Binding of recombinant SpaFED-piliated lactococcal cells to ECM proteins.

<p><i>In vitro</i> binding specificities between the normalized (OD600 = 0.5) cultures of recombinant WT (GRS1189) and SpaF pilin-deleted (GRS1126) SpaFED-piliated lactococci (along with GRS71 and GRS1052 cells as controls) and the fibronectin (<b>A</b>), collagen I (<b>B</b>), and collagen IV (<b>C</b>) proteins were evaluated using the procedure described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113922#s4" target="_blank">Materials and Methods</a>. Triplicate measurements were made for each of the ECM protein experiments, each of which was performed independently twice. SEM is indicated as error bars. For all ECM proteins tested, differences between the GRS1189 and GRS1226 binding results from pairwise comparisons against the GRS71 data are regarded very significant (<i>P</i>≤0.005).</p

Immuno-electron microscopy of recombinant SpaFED-piliated lactococcal cells.

<p>Immunogold pilin protein-labeling and electron microscopic analysis of recombinant WT (GRS1189) and SpaF pilin-deleted (GRS1126) SpaFED-piliated lactococci (including GRS71 cells as a control) were done using the techniques described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113922#s4" target="_blank">Materials and Methods</a>. GRS1189 (<b>A and inset</b>) and GRS71 (<b>B</b>) cells are single-labeled with anti-SpaD serum and protein A-10-nm gold particles. GRS1189 cells are double-labeled either with SpaD (10-nm; white arrowhead) and SpaF (15-nm; gray arrowhead) antisera (<b>C</b>) or with SpaD (10-nm; white arrowhead) and SpaE (15-nm; black arrowhead) antisera (<b>D</b>). GRS1226 cells are single-labeled with SpaD antiserum (10-nm) (<b>E</b>) as well as double-labeled either with SpaD (10-nm) and SpaF (15-nm) antisera (<b>F</b>) or with SpaD (10-nm; white arrowhead) and SpaE (15-nm; black arrowhead) antisera (<b>G</b>). A scale bar with dimensions is included in each panel.</p

Stimulation of TLR2-dependent NF-κB activation by recombinant SpaFED-piliated lactococci.

<p>The HEK-TLR2 cell line was treated with live (−) or heat-treated (100°C for 10 minutes) (+) normalized cultures of recombinant WT (GRS1189) and SpaF pilin-deleted (GRS1126) SpaFED-piliated lactococci (MOI 100). Levels of TLR2-dependent NF-κB activation were assessed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113922#s4" target="_blank">Materials and Methods</a>. Testing of the GRS71 and GRS1052 control strains was as well conducted. DMEM cell-culture medium and a TLR2-agonist lipopeptide (Pam3CSK4; 1 ng/ml) served as negative and positive controls, respectively. Quadruplicate measurements were taken for two independent experiments. SEM is shown as error bars. Pairwise differences between the GRS1189 and GRS1226 data (without heat inactivation) are considered very significant (<i>P</i>≤0.005).</p

Effect of cell-to-cell contacts on TLR2-induced NF-κB activation by recombinant SpaFED-piliated lactococci.

<p>Using a Transwell membrane-segregated system, HEK-TLR2 cells were treated with non-partitioned (−) and partitioned (+) normalized cultures of recombinant WT (GRS1189) and SpaF pilin-deleted (GRS1126) SpaFED-piliated lactococci (MOI 100). Monitoring of TLR2-dependent NF-κB activation was carried out as outlined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113922#s4" target="_blank">Materials and Methods</a>. Included as controls were GRS71 and GRS1052 cells (MOI 100), DMEM cell-culture medium, and Pam3CSK4 (1 ng/ml). Triplicate measurements were taken for a single experiment. SEM is indicated as error bars.</p

Stimulation of DC-cytokine production by recombinant SpaFED-piliated lactococci.

<p>Human monocyte-derived dendritic cells (moDCs) were treated with normalized cultures of recombinant WT (GRS1189) and SpaF pilin-deleted (GRS1126) SpaFED-piliated lactococci (MOI 50). Stimulated TNF-α (<b>A</b>), IL-12 (<b>B</b>), and IL-10 (<b>C</b>) cytokine production was measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113922#s4" target="_blank">Materials and Methods</a>. GRS71 and GRS1052 cells (MOI 50) and DMEM cell-culture medium were included as controls. Measurements were performed in triplicate using moDCs from four new and different donors each time. SEM is shown as error bars. For all tested cytokines, differences in a pairwise comparison between the GRS1189 and GRS1226 data are judged not significant (<i>P</i>≥0.05).</p

Immunoblot analysis of recombinant SpaFED-piliated lactococcal cells.

