The Crohn's disease-associated adherent-invasive Escherichia coli strain LF82 replicates in mature phagolysosomes within J774 macrophages

Adherent-invasive Escherichia coli (AIEC) bacteria isolated from Crohn's disease patients are able to extensively replicate within macrophages in large vacuoles. The mechanism by which AIEC bacteria survive within phagocytic cells is unknown. This report describes the maturation of AIEC LF82-containing phagosomes within J774 macrophages. LF82-containing phagosomes traffic through the endocytic pathway as shown by the sequential acquisition and loss of EEA1 and Rab7 and by accumulation of Lamp-1, Lamp-2 and cathepsin D. We demonstrated that AIEC LF82-containing phagosomes mature into active phagolysosomes where bacteria are exposed to low pH and to the degradative activity of cathepsin D. Finally, we showed that an acidic environment is necessary for replication of AIEC LF82 bacteria within J774 macrophages. Thus, evidence is provided that AIEC LF82 bacteria do not escape from the endocytic pathway but undergo normal interaction with host endomembrane organelles and replicate within acidic and cathepsin D-positive vacuolar phagolysosomes.     

Role of meprins to protect ileal mucosa of Crohn's disease patients from colonization by adherent-invasive E. coli

Ileal lesions in Crohn's disease (CD) patients are colonized by pathogenic adherent-invasive Escherichia coli (AIEC) able to adhere to and invade intestinal epithelial cells (IEC), and to survive within macrophages. The interaction of AIEC with IEC depends on bacterial factors mainly type 1 pili, flagella, and outer membrane proteins. In humans, proteases can act as host defence mechanisms to counteract bacterial colonization. The protease meprin, composed of multimeric complexes of the two subunits alpha and beta, is abundantly expressed in IECs. Decreased levels of this protease correlate with the severity of the inflammation in patients with inflammatory bowel disease. The aim of the present study was to analyze the ability of meprin to modulate the interaction of AIEC with IECs. In patients with ileal CD we observed decreased levels of meprins, in particular that of meprin β. Dose-dependent inhibition of the abilities of AIEC strain LF82 to adhere to and invade intestinal epithelial T84 cells was observed when bacteria were pre-treated with both exogenous meprin α and meprin β. Dose-dependent proteolytic degradation of type 1 pili was observed in the presence of active meprins, but not with heat-inactivated meprins, and pretreatment of AIEC bacteria with meprins impaired their ability to bind mannosylated host receptors and led to decreased secretion of the pro-inflammatory cytokine IL-8 by infected T84 cells. Thus, decreased levels of protective meprins as observed in CD patients may contribute to increased AIEC colonization.     

OmpC and the sigma(E) regulatory pathway are involved in adhesion and invasion of the Crohn's disease-associated Escherichia coli strain LF82

Ileal lesions of 36.4% of patients with Crohn's disease (CD), an inflammatory bowel disease in humans, are colonized by pathogenic adherent-invasive Escherichia coli (AIEC), and high levels of antibodies directed against E. coli OmpC are present in 37-55% of CD patients. We therefore investigated the expression of OmpC and its role in the interaction of CD-associated adherent-invasive E. coli strain LF82 with intestinal epithelial cells. High osmolarity induced a significant increase in the ability of LF82 bacteria to interact with Intestine-407 cells, which correlates with increased OmpC expression. Deletion of ompC gene markedly decreased the adhesion and invasion levels of the corresponding mutant. A LF82-DeltaompR mutant impaired in OmpC and OmpF expression, showed decreased adhesion and invasion, and unlike a K-12-negative OmpR mutant did not express flagella and type 1 pili. Interestingly, the wild-type phenotype was restored when OmpC or OmpF expression was induced in the LF82-DeltaompR mutant. Overexpression of RpoE in the LF82-DeltaompR isogenic mutant restored a full wild-type phenotype without restoring OmpC expression. Increased expression of RpoE was observed in wild-type strain LF82 at high osmolarity. Hence, the role of OmpC in the AIEC LF82 adhesion and invasion is indirect and involves the sigma(E) regulatory pathway.     

