- Email: [email protected]
Sa1804 Quorum Sensing Driven by N-Acyl-Homoserine Lactone in Inflammatory Bowel Diseases Associated Dysbiosis
in vivo. It is our hope that these will yield novel targets for therapeutic intervention in Crohn's disease. Sa1806
AGA Abstracts
Molecular and Functional Characterization of Fimh in Crohn's Disease Associated E. Coli Brendan Chandler, Belgin Dogan, Ellen J. Scherl, Kenneth W. Simpson Background: The minor fimbrial subunit fimH is a mannose sensitive adhesin that has been implicated as a virulence factor of Adherent and Invasive E. coli (AIEC) in Crohn's disease (CD). The interaction of fimH and inflammation induced CEACAM-6 is thought to promote adhesion and invasion in the ileum. fimH polymorphisms in uropathogenic E. coli (UPEC) modulate binding affinity. We sought to investigate the effect of fimH polymorphisms on adhesion and invasion of intestinal epithelial cells by E. coli associated with CD. Methods: We evaluated a collection of 49 E. coli strains isolated from the ileal mucosa of patients with CD (n=36) and non IBD controls (n=13). Strains were classified as AIEC if they survived in macrophages and were able to adhere and invade more successfully than DH5 α. Triplex PCR was used to determine phylogroup. fimH was sequenced and aligned to K-12 and examined for polymorphisms. fimH expression was determined by yeast agglutination and adhesion to cultured CaCo-2 cells in the presence and absence of mannose. Invasion of CaCo-2 was evaluated by gentamicin protection. The impact of multiplicity of infection (MOI, 1-200) on adhesion and invasion of representative AIEC strains was determined in the presence and absence of mannose. Results: Of the 34 CD strains, 20 were AIEC and 14 non-AIEC. Of 13 control strains 5 were AIEC and 8 non-AIEC E.coli spanned all 4 phylogenetic groups (15A,15B1, 11B2, 8d) with no difference between CD and control, and AIEC and non-AIEC. fimH sequences clustered by phylogroup independent of disease association, and E.coli pathotype. There was no significant correlation between fimH polymorphisms and CaCo-2 adhesion or invasion. fimH was present in 44/49 strains and mannose inhibited agglutination of all 44 strains. In CaCo-2 adhesion assays, 15/44 strains had .75% residual adhesion in the presence of mannose. Mannose had little impact on invasion of CaCo2. Additional evaluation of AIEC strains from different phylogroups in the presence of mannose confirmed a much larger effect on adhesion than invasion across a range of MOI e.g. ,1% residual adhesion in the presence of mannose, compared to .90% residual invasion for LF82. Conclusions: These findings link fimH polymorphisms to phylogroup in ileal E.coli. We found no association between fimH polymorphisms and disease status or the AIEC pathotype. The persistence of CaCo2 adhesion and invasion in the face of mannose supports the presence of fimH independent mechanisms. The discordance between adhesion and invasion in the presence of mannose suggests that therapeutic strategies directed at decreasing fimH adhesion may have little impact on invasion.
