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1.
BMC Microbiol ; 20(1): 178, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32576138

RESUMO

BACKGROUND: Crohn's disease (CD) is characterized by chronic inflammation of the human intestine. Several studies have demonstrated that the intestinal mucosa of CD patients in Western countries is abnormally colonized by adherent-invasive Escherichia coli (AIEC) strains. However, no studies to date have focused on the involvement of such E. coli strains in CD patients in Brazil. Here, we characterized E. coli strains associated with the ileal mucosa of Brazilian CD patients (ileal biopsies from 35 subjects, 24 CD patients and 11 controls). RESULTS: The colonization level of adherent Enterobacteriaceae associated with the ileal mucosa of CD patients was significantly higher than that of the controls. The proportions of E. coli strains belonging to phylogroups B1 and B2 were two-fold higher in strains isolated from CD patients than in those isolated from controls. CD patients in the active phase harbored 10-fold more E. coli belonging to group B2 than CD patients in remission. Only a few E. coli isolates had invasive properties and the ability to survive within macrophages, but 25% of CD patients in Brazil (6/24) harbored at least one E. coli strain belonging to the AIEC pathobiont. However, fimH sequence analysis showed only a few polymorphisms in the FimH adhesin of strains isolated in this study compared to the FimH adhesin of AIEC collections isolated from European patients. CONCLUSIONS: Mucosa-associated E. coli strains colonize the intestinal mucosa of Brazilian CD patients. However, the strains isolated from Brazilian CD patients have probably not yet co-evolved with their hosts and therefore have not fully developed a strong adherent-invasive phenotype. Thus, it will be crucial to follow in the future the emergence and evolution of AIEC pathobionts in the Brazilian population.


Assuntos
Doença de Crohn/microbiologia , Escherichia coli/classificação , Escherichia coli/isolamento & purificação , Mucosa Intestinal/microbiologia , Adesinas de Escherichia coli/genética , Adulto , Idoso , Aderência Bacteriana , Brasil , Estudos de Casos e Controles , Linhagem Celular , Escherichia coli/genética , Feminino , Proteínas de Fímbrias/genética , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Filogenia , Células THP-1
2.
Gut ; 66(8): 1382-1389, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-27196580

RESUMO

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.


Assuntos
Doença de Crohn/microbiologia , Citocinas/metabolismo , DNA Bacteriano/análise , Escherichia coli/genética , Aderência Bacteriana , Linhagem Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Células Epiteliais/microbiologia , Escherichia coli/crescimento & desenvolvimento , Infecções por Escherichia coli/complicações , Genoma , Interações Hospedeiro-Patógeno , Humanos , Interleucina-10/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiologia , Fenótipo , Filogenia , Análise de Sequência de DNA , Fator de Necrose Tumoral alfa/metabolismo
3.
Cell Microbiol ; 18(5): 617-31, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26499863

RESUMO

The aetiology of Crohn's disease (CD) involves disorders in host genetic factors and intestinal microbiota. Adherent-invasive Escherichia coli (AIEC) are receiving increased attention because in studies of mucosa-associated microbiota, they are more prevalent in CD patients than in healthy subjects. AIEC are associated both with ileal and colonic disease phenotypes. In this study, we reported a protease called Vat-AIEC from AIEC that favours the mucosa colonization. The deletion of the Vat-AIEC-encoding gene resulted in an adhesion-impaired phenotype in vitro and affected the colonization of bacteria in contact with intestinal epithelial cells in a murine intestinal loop model, and also their gut colonization in vivo. Furthermore, unlike LF82Δvat-AIEC, wild-type AIEC reference strain LF82 was able to penetrate a mucus column extensively and promoted the degradation of mucins and a decrease in mucus viscosity. Vat-AIEC transcription was stimulated by several chemical conditions found in the ileum environment. Finally, the screening of E. coli strains isolated from CD patients revealed a preferential vat-AIEC association with AIEC strains belonging to the B2 phylogroup. Overall, this study revealed a new component of AIEC virulence that might favour their implantation in the gut of CD patients.


Assuntos
Toxinas Bacterianas/genética , Doença de Crohn/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Microbioma Gastrointestinal/genética , Animais , Aderência Bacteriana/genética , Toxinas Bacterianas/metabolismo , Doença de Crohn/microbiologia , Doença de Crohn/patologia , Células Epiteliais/microbiologia , Escherichia coli/patogenicidade , Infecções por Escherichia coli/genética , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/patologia , Proteínas de Escherichia coli/metabolismo , Humanos , Íleo/microbiologia , Íleo/patologia , Mucosa Intestinal/microbiologia , Camundongos , Muco/microbiologia
4.
Gut ; 65(2): 278-85, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25588406

