associated features may convey metabolic advantages (e.g., iron acquisition and carbohydrate consumption) to promote efficient mucosal colonization. Our findings establish the in-vivo mucosal colonizer, not necessarily AIEC, as a principal dysbiosis driver through crosstalk with host and associated microbes. Furthermore, we highlight the utility of high-throughput screens to decode the in-vivo colonization dynamics of patient-derived bacteria in murine models.

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 consortia of clinical E. coli strains with distinct in vitro adherent/invasive properties establish their own co-colonization niche and shape the intestinal microbiota in inflammation-susceptible mice

BackgroundInflammatory bowel disease (IBD) patients experience recurrent episodes of intestinal inflammation and often follow an unpredictable disease course. Mucosal colonization with adherent-invasive Escherichia coli (AIEC) are believed to perpetuate intestinal inflammation. However, it remains unclear if the 24-year-old AIEC in vitro definition fully predicts mucosal colonization in vivo. To fill this gap, we have developed a novel molecular barcoding approach to distinguish strain variants in the gut and have integrated this approach to explore mucosal colonization of distinct patient-derived E. coli isolates in gnotobiotic mouse models of colitis.ResultsGerm-free inflammation-susceptible interleukin-10-deficient (Il10−/−) and inflammation-resistant WT mice were colonized with a consortium of AIEC and non-AIEC strains, then given a murine fecal transplant to provide niche competition. E. coli strains isolated from human intestinal tissue were each marked with a unique molecular barcode that permits identification and quantification by barcode-targeted sequencing. 16S rRNA sequencing was used to evaluate the microbiome response to E. coli colonization. Our data reveal that specific AIEC and non-AIEC strains reproducibly colonize the intestinal mucosa of WT and Il10−/− mice. These E. coli expand in Il10−/− mice during inflammation and induce compositional dysbiosis to the microbiome in an inflammation-dependent manner. In turn, specific microbes co-evolve in inflamed mice, potentially diversifying E. coli colonization patterns. We observed no selectivity in E. coli colonization patterns in the fecal contents, indicating minimal selective pressure in this niche from host-microbe and interbacterial interactions. Because select AIEC and non-AIEC strains colonize the mucosa, this suggests the in vitro AIEC definition may not fully predict in vivo colonization potential. Further comparison of seven E. coli genomes pinpointed unique genomic features contained only in highly colonizing strains (two AIEC and two non-AIEC). Those colonization-associated features may convey metabolic advantages (e.g., iron acquisition and carbohydrate consumption) to promote efficient mucosal colonization.ConclusionsOur findings establish the in vivo mucosal colonizer, not necessarily AIEC, as a principal dysbiosis driver through crosstalk with host and associated microbes. Furthermore, we highlight the utility of high-throughput screens to decode the in vivo colonization dynamics of patient-derived bacteria in murine models.6LG4zbnj4e64eDS25av5ePVideo

Identification of differences in gene expression implicated in the Adherent-Invasive Escherichia coli phenotype during in vitro infection of intestinal epithelial cells.

Adherent-invasive Escherichia coli (AIEC) is strongly associated with the pathogenesis of Crohn's disease (CD). However, no molecular markers currently exist for AIEC identification. This study aimed to identify differentially expressed genes (DEGs) between AIEC and non-AIEC strains that may contribute to AIEC pathogenicity and to evaluate their utility as molecular markers. Comparative transcriptomics was performed on two closely related AIEC/non-AIEC strain pairs during Intestine-407 cell infection. DEGs were quantified by RT-qPCR in the same RNA extracts, as well as in 14 AIEC and 23 non-AIEC strains to validate the results across a diverse strain collection. Binary logistical regression was performed to identify DEGs whose quantification could be used as AIEC biomarkers. Comparative transcriptomics revealed 67 differences in expression between the two phenotypes in the strain pairs, 50 of which (81.97%) were corroborated by RT-qPCR. When explored in the whole strain collection, 29 DEGs were differentially expressed between AIEC and non-AIEC phenotypes (p-value < 0.042), and 42 genes between the supernatant fraction of infected cell cultures and the cellular fraction containing adhered and intracellular bacteria (p-value < 0.049). Notably, six DEGs detected in the strain collection were implicated in arginine biosynthesis and five in colanic acid synthesis. Furthermore, two biomarkers based on wzb and cueR gene expression were proposed with an accuracy of ≥ 85% in our strain collection. This is the first transcriptomic study conducted using AIEC-infected cell cultures. We have identified several genes that may be involved in AIEC pathogenicity, two of which are putative biomarkers for identification.