<p>Immunoblots of lactococcal cells corresponding to the empty vector GRS1052 clone (lane 1) and those to the nisin-induced WT (GRS1189; lane 2) and SpaF pilin-deleted (GRS1126; lane 3) SpaFED-piliated clones were probed separately with polyclonal anti-SpaD (<b>A</b>), anti-SpaE (<b>B</b>), and anti-SpaF (<b>C</b>) sera as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113922#s4" target="_blank">Materials and Methods</a>. Apparent positioning of monomeric SpaD, SpaE, and SpaF proteins is indicated on the right of each immunoblot by an asterisk. A dense ladder-like smear of high-molecular-weight (HMW) protein bands represents the longest lengths of pili and these are indicated on the top left of the immunoblot. The positions and sizes of the molecular weight markers are shown along the left side of the immunoblot.</p

Upstream sequence alignment comparison of the <i>spaCBA</i> pilus operon promoter region.

<p>Shown is a comparative alignment of an upstream stretch of nucleotide sequence preceding the coding region of the <i>spaC</i> pilus gene in the <i>L. rhamnosus</i> GG, LMS2-1, and E800 strains, and as well, the <i>L. casei</i> BL23 strain. The −10/−35 promoter elements predicted previously for the <i>L. rhamnosus</i> GG fimbrial <i>spaCBA</i> operon <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113922#pone.0113922-Douillard1" target="_blank">[15]</a> are indicated in red. Nucleotides identical to this consensus region in the LMS2-1, E800, and BL23 strains are underlined. The nucleotide so designated as the transcriptional start site (TSS) for the <i>L. rhamnosus</i> GG <i>spaCBA</i> pilus locus <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113922#pone.0113922-Douillard1" target="_blank">[15]</a> is indicated. Two hexanucleotide sequences more resembling the typical canonical −10 and −35 consensus promoter elements (as so specified), including a candidate transcriptional initiation nucleotide, are shown in blue. Nucleotides matching the canonical consensus regions are underlined. Nucleotide sequences for the ribosomal binding site (RBS) and the first five codons of the <i>spaC</i> gene are in uppercase black boldface lettering.</p

Induction of IL-8 cytokine production in Caco-2 cells by recombinant SpaFED-piliated lactococci.

<p>Caco-2 cells were treated with normalized cultures of recombinant WT (GRS1189) and SpaF pilin-deleted (GRS1126) SpaFED-piliated lactococci (MOI 100). GRS71 and GRS1052 cells (MOI 100) were used as controls. Endogenous IL-8 cytokine production levels in spent cell culture supernatants were measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113922#s4" target="_blank">Materials and Methods</a>. Triplicate measurements were taken for the experiments, which were repeated independently four times. SEM is displayed as error bars. Pairwise differences between GRS1189 and GRS1226 data are deemed significant (<i>P</i>≤0.05).</p

Adhesive interactions between recombinant SpaFED-piliated lactococci and human intestinal cell lines.

<p>Cell-to-cell adhesion assays involving the Caco-2 (<b>A</b>) and HT-29 (<b>B</b>) gut-related epithelial cell lines and the normalized (OD600 = 0.5) cultures of recombinant WT (GRS1189) and SpaF pilin-deleted (GRS1126) SpaFED-piliated lactococci, including GRS71 and GRS1052 cells as controls, were performed as outlined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113922#s4" target="_blank">Materials and Methods</a>. Triplicate measurements were taken for both sets of experiments. Caco-2 and HT-29 binding experiments were carried out independently four and three times, respectively. SEM is displayed as error bars. For both of the intestinal cell lines, pairwise comparisons made between GRS1189 or GRS1226 cells and the GRS71 control strain indicate the differences in binding data are statistically extremely significant (<i>P</i>≤0.0001).</p

Coding regions of the native fimbrial <i>spaFED</i> operon and the corresponding recombinant SpaFED-piliated lactococcal constructs.

<p>A schematic representation of the native coding region of the <i>L. rhamnosus</i> GG fimbrial <i>spaFED</i> operon, including a depiction of the corresponding lactococcal <i>nisA</i> promoter (P<i>nisA</i>) constructs for expressing WT and SpaF pilin-deleted SpaFED pili, is shown. Names of loci that encode for three different pilin subunits (<i>spaF</i>, <i>spaE</i>, and <i>spaD</i>) and a single pilin-specific sortase (<i>srtC2</i>) are given. Deletion of the <i>spaF</i> gene is indicated by “×”. WT and SpaF pilin-deleted SpaFED-piliated lactococcal clones (GRS1189 and GRS1226, respectively), and the <i>spaFED</i> operon expression plasmids they contain (pKTH5393 and pKTH5443, respectively), are specified.</p