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 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

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

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