Main muropeptides from <i>L. casei</i> BL23 PG hydrolyzed by Lc-p75 and main products of digestion.

a<p>Peak numbers refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032301#pone-0032301-g002" target="_blank">Figure 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032301#pone.0032301.s004" target="_blank">Figure S3</a>. New peaks obtained after Lc-p75 digestion, are indicated by letters.</p>b<p>Di, disaccharide dipeptide (L-Ala-D-iGln); Tri, disaccharide tripeptide (L-Ala-D-iGln-L-Lys); Tetra, disaccharide tetrapeptide (L-Ala-D-iGln-L-Lys-D-Ala); Disaccharide, GlcNAc-MurNAc; Ac, acetylation on MurNAc, iGln, isoglutamine; N, D-Asn; A, D-Ala; K, L-Lys.</p>c<p>Sodiated molecular ions were the most abundant ones on MALDI-TOF mass spectra for all muropeptides.</p>d<p>Percentage of each peak was calculated as the ratio of the peak area over the sum of areas of all the peaks identified in the corresponding chromatogram (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032301#pone.0032301.s008" target="_blank">Table S3</a>).</p>e<p>ND, non detected.</p

RP-HPLC separation profile of muropeptides obtained from <i>L. casei</i> BL23 PG.

<p>PG was digested by mutanolysin (A) or by mutanolysin and recombinant Lc-p75 (B). Numbers correspond to the main muropeptides which amounts changed between Panel A and B. Letters indicate new peaks present in Panel B and absent in Panel A. Complete annotation of the chromatograms is presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032301#pone.0032301.s004" target="_blank">Figure S3</a>.</p

Detection of Lc-p75 in culture supernatant.

<p>Analysis by SDS-PAGE (A, B) and zymogram assay (C, D) of the culture supernatant of wild type BL23 (A, C) and negative mutant (B, D) grown on AOAC medium. Lc-p75 is indicated by an arrow.</p

Indirect immunofluorescence localization of Strep-tagged Lc-p75 in <i>L. casei</i>.

<p>Strep-tagged Lc-p75 was localized in overexpressing strain (A) and in complemented negative mutant (B) with monoclonal antibody directed against Strep-tag as first antibody.</p

Lectin-Like Molecules of <i>Lactobacillus rhamnosus</i> GG Inhibit Pathogenic <i>Escherichia coli</i> and <i>Salmonella</i> Biofilm Formation - Fig 8

<p>(A) Functional analysis of <i>llp1</i> (CMPG10701<i>)</i>, <i>llp2</i> (CMPG10706) and double (CMPG10707) mutant of <i>L</i>. <i>rhamnosus</i> GG for adhesion to gastrointestinal (CaCo2) and vaginal (VK2/E6E7) epithelial cells. The results are expressed relatively to the adhesion of <i>L</i>. <i>rhamnosus</i> GG wild-type, which was set at 100%. (B) Binding of FITC-labeled lectin domains of Llp1 and Llp2 to CaCo2 and VK2/E6E7 cells. (C) Functional analysis of <i>llp1</i> (CMPG10701<i>)</i>, <i>llp2</i> (CMPG10706) and double (CMPG10707) mutant of <i>L</i>. <i>rhamnosus</i> GG for biofilm formation. The error bars represent standard deviation of three independent experiments. The dataset comparisons (mutant pairwise to wild-type) are considered significant (p < 0.05 indicated with one asterisk in the picture or p < 0.01 indicated with two asterisks in the figure).</p

Antibiofilm activity of Llp1 and Llp2 against <i>S</i>. Typhimurium ATCC14028.

