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. 2013 Jan;195(1):76-84.
doi: 10.1128/JB.01079-12. Epub 2012 Oct 26.

VSports注册入口 - The σE pathway is involved in biofilm formation by Crohn's disease-associated adherent-invasive Escherichia coli

Benoit Chassaing  1 , Arlette Darfeuille-Michaud
Affiliations

The σE pathway is involved in biofilm formation by Crohn's disease-associated adherent-invasive Escherichia coli

Benoit Chassaing et al. J Bacteriol. 2013 Jan.

Abstract

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 VSports手机版. 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. .

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Figures

Fig 1
Fig 1
(A, B) Activation of the σE pathway in AIEC strain LF82 and nonpathogenic E. coli strain MG1655. Fold variation of rpoE mRNA levels in wild-type strains LF82 (A) and MG1655 (B) grown in medium at pH 6, in medium with 2% bile salts, or in medium with 20 g · liter−1 NaCl or adherent to I-407 epithelial cells, relative to that in wild-type strains grown in classic medium. 16S rRNA levels were measured as controls. Data are the mean ± the SEM of three separate experiments. *, P < 0.05. (C) Adhesion of AIEC strain LF82, nonpathogenic E. coli K-12 strain MG1655, and isogenic mutant LF82-ΔfimA to I-407 cells. Cell-associated bacteria were quantified after a 3-h infection period. Each value is the mean number of CFU ± the SEM of at least four separate experiments. (D) Activation of the σE pathway in AIEC strain LF82 associated with I-407 cells after anti-Gp96 antibody pretreatment. Fold variation of rpoE mRNA levels in I-407 epithelial-cell-adhering bacteria of wild-type strain LF82 with or without a 30-min pretreatment of cell monolayers with anti-Gp96 antibody. N.S., not statistically significant.
Fig 2
Fig 2
(A, B) Fold variation of rseA (A) and rpoE (B) mRNAs levels in strains LF82, LF82/pBAD24, and LF82/pBAD24-rseAB in the presence of various doses of arabinose. Results are expressed as relative expression compared to that of wild-type strain LF82 in the absence of arabinose. 16S rRNA levels were measured as controls. Data are the mean ± the SEM of three separate experiments. (C, D) Activation of the rpoE (C) and rpoH (D) promoters in strains LF82, LF82/pBAD24, and LF82/pBAD24-rseAB in the presence of various doses of arabinose. Shown is the β-galactosidase activity per OD620 unit resulting from the expression of lacZ driven by the DNA sequence upstream of the rpoE or rpoH gene. Data are the mean ± the SEM of four separate experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Fig 3
Fig 3
(A, B) Abilities of strains LF82, LF82/pBAD24-rseAB, MG1655, and MG1655/pBAD24-rseAB to adhere to (A) and invade (B) I-407 IEC. Each value is the mean ± the SEM of at least four separate experiments. **, P < 0.01; N.S., not statistically significant.
Fig 4
Fig 4
(A) Motility assay of wild-type strain LF82 and strain LF82/pBAD24-rseAB on 0.3% agar at 37°C. (B) Regulation of type 1 pili in strain LF82/pBAD24-rseAB. PCR analysis was used to determine the invertible element orientation of the fim operon in strains LF82, LF82/pBAD24, and LF82/pBAD24-rseAB. A 450-bp product revealed the ON orientation of the invertible element, and a 750-bp product revealed its OFF orientation. (C, D) The abilities of strains LF82 + pHSG575-fim, LF82/pBAD24-rseAB, and LF82/pBAD24-rseAB/pHSG575-fim to adhere to (C) and invade (D) I-407 IEC. Centrifugation was performed to force contact between bacteria and I-407 IEC. Each value is the mean ± the SEM of at least four separate experiments. *, P < 0.05; **, P < 0.01.
Fig 5
Fig 5
(A) SBF indexes of AIEC strain LF82 and nonpathogenic E. coli strain MG1655 with or without RseAB overexpression. Data are the mean ± the SEM of three separate experiments. (B) SBF index of isogenic mutant LF82-ΔrpoE transcomplemented with pBAD30-rpoE and grown in the presence of 0.00, 0.31, 0.63, 1.25, 2.50, 5.00, or 10.00 g · liter−1 arabinose. Data are the mean ± the SEM of three separate experiments. **, P < 0.01; ***, P < 0.001; N.S., not statistically significant.
Fig 6
Fig 6
(A) Confocal analysis of LF82, LF82/pBAD24, LF82/pBAD24-rseAB, and MG1655 biofilm formation at the surface of a PFA-fixed monolayer of I-407 IEC. Bacteria expressing GFP were used, actin is stained red with phalloidin-TRITC, and nuclei are stained blue with Hoechst. Representative z sections were visualized under each confocal slice. Bars, 50 μm. (B) CI of strain LF82/pBAD24-rseAB compared to that of wild-type strain LF82. Intestinal ileal loops were inoculated with mixed inoculums comprising equivalent numbers of the wild-type and LF82 pBAD24-rseAB strains, and their presence was compared by CI analysis, which provides a sensitive measurement of the relative degree of attenuation. **, P < 0.01; ***, P < 0.001.
Fig 7
Fig 7
Activation of the σE pathway in AIEC strain LF82 during the biofilm formation process. Shown is the n-fold variation of rpoE mRNA levels in wild-type strain LF82 during biofilm formation (4, 16, and 24 h) relative to that of the wild-type strain grown for 4 h in classic medium. 16S rRNA levels were measured as controls. Data are the mean ± the SEM of three separate experiments. *, P < 0.05; **, P < 0.01.
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