Sa1804 Quorum Sensing Driven by N-Acyl-Homoserine Lactone in Inflammatory Bowel Diseases Associated Dysbiosis Cecilia Landman, Alison Besse, Marie-anne Maubert, Loic Brot, Lydie Humbert, Jacques Cosnes, Laurent Beaugerie, Germain Trugnan, Harry Sokol, Dominique Rainteau, Elodie Quévrain, Philippe Seksik Background Gut microbiota imbalance, namely dysbiosis, is a key factor in inflammatory bowel diseases (IBD) physiopathology. Auto-inducer quorum sensing (QS) driven by N-acylhomoserine lactone (acyl-HSL), a bacterial communication network, has not been studied in the human gut microbiota yet and could be involved in dysbiosis. The aims of this study were to look for acyl-HSLs in human gut microbiota and to investigate their features in IBD associated dysbiosis. Methods Fecal samples from 19 IBD patients in remission (n=10) and during flare (n= 9) and from 11 healthy subjects (HS) were analyzed. After extraction and purification, an acyl-HSL profile was determined for each sample using HPLC coupled with tandem mass spectrometry (LC-MS/MS). Concentrations were estimated by AUC of each peak (arbitrary units +/- SEM). Acyl-HSL biologic activity was validated on a bacterial biosensor system (Agrobacterium tumefasciens NTL4). In parallel, fecal microbiota composition was assessed by real-time quantitative PCR. For statistical analysis, a non parametric test was used. Results Nine acyl-HSLs were identified in fecal samples while 2 were prominent: acyl-HSL at m/z 216.1 (3-OH-C6-HSL) and acyl-HSL at m/z 294.2 (3-oxo-C12:2-HSL or C13:2-HSL) recognized by NTL4 biosensor. Prominent acyl-HSLs concentrations differed between IBD patients and HS: acyl-HSL at m/z 216.1 was significantly increased in inactive IBD compared to HS (AUC = 2897 +/- 572 vs 1242 +/- 353, P=0.04) and acyl-HSL at m/ z 294.2 was significantly decreased in active IBD (AUC = 2726 +/- 2726) compared to HS (AUC = 30532 +/- 12230, P=0.007) and to inactive IBD (AUC = 26409 +/- 12291, P=0.02). Overall, IBD patients tended to have higher acyl-HSL at m/z 216.1 and lower acyl-HSL at m/z 294.2. In parallel, dysbiosis was observed in active IBD characterized by low counts in Firmicutes (C. coccoïdes group P=0.01 and C. leptum group P=0.003 including F. prausnitzii P=0.04), significant increase in Lactobacilli (P=0.002) and non significant increase in E. coli (P=0.6). In samples with high acyl-HSL at m/z 294.2 (concentration above median), C. leptum group was significantly more represented (10.22 +/-0.07 vs 9.72 +/- 0.19 log/g of feces, P=0.046). In these samples, there was also a trend towards higher counts in C. coccoïdes group (P=0.06) and lower counts in E. coli (P=0.09). Conclusion Our study showed for the first time that QS driven by acyl-HSLs occurs in human gut microbiota. Moreover, in IBD, acyl-HSLs profile characterized by prominent acyl-HSL at m/z 216.1 and a decrease in acyl-HSL at m/z 294.2 during flare differs from HS. The lack of this acyl-HSL was associated with low counts in Firmicutes. These results invite us to investigate acylHSL functional role in dysbiosis onset and in host physiology.
Sa1807 Fermentation Products and Fecal Microbiota in Patients With Quiescent Ulcerative Colitis Michael Conlon, Claus T. Christophersen, Ian C. Roberts-Thomson There is only limited information on fermentation products and fecal microbiota in patients with ulcerative colitis in remission. Methods Fecal samples were collected from 53 adults with ulcerative colitis in remission and from 45 healthy adult controls. Patients had been in remission and had not received antibiotics for at least 3 months. Ten patients were in long-term remission and had ceased all medication. Samples were immediately placed in portable freezers and stored at -80C. Samples were analyzed for fecal moisture, pH, ammonia, phenols, p-cresol and short chain fatty acids and DNA was extracted for analysis of microbial diversity using quantitative real-time PCR. The abundance of bacterial targets was expressed per g (wet weight) of stool and as a proportion of the total number of bacteria. Results Fecal samples from patients and controls had a similar pH but specimens from UC had more fecal moisture. Both groups had similar concentrations of ammonia, total phenols and short chain fatty acids (acetate, butyrate and propionate). However, the concentration of p-cresol was significantly lower in the UC group (p=0.014). Total numbers of bacteria were significantly lower for the UC group as were numbers of Akkermansia muciniphila, Faecalibacterium prausnitzii, Roseburia intestinalis and Bifidobacterium spp. In contrast, there was an increase in bacteria belonging to the C. coccoides group. When bacterial numbers were expressed as a proportion of the total, A. muciniphilia and Lactobacillus spp. were significantly lower in the UC group. Conclusion Fecal samples from patients with UC in remission have lower concentrations of fecal bacteria and a relative deficiency of A. muciniphilia and Lactobacillus spp. Although there is no apparent deficiency of short chain fatty acids numbers of the key butyrate-producers, F. prausnitzii and R. intestinalis, are low. Low levels of p-cresol are consistent with a defect in protein fermentation but the microbes responsible for this effect remain unclear.