<p>(A) Effect of the lectin domains (LD) of Llp1 and Llp2 on <i>S</i>. Typhimurium ATCC14028 biofilms added at different concentrations at the start of the biofilm formation. The lowest concentration in which both of the lectins showed significant reduction in the biofilm is indicated with a shaded bow. (B) Effect of LD on <i>S</i>. Typhimurium ATCC14028 biofilms added after 0, 1.5, 8h, 24 h and after 0 and 24 h with fresh medium to the biofilms at a concentration of 50 μg/ml. (C) Absolute CFU <i>S</i>. Typhimurium ATCC14028 biofilms grown for 48h with lectins added at zero-time point at a concentration of 50 μg/ml. Absolute CFU counts of the treatments was normalized towards the control, which was grown in medium without lectins. (D) Effect of full length (FL) lectin (50 μg/ml) on <i>S</i>. Typhimurium ATCC14028 biofilms added at zero-time point to the biofilms. (E) Growth of <i>S</i>. Typhimurium ATCC14028 in presence of lectin domain of Llp1 and Llp2 (200 μg/ml) in 1/20 TSB medium. (F) Effect of lectin domains (50 μg/ml) on various <i>Salmonella</i> biofilms added at zero-time point. The error bars represent standard deviation of three independent experiments. The dataset comparisons are considered significant (p < 0.05 indicated with one asterisk in the picture, p < 0.01 indicated with two asterisks in the picture or p< 0.001 indicated with three asterisks on the picture).</p

Effect of the lectin domains of Llp1 and Llp2 of <i>L</i>. <i>rhamnosus</i> GG on <i>E</i>. <i>coli</i> UTI89 biofilms.

<p>(A) Effect of the lectin domains (LD) of Llp1 and Llp2 on <i>E</i>. <i>coli</i> UTI89 biofilms added at different concentration at beginning of the biofilm formation. The lowest concentration in which both of the lectins showed significant reduction in the biofilm is indicated with a shaded bow. (B) The purified lectin domains of Llp1 and Llp2 were added after 0, 1.5, 8 and 24 hours to the biofilms. (C) Biofilm formation of <i>E</i>. <i>coli</i> UTI89 based on absolute cell counts. Biofilms were grown for 48h in 1/20 TSB without (control) for or with 50 μg/ml of lectin domain of Llp1 and Llp2. (D) Growth of <i>E</i>. <i>coli</i> UTI89 in the presence of lectin domain of Llp1 and Llp2 added at concentrations of 200 μg/ml. The error bars represent standard deviation of three independent experiments. The dataset comparisons are considered significant (p < 0.01 indicated with two asterisks or p< 0.001 indicated with three asterisks). (E) Biofilms of wild type <i>E</i>. <i>coli</i> UTI89 grown in 1/20 TSB medium containing 50 μg/ml FITC labeled Llp1 (F) and FITC labeled Llp2 (G). Holes in the biofilm are indicated with arrows. The zoomed images show single non-fluorescent bacterial cells, suggesting that lectins bind biofilm matrix and not the pathogenic cells.</p

Lectin-Like Molecules of <i>Lactobacillus rhamnosus</i> GG Inhibit Pathogenic <i>Escherichia coli</i> and <i>Salmonella</i> Biofilm Formation - Fig 3

<p>Biofilms of GFP-expressing <i>S</i>. Typhimurium ATCC14028 (A) or wild type <i>S</i>. Typhimurium ATCC14028 (E) grown in 1/20 TSB medium containing 50 μg/ml lectin domains of Llp1 (B) or FITC labeled Llp1 (F) and Llp2 (C) or FITC labeled Llp2 (G). Holes in the biofilm are indicated with arrows. (D) Fluorescent signal histogram of GFP-expressing <i>S</i>. Typhimurium ATCC14028 biofilm alone or treated with lectin domains of Llp1 and Llp2.</p

Lc-p75 activity on purified muropeptides selected as substrates.

a<p>Di, disaccharide dipeptide; Tri, disaccharide tripeptide (L-Ala-D-iGln-L-Lys); Tetra, disaccharide tetrapeptide (L-Ala-D-iGln-L-Lys-D-Ala); Disaccharide, GlcNAc-MurNAc; iGln, isoglutamine; N, Asn; Ac, acetylation on MurNAc.</p>b<p>Similar amounts of each muropeptide were used for each test.</p>c<p>Percentage of each peak was calculated as the ratio of the peak area over the sum of areas of all the peaks identified in the corresponding chromatogram.</p>d<p>Other forms of muropeptides resulting from partial digestion of the substrate.</p

Schematic structure of <i>L. casei</i> BL23 peptidoglycan and site of cleavage determined for Lc-p75.

<p>GlcNAc, N-acetylglucosamine; MurNAc, N-acetylmuramic acid. MurNAc may be O-acetylated (O-Ac) or not in PG.</p