RESUMO

OBJECTIVE: Colorectal cancers (CRCs) are frequently colonised by colibactin toxin-producing Escherichia coli bacteria that induce DNA damage in host cells and exhibit protumoural activities. Our objective was to identify small molecules inhibiting the toxic effects induced by these colibactin-producing bacteria. DESIGN: A structural approach was adopted for the identification of a putative ligand for the ClbP enzyme involved in the synthesis of colibactin. Intestinal epithelial cells and a CRC mouse model were used to assess the activity of the selected compounds in vitro and in vivo. RESULTS: Docking experiments identified two boron-based compounds with computed ligand efficiency values (-0.8 and -0.9 kcal/mol/atom) consistent with data expected for medicinal chemistry leads. The crystalline structure of ClbP in complex with the compounds confirmed that the compounds were binding to the active site of ClbP. The two compounds (2 mM) suppressed the genotoxic activity of colibactin-producing E coli both in vitro and in vivo. The mean degree of suppression of DNA damage for the most efficient compound was 98±2% (95% CI). This compound also prevented cell proliferation and colibactin-producing E coli-induced tumourigenesis in mice. In a CRC murine model colonised by colibactin-producing E coli, the number of tumours decreased by 3.5-fold in animals receiving the compound in drinking water (p<0.01). CONCLUSIONS: These results demonstrate that targeting colibactin production controls the genotoxic and protumoural effects induced by this toxin.


Assuntos
Ácidos Borônicos/farmacologia , Neoplasias Colorretais/prevenção & controle , Escherichia coli/efeitos dos fármacos , Peptídeos/metabolismo , Policetídeos/metabolismo , Animais , Proteínas de Ligação ao Cálcio/farmacologia , Neoplasias Colorretais/microbiologia , Dano ao DNA/fisiologia , Escherichia coli/metabolismo , Ligantes , Camundongos , Camundongos Endogâmicos BALB C , Mutagênicos
5.
Gut ; 64(3): 428-37, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24898815

RESUMO

OBJECTIVE: Adherent-invasive Escherichia coli (AIEC) are abnormally predominant on Crohn's disease (CD) ileal mucosa. AIEC strains adhere to enterocytes via interaction between type 1 pili and CEACAM6 receptors abnormally expressed on CD ileal mucosa, leading to gut inflammation. We analysed whether epigenetic mechanisms are involved in the upregulation of CEACAM6 expression in intestinal epithelial cells (IECs). DESIGN: Methylation of CEACAM6 promoter was analysed using bisulfite sequencing and site-specific methylation by SnapShot. pCpGfree reporter system was used to analyse CEACAM6 promoter activity. Transgenic mice expressing human CEACAM6 fed either standard food or a low-methyl diet (LMD) were orally challenged with 10(9) AIEC LF82. After 3 days, gut-associated AIEC and proinflammatory cytokines were quantified. RESULTS: Analysis of CEACAM6 gene promoter revealed potentially methylated dinucleotide CpGs within HIF-1-responsive elements (HREs). Methylation levels of CpG within CEACAM6 promoter were inversely correlated with CEACAM6 expression in IEC expressing various levels of CEACAM6. We show the critical role of HRE methylation and transcription factor HIF-1 in the regulation of CEACAM6 gene in IEC. This was confirmed in transgenic mice expressing human CEACAM6 fed a LMD. LMD-dependent HRE demethylation led to abnormal gut expression of CEACAM6, favouring AIEC colonisation and subsequent inflammation. CONCLUSIONS: HRE hypomethylation in CEACAM6 promoter correlates with high expression in IEC. Our findings suggest that abnormal DNA methylation leading to CEACAM6 increased expression and AIEC-mediated gut inflammation can be related to changes in nutritional habits, such as low intake in methyl donor molecules, leading to abnormal epigenetic marks in mouse model mimicking CD susceptibility.


Assuntos
Antígenos CD/metabolismo , Moléculas de Adesão Celular/metabolismo , Doença de Crohn/etiologia , Dieta/efeitos adversos , Infecções por Escherichia coli/complicações , Proteínas Ligadas por GPI/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Animais , Antígenos CD/fisiologia , Células CACO-2 , Moléculas de Adesão Celular/fisiologia , Doença de Crohn/metabolismo , Doença de Crohn/microbiologia , Metilação de DNA , Epigênese Genética , Infecções por Escherichia coli/metabolismo , Proteínas Ligadas por GPI/fisiologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Camundongos , Camundongos Transgênicos
6.
J Bacteriol ; 197(8): 1451-65, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25666140

RESUMO

UNLABELLED: Ileal lesions of patients with Crohn's disease are colonized by adherent-invasive Escherichia coli (AIEC), which is able to adhere to and to invade intestinal epithelial cells (IEC), to replicate within macrophages, and to form biofilms on the surface of the intestinal mucosa. Previous analyses indicated the involvement of the σ(E) pathway in AIEC-IEC interaction, as well as in biofilm formation, with σ(E) pathway inhibition leading to an impaired ability of AIEC to colonize the intestinal mucosa and to form biofilms. The aim of this study was to characterize the σ(E) regulon of AIEC strain LF82 in order to identify members involved in AIEC phenotypes. Using comparative in silico analysis of the σ(E) regulon, we identified the waaWVL operon as a new member of the σ(E) regulon in reference AIEC strain LF82. We determined that the waaWVL operon is involved in AIEC lipopolysaccharide structure and composition, and the waaWVL operon was found to be essential for AIEC strains to produce biofilm and to colonize the intestinal mucosa. IMPORTANCE: An increased prevalence of adherent-invasive Escherichia coli (AIEC) bacteria was previously observed in the intestinal mucosa of Crohn's disease (CD) patients, and clinical observations revealed bacterial biofilms associated with the mucosa of CD patients. Here, analysis of the σ(E) regulon in AIEC and commensal E. coli identified 12 genes controlled by σ(E) only in AIEC. Among them, WaaWVL factors were found to play an essential role in biofilm formation and mucosal colonization by AIEC. In addition to identifying molecular tools that revealed a pathogenic population of E. coli colonizing the mucosa of CD patients, these results indicate that targeting the waaWVL operon could be a potent therapeutic strategy to interfere with the ability of AIEC to form biofilms and to colonize the gut mucosa.