Heterogeneity among Clinical Intestinal Escherichia coli Isolates upon Acquired Streptomycin Resistance.

Escherichia coli isolates from inflammatory bowel disease (IBD) patients are often multidrug resistant, including to streptomycin. Streptomycin resistance (StrR) mutations can alter bacterial behavior, which may influence intestinal disease. We generated a spontaneous StrR strain of the intestinal adherent-invasive E. coli (AIEC) strain NC101. Whole-genome sequencing revealed a single missense mutation in rpsL that commonly confers StrR, rpsL-K43N. StrR NC101 exhibited a striking loss of aggregation and significantly increased motility, behaviors that can impact host-microbe interactions. Behavioral changes were associated with reduced transcription of csgA, encoding the biofilm component curli, and increased transcription of fliC, encoding flagellin. Scanning electron microscopy (SEM) detailed morphologic changes consistent with the observed alterations in multicellular behavior. Because intestinal E. coli isolates exhibit remarkable strain-specific differences, we generated spontaneous StrR mutants of 10 clinical E. coli phylotype B2 strains from patients with IBD, colorectal cancer, and urinary tract infection. Out of these 10 StrR clinical strains, two had altered colony morphology on Congo red agar (suggesting changes in extracellular products), and three had significant changes in motility. These changes were not associated with a particular rpsL mutation nor with the presence of virulence genes encoding the inflammation-associated E. coli metabolites yersiniabactin or colibactin. We conclude that common mutations in rpsL, which confer StrR, can differentially alter disease-associated phenotypes across intestinal E. coli strains. These findings highlight the heterogeneity among seemingly similar intestinal E. coli strains and reveal the need to carefully study the strain-specific effects of antibiotic resistance mutations, particularly when using these mutations during strain selection studies. IMPORTANCE We demonstrate that StrR, commonly acquired through a single point mutation in rpsL (a gene encoding part of the 30S bacterial ribosome), strikingly alters the morphology and behavior of a key intestinal AIEC strain, NC101. These changes include remarkably diminished aggregation and significantly increased motility, traits that are linked to AIEC-defining features and disease development. Phenotypic changes were heterogeneous among other StrR clinical E. coli strains, underscoring the need to evaluate the strain-specific effects of commonly acquired antibiotic resistance mutations. This is important, as the results of studies using mutant StrR Enterobacteriaceae strains (e.g., for cloning or in vivo selection) may be confounded beyond our demonstrated effects. Long term, these findings can help researchers better distinguish the contribution of specific E. coli traits to functional changes in the microbiota. Evaluating these strain-level differences could provide insight into the diversity of IBD symptoms and lead to improved therapies for microbiota-driven intestinal disorders.

Evidence for a Causal Role for Escherichia coli Strains Identified as Adherent-Invasive (AIEC) in Intestinal Inflammation.

