Interactions with M cells and macrophages as key steps in the pathogenesis of enterohemorrhagic Escherichia coli infections

Enterohemorrhagic Escherichia coli (EHEC) are food-borne pathogens that can cause serious infections ranging from diarrhea to hemorrhagic colitis (HC) and hemolytic-uremic syndrome (HUS). Translocation of Shiga-toxins (Stx) from the gut lumen to underlying tissues is a decisive step in the development of the infection, but the mechanisms involved remain unclear. Many bacterial pathogens target the follicle-associated epithelium, which overlies Peyer's patches (PPs), cross the intestinal barrier through M cells and are captured by mucosal macrophages. Here, translocation across M cells, as well as survival and proliferation of EHEC strains within THP-1 macrophages were investigated using EHEC O157:H7 reference strains, isogenic mutants, and 15 EHEC strains isolated from HC/HUS patients. We showed for the first time that E. coli O157:H7 strains are able to interact in vivo with murine PPs, to translocate ex vivo through murine ileal mucosa with PPs and across an in vitro human M cell model. EHEC strains are also able to survive and to produce Stx in macrophages, which induce cell apoptosis and Stx release. In conclusion, our results suggest that the uptake of EHEC by M cells and underlying macrophages in the PP may be a critical step in Stx translocation and release in vivo. A new model for EHEC infection in humans is proposed that could help in a fuller understanding of EHEC-associated diseases

Tour d'horizon du potentiel de développement de l'aquaponie en France : présentation et regard critique sur cette voie de développement alternative pour les productions piscicoles et horticoles

L'aquaponie repose sur l'intégration de process de production aquacole et hydroponique permettant le recyclage et la valorisation des nutriments émis par l'élevage aquacole par des cultures végétales. Cette démarche innovante attire à la fois les filières aquacoles, car la co-production permet de réutiliser l'eau en permanence pour l'élevage ; mais également les filières horticoles en réduisant l'emploi d'intrants chimiques dans la conduite de production végétale. Le projet APIVA® (Aquaponie Innovation Végétale et Aquaculture) vise à développer et caractériser des pilotes aquaponiques fonctionnels, d'étudier leur faisabilité économique, leur impact environnemental et la qualité des produits obtenus, tout en modélisant les flux se produisant entre les compartiments (bassins d'élevage, surfaces de culture, filtre biologique). Après une rapide présentation de l'origine de l'aquaponie, cet article vise à définir les avantages et inconvénients de cette pratique innovante, les différentes formes qu'elle peut prendre, les diverses modalités de conception envisageables, ainsi que les aspects techniques à appréhender. En France, très peu de systèmes d'aquaponie commerciale sont actifs, mais beaucoup de projets émergent depuis quelques années, notamment en milieu urbain. Des exemples d'entreprises d'aquaponie commerciale à l'international sont présentés, avant de définir une typologie des porteurs de projet français. (Résumé d'auteur

Exploring TAR–RNA aptamer loop–loop interaction by X-ray crystallography, UV spectroscopy and surface plasmon resonance

In HIV-1, trans-activation of transcription of the viral genome is regulated by an imperfect hairpin, the trans-activating responsive (TAR) RNA element, located at the 5′ untranslated end of all viral transcripts. TAR acts as a binding site for viral and cellular proteins. In an attempt to identify RNA ligands that would interfere with the virus life-cycle by interacting with TAR, an in vitro selection was previously carried out. RNA hairpins that formed kissing-loop dimers with TAR were selected [Ducongé F. and Toulmé JJ (1999) RNA, 5:1605–1614]. We describe here the crystal structure of TAR bound to a high-affinity RNA aptamer. The two hairpins form a kissing complex and interact through six Watson–Crick base pairs. The complex adopts an overall conformation with an inter-helix angle of 28.1°, thus contrasting with previously reported solution and modelling studies. Structural analysis reveals that inter-backbone hydrogen bonds between ribose 2′ hydroxyl and phosphate oxygens at the stem-loop junctions can be formed. Thermal denaturation and surface plasmon resonance experiments with chemically modified 2′-O-methyl incorporated into both hairpins at key positions, clearly demonstrate the involvement of this intermolecular network of hydrogen bonds in complex stability

Crohn's disease adherent-invasive Escherichia coli colonize and induce strong gut inflammation in transgenic mice expressing human CEACAM