Assuntos
Biofilmes , Doença de Crohn/microbiologia , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Fator sigma/metabolismo , Aderência Bacteriana/fisiologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Humanos , Família Multigênica , Óperon , Regulon , Fator sigma/genética
7.
Infect Immun ; 83(4): 1305-17, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25605769

RESUMO

A critical step in the life cycle of all organisms is the duplication of the genetic material during cell division. Ribonucleotide reductases (RNRs) are essential enzymes for this step because they control the de novo production of the deoxyribonucleotides required for DNA synthesis and repair. Enterobacteriaceae have three functional classes of RNRs (Ia, Ib, and III), which are transcribed from separate operons and encoded by the genes nrdAB, nrdHIEF, and nrdDG, respectively. Here, we investigated the role of RNRs in the virulence of adherent-invasive Escherichia coli (AIEC) isolated from Crohn's disease (CD) patients. Interestingly, the LF82 strain of AIEC harbors four different RNRs (two class Ia, one class Ib, and one class III). Although the E. coli RNR enzymes have been extensively characterized both biochemically and enzymatically, little is known about their roles during bacterial infection. We found that RNR expression was modified in AIEC LF82 bacteria during cell infection, suggesting that RNRs play an important role in AIEC virulence. Knockout of the nrdR and nrdD genes, which encode a transcriptional regulator of RNRs and class III anaerobic RNR, respectively, decreased AIEC LF82's ability to colonize the gut mucosa of transgenic mice that express human CEACAM6 (carcinoembryonic antigen-related cell adhesion molecule 6). Microarray experiments demonstrated that NrdR plays an indirect role in AIEC virulence by interfering with bacterial motility and chemotaxis. Thus, the development of drugs targeting RNR classes, in particular NrdR and NrdD, could be a promising new strategy to control gut colonization by AIEC bacteria in CD patients.


Assuntos
Antígenos CD/biossíntese , Aderência Bacteriana/genética , Moléculas de Adesão Celular/biossíntese , Quimiotaxia/genética , Proteínas de Escherichia coli/genética , Escherichia coli/patogenicidade , Animais , Antígenos CD/genética , Moléculas de Adesão Celular/genética , Doença de Crohn/microbiologia , Escherichia coli/genética , Escherichia coli/imunologia , Infecções por Escherichia coli/genética , Infecções por Escherichia coli/patologia , Proteínas de Escherichia coli/biossíntese , Proteínas Ligadas por GPI/biossíntese , Proteínas Ligadas por GPI/genética , Regulação Bacteriana da Expressão Gênica , Humanos , Mucosa Intestinal/microbiologia , Masculino , Camundongos , Camundongos Transgênicos , Análise em Microsséries , Regiões Promotoras Genéticas , Ribonucleotídeo Redutases/genética , Fatores de Virulência/biossíntese , Fatores de Virulência/genética
8.
Lab Invest ; 95(3): 296-307, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25545478

RESUMO

Intestinal dysbiosis has been reported in patients with colorectal cancer, and there is a high prevalence of Escherichia coli belonging to B2 phylogroup and producing a genotoxin, termed colibactin. Macrophages are one of the predominant tumor-infiltrating immune cells supporting key processes in tumor progression by producing protumoral factors such as cyclooxygenase-2 (COX-2). Here, we investigated whether B2 E. coli colonizing colon tumors could influence protumoral activities of macrophages. In contrast to commensal or nonpathogenic E. coli strains that were efficiently and rapidly degraded by macrophages at 24 h after infection, colon cancer-associated E. coli were able to resist killing by human THP-1 macrophages, to replicate intracellularly, and to persist inside host cells until at least 72 h after infection. Significant increases in COX-2 expression were observed in macrophages infected with colon cancer E. coli compared with macrophages infected with commensal and nonpathogenic E. coli strains or uninfected cells at 72 h after infection. Induction of COX-2 expression required live bacteria and was not due to colibactin production, as similar COX-2 levels were observed in macrophages infected with the wild-type colon cancer-associated E. coli 11G5 strain or a clbQ mutant unable to produce colibactin. Treatment of macrophages with ofloxacin, an antibiotic with intracellular tropism, efficiently decreased the number of intracellular bacteria and suppressed bacteria-induced COX-2 expression. This study provides new insights into the understanding of how tumor- infiltrating bacteria could influence cancer progression through their interaction with immune cells. Manipulation of microbes associated with tumors could have a deep influence on the secretion of protumoral molecules by infiltrating macrophages.