Enrichment of adherent-invasive Escherichia coli (AIEC) has been consistently detected in subsets of inflammatory bowel disease (IBD) patients. Although some AIEC strains cause colitis in animal models, these studies did not systematically compare AIEC with non-AIEC strains, and causal links between AIEC and disease are still disputed. Specifically, it remains unclear whether AIEC shows enhanced pathogenicity compared to that of commensal E. coli found in the same ecological microhabitat and if the in vitro phenotypes used to classify strains as AIEC are pathologically relevant. Here, we utilized in vitro phenotyping and a murine model of intestinal inflammation to systematically compare strains identified as AIEC with those identified as non-AIEC and relate AIEC phenotypes to pathogenicity. Strains identified as AIEC caused, on average, more severe intestinal inflammation. Intracellular survival/replication phenotypes routinely used to classify AIEC positively correlated with disease, while adherence to epithelial cells and tumor necrosis factor alpha production by macrophages did not. This knowledge was then applied to design and test a strategy to prevent inflammation by selecting E. coli strains that adhered to epithelial cells but poorly survived/replicated intracellularly. Two E. coli strains that ameliorated AIEC-mediated disease were subsequently identified. In summary, our results show a relationship between intracellular survival/replication in E. coli and pathology in murine colitis, suggesting that strains possessing these phenotypes might not only become enriched in human IBD but also contribute to disease. We provide new evidence that specific AIEC phenotypes are pathologically relevant and proof of principle that such mechanistic information can be therapeutically exploited to alleviate intestinal inflammation. IMPORTANCE Inflammatory bowel disease (IBD) is associated with an altered gut microbiota composition, including expansion of Proteobacteria. Many species in this phylum are thought to contribute to disease under certain conditions, including adherent-invasive Escherichia coli (AIEC) strains, which are enriched in some patients. However, whether this bloom contributes to disease or is just a response to IBD-associated physiological changes is unknown. Although assigning causality is challenging, appropriate animal models can test the hypothesis that AIEC strains have an enhanced ability to cause colitis in comparison to other gut commensal E. coli strains and to identify bacterial traits contributing to virulence. We observed that AIEC strains are generally more pathogenic than commensal E. coli and that bacterial intracellular survival/replication phenotypes contributed to disease. We also found that E. coli strains lacking primary virulence traits can prevent inflammation. Our findings provide critical information on E. coli pathogenicity that may inform development of IBD diagnostic tools and therapies.

Motility increase of adherent invasive Escherichia coli (AIEC) induced by a sub-inhibitory concentration of recombinant endolysin LysPA90.

Endolysins are bacteriophage enzymes required for the eruption of phages from inside host bacteria via the degradation of the peptidoglycan cell wall. Recombinant endolysins are increasingly being seen as potential antibacterial candidates, with a number currently undergoing clinical trials. Bacteriophage PBPA90 infecting Pseudomonas aeruginosa harbors a gene encoding an endolysin, lysPA90. Herein, recombinant LysPA90 demonstrated an intrinsic antibacterial activity against Escherichia coli in vitro. It was observed that a sub-inhibitory concentration of the recombinant protein induced the upregulation of genes related to flagella biosynthesis in a commensal E. coli strain. Increases in the number of bacterial flagella, and in motility, were experimentally substantiated. The treatment caused membrane stress, leading to the upregulation of genes rpoE, rpoH, dnaK, dnaJ, and flhC, which are upstream regulators of flagella biosynthesis. When adherent invasive Escherichia coli (AIEC) strains were treated with subinhibitory concentrations of the endolysin, bacterial adhesion and invasion into intestinal epithelial Caco-2 cells was seen to visibly increase under microscopic examination. Bacterial counting further corroborated this adhesion and invasion of AIEC strains into Caco-2 cells, with a resultant slight decrease in the viability of Caco-2 cells then being observed. Additionally, genes related to flagella expression were also upregulated in the AIEC strains. Finally, the enhanced expression of the proinflammatory cytokine genes TNF-α, IL-6, IL-8, and MCP1 in Caco-2 cells was noted after the increased invasion of the AIEC strains. While novel treatments involving endolysins offer great promise, these results highlight the need for the further exploration of possible unanticipated and unintended effects.

Efficacy of Selected Live Biotherapeutic Candidates to Inhibit the Interaction of an Adhesive-Invasive Escherichia coli Strain with Caco-2, HT29-MTX Cells and Their Co-Culture.

