Intestinal meprin α and meprin β mRNA.

<p>A and B, Mep1A (A) and Mep1B (B) mRNA levels were determined by TaqMan quantitative real time PCR and are displayed as amounts relative to the intestinal epithelial marker villin-1. Healthy controls (hc) were compared with ulcerative colitis (UC) and Crohn's disease (CD) patients. CD patient biopsies were separated into groups with normal appearance or with macroscopic inflammation, as determined by an experienced endoscopist. Statistics were performed using GraphPad Prism 5.0 Software, and <i>P</i> values were calculated with the non-parametric Mann-Whitney test. C and D, Mep1A (C) and Mep1B (D) mRNA levels in C57Bl/6J mouse ileum and colon uninfected or infected with AIEC LF82 bacteria. The effect of AIEC LF82 infection on meprin expression was determined in mouse ileum and colon by quantitative real time PCR. Data are displayed as meprin amounts relative to the housekeeping TATA box binding protein (TBP) gene. Statistics were performed using GraphPad Prism 5.0 Software, and <i>P</i> values were calculated with the one-way ANOVA test. Dot plots show individual samples with relative mRNA (cDNA) amounts on a linear scale. Horizontal bars represent the median.</p

Meprin treatment affect mannose residue recognition by AIEC and AIEC-induced IL-8 secretion by T84 cells.

<p>A, ability of type 1 pili to bind D-mannose residues as determined by a yeast aggregation test. AIEC LF82 bacteria were treated with 100 µg/ml of meprin α or β at 37°C for 120 min. A fixed amount of inactivated yeast cells (<i>Saccharomyces cerevisiae</i>) suspension and decreasing concentrations of treated and untreated bacteria were mixed, and the loss of the ability to form homogenous aggregation was used as the read-out for impaired type 1 pili-yeast interaction. B, Amount of IL-8 secreted by uninfected or AIEC LF82- or type 1 pili negative mutant LF82-Δ<i>fimA</i>-infected T84 cells, at 24 h post-infection. AIEC LF82 and LF82-Δ<i>fimA</i> bacteria were treated with 100 µg/ml of meprins. Il-8 secretion was determined by ELISA. Data are expressed as fold increase in the amount of secreted IL-8 ± SEM by T84 cells infected with untreated or treated bacteria relative to non infected cells. Student's <i>t</i>-test, * <i>P</i><0.05 for comparison between IL-8 secretion induced by untreated versus meprin-treated AIEC LF82 or LF82-Δ<i>fimA</i> bacteria. C, LF82-Δ<i>fimA</i> bacteria were pretreated with exogenous meprin α or meprin β at 100 µg/ml and undifferentiated T84 cells were infected at a MOI of 10. The number of associated bacteria was determined. Results are expressed as the percentage of cell-associated bacteria pretreated with exogenous meprins relative to untreated bacteria, defined as 100%.D, effect of meprins on recombinant human IL-8. Recombinant human IL-8 (110 ng/ml) was treated with meprin α or β (100 µg/ml), electroblotted and detected with mouse anti-human IL-8.</p

Proteolytic activity of meprins α and β on AIEC LF82 outer membrane proteins and flagellin.

<p>Total protein extracts from untreated or meprin-treated (100 µg/mL) whole bacteria were immunoblotted for OmpA and OmpC/F (A) or Flagellin (B). The inner membrane protein Lep was used as internal control. Amounts of proteins were quantified by using <i>Image J</i> software. Results are expressed as protein amount relative to Lep. Data are mean ± SEM for at least three independent experiments. Student's <i>t</i>-test, * <i>P</i><0.05.</p

Meprins impair AIEC LF82 ability to adhere to and to invade differentiated intestinal epithelial cells.

<p>AIEC LF82 bacteria were treated before infection with increased concentrations of exogenous meprins α and β (from 0.1 µg/ml to 100 µg/ml, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021199#s4" target="_blank">Materials and Methods</a> section). Differentiated T84 cells were infected at a MOI of 10 with untreated or meprin pretreated bacteria. A and D, the number of cell-associated bacteria was determined after a 3 h infection period. B and E, the number of internalized bacteria was determined after a 3 h infection period followed by gentamicin treatment for 1 h. Results are expressed as percentage of cell-associated (adherent + intracellular) (A and D), or intracellular (B and E) bacteria relative to initial inoculum. C and F, effect of meprin treatment on AIEC bacteria viability. Equal amounts of bacteria were exposed or not with a dose of 100 µg/ml of meprin α or β for 120 min. Thereafter, the number of viable bacteria was determined by plating on agar plate. Results are expressed as colony forming units (CFU) per ml (C and F). Data are mean ± SEM for at least three independent experiments. Student's <i>t</i>-test, * <i>P</i><0.05.</p

Proteolytic activity of meprins α and β on AIEC LF82 type 1 pili.

<p>A, proteolytic effect of various concentrations (from 1 µg/ml to 100 µg/ml) of meprins on purified AIEC LF82 type 1 pili. Proteins were loaded on 15% polyacrylamid gel for SDS-PAGE and stained with Coomassie brilliant blue. Results are expressed as FimA protein amount for meprin-treated type 1 pili relative to that of untreated type 1 pili. B, proteolytic effect of meprins on type 1 pili expressed at the surface of whole AIEC LF82 bacteria. AIEC bacteria were untreated or treated with active or heat-inactivated meprins at 100 µg/mL. Total proteins were immunoblotted with rabbit antiserum raised against purified type 1 pili and the inner membrane protein Lep, as an internal control. Results are expressed as FimA amount relative to Lep. Amounts of proteins were quantified by using <i>Image J</i> software. Data are mean ± SEM for at least three independent experiments. Student's <i>t</i>-test, * <i>P</i><0.05. C and D, MALDI-TOF MS profile of purified AIEC LF82 type 1 pili treated with active or heat-inactivated meprin α or meprin β. Spectra were acquired in a mass spectrometer MALDI-TOF from LF82 type 1 pili treated with 100 µg/ml of meprin α (C) or with meprin β (D).</p

Effect of meprins on the ability of AIEC strains and <i>Salmonella</i> Typhimurium strain LT2 to adhere to and to invade intestinal epithelial cells.

<p>Bacteria were pretreated with exogenous meprin α or meprin β at 10 µg/ml. A and B, undifferentiated Intestine-407 (I407), Caco-2 and T84 cells infected with AIEC LF82 at a MOI of 10. The number of associated (A) and internalized (B) bacteria was determined. Results are expressed as the percentage of cell-associated (A) or intracellular bacteria (B) relative to initial inoculum. C and D, undifferentiated T84 cells were infected at a MOI of 10 with <i>Salmonella</i> Typhimurium and AIEC strains LF82, LF9, LF15 and LF31. The number of associated (C) and internalized (D) bacteria was determined. Results are expressed as the percentage of cell-associated (C) or intracellular bacteria (D) relative to untreated bacteria, defined as 100% (bar, C and D). Data are mean ± SEM for at least three independent experiments. Student's <i>t</i>-test, * <i>P</i><0.05.</p