Sa1805 Identification of Genes Required for Epithelial Cell Invasion in Adherent and Invasive E. Coli NC101 by Signature-Tagged Mutagenesis Belgin Dogan, Alyssa Chandler, Yasemin Araz, Brendan Chandler, Ryan B. Sartor, Kenneth W. Simpson BACKGROUND: Adherent and invasive E. coli (AIEC), which can persist and replicate in epithelial cells and macrophages, is increasingly implicated in the pathogenesis of ileal Crohn's disease (CD). AIEC strain NC101 isolated from specific pathogen free wild type mice has been shown to induce ileitis, colitis and the secretion of 2 CD related cytokines, IFN-γ and IL-17, in a monocolonized germ-free IL-10 knockout mouse. The bacterial factors underlying the virulence of AIEC NC101 remain to be elucidated. The aim of this study is to identify genes in AIEC NC101 that are involved in the invasion of intestinal epithelial cells. METHODS: We used a strategy of Signature-Tagged Mutagenesis (STM) and sequencebased analysis of invasion deficient mutants. A library of 1800 transposon-tagged NC101 mutants was created by 90 different conjugation reaction between nalidixic acid-resistant NC101 and plasmid-containing donor E. coli strains. Mutants were screened for their ability to invade Caco-2 epithelial cells. The chromosomal location of transposon insertions in strains with decreased invasion was identified by sequencing PCR products. Mutant strains with decreased ability to invade Caco-2 cells were further characterized for growth, motility and type 1 pili expression. RESULTS: We identified 25 strains with a decreased ability to invade Caco-2. Sequence based analysis identified insertions in a variety of genes involved in flagellar synthesis, type 1 pili regulation, colanic acid biosynthesis, lipopolysaccharide biosynthesis (genes necessary for assembly and modification of core oligosaccharide and O antigen), glucan biosynthesis, inner and outer membrane proteins and lipoproteins, membrane transporter, transcriptional regulator (sirB1), DNA metabolism (DNA binding protein stpA, SMC domain containing protein) and rRNA methyltransferase. Seven mutants were not motile and centrifugation partially restored invasion defects. All mutants agglutinated yeast cells, indicating expression of type 1 pili. CONCLUSION: We have identified 25 candidate genes in 8 functional categories that enable AIEC NC101 to invade epithelial cells. These results will be extended by complementation and functional analyses in vitro and
AGA Abstracts
Sa1808 Crohn's Disease-Associated Escherichia coli (AIEC) Invade Macrophages by Suppressing NFκB Signaling Khalidur Rahman, Maiko Sasaki, Jan-Michael A. Klapproth Background: Epidemiological and genetic studies suggest a role for enteric flora in the pathogenesis of Crohn's disease (CD), specifically, adherent invasive Escherichia coli (AIEC) representative strain LF82. Recently, we reported a CD-associated AIEC strain, 13I, which can survive in macrophages and induce high concentrations of TNF- α. Aim: The aim of this study was to determine the mechanisms by which 13I evades detection and clearance by macrophages. Methods: 13I was isolated from inflamed colonic tissue of a CD patient. Intracellular survival of AIEC and control E. coli strains was tested in the murine macrophage cell line, J774A.1 by gentamicin protection assay. Modulation of intracellular cell signaling pathways by E. coli strains were assessed by western blot analysis and confocal microscopy. Results: 13I demonstrated increased survival in macrophages with 33-fold higher intracellular bacterial colonies compared to LF82. Despite the increased survival of 13I in macrophages,