Adherent-invasive Escherichia coli (AIEC) has been implicated as a microbiological factor in the pathogenesis of inflammatory bowel disease (IBD). We evaluated the ability of six live biotherapeutic products (LBPs) to inhibit the interaction of an AIEC strain to three cell lines representing human gut epithelium. Co-inoculation of LBPs with AIEC showed a reduction in adhesion (up to 73%) and invasion of AIEC (up to 89%). Pre-inoculation of LBPs in HT-29-MTX and Caco-2 cells before challenging with AIEC further reduced the adhesion and invasion of the AIEC, with three LBPs showing significantly (p < 0.0001) higher efficiency in reducing the adhesion of AIEC. In co-inoculation experiments, the highest reduction in adhesion (73%) of AIEC was observed in HT-29-MTX cells, whereas the highest reduction in invasion (89%) was seen in HT-29-MTX and the co-culture of cells. Pre-inoculation of LBPs further reduced the invasion of AIEC with highest reduction (97%) observed in co-culture of cells. Our results indicated that whilst there were differences in the efficacy of LBPs, they all reduced interaction of AIEC with cell lines representing gut epithelium. Their efficiency was higher when they were pre-inoculated onto the cells, suggesting their potential as candidates for alleviating pathogenesis of AIEC in patients with IBD.

Characterization of Adherent-Invasive Escherichia coli (AIEC) Outer Membrane Proteins Provides Potential Molecular Markers to Screen Putative AIEC Strains.

Adherent-invasive E. coli (AIEC) is a pathotype associated with the etiopathogenesis of Crohn’s disease (CD), albeit with an as-yet unclear role. The main pathogenic mechanisms described for AIEC are adherence to epithelial cells, invasion of epithelial cells, and survival and replication within macrophages. A few virulence factors have been described as participating directly in these phenotypes, most of which have been evaluated only in AIEC reference strains. To date, no molecular markers have been identified that can differentiate AIEC from other E. coli pathotypes, so these strains are currently identified based on the phenotypic characterization of their pathogenic mechanisms. The identification of putative AIEC molecular markers could be beneficial not only from the diagnostic point of view but could also help in better understanding the determinants of AIEC pathogenicity. The objective of this study was to identify molecular markers that contribute to the screening of AIEC strains. For this, we characterized outer membrane protein (OMP) profiles in a group of AIEC strains and compared them with the commensal E. coli HS strain. Notably, we found a set of OMPs that were present in the AIEC strains but absent in the HS strain. Moreover, we developed a PCR assay and performed phylogenomic analyses to determine the frequency and distribution of the genes coding for these OMPs in a larger collection of AIEC and other E. coli strains. As result, it was found that three genes (chuA, eefC, and fitA) are widely distributed and significantly correlated with AIEC strains, whereas they are infrequent in commensal and diarrheagenic E. coli strains (DEC). Additional studies are needed to validate these markers in diverse strain collections from different geographical regions, as well as investigate their possible role in AIEC pathogenicity.

Inactivation of the Pyrimidine Biosynthesis pyrD Gene Negatively Affects Biofilm Formation and Virulence Determinants in the Crohn's Disease-Associated Adherent Invasive Escherichia coli LF82 Strain.

In Crohn’s disease (CD) patients, the adherent-invasive Escherichia coli (AIEC) pathovar contributes to the chronic inflammation typical of the disease via its ability to invade gut epithelial cells and to survive in macrophages. We show that, in the AIEC strain LF82, inactivation of the pyrD gene, encoding dihydroorotate dehydrogenase (DHOD), an enzyme of the de novo pyrimidine biosynthetic pathway, completely abolished its ability of to grow in a macrophage environment-mimicking culture medium. In addition, pyrD inactivation reduced flagellar motility and strongly affected biofilm formation by downregulating transcription of both type 1 fimbriae and curli subunit genes. Thus, the pyrD gene appears to be essential for several cellular processes involved in AIEC virulence. Interestingly, vidofludimus (VF), a DHOD inhibitor, has been proposed as an effective drug in CD treatment. Despite displaying a potentially similar binding mode for both human and E. coli DHOD in computational molecular docking experiments, VF showed no activity on either growth or virulence-related processes in LF82. Altogether, our results suggest that the crucial role played by the pyrD gene in AIEC virulence, and the presence of structural differences between E. coli and human DHOD allowing for the design of specific inhibitors, make E. coli DHOD a promising target for therapeutical strategies aiming at counteracting chronic inflammation in CD by acting selectively on its bacterial triggers.

