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 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