Abnormal expression of CEACAM6 is observed at the apical surface of the ileal epithelium in Crohn's disease (CD) patients, and CD ileal lesions are colonized by pathogenic adherent-invasive Escherichia coli (AIEC). We investigated the ability of AIEC reference strain LF82 to colonize the intestinal mucosa and to induce inflammation in CEABAC10 transgenic mice expressing human CEACAMs. AIEC LF82 virulent bacteria, but not nonpathogenic E. coli K-12, were able to persist in the gut of CEABAC10 transgenic mice and to induce severe colitis with reduced survival rate, marked weight loss, increased rectal bleeding, presence of erosive lesions, mucosal inflammation, and increased proinflammatory cytokine expression. The colitis depended on type 1 pili expression by AIEC bacteria and on intestinal CEACAM expression because no sign of colitis was observed in transgenic mice infected with type 1 pili–negative LF82-ΔfimH isogenic mutant or in wild-type mice infected with AIEC LF82 bacteria. These findings strongly support the hypothesis that in CD patients having an abnormal intestinal expression of CEACAM6, AIEC bacteria via type 1 pili expression can colonize the intestinal mucosa and induce gut inflammation. Thus, targeting AIEC adhesion to gut mucosa represents a new strategy for clinicians to prevent and/or to treat ileal CD

Interactions with M Cells and Macrophages as Key Steps in the Pathogenesis of Enterohemorragic Escherichia coli Infections

Enterohemorrhagic Escherichia coli (EHEC) are food-borne pathogens that can cause serious infections ranging from diarrhea to hemorrhagic colitis (HC) and hemolytic-uremic syndrome (HUS). Translocation of Shiga-toxins (Stx) from the gut lumen to underlying tissues is a decisive step in the development of the infection, but the mechanisms involved remain unclear. Many bacterial pathogens target the follicle-associated epithelium, which overlies Peyer's patches (PPs), cross the intestinal barrier through M cells and are captured by mucosal macrophages. Here, translocation across M cells, as well as survival and proliferation of EHEC strains within THP-1 macrophages were investigated using EHEC O157:H7 reference strains, isogenic mutants, and 15 EHEC strains isolated from HC/HUS patients. We showed for the first time that E. coli O157:H7 strains are able to interact in vivo with murine PPs, to translocate ex vivo through murine ileal mucosa with PPs and across an in vitro human M cell model. EHEC strains are also able to survive and to produce Stx in macrophages, which induce cell apoptosis and Stx release. In conclusion, our results suggest that the uptake of EHEC by M cells and underlying macrophages in the PP may be a critical step in Stx translocation and release in vivo. A new model for EHEC infection in humans is proposed that could help in a fuller understanding of EHEC-associated diseases

Comparative genomics of Crohn's disease-associated adherent-invasive Escherichia coli

OBJECTIVE Adherent-invasive Escherichia coli (AIEC) are a leading candidate bacterial trigger for Crohn's disease (CD). The AIEC pathovar is defined by in vitro cell-line assays examining specific bacteria/cell interactions. No molecular marker exists for their identification. Our aim was to identify a molecular property common to the AIEC phenotype. DESIGN 41 B2 phylogroup E. coli strains were isolated from 36 Australian subjects: 19 patients with IBD and 17 without. Adherence/invasion assays were conducted using the I-407 epithelial cell line and survival/replication assays using the THP-1 macrophage cell line. Cytokine secretion tumour necrosis factor ((TNF)-α, interleukin (IL) 6, IL-8 and IL-10) was measured using ELISA. The genomes were assembled and annotated, and cluster analysis performed using CD-HIT. The resulting matrices were analysed to identify genes unique/more frequent in AIEC strains compared with non-AIEC strains. Base composition differences and clustered regularly interspaced palindromic repeat (CRISPR) analyses were conducted. RESULTS Of all B2 phylogroup strains assessed, 79% could survive and replicate in macrophages. Among them, 11/41 strains (5 CD, 2 UCs, 5 non-IBD) also adhere to and invade epithelial cells, a phenotype assigning them to the AIEC pathovar. The AIEC strains were phylogenetically heterogeneous. We did not identify a gene (or nucleic acid base composition differences) common to all, or the majority of, AIEC. Cytokine secretion and CRISPRs were not associated with the AIEC phenotype. CONCLUSIONS Comparative genomic analysis of AIEC and non-AIEC strains did not identify a molecular property exclusive to the AIEC phenotype. We recommend a broader approach to the identification of the bacteria-host interactions that are important in the pathogenesis of Crohn's disease.This research was supported by an Australian Academy of Science France-Australia Science Innovation Collaboration early career fellowship; a Gastroenterological Society of Australia (GESA) Clinical Research grant; and funding from the Ministère de la Recherche et de la Technologie, Inserm (UMR1071), INRA (USC-2018); and Nouveau Chercheur EPST from Conseil Régional Auvergne

Abnormally expressed ER stress response chaperone Gp96 in CD favours adherent-invasive Escherichia coli invasion