Assuntos
Ciclo-Oxigenase 2/imunologia , Escherichia coli/imunologia , Macrófagos/imunologia , Viabilidade Microbiana/imunologia , Linhagem Celular Tumoral , Neoplasias do Colo/imunologia , Neoplasias do Colo/microbiologia , Neoplasias do Colo/patologia , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/imunologia , Dinoprostona/metabolismo , Escherichia coli/genética , Escherichia coli/fisiologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Immunoblotting , Macrófagos/metabolismo , Macrófagos/microbiologia , Viabilidade Microbiana/genética , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Peptídeos/genética , Peptídeos/imunologia , Peptídeos/metabolismo , Policetídeos/imunologia , Policetídeos/metabolismo , Vacúolos/microbiologia , Vacúolos/ultraestrutura , Proteínas Quinases p38 Ativadas por Mitógeno/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
9.
Gastroenterology ; 146(2): 508-19, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24148619

RESUMO

BACKGROUND & AIMS: Levels of microRNAs are altered in intestinal tissues of patients with Crohn's disease (CD). The adherent-invasive Escherichia coli (AIEC), which colonize the ileal mucosa of patients with CD, adhere to and invade intestinal epithelial cells. We investigated the mechanism by which AIEC infection alters the expression of microRNAs and the host immune response. METHODS: Levels of microRNAs in human intestinal epithelial T84 cells and in mouse enterocytes were measured using quantitative reverse-transcription polymerase chain reaction. Luciferase assays were used to measure binding of microRNAs to the 3'-untranslated region of messenger RNA targets. Binding of nuclear factor-κB to promoters of genes encoding microRNAs was assessed by chromatin immunoprecipitation assays. Autophagy was measured by immunoblot analyses and immunofluorescent labeling of LC3. Anti-microRNAs were transferred to mice using ileal loops. Biopsy specimens from the terminal ileum of patients with ulcerative colitis (n = 20), CD (n = 20), or individuals without inflammatory bowel disease undergoing surveillance colonoscopies (controls, n = 13) were collected during endoscopic examination. RESULTS: AIEC infection up-regulated levels of microRNA (MIR) 30C and MIR130A in T84 cells and in mouse enterocytes by activating nuclear factor-κB. Up-regulation of these microRNAs reduced the levels of ATG5 and ATG16L1 and inhibited autophagy, leading to increased numbers of intracellular AIEC and an increased inflammatory response. In ileal biopsy samples of patients with CD, there was an inverse correlation between levels of MIR30C and MIR130A and those of ATG5 and ATG16L1, supporting in vitro findings. Inhibition of MIR30C and MIR130A in cultured intestinal epithelial cells and in mouse enterocytes blocked AIEC-induced inhibition of ATG5 and ATG16L1 expression and restored functional autophagy. This resulted in more effective clearance of intracellular AIEC and reduced AIEC-induced inflammation. CONCLUSIONS: Infection with AIEC up-regulates microRNAs to reduce expression of proteins required for autophagy and autophagy response in intestinal epithelial cells. Ileal samples from patients with CD have increased levels of these same microRNAs and reduced levels of ATG5 and ATG16L1.


Assuntos
Autofagia/fisiologia , Doença de Crohn/microbiologia , Infecções por Escherichia coli/metabolismo , Ileíte/microbiologia , Íleo/metabolismo , Mucosa Intestinal/metabolismo , MicroRNAs/metabolismo , Animais , Proteína 5 Relacionada à Autofagia , Proteínas Relacionadas à Autofagia , Biomarcadores/metabolismo , Biópsia , Western Blotting , Proteínas de Transporte/metabolismo , Linhagem Celular , Colite Ulcerativa/metabolismo , Colite Ulcerativa/microbiologia , Colite Ulcerativa/patologia , Doença de Crohn/metabolismo , Doença de Crohn/patologia , Infecções por Escherichia coli/etiologia , Infecções por Escherichia coli/patologia , Humanos , Ileíte/metabolismo , Ileíte/patologia , Íleo/microbiologia , Íleo/patologia , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , NF-kappa B/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para Cima
10.
PLoS Pathog ; 9(1): e1003141, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23358328

RESUMO

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.


Assuntos
Adesinas de Escherichia coli/genética , Doença de Crohn/microbiologia , Infecções por Escherichia coli/microbiologia , Escherichia coli/patogenicidade , Proteínas de Fímbrias/genética , Mutação Puntual , Adesinas de Escherichia coli/metabolismo , Animais , Antígenos CD/metabolismo , Aderência Bacteriana , Moléculas de Adesão Celular/metabolismo , Linhagem Celular Tumoral , Colite/metabolismo , Colite/patologia , Doença de Crohn/metabolismo , Enterócitos/metabolismo , Enterócitos/microbiologia , Enterócitos/patologia , Escherichia coli/genética , Escherichia coli/metabolismo , Infecções por Escherichia coli/metabolismo , Proteínas de Fímbrias/metabolismo , Proteínas Ligadas por GPI/metabolismo , Genes Bacterianos/genética , Humanos , Íleo/metabolismo , Íleo/microbiologia , Íleo/patologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Masculino , Camundongos , Camundongos Transgênicos , Fatores de Virulência
11.
Biomacromolecules ; 16(6): 1827-36, 2015 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-25961760