S-310
AGA Abstracts
Molecular and Functional Characterization of Fimh in Crohn's Disease Associated E. Coli Brendan Chandler, Belgin Dogan, Ellen J. Scherl, Kenneth W. Simpson Background: The minor fimbrial subunit fimH is a mannose sensitive adhesin that has been implicated as a virulence factor of Adherent and Invasive E. coli (AIEC) in Crohn's disease (CD). The interaction of fimH and inflammation induced CEACAM-6 is thought to promote adhesion and invasion in the ileum. fimH polymorphisms in uropathogenic E. coli (UPEC) modulate binding affinity. We sought to investigate the effect of fimH polymorphisms on adhesion and invasion of intestinal epithelial cells by E. coli associated with CD. Methods: We evaluated a collection of 49 E. coli strains isolated from the ileal mucosa of patients with CD (n=36) and non IBD controls (n=13). Strains were classified as AIEC if they survived in macrophages and were able to adhere and invade more successfully than DH5 α. Triplex PCR was used to determine phylogroup. fimH was sequenced and aligned to K-12 and examined for polymorphisms. fimH expression was determined by yeast agglutination and adhesion to cultured CaCo-2 cells in the presence and absence of mannose. Invasion of CaCo-2 was evaluated by gentamicin protection. The impact of multiplicity of infection (MOI, 1-200) on adhesion and invasion of representative AIEC strains was determined in the presence and absence of mannose. Results: Of the 34 CD strains, 20 were AIEC and 14 non-AIEC. Of 13 control strains 5 were AIEC and 8 non-AIEC E.coli spanned all 4 phylogenetic groups (15A,15B1, 11B2, 8d) with no difference between CD and control, and AIEC and non-AIEC. fimH sequences clustered by phylogroup independent of disease association, and E.coli pathotype. There was no significant correlation between fimH polymorphisms and CaCo-2 adhesion or invasion. fimH was present in 44/49 strains and mannose inhibited agglutination of all 44 strains. In CaCo-2 adhesion assays, 15/44 strains had .75% residual adhesion in the presence of mannose. Mannose had little impact on invasion of CaCo2. Additional evaluation of AIEC strains from different phylogroups in the presence of mannose confirmed a much larger effect on adhesion than invasion across a range of MOI e.g. ,1% residual adhesion in the presence of mannose, compared to .90% residual invasion for LF82. Conclusions: These findings link fimH polymorphisms to phylogroup in ileal E.coli. We found no association between fimH polymorphisms and disease status or the AIEC pathotype. The persistence of CaCo2 adhesion and invasion in the face of mannose supports the presence of fimH independent mechanisms. The discordance between adhesion and invasion in the presence of mannose suggests that therapeutic strategies directed at decreasing fimH adhesion may have little impact on invasion.