P010 Adherent-Invasive Escherichia Coli strain O83:H1 induces inflammatory response in Crohn’s disease

Abstract Background Several studies suggest that Adherent-Invasive Escherichia Coli (AIEC) colonize the ileal mucosa of patients with Crohn’s disease (CD). This strain is able to adhere to and invade intestinal epithelial cells, and survive within macrophages, prompting an inflammatory response. The aim of this study was to investigate, by using an organ culture model, the ability of AIEC strain O83:H1 to colonize intestinal epithelial cells of CD patients, inducing chronic inflammation. Methods An organ culture model conducted with colonic biopsies derived from CD patients was set up to evaluate the ability of AIEC strain 083:H1, compared to a non-pathogenic (NP) E. Coli strain, to upregulate CEACAM 6 (CarcinoEmbryonic Antigen-related Cellular Adhesion Molecule 6), LAMP-1 (Lysosome Associated Membrane Protein 1), ICAM-1 (Intracellular Adhesion Molecule) and HLA-DR antigen expression, by immunohistochemistry, and to induce IFN-γ, TNF-α and IL-8 production, by RT-qPCR. The staining of epithelial cells that expressed CEACAM6 and LAMP1 as well as the expression of ICAM1 on blood vessels was evaluated in terms of staining intensity. The number of LAMP1 and HLA-DR lamina propria mononuclear cells (LPMNC) was evaluated within a total area of 1 mm2 of lamina propria. Results Expression of CEACAM6 on intestinal epithelial cells, and the expression of LAMP-1 either on epithelium as well as in the LPMNCs, were significantly increased (P&amp;lt;0,05) in the biopsies cultured with the AIEC strain O83:H1 compared to the biopsies cultured with NP strain. ICAM-1 and HLA-DR were significantly increased (P&amp;lt;0,05) on blood vessels and on LPMNCs, respectively, in presence of AIEC strain O83:H1 as compared with NP strain. Moreover we observed a higher level of IFN-γ, TNF-α and IL-8 mRNA trascripts in biopsies cultured with AIEC strain 083:H1 than in biopsies cultured with NP strain. Conclusion Our data suggest that AIEC strain O83:H1 in CD is able to adhere to and invade the intestinal epithelial cells, and to play an important role in activating chronic inflammatory responses observed in CD patients.

Long-read sequencing to interrogate strain-level variation among adherent-invasive Escherichia coli isolated from human intestinal tissue.

Adherent-invasive Escherichia coli (AIEC) is a pathovar linked to inflammatory bowel diseases (IBD), especially Crohn’s disease, and colorectal cancer. AIEC are genetically diverse, and in the absence of a universal molecular signature, are defined by in vitro functional attributes. The relative ability of difference AIEC strains to colonize, persist, and induce inflammation in an IBD-susceptible host is unresolved. To evaluate strain-level variation among tissue-associated E. coli in the intestines, we develop a long-read sequencing approach to identify AIEC by strain that excludes host DNA. We use this approach to distinguish genetically similar strains and assess their fitness in colonizing the intestine. Here we have assembled complete genomes using long-read nanopore sequencing for a model AIEC strain, NC101, and seven strains isolated from the intestinal mucosa of Crohn’s disease and non-Crohn’s tissues. We show these strains can colonize the intestine of IBD susceptible mice and induce inflammatory cytokines from cultured macrophages. We demonstrate that these strains can be quantified and distinguished in the presence of 99.5% mammalian DNA and from within a fecal population. Analysis of global genomic structure and specific sequence variation within the ribosomal RNA operon provides a framework for efficiently tracking strain-level variation of closely-related E. coli and likely other commensal/pathogenic bacteria impacting intestinal inflammation in experimental settings and IBD patients.