RESUMO

n-Heptyl α-d-mannose (HM) is a nanomolar antagonist of FimH, a virulence factor of E. coli. Herein we report on the construction of multivalent HM-based glycopolymers as potent antiadhesives of type 1 piliated E. coli. We investigate glycopolymer/FimH and glycopolymer/bacteria interactions and show that HM-based glycopolymers efficiently inhibit bacterial adhesion and disrupt established cell-bacteria interactions in vitro at very low concentration (0.1 µM on a mannose unit basis). On a valency-corrected basis, HM-based glycopolymers are, respectively, 10(2) and 10(6) times more potent than HM and d-mannose for their capacity to disrupt the binding of adherent-invasive E. coli to T84 intestinal epithelial cells. Finally, we demonstrate that the antiadhesive capacities of HM-based glycopolymers are preserved ex vivo in the colonic loop of a transgenic mouse model of Crohn's disease. All together, these results underline the promising scope of HM-based macromolecular ligands for the antiadhesive treatment of E. coli induced inflammatory bowel diseases.


Assuntos
Proteínas de Fímbrias/antagonistas & inibidores , Mucosa Intestinal/efeitos dos fármacos , Polissacarídeos Bacterianos/farmacologia , Adesinas de Escherichia coli , Animais , Adesão Celular/efeitos dos fármacos , Escherichia coli/patogenicidade , Células HeLa , Heptanol/química , Humanos , Doenças Inflamatórias Intestinais/microbiologia , Manose/química , Camundongos , Polissacarídeos Bacterianos/química
12.
Gut ; 63(1): 116-24, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23598352

RESUMO

OBJECTIVE: Western diet is a risk factor for Crohn's disease (CD). Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is abnormally expressed in CD patients. This allows adherent-invasive Escherichia coli (AIEC) to colonise the gut mucosa and leads to inflammation. We assessed the effects of a high fat/high sugar (HF/HS) Western diet on gut microbiota composition, barrier integrity and susceptibility to infection in transgenic CEABAC10 mice expressing human CEACAMs. DESIGN: Colonic microbiota composition and susceptibility of CEABAC10 mice to AIEC LF82 bacteria infection were determined in mice fed a conventional or HF/HS diet. Barrier function and inflammatory response were assessed by studying intestinal permeability, tight junction protein and mucin expression and localisation, and by determining histological score and levels of cytokine release. RESULTS: HF/HS diet led to dysbiosis in WT and transgenic CEABAC10 mice, with a particular increase in E coli population in HF/HS-fed CEABAC10 mice. These mice showed decreased mucus layer thickness, increased intestinal permeability, induction of Nod2 and Tlr5 gene transcription, and increased TNFα secretion. These modifications led to a higher ability of AIEC bacteria to colonise the gut mucosa and to induce inflammation. CONCLUSIONS: Western diet induces changes in gut microbiota composition, alters host homeostasis and promotes AIEC gut colonisation in genetically susceptible mice. These results support the multifactorial aetiology of CD and highlight the importance of diet in CD pathogenesis.


Assuntos
Colo/microbiologia , Dieta Hiperlipídica/efeitos adversos , Sacarose Alimentar/efeitos adversos , Disbiose/etiologia , Escherichia coli/fisiologia , Interações Hospedeiro-Patógeno , Mucosa Intestinal/microbiologia , Animais , Biomarcadores/metabolismo , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Colo/metabolismo , Colo/patologia , Doença de Crohn/etiologia , Citocinas/metabolismo , Disbiose/metabolismo , Disbiose/microbiologia , Disbiose/patologia , Infecções por Escherichia coli/etiologia , Feminino , Predisposição Genética para Doença , Homeostase , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microbiota
13.
Gut ; 63(12): 1932-42, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24658599

RESUMO

BACKGROUND: Escherichia coli strains harbouring the pks island (pks+ E. coli) are often seen in human colorectal tumours and have a carcinogenic effect independent of inflammation in an AOM/IL-10(-/-) (azoxymethane/interleukin) mouse model. OBJECTIVE: To investigate the mechanism sustaining pks+ E. coli-induced carcinogenesis. METHOD: Underlying cell processes were investigated in vitro and in vivo (xenograft model) using intestinal epithelial cells infected by pks+ E. coli or by an isogenic mutant defective for pks (pks- E. coli). The results were supported by data obtained from an AOM/DSS (azoxymethane/dextran sodium sulphate) colon cancer mouse model and from human colon cancer biopsy specimens colonised by pks+ E. coli or pks- E. coli. RESULTS: Colibactin-producing E. coli enhanced tumour growth in both xenograft and AOM/DSS models. Growth was sustained by cellular senescence (a direct consequence of small ubiquitin-like modifier (SUMO)-conjugated p53 accumulation), which was accompanied by the production of hepatocyte growth factor (HGF). The underlying mechanisms involve microRNA-20a-5p, which targets SENP1, a key protein regulating p53 deSUMOylation. These results are consistent with the expression of SENP1, microRNA-20a-5p, HGF and phosphorylation of HGF receptor found in human and mouse colon cancers colonised by pks+ E. coli. CONCLUSION: These data reveal a new paradigm for carcinogenesis, in which colibactin-induced senescence has an important role.