Crohn's disease-associated adherent-invasive E. coli are selectively favoured by impaired autophagy to replicate intracellularly

Ileal lesions in Crohn's disease (CD) patients are colonized by pathogenic adherent-invasive Escherichia coli (AIEC) able to invade and to replicate within intestinal epithelial cells. Recent genome-wide association studies have highlighted the autophagy pathway as being associated with CD risk. In the present study we investigated whether defects in autophagy enhance replication of commensal and pathogenic Escherichia coli and CD-associated AIEC. We show that functional autophagy limits intracellular AIEC replication and that a subpopulation of the intracellular bacteria is located within LC3-positive autophagosomes. In IRGM and ATG16L1 deficient cells intracellular AIEC LF82 bacteria have enhanced replication. Surprisingly autophagy deficiency did not interfere with the ability of intracellular bacteria to survive and/or replicate for any other E. coli strains tested, including non-pathogenic, environmental, commensal, or pathogenic strains involved in gastro enteritis. Together these findings demonstrate a central role for autophagy restraining Adherent-Invasive E. coli strains associated with ileal CD. AIEC infection in patients with polymorphisms in autophagy genes may have a significant impact on the outcome of intestinal inflammation.     

Adherent-invasive Escherichia coli isolated from Crohn's disease patients induce granulomas in vitro

Adherent-invasive Escherichia coli (AIEC) have been shown to be highly associated with ileal Crohn's disease (CD). AIEC survive within infected macrophages, residing within the phagolysosomal compartment where they take advantage of the low pH to replicate extensively. We investigated whether, like the tuberculous bacillus which also persists within macrophages, AIEC LF82 induces the formation of granulomas, which are a common histopathological feature of CD. For this purpose, we have taken advantage of an in vitro model of human granulomas that we recently developed, based on blood-derived mononuclear cells. We demonstrated that AIEC LF82 induces aggregation of infected macrophages, fusion of some of them to form multinucleated giant cells and subsequent recruitment of lymphocytes. Light microscopy and scanning electron microscopy analysis of the cell aggregates confirmed their granuloma features. This was further confirmed by histological analysis of granuloma sections. Noteworthy, this phenomenon can be reproduced by soluble protein extracts of AIEC LF82 coated onto beads. Although the cell aggregates not completely mimic natural CD-associated granulomas, they are very similar to early stages of epithelioid granulomas.     

Point Mutations in FimH Adhesin of Crohn's Disease-Associated Adherent-Invasive Escherichia coli Enhance Intestinal Inflammatory Response

Adherent-invasive Escherichia coli (AIEC) are abnormally predominant on Crohn''s disease (CD) ileal mucosa. AIEC reference strain LF82 adheres to ileal enterocytes via the common type 1 pili adhesin FimH and recognizes CEACAM6 receptors abnormally expressed on CD ileal epithelial cells. The fimH genes of 45 AIEC and 47 non-AIEC strains were sequenced. The phylogenetic tree based on fimH DNA sequences indicated that AIEC strains predominantly express FimH with amino acid mutations of a recent evolutionary origin - a typical signature of pathoadaptive changes of bacterial pathogens. Point mutations in FimH, some of a unique AIEC-associated nature, confer AIEC bacteria a significantly higher ability to adhere to CEACAM-expressing T84 intestinal epithelial cells. Moreover, in the LF82 strain, the replacement of fimH _LF82 (expressing FimH with an AIEC-associated mutation) with fimH _K12 (expressing FimH of commensal E. coli K12) decreased the ability of bacteria to persist and to induce severe colitis and gut inflammation in infected CEABAC10 transgenic mice expressing human CEACAM receptors. Our results highlight a mechanism of AIEC virulence evolution that involves selection of amino acid mutations in the common bacterial traits, such as FimH protein, and leads to the development of chronic inflammatory bowel disease (IBD) in a genetically susceptible host. The analysis of fimH SNPs may be a useful method to predict the potential virulence of E. coli isolated from IBD patients for diagnostic or epidemiological studies and to identify new strategies for therapeutic intervention to block the interaction between AIEC and gut mucosa in the early stages of IBD.     

