8 research outputs found

    LrpCBA pilus-mediated cellular variation of endogenous IL-8 production in Caco-2 intestinal cells.

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    <p>Caco-2 cells were exposed to normalized cultures of <i>L</i>. <i>ruminis</i> cells and the recombinant GRS1224 (WT) and GRS1225 (LrpC-deleted) lactococci (MOI 100), with spent cell culture supernatants then measured for any variation in the levels of endogenous IL-8 cytokine production. GRS71 and GRS1052 cells were included as controls and treated likewise. Measurements were done in triplicate and limit bars indicate the SEM. Statistical differences for individual pairwise comparisons against the GRS71 control are designated as * = <i>P</i> ‚ȧ 0.05 (significant) or <i>ns</i> = <i>P</i> > 0.05 (not significant).</p

    LrpCBA pilus-mediated effects on cellular biofilm formation.

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    <p>Normalized cultures of <i>L</i>. <i>ruminis</i> cells and the recombinant GRS1224 (WT) and GRS1225 (LrpC-deleted) lactococci were assessed for static biofilm formation using a crystal violet staining assay. Various cellular controls were used and treated in the same manner. These included the pilus-less GRS71 and GRS1052 lactococci, the recombinant WT and SpaC-deleted SpaCBA-piliated (GRS1185 and GRS1211, respectively) and WT and SpaF-deleted SpaFED-piliated (GRS1189 and GRS1226, respectively) lactococcal clones, and the SpaCBA-piliated <i>L</i>. <i>rhamnosus</i> GG strain. Multiple measurements were taken and an average value calculated (<i>n</i> = 7). Limit bars show the SEM. Statistical differences for individual pairwise comparisons against the GRS71 control are indicated as *** = <i>P</i> ‚ȧ 0.001 (highly significant) or <i>ns</i> = <i>P</i> > 0.05 (not significant).</p

    Influence of cell-to-cell interactions on the LrpCBA pilus-mediated cellular variation of TLR2-regulated NF-őļB responses and endogenous IL-8 production in HEK-TLR2 cells.

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    <p>Normalized cultures of <i>L</i>. <i>ruminis</i> cells and the recombinant GRS1224 (WT) and GRS1225 (LrpC-deleted) lactococci (MOI 100), either non-partitioned (-) or Transwell-partitioned (+), were added to HEK-TLR2 cells, with the levels of TLR2-dependent NF-őļB activation <b>(A)</b> and endogenous IL-8 production <b>(B)</b> then quantified. GRS71 cells (MOI 100), DMEM cell-culture medium, and Pam3CSK4 (1 ng/ml) were also included as controls. Measurements for both sets of experiments were done in triplicate, with limit bars representing the SEM. Statistical differences for individual pairwise comparisons against the GRS71 control (without Transwell membranes) as well as the individual pairwise data comparisons of samples with or without Transwell membranes are denoted as *** = <i>P</i> ‚ȧ 0.001 (highly significant) or ** = <i>P</i> ‚ȧ 0.01 (very significant).</p

    LrpCBA pilus-mediated cellular adhesion to gut epithelial cells.

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    <p>Assessment of LrpCBA pilus-mediated cell-to-cell adhesion between normalized (OD600 = 0.5) cultures of <i>L</i>. <i>ruminis</i>, and as well recombinant GRS1224 (WT) and GRS1225 (LrpC-deleted) lactococci, and each of the human intestinal Caco-2 <b>(A)</b> and HT-29 <b>(B)</b> cell lines was performed. GRS71 and GRS1052 cells were included as controls. Measurements were performed in triplicate for both sets of experiments. Limit bars indicate the SEM. Statistical differences for individual pairwise comparisons against the GRS71 control are designated as *** = <i>P</i> ‚ȧ 0.001 (highly significant), * = <i>P</i> ‚ȧ 0.05 (significant), or <i>ns</i> = <i>P</i> > 0.05 (not significant).</p

    Schematic representation of the coding regions of the fimbrial <i>LrpCBA</i> operon from <i>L</i>. <i>ruminis</i> ATCC 25644.

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    <p>The clustered genomic arrangement of the <i>lrpC</i>, <i>lrpB</i>, <i>lrpA</i>, and <i>srtC</i> genes within the <i>LrpCBA</i> pilus operon is shown. Numbers indicate the beginning and ending nucleotide (nt) positions of the coding region for the operon. Arrows for the genes point in the direction of transcription and translation predicted from the DNA. Shown for each gene is nt sequence that resembles the ribosomal binding site (RBS). Deduced amino acid sequences for the N-terminal secretion signal and C-terminal sortase recognition domains in the predicted LrpC, LrpB, and LrpA pilin-proteins are provided. Residues for the LPXTG-like motifs in each pilin-protein are underlined. For each pilin-protein, predicted residues sharing similarity with the so-called pilin motif (WxxxVxVYPKN) and E-box domain (YxLxETxAPxGY) are indicated.</p

    Evidence of <i>lrpCBA</i> operon-encoded pilus expression and surface localization in <i>L</i>. <i>ruminis</i>.