Background and aims Crohn's disease (CD) ileal lesions are colonised by pathogenic adherent-invasive Escherichia coli (AIEC) producing outer membrane vesicles (OMVs) that contribute to the bacterial invasion process. In addition, increased expression of endoplasmic reticulum (ER)-localised stress response proteins, due to ER stress, is observed in patients with CD. The expression of the ER-localised stress response protein Gp96 in patients with CD and its biological role with regards to the ability of AIEC to invade intestinal epithelial cells were analysed.Methods and results Immunohistochemistry on tissue arrays showed that, together with CEACAM6 (carcinoembryonic antigen-related cell adhesion molecule 6) or the ER stress protein Grp78, Gp96 is also strongly expressed at the apical plasma membrane of the ileal epithelial cells of 50% of patients with CD. Invasion experiments in the presence of antibodies raised against Gp96, or after transfection of Intestine-407 cells with gp96 small interfering RNA (siRNA), indicated that Gp96 is essential to promote AIEC LF82 invasion, allowing, via the recognition of the outer membrane protein OmpA, OMVs to fuse with intestinal epithelial cells.Conclusions Gp96 is overexpressed on the apical surface of ileal epithelial cells in patients with CD and acts as a host cell receptor for OMVs, promoting AIEC invasion. From the results shown here, it is speculated that AIEC could take advantage of the abnormal expression of Gp96 in patients with CD to invade the ileal mucosa

Locked nucleic acids: Promising nucleic acid analogs for therapeutic applications

Locked Nucleic Acid (LNA) is a unique nucleic-acid modification possessing very high binding affinity and excellent specificity toward complementary RNA or DNA oligonucleotides. The remarkable properties exhibited by LNA oligonucleotides have been employed in different nucleic acid-based therapeutic strategies both in vitro and in vivo. Herein, we highlight the applications of LNA nucleotides for controlling gene expression

Biofilm formation as a novel phenotypic feature of adherent-invasive Escherichia coli (AIEC)

<p>Abstract</p> <p>Background</p> <p>Crohn's disease (CD) is a high morbidity chronic inflammatory disorder of unknown aetiology. Adherent-invasive <it>Escherichia coli </it>(AIEC) has been recently implicated in the origin and perpetuation of CD. Because bacterial biofilms in the gut mucosa are suspected to play a role in CD and biofilm formation is a feature of certain pathogenic <it>E. coli </it>strains, we compared the biofilm formation capacity of 27 AIEC and 38 non-AIEC strains isolated from the intestinal mucosa. Biofilm formation capacity was then contrasted with the AIEC phenotype, the serotype, the phylotype, and the presence of virulence genes.</p> <p>Results</p> <p>Specific biofilm formation (SBF) indices were higher amongst AIEC than non-AIEC strains (P = 0.012). In addition, 65.4% of moderate to strong biofilms producers were AIEC, whereas 74.4% of weak biofilm producers were non-AIEC (P = 0.002). These data indicate that AIEC strains were more efficient biofilm producers than non-AIEC strains. Moreover, adhesion (P = 0.009) and invasion (P = 0.003) indices correlated positively with higher SBF indices. Additionally, motility (100%, P < 0.001), H1 type flagellin (53.8%, P < 0.001), serogroups O83 (19.2%, P = 0.008) and O22 (26.9%, P = 0.001), the presence of virulence genes such as <it>sfa/focDE </it>(38.5%, P = 0.003) and <it>ibeA </it>(26.9%, P = 0.017), and B2 phylotype (80.8%, P < 0.001) were frequent characteristics amongst biofilm producers.</p> <p>Conclusion</p> <p>The principal contribution of the present work is the finding that biofilm formation capacity is a novel, complementary pathogenic feature of the recently described AIEC pathovar. Characterization of AIEC specific genetic determinants, and the regulatory pathways, involved in biofilm formation will likely bring new insights into AIEC pathogenesis.</p

Increased S-nitrosylation and proteasomal degradation of caspase-3 during infection contribute to the persistence of adherent invasive escherichia coli (AIEC) in immune cells

Adherent invasive Escherichia coli (AIEC) have been implicated as a causative agent of Crohn's disease (CD) due to their isolation from the intestines of CD sufferers and their ability to persist in macrophages inducing granulomas. The rapid intracellular multiplication of AIEC sets it apart from other enteric pathogens such as Salmonella Typhimurium which after limited replication induce programmed cell death (PCD). Understanding the response of infected cells to the increased AIEC bacterial load and associated metabolic stress may offer insights into AIEC pathogenesis and its association with CD. Here we show that AIEC persistence within macrophages and dendritic cells is facilitated by increased proteasomal degradation of caspase-3. In addition S-nitrosylation of pro- and active forms of caspase-3, which can inhibit the enzymes activity, is increased in AIEC infected macrophages. This S-nitrosylated caspase-3 was seen to accumulate upon inhibition of the proteasome indicating an additional role for S-nitrosylation in inducing caspase-3 degradation in a manner independent of ubiquitination. In addition to the autophagic genetic defects that are linked to CD, this delay in apoptosis mediated in AIEC infected cells through increased degradation of caspase-3, may be an essential factor in its prolonged persistence in CD patients