Assuntos
Carcinogênese/metabolismo , Neoplasias do Colo , Escherichia coli , Peptídeos/genética , Animais , Senescência Celular , Neoplasias do Colo/microbiologia , Neoplasias do Colo/patologia , Cisteína Endopeptidases , Endopeptidases/metabolismo , Escherichia coli/genética , Escherichia coli/patogenicidade , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Camundongos , Mutagênicos , Mutação , Neoplasias Experimentais , Proteínas Nucleares/metabolismo , Policetídeos , Proteínas Proto-Oncogênicas c-met
14.
Gut ; 63(8): 1265-74, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24092863

RESUMO

OBJECTIVE: Altered microbiota composition, changes in immune responses and impaired intestinal barrier functions are observed in IBD. Most of these features are controlled by proteases and their inhibitors to maintain gut homeostasis. Unrestrained or excessive proteolysis can lead to pathological gastrointestinal conditions. The aim was to validate the identified protease IBD candidates from a previously performed systematic review through a genetic association study and functional follow-up. DESIGN: We performed a genetic association study in a large multicentre cohort of patients with Crohn's disease (CD) and UC from five European IBD referral centres in a total of 2320 CD patients, 2112 UC patients and 1796 healthy controls. Subsequently, we did an extensive functional assessment of the candidate genes to explore their causality in IBD pathogenesis. RESULTS: Ten single nucleotide polymorphisms (SNPs) in four genes were significantly associated with CD: CYLD, USP40, APEH and USP3. CYLD was the most significant gene with the intronically located rs12324931 the strongest associated SNP (p(FDR)=1.74e-17, OR=2.24 (1.83 to 2.74)). Five SNPs in four genes were significantly associated with UC: USP40, APEH, DAG1 and USP3. CYLD, as well as some of the other associated genes, is part of the ubiquitin proteasome system (UPS). We therefore determined if the IBD-associated adherent-invasive Escherichia coli (AIEC) can modulate the UPS functioning. Infection of intestinal epithelial cells with the AIEC LF82 reference strain modulated the UPS turnover by reducing poly-ubiquitin conjugate accumulation, increasing 26S proteasome activities and decreasing protein levels of the NF-κB regulator CYLD. This resulted in IκB-α degradation and NF-κB activation. This activity was very important for the pathogenicity of AIEC since decreased CYLD resulted in increased ability of AIEC LF82 to replicate intracellularly. CONCLUSIONS: Our results reveal the UPS, and CYLD specifically, as an important contributor to IBD pathogenesis, which is favoured by both genetic and microbial factors.


Assuntos
Colite Ulcerativa/genética , Doença de Crohn/genética , Células Epiteliais/enzimologia , Proteínas Supressoras de Tumor/metabolismo , Aderência Bacteriana , Estudos de Casos e Controles , Sobrevivência Celular , Células Cultivadas , Colite Ulcerativa/enzimologia , Colite Ulcerativa/microbiologia , Doença de Crohn/enzimologia , Doença de Crohn/microbiologia , Enzima Desubiquitinante CYLD , Distroglicanas/genética , Células Epiteliais/microbiologia , Escherichia coli/patogenicidade , Estudos de Associação Genética , Humanos , Proteínas I-kappa B/metabolismo , Mucosa Intestinal/microbiologia , NF-kappa B/metabolismo , Peptídeo Hidrolases/genética , Polimorfismo de Nucleotídeo Único , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Supressoras de Tumor/genética , Ubiquitina Tiolesterase/genética , Proteases Específicas de Ubiquitina/genética
15.
Gastroenterology ; 145(3): 602-12.e9, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23684751

RESUMO

BACKGROUND & AIMS: Inducible chitinase 3-like-1 is expressed by intestinal epithelial cells (IECs) and adheres to bacteria under conditions of inflammation. We performed a structure-function analysis of the chitin-binding domains encoded by the chiA gene, which mediates the pathogenic effects of adherent invasive Escherichia coli (AIEC). METHODS: We created AIEC (strain LF82) with deletion of chiA (LF82-ΔchiA) or that expressed chiA with specific mutations. We investigated the effects of infecting different IEC lines with these bacteria compared with nonpathogenic E coli; chitinase activities were measured using the colloidal chitin-azure method. Colitis was induced in C57/Bl6 mice by administration of dextran sodium sulfate, and mice were given 10(8) bacteria for 15 consecutive days by gavage. Stool/tissue samples were collected and analyzed. RESULTS: LF82-ΔchiA had significantly less adhesion to IEC lines than LF82. Complementation of LF82-ΔchiA with the LF82 chiA gene, but not chiA from nonpathogenic (K12) E coli, increased adhesion. We identified 5 specific polymorphisms in the chitin-binding domain of LF82 chiA (at amino acids 362, 370, 378, 388, and 548) that differ from chiA of K12 and were required for LF82 to interact directly with IECs. This interaction was mediated by an N-glycosylated asparagine in chitinase 3-like-1 (amino acid 68) on IECs. Mice infected with LF82, or LF82-ΔchiA complemented with LF82 chiA, developed more severe colitis after administration of dextran sodium sulfate than mice infected with LF82-ΔchiA or LF82 that expressed mutant forms of chiA. CONCLUSIONS: AIEC adheres to an N-glycosylated chitinase 3-like-1 on IECs via the chitin-binding domain of chiA. This mechanism promotes the pathogenic effects of AIEC in mice with colitis.