Crohn's Disease-Associated Adherent-Invasive Escherichia coli Adhesion Is Enhanced by Exposure to the Ubiquitous Dietary Polysaccharide Maltodextrin

Crohn''s disease (CD) is associated with intestinal dysbiosis evidenced by an altered microbiome forming thick biofilms on the epithelium. Additionally, adherent-invasive E. coli (AIEC) strains are frequently isolated from ileal lesions of CD patients indicating a potential role for these strains in disease pathogenesis. The composition and characteristics of the host microbiome are influenced by environmental factors, particularly diet. Polysaccharides added to food as emulsifiers, stabilizers or bulking agents have been linked to bacteria-associated intestinal disorders. The escalating consumption of polysaccharides in Western diets parallels an increased incidence of CD during the latter 20^th century. In this study, the effect of a polysaccharide panel on adhesiveness of the CD-associated AIEC strain LF82 was analyzed to determine if these food additives promote disease-associated bacterial phenotypes. Maltodextrin (MDX), a polysaccharide derived from starch hydrolysis, markedly enhanced LF82 specific biofilm formation. Biofilm formation of multiple other E. coli strains was also promoted by MDX. MDX-induced E. coli biofilm formation was independent of polysaccharide chain length indicating a requirement for MDX metabolism. MDX exposure induced type I pili expression, which was required for MDX-enhanced biofilm formation. MDX also increased bacterial adhesion to human intestinal epithelial cell monolayers in a mechanism dependent on type 1 pili and independent of the cellular receptor CEACAM6, suggesting a novel mechanism of epithelial cell adhesion. Analysis of mucosa-associated bacteria from individuals with and without CD showed increased prevalence of malX, a gene essential for MDX metabolism, uniquely in the ileum of CD patients. These findings demonstrate that the ubiquitous dietary component MDX enhances E. coli adhesion and suggests a mechanism by which Western diets rich in specific polysaccharides may promote dysbiosis of gut microbes and contribute to disease susceptibility.     

A potential role of Escherichia coli pathobionts in the pathogenesis of pediatric inflammatory bowel disease

Through genomic analysis of mucosa-associated Escherichia coli strains, we found a close genetic association among isolates from pediatric inflammatory bowel disease (IBD) patients. A specific E. coli pathovar, adherent-invasive E. coli (AIEC), was found in Crohn's disease (CD) adult patients - this pathovar has enhanced adhesive and invasive properties, mainly due to the mannose-bonding FimH protein. We aimed to characterize 52 mucosa-associated E. coli strains isolated from pediatric IBD and non-IBD patients. Eleven E. coli strains, showing a strong similarity in fimH gene sequence to that of E. coli AIEC LF82, were characterized for fimH gene sequence, genomic profiling, adhesive and invasive ability, and phylogrouping. The results were compared with E. coli strains AIEC LF82 and MG1655. The 11 E. coli isolates showed 82.4% ± 1.4% fimH sequence similarity and 80.6% ± 1.3% genomic similarity to strain AIEC LF82. All these strains harbored V27A and S78N FimH mutations, as found in LF82. Nine of them belonged to the more virulent B2 and D phylogroups. Neuraminidase treatment, mimicking inflamed mucosa, enhanced adhesion of all 11 strains by 3.5-fold, but none showed invasion ability. It could be argued that the 11 selected strains could be a branch of an E. coli subpopulation (pathobionts), that could take advantage in an inflamed context because of a suitable genomic and (or) genetic backdrop.     

Adherent-invasive Escherichia coli, strain LF82 disrupts apical junctional complexes in polarized epithelia

Background  

Although bacteria are implicated in the pathogenesis of chronic inflammatory bowel diseases (IBD), mechanisms of intestinal injury and immune activation remain unclear. Identification of adherent-invasive Escherichia coli (AIEC) strains in IBD patients offers an opportunity to characterize the pathogenesis of microbial-induced intestinal inflammation in IBD. Previous studies have focused on the invasive phenotype of AIEC and the ability to replicate and survive in phagocytes. However, the precise mechanisms by which these newly identified microbes penetrate the epithelial lining remain to be clarified. Therefore, the aim of this study was to delineate the effects of AIEC, strain LF82 (serotype O83:H1) on model polarized epithelial monolayers as a contributor to intestinal injury in IBD.     

The probiotic Escherichia coli Nissle 1917 reduces pathogen invasion and modulates cytokine expression in Caco-2 cells infected with Crohn's disease-associated E. coli LF82

Increased numbers of adherent invasive Escherichia coli (AIEC) have been found in Crohn's disease (CD) patients. In this report, we investigate the potential of the probiotic Escherichia coli Nissle 1917 (EcN) to reduce features associated with AIEC pathogenicity in an already established infection with AIEC reference strain LF82.     