Sa1804 Quorum Sensing Driven by N-Acyl-Homoserine Lactone in Inflammatory Bowel Diseases Associated Dysbiosis Cecilia Landman, Alison Besse, Marie-anne Maubert, Loic Brot, Lydie Humbert, Jacques Cosnes, Laurent Beaugerie, Germain Trugnan, Harry Sokol, Dominique Rainteau, Elodie Quévrain, Philippe Seksik Background Gut microbiota imbalance, namely dysbiosis, is a key factor in inflammatory bowel diseases (IBD) physiopathology. Auto-inducer quorum sensing (QS) driven by N-acylhomoserine lactone (acyl-HSL), a bacterial communication network, has not been studied in the human gut microbiota yet and could be involved in dysbiosis. The aims of this study were to look for acyl-HSLs in human gut microbiota and to investigate their features in IBD associated dysbiosis. Methods Fecal samples from 19 IBD patients in remission (n=10) and during flare (n= 9) and from 11 healthy subjects (HS) were analyzed. After extraction and purification, an acyl-HSL profile was determined for each sample using HPLC coupled with tandem mass spectrometry (LC-MS/MS). Concentrations were estimated by AUC of each peak (arbitrary units +/- SEM). Acyl-HSL biologic activity was validated on a bacterial biosensor system (Agrobacterium tumefasciens NTL4). In parallel, fecal microbiota composition was assessed by real-time quantitative PCR. For statistical analysis, a non parametric test was used. Results Nine acyl-HSLs were identified in fecal samples while 2 were prominent: acyl-HSL at m/z 216.1 (3-OH-C6-HSL) and acyl-HSL at m/z 294.2 (3-oxo-C12:2-HSL or C13:2-HSL) recognized by NTL4 biosensor. Prominent acyl-HSLs concentrations differed between IBD patients and HS: acyl-HSL at m/z 216.1 was significantly increased in inactive IBD compared to HS (AUC = 2897 +/- 572 vs 1242 +/- 353, P=0.04) and acyl-HSL at m/ z 294.2 was significantly decreased in active IBD (AUC = 2726 +/- 2726) compared to HS (AUC = 30532 +/- 12230, P=0.007) and to inactive IBD (AUC = 26409 +/- 12291, P=0.02). Overall, IBD patients tended to have higher acyl-HSL at m/z 216.1 and lower acyl-HSL at m/z 294.2. In parallel, dysbiosis was observed in active IBD characterized by low counts in Firmicutes (C. coccoïdes group P=0.01 and C. leptum group P=0.003 including F. prausnitzii P=0.04), significant increase in Lactobacilli (P=0.002) and non significant increase in E. coli (P=0.6). In samples with high acyl-HSL at m/z 294.2 (concentration above median), C. leptum group was significantly more represented (10.22 +/-0.07 vs 9.72 +/- 0.19 log/g of feces, P=0.046). In these samples, there was also a trend towards higher counts in C. coccoïdes group (P=0.06) and lower counts in E. coli (P=0.09). Conclusion Our study showed for the first time that QS driven by acyl-HSLs occurs in human gut microbiota. Moreover, in IBD, acyl-HSLs profile characterized by prominent acyl-HSL at m/z 216.1 and a decrease in acyl-HSL at m/z 294.2 during flare differs from HS. The lack of this acyl-HSL was associated with low counts in Firmicutes. These results invite us to investigate acylHSL functional role in dysbiosis onset and in host physiology.
Sa1807 Fermentation Products and Fecal Microbiota in Patients With Quiescent Ulcerative Colitis Michael Conlon, Claus T. Christophersen, Ian C. Roberts-Thomson There is only limited information on fermentation products and fecal microbiota in patients with ulcerative colitis in remission. Methods Fecal samples were collected from 53 adults with ulcerative colitis in remission and from 45 healthy adult controls. Patients had been in remission and had not received antibiotics for at least 3 months. Ten patients were in long-term remission and had ceased all medication. Samples were immediately placed in portable freezers and stored at -80C. Samples were analyzed for fecal moisture, pH, ammonia, phenols, p-cresol and short chain fatty acids and DNA was extracted for analysis of microbial diversity using quantitative real-time PCR. The abundance of bacterial targets was expressed per g (wet weight) of stool and as a proportion of the total number of bacteria. Results Fecal samples from patients and controls had a similar pH but specimens from UC had more fecal moisture. Both groups had similar concentrations of ammonia, total phenols and short chain fatty acids (acetate, butyrate and propionate). However, the concentration of p-cresol was significantly lower in the UC group (p=0.014). Total numbers of bacteria were significantly lower for the UC group as were numbers of Akkermansia muciniphila, Faecalibacterium prausnitzii, Roseburia intestinalis and Bifidobacterium spp. In contrast, there was an increase in bacteria belonging to the C. coccoides group. When bacterial numbers were expressed as a proportion of the total, A. muciniphilia and Lactobacillus spp. were significantly lower in the UC group. Conclusion Fecal samples from patients with UC in remission have lower concentrations of fecal bacteria and a relative deficiency of A. muciniphilia and Lactobacillus spp. Although there is no apparent deficiency of short chain fatty acids numbers of the key butyrate-producers, F. prausnitzii and R. intestinalis, are low. Low levels of p-cresol are consistent with a defect in protein fermentation but the microbes responsible for this effect remain unclear.