Assuntos
Adesinas de Escherichia coli/metabolismo , Aderência Bacteriana/fisiologia , Quitinases/metabolismo , Colite/microbiologia , Células Epiteliais/microbiologia , Escherichia coli/patogenicidade , Mucosa Intestinal/microbiologia , Adesinas de Escherichia coli/química , Adesinas de Escherichia coli/genética , Animais , Aderência Bacteriana/genética , Biomarcadores/metabolismo , Linhagem Celular , Proteína 1 Semelhante à Quitinase-3 , Quitinases/química , Quitinases/genética , Colite/induzido quimicamente , Colite/enzimologia , Sulfato de Dextrana , Células Epiteliais/enzimologia , Escherichia coli/enzimologia , Escherichia coli/genética , Infecções por Escherichia coli/enzimologia , Infecções por Escherichia coli/microbiologia , Glicoproteínas/metabolismo , Glicosilação , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Polimorfismo Genético
16.
J Bacteriol ; 195(1): 76-84, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23104802

RESUMO

Ileal lesions of patients with Crohn's disease are colonized by adherent-invasive Escherichia coli (AIEC) bacteria that are able to adhere to and invade intestinal epithelial cells (IEC), to replicate within macrophages, and to form biofilm. Clinical observations showed that bacterial biofilms were associated with the mucosa of inflammatory bowel disease patients. In the present study, we analyzed the relationship between AIEC colonization of the gut and the formation of biofilm, focusing on the involvement of the σ(E) pathway in the AIEC-IEC interaction. We observed that σ(E) pathway inhibition in AIEC reference strain LF82 led to an impaired ability to adhere to and invade IEC but also induced a large decrease in the abilities to colonize the intestinal mucosa and form biofilm. This indicates that targeting of the σ(E) pathway could be a very potent therapeutic strategy by which to interfere with the ability of AIEC to form biofilm on the gut mucosa of Crohn's disease patients.


Assuntos
Biofilmes/crescimento & desenvolvimento , Doença de Crohn/microbiologia , Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiologia , Fator sigma/metabolismo , Animais , Aderência Bacteriana/fisiologia , Linhagem Celular , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Humanos , Masculino , Camundongos , Mutação , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator sigma/genética , Transdução de Sinais
17.
Environ Microbiol ; 15(2): 355-71, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22789019

RESUMO

Ileal lesions of patients with Crohn's disease are colonized by adherent-invasive Escherichia coli (AIEC). The earliest lesions of recurrent Crohn's disease are erosions of Peyer's patches (PP). We recently reported the presence of a functional lpf operon in AIEC, encoding long polar fimbriae (LPF), that allows AIEC bacteria to interact with PP and to translocate across M cells. The aim of this study was to analyse the effect of gastrointestinal conditions on LPF expression in AIEC strains. The LF82 bacterial growth in an acid pH medium or at high osmolarity medium had no effect on lpf transcription level, in contrast to bacterial growth in the presence of bile salts, which promoted activation of lpf transcription. When cultured in the presence of bile salt, LF82 wild-type bacteria, but not the isogenic mutant deleted for lpfA, exhibited a higher level of interaction with PP and a higher level of translocation through M cell monolayers. The FhlA transcriptional factor was found to be a key bacterial regulator at the origin of LPF expression in the presence of bile salts.


Assuntos
Ácidos e Sais Biliares/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/fisiologia , Fímbrias Bacterianas/genética , Regulação Bacteriana da Expressão Gênica , Nódulos Linfáticos Agregados/microbiologia , Animais , Aderência Bacteriana/fisiologia , Sequência de Bases , Células CACO-2 , Doença de Crohn/microbiologia , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Infecções por Escherichia coli/patologia , Proteínas de Escherichia coli/metabolismo , Fímbrias Bacterianas/metabolismo , Humanos , Mucosa Intestinal/microbiologia , Masculino , Camundongos , Dados de Sequência Molecular , Óperon , Regiões Promotoras Genéticas/genética , Transativadores/metabolismo
18.
J Antimicrob Chemother ; 68(7): 1558-61, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23447140