Adherent-Invasive and Non-Invasive Escherichia coli Isolates Differ in Their Effects on Caenorhabditis elegans' Lifespan.

The adherent-invasive Escherichia coli (AIEC) pathotype has been implicated in the pathogenesis of inflammatory bowel diseases in general and in Crohn’s disease (CD) in particular. AIEC strains are primarily characterized by their ability to adhere to and invade intestinal epithelial cells. However, the genetic and phenotypic features of AIEC isolates vary greatly as a function of the strain’s clonality, host factors, and the gut microenvironment. It is thus essential to identify the determinants of AIEC pathogenicity and understand their role in intestinal epithelial barrier dysfunction and inflammation. We reasoned that soil nematode Caenorhabditis elegans (a simple but powerful model of host-bacterium interactions) could be used to study the virulence of AIEC vs. non- AIEC E. coli strains. Indeed, we found that the colonization of C. elegans (strain N2) by E. coli impacted survival in a strain-specific manner. Moreover, the AIEC strains’ ability to invade cells in vitro was linked to the median lifespan in C. elegans (strain PX627). However, neither the E. coli intrinsic invasiveness (i.e., the fact for an individual strain to be characterized as invasive or not) nor AIEC’s virulence levels (i.e., the intensity of invasion, established in % from the infectious inoculum) in intestinal epithelial cells was correlated with C. elegans’ lifespan in the killing assay. Nevertheless, AIEC longevity of C. elegans might be a relevant model for screening anti-adhesion drugs and anti-invasive probiotics.

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.

A nadA Mutation Confers Nicotinic Acid Auxotrophy in Pro-carcinogenic Intestinal Escherichia coli NC101.

Inflammatory bowel diseases (IBDs) and inflammation-associated colorectal cancer (CRC) are linked to blooms of adherent-invasive Escherichia coli (AIEC) in the intestinal microbiota. AIEC are functionally defined by their ability to adhere/invade epithelial cells and survive/replicate within macrophages. Changes in micronutrient availability can alter AIEC physiology and interactions with host cells. Thus, culturing AIEC for mechanistic investigations often involves precise nutrient formulation. We observed that the pro-inflammatory and pro-carcinogenic AIEC strain NC101 failed to grow in minimal media (MM). We hypothesized that NC101 was unable to synthesize a vital micronutrient normally found in the host gut. Through nutrient supplementation studies, we identified that NC101 is a nicotinic acid (NA) auxotroph. NA auxotrophy was not observed in the other non-toxigenic E. coli or AIEC strains we tested. Sequencing revealed NC101 has a missense mutation in nadA, a gene encoding quinolinate synthase A that is important for de novo nicotinamide adenine dinucleotide (NAD) biosynthesis. Correcting the identified nadA point mutation restored NC101 prototrophy without impacting AIEC function, including motility and AIEC-defining survival in macrophages. Our findings, along with the generation of a prototrophic NC101 strain, will greatly enhance the ability to perform in vitro functional studies that are needed for mechanistic investigations on the role of intestinal E. coli in digestive disease.

Norepinephrine affects the interaction of adherent-invasive Escherichia coli with intestinal epithelial cells