Sa1805 Identification of Genes Required for Epithelial Cell Invasion in Adherent and Invasive E. Coli NC101 by Signature-Tagged Mutagenesis Belgin Dogan, Alyssa Chandler, Yasemin Araz, Brendan Chandler, Ryan B. Sartor, Kenneth W. Simpson BACKGROUND: Adherent and invasive E. coli (AIEC), which can persist and replicate in epithelial cells and macrophages, is increasingly implicated in the pathogenesis of ileal Crohn's disease (CD). AIEC strain NC101 isolated from specific pathogen free wild type mice has been shown to induce ileitis, colitis and the secretion of 2 CD related cytokines, IFN-γ and IL-17, in a monocolonized germ-free IL-10 knockout mouse. The bacterial factors underlying the virulence of AIEC NC101 remain to be elucidated. The aim of this study is to identify genes in AIEC NC101 that are involved in the invasion of intestinal epithelial cells. METHODS: We used a strategy of Signature-Tagged Mutagenesis (STM) and sequencebased analysis of invasion deficient mutants. A library of 1800 transposon-tagged NC101 mutants was created by 90 different conjugation reaction between nalidixic acid-resistant NC101 and plasmid-containing donor E. coli strains. Mutants were screened for their ability to invade Caco-2 epithelial cells. The chromosomal location of transposon insertions in strains with decreased invasion was identified by sequencing PCR products. Mutant strains with decreased ability to invade Caco-2 cells were further characterized for growth, motility and type 1 pili expression. RESULTS: We identified 25 strains with a decreased ability to invade Caco-2. Sequence based analysis identified insertions in a variety of genes involved in flagellar synthesis, type 1 pili regulation, colanic acid biosynthesis, lipopolysaccharide biosynthesis (genes necessary for assembly and modification of core oligosaccharide and O antigen), glucan biosynthesis, inner and outer membrane proteins and lipoproteins, membrane transporter, transcriptional regulator (sirB1), DNA metabolism (DNA binding protein stpA, SMC domain containing protein) and rRNA methyltransferase. Seven mutants were not motile and centrifugation partially restored invasion defects. All mutants agglutinated yeast cells, indicating expression of type 1 pili. CONCLUSION: We have identified 25 candidate genes in 8 functional categories that enable AIEC NC101 to invade epithelial cells. These results will be extended by complementation and functional analyses in vitro and
AGA Abstracts
Sa1808 Crohn's Disease-Associated Escherichia coli (AIEC) Invade Macrophages by Suppressing NFκB Signaling Khalidur Rahman, Maiko Sasaki, Jan-Michael A. Klapproth Background: Epidemiological and genetic studies suggest a role for enteric flora in the pathogenesis of Crohn's disease (CD), specifically, adherent invasive Escherichia coli (AIEC) representative strain LF82. Recently, we reported a CD-associated AIEC strain, 13I, which can survive in macrophages and induce high concentrations of TNF- α. Aim: The aim of this study was to determine the mechanisms by which 13I evades detection and clearance by macrophages. Methods: 13I was isolated from inflamed colonic tissue of a CD patient. Intracellular survival of AIEC and control E. coli strains was tested in the murine macrophage cell line, J774A.1 by gentamicin protection assay. Modulation of intracellular cell signaling pathways by E. coli strains were assessed by western blot analysis and confocal microscopy. Results: 13I demonstrated increased survival in macrophages with 33-fold higher intracellular bacterial colonies compared to LF82. Despite the increased survival of 13I in macrophages,
S-310