RESUMO

OBJECTIVES: Bacteria multiresistant to antibiotics are widely supposed to be weakly virulent. However, the virulence traits of carbapenem-resistant Enterobacteriaceae have not been investigated. In this work, we investigated the virulence and resistance mechanism of an extraintestinal pathogenic Escherichia coli (ExPEC) strain (LEB15) that exhibited decreased susceptibility to carbapenems. METHODS: The MICs were determined by a microdilution method. The ß-lactamase-encoding gene was identified by PCR and sequencing, and the genetic environment was analysed by PFGE and PCR mapping. The genetic background was investigated by multilocus sequence typing (MLST). Virulence-factor-encoding genes and pathogenic islands (PAIs) were detected by multiplex PCR. Virulence was assessed in a mouse sepsis model. RESULTS: Strain LEB15 produced a chromosomal OXA-48 carbapenemase. The complete bla(OXA-48)-encoding Tn1999.2 transposon was inserted in the LEB15 chromosome. The strain belonged to an MLST cluster of emerging ExPEC strains (ST-127/ST-22). It had a high pathogenic score and eight PAIs (I536, II536, III536, IV536, VI536, I(CFT073), II(CFT073) and II(J96)) and induced an unusually high lethality in the mouse sepsis model. CONCLUSIONS: Strain LEB15 combines both an atypical broad accumulation of virulence factors, which confers a strong killer phenotype, and a decrease in susceptibility to carbapenems following the chromosomal acquisition of bla(OXA-48). This association of virulence and carbapenemase in E. coli strains might pose major problems in the future for E. coli infection management.


Assuntos
Proteínas de Bactérias/genética , Cromossomos Bacterianos , Proteínas de Escherichia coli/genética , Escherichia coli/enzimologia , Escherichia coli/patogenicidade , Genes Bacterianos , Fatores de Virulência/genética , beta-Lactamases/genética , Animais , Antibacterianos/farmacologia , DNA Bacteriano/química , DNA Bacteriano/genética , Modelos Animais de Doenças , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/patologia , Feminino , Humanos , Recém-Nascido , Masculino , Camundongos , Testes de Sensibilidade Microbiana , Tipagem Molecular , Reação em Cadeia da Polimerase , Sepse/microbiologia , Sepse/patologia , Análise de Sequência de DNA
19.
Cell Microbiol ; 14(6): 791-807, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22309232

RESUMO

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.


Assuntos
Autofagia , Citocinas/metabolismo , Escherichia coli/fisiologia , Mediadores da Inflamação/metabolismo , Macrófagos/microbiologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas Relacionadas à Autofagia , Aderência Bacteriana , Carga Bacteriana , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Sobrevivência Celular , Doença de Crohn/imunologia , Doença de Crohn/microbiologia , Doença de Crohn/patologia , Escherichia coli/imunologia , Escherichia coli/metabolismo , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/patologia , Feminino , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Técnicas de Silenciamento de Genes , Proteínas de Fluorescência Verde/biossíntese , Interações Hospedeiro-Patógeno , Humanos , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/fisiologia , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/metabolismo , Proteína Adaptadora de Sinalização NOD2/deficiência , Proteína Adaptadora de Sinalização NOD2/genética , Proteína Adaptadora de Sinalização NOD2/metabolismo , Interferência de RNA , Proteínas Recombinantes/biossíntese , Proteína Sequestossoma-1
20.
Lab Invest ; 92(3): 411-9, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22042084

RESUMO

Adherent and invasive Escherichia coli (AIEC) associated with Crohn's disease are able to survive and to replicate extensively in active phagolysosomes within macrophages. AIEC-infected macrophages release large amounts of tumour necrosis factor-alpha (TNF-α) and do not undergo cell death. The aim of the present study was to determine what benefit AIEC bacteria could gain from inducing the release of large amounts of TNF-α by infected macrophages and to what extent the neutralization of TNF-α could affect AIEC intramacrophagic replication. Our results showed that the amount of TNF-α released by infected macrophages is correlated with the load of intramacrophagic AIEC bacteria and their intracellular replication. TNF-α secretion was not related to the number of bacteria entering host cells because when the number of bacteria internalized in macrophage was decreased by blocking lipid raft-dependent and clathrin-coated pits-dependent endocytosis, the amount of TNF-α secreted by infected macrophages was not modified. Interestingly, dose-dependent increases in the number of intracellular AIEC LF82 bacteria were observed when infected macrophages were stimulated with exogenous TNF-α, and neutralization of TNF-α secreted by AIEC-infected macrophages using anti-TNF-α antibodies induced a significant decrease in the number of intramacrophagic bacteria. These results indicate that AIEC bacteria use TNF-α as a Trojan horse to ensure their intracellular replication because replication of AIEC bacteria within macrophages induces the release of TNF-α, which in turn increases the intramacrophagic replication of AIEC. Neutralizing TNF-α secreted by infected macrophages may represent an effective strategy to control AIEC intracellular replication.


Assuntos
Doença de Crohn/microbiologia , Escherichia coli Enteropatogênica/fisiologia , Interações Hospedeiro-Patógeno , Macrófagos/microbiologia , Fator de Necrose Tumoral alfa/metabolismo , Animais , Linhagem Celular , Endocitose , Macrófagos/metabolismo , Camundongos
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