ABSTRACT Norepinephrine (NE), the stress hormone, stimulates many bacterial species’ growth and virulence, including Escherichia coli. However, the hormone’s impact on the adherent-invasive E. coli (AIEC) implicated in Crohn’s disease is poorly understood. In the study, we have investigated the effect of NE on the interaction of six AIEC strains isolated from an intestinal biopsy from 6 children with Crohn’s disease with Caco-2 cells. Our study focused on type 1 fimbria and CEACAM6 molecules serving as docking sites for these adhesins. The study results demonstrated that the hormone significantly increased the adherence and invasion of AIEC to Caco-2 cells in vitro. However, the effect was not associated with the impact of NE on the increased proliferation rate of AIEC or the fimA gene expression vital for their interaction with intestinal epithelial cells. Instead, the carcinoembryonic antigen-related cell-adhesion-molecule-6 (CEACAM6) level was increased significantly in NE-treated Caco-2 cells infected with AIEC in contrast to control uninfected NE-treated cells. These results indicated that NE influenced the interaction of AIEC with intestinal epithelium by increasing the level of CEACAM6 in epithelial cells, strengthening their adherence and invasion.

Adherent invasive Escherichia coli (AIEC) strain LF82, but not Candida albicans, plays a profibrogenic role in the intestine

BackgroundIntestinal fibrosis is a frequent complication of Crohn’s disease. However, the factors that cause chronicity and promote fibrogenesis are not yet understood.AimsIn the present study, we evaluated the profibrotic effects of adherent-invasive Escherichia coli (AIEC) LF82 strain and Candida albicans in the gut.MethodsColonic fibrosis was induced in C57BL/6 mice by administration of three cycles of 2.5% (w/v) dextran sulfate sodium (DSS) for 5 weeks. LF82 and C. albicans were administered orally once at the start of each week or each cycle, respectively. Expression of markers of myofibroblast activation was determined in TGF-β1-stimulated human intestinal epithelial cells (IECs).ResultsLF82 administration exacerbated fibrosis in DSS-treated mice, revealed by increased colonic collagen deposition and expression of the profibrotic genes Col1a1, Col3a1, Fn1 and Vim. This was accompanied by enhanced gene expression of proinflammatory cytokines and chemokines, as well as more recruited inflammatory cells into the intestine. LF82 also potentiated TGF-β1-stimulated epithelial–mesenchymal transition and myofibroblast activation in IECs, by further inducing gene expression of the main mesenchymal cell markers FN1 and VIM and downregulating the IEC marker OCLN. Proinflammatory cytokines were overexpressed with LF82 in TGF-β1-stimulated IECs. Conversely, C. albicans did not affect intestinal fibrosis progression in DSS-treated mice or myofibroblast activation in TGF-β1-stimulated IECs.ConclusionsThese results demonstrate that AIEC strain LF82, but not C. albicans, may play a major profibrogenic role in the gut.

Commonality of adherent-invasive Escherichia coli isolated from patients with extraintestinal infections, healthy individuals and the environment.

Adherent-invasive Escherichia coli (AIEC) has been implicated as a microbiological factor in inflammatory bowel disease (IBD) pathogenesis. These strains are defined by their ability to adhere to and invade intestinal epithelial cells, and to survive and replicate in macrophages. We postulated that AIEC strains may commonly inhabit the gut of healthy individuals (HI), cause extraintestinal infections, and be found in sewage treatment plants (STP) and surface waters (SW). A total of 808 E. coli strains isolated from HI; patients with community-acquired urinary tract infection (CA-UTI), septicaemia and urosepsis; STP; and SW, showing a diffuse adhesion pattern to Caco-2 cells were included in this study. Typing of the strains using a combination of RAPD-PCR and PhPlate fingerprinting grouped them into 48 common clones (CCs). Representatives of each CC were tested for the ability to invade Caco-2 cells, survive and replicate in macrophages, and for the presence of six virulence genes commonly found among AIEC strains. Twenty CCs were deemed AIEC based on their ability to survive and replicate in macrophages, while encoding htrA, dsbA and clbA genes. These CCs primarily originated from HI and CA-UTI patients but were also detected in secondary locations including STP and SW. Strains lacking intramacrophagic survival and replication abilities were regarded as diffusely adhering E. coli (DAEC). Certain clones of AIEC are common in the gut of HI whilst promoting CA-UTI. The survival and persistence of AIEC in STP and SW may have serious public health ramifications for individuals predisposed to IBD.