Type 1 pili-mediated adherence of Escherichia coli strain LF82 isolated from Crohn's disease is involved in bacterial invasion of intestinal epithelial cells

We previously characterized the invasive ability of Escherichia coli strain LF82, isolated from an ileal biopsy of a patient with Crohn's disease. In the present study, we performed TnphoA insertion mutagenesis to identify genes involved in LF82 invasion of intestinal epithelial cells. Most of the non-invasive mutants had an insertion mutation within the type 1 pili-encoding operon. Two non-invasive fim mutants, which harboured an insertion within the fimI and fimF genes, still adhered but had lost the ability to induce host cell membrane elongations at the sites of contact with the epithelial cells. Transcomplementation experiments with a fim operon cloned from E. coli K-12 restored both invasive ability and the ability to induce host cell membrane elongations. Expression of the cloned LF82 or K-12 fim operon into the non-invasive laboratory strain JM109 did not confer invasive properties. Thus, these findings showed that: (i) type 1 pili-mediated adherence is involved in LF82-induced perturbation of host cell signalling responsible for membrane elongations; (ii) native shafts are required for type 1 pilus-mediated induction of membrane elongations; (iii) this active phenomenon is a key step in the establishment of the invasive process; and (iv) type 1 pili alone are not sufficient to trigger bacterial internalization.     

Defects in autophagy favour adherent-invasive Escherichia coli persistence within macrophages leading to increased pro-inflammatory response

Ileal lesions in Crohn's disease (CD) patients are abnormally colonized by pathogenic adherent-invasive Escherichia coli (AIEC). AIEC bacteria are able to replicate within epithelial cells after lysis of the endocytic vacuole and within macrophages in a large vacuole. CD-associated polymorphisms in NOD2, ATG16L1 and IRGM affect bacterial autophagy, a crucial innate immunity mechanism. We previously determined that defects in autophagy impaired the ability of epithelial cells to control AIEC replication. AIEC behave differently within epithelial cells and macrophages and so we investigated the impact of defects in autophagy on AIEC intramacrophagic replication and pro-inflammatory cytokine response. AIEC bacteria induced the recruitment of the autophagy machinery at the site of phagocytosis, and functional autophagy limited AIEC intramacrophagic replication. Impaired ATG16L1, IRGM or NOD2 expression induced increased intramacrophagic AIEC and increased secretion of IL-6 and TNF-α in response to AIEC infection. In contrast, forced induction of autophagy decreased the numbers of intramacrophagic AIEC and pro-inflammatory cytokine release, even in a NOD2-deficient context. On the basis of our findings, we speculate that stimulating autophagy in CD patients would be a powerful therapeutic strategy to concomitantly restrain intracellular AIEC replication and slow down the inflammatory response.     

IL-17在黏附侵袭性大肠杆菌感染小鼠结肠过程中的作用机制研究

目的:评价IL-17在黏附侵袭性大肠杆菌感染小鼠结肠过程中的作用及其可能机制。方法:选择野生型及IL-17基因敲除的SPF级C57BL/6小鼠并随机分成4组,分别给予不同的处理:(1)野生小鼠+单纯蒸馏水灌胃处理组;(2)野生小鼠+E.coli LF82(1×109/CFU/只)灌胃10天处理组;(3)IL-17敲除小鼠+单纯蒸馏水灌胃处理组;(4)IL-17敲除小鼠+E.coli LF82灌胃10天处理组。从以下5个方面评价各组小鼠的炎症反应和IL-17水平:(1)组织病理评分评估炎症反应严重程度;(2)透射电镜下观察结肠上皮细胞的超微结构;(3)免疫组织化学检测结肠分泌的IL-17;(4)PCR检测小鼠结肠中IL-17m RNA表达;(5)ELISA检测结肠组织中IL-17的含量。结果:定植了E.coli LF82的IL-17敲除小鼠肠道炎症程度和超微结构损伤较野生型小鼠更加严重(P0.05)。与未经E.coli LF82处理组相比,定植了E.coli LF82的野生小鼠肠道中IL-17m RNA和IL-17含量明显升高(P0.05)。结论:IL-17在E.coli LF82在黏附侵袭结肠粘膜过程中的保护作用,IL-17是针对AIEC菌株E.coli LF82免疫的重要效应物,而结肠局部分泌增加的IL-17会改善感染的结果。