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    <p><b>(A)</b> Immunoblot analysis of LrpCBA piliation in <i>L</i>. <i>ruminis</i>. Whole-cell preparates of <i>L</i>. <i>ruminis</i> ATCC 25644 (lane 1) and <i>L</i>. <i>rhamnosus</i> GG (lane 2) were each immunoblotted and probed with polyclonal anti-LrpA (left panel), anti-LrpB (middle panel), and anti-LrpC (right panel) sera. A condensed laddering of high-molecular-weight (HMW) protein bands, which are indicated along the top left of the immunoblot represents the lengthiest fragments of assembled pili. Protein bands approximating the molecular size of the LrpA (~49 kDa), LrpB (~39 kDa), and LrpC (~123 kDa) monomers are identified by an asterisk on the right side of the immunoblot, whereas the sizing markers for molecular weight (kDa) are shown to the left. <b>(B)</b> Immuno-electron microscopic visualization of LrpCBA piliation in <i>L</i>. <i>ruminis</i>. Immunogold pilin-protein labeling and electron microscopy of <i>L</i>. <i>ruminis</i> (ATCC 25644) were performed accordingly. <i>L</i>. <i>ruminis</i> cells were labeled with antiserum against LrpA pilin subunits and protein A-10-nm gold particles (top left panel). The <i>L</i>. <i>rhamnosus</i> GG strain is used as a control and was treated the same (top right panel). Double labeling of <i>L</i>. <i>ruminis</i> cells was performed with combined antisera against either LrpA (10-nm; white arrowhead) and LrpC (15-nm; gray arrowhead) pilins (bottom left panel) or LrpA (10-nm; white arrowhead) and LrpB (15-nm; black arrowhead) pilins (bottom right panel). Shown is a representative image from each EM experiment. A scale bar is included at the bottom of each panel.</p

    LrpCBA pilus-mediated cellular variation of TLR2-regulated NF-őļB responses and endogenous IL-8 production in live and heat-treated HEK-TLR2 cells.

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    <p>Live (-) and heat-treated (+) normalized cultures of <i>L</i>. <i>ruminis</i> cells and the recombinant GRS1224 (WT) and GRS1225 (LrpC-deleted) lactococci (MOI 100) were combined with HEK-TLR2 cells. TLR2-dependent NF-őļB activation <b>(A)</b> and endogenous IL-8 production <b>(B)</b> were measured. GRS71 and GRS1052 cells were used as controls and treated similarly. Measurements also taken for DMEM cell-culture medium and a TLR2-agonist lipopeptide (Pam3CSK4; 1 ng/ml) were used as negative and positive controls, respectively. Triplicate measurements were made for both sets of experiments. Limit bars show SEM. Statistical differences for individual pairwise comparisons against the GRS71 control (unheated) are specified as *** = <i>P</i> ‚ȧ 0.001 (highly significant), ** = <i>P</i> ‚ȧ 0.01 (very significant), or <i>ns</i> = <i>P</i> > 0.05 (not significant). Individual pairwise comparisons of data between the heated and unheated samples are indicated as *** = <i>P</i> ‚ȧ 0.001 (highly significant), ** = <i>P</i> ‚ȧ 0.01 (very significant), * = <i>P</i> ‚ȧ 0.05 (significant), or <i>ns</i> = <i>P</i> > 0.05 (not significant).</p

    LrpCBA pilus-mediated cellular adhesion to ECM-related proteins.

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    <p>Normalized (OD600 = 0.5) cultures of <i>L</i>. <i>ruminis</i> cells and the recombinant WT and LrpC-deleted LrpCBA-piliated lactococci (GRS1224 and GRS1225, respectively) were assayed for <i>in vitro</i> binding to collagen type I <b>(A)</b>, type IV <b>(B)</b>, and fibronectin <b>(C)</b>. Vectorless (GRS71) and empty vector (GRS1052) lactococci were included as controls in these experiments. Measurements were normally made in triplicate, with the standard error of mean (SEM) shown by limit bars. Statistical differences for individual pairwise comparisons against the GRS71 control are denoted as *** = <i>P</i> ‚ȧ 0.001 (highly significant) or ** = <i>P</i> ‚ȧ 0.01 (very significant).</p
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