29 research outputs found

    TLR2-mediated immune responses induced by recombinant SpaCBA-piliated lactococcal cells.

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    <p>Stimulation of TLR2-dependent NF-ÎșB activation in the HEK-TLR2 cell line by normalized cultures of recombinant SpaCBA-piliated lactococcal cells (GRS1185, GRS1195, GRS1211, and GRS1223) was carried out according to the method described in Materials and Methods. GRS71 and GRS1052 lactococcal cells as well as DMEM cell culture media were included as negative controls. TLR2-agonist lipopeptide Pam3CSK4 (1 ng/ml) was used a positive control. Measurements were done in quadruplicate and the experiment was performed three times. Error bars are standard error of mean (SEM). Differences between data (GRS1185, GRS1195, GRS1211, or GRS1223) from pairwise comparisons against GRS71 data are considered extremely significant (<i>P</i><0.001).</p

    Uncovering Surface-Exposed Antigens of <i>Lactobacillus rhamnosus</i> by Cell Shaving Proteomics and Two-Dimensional Immunoblotting

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    The present study reports the identification and comparison of all expressed cell-surface exposed proteins from the well-known probiotic <i>L. rhamnosus</i> GG and a related dairy strain, Lc705. To obtain this information, the cell-surface bound proteins were released from intact cells by trypsin shaving under hypertonic conditions with and without DTT. Liquid chromatography tandem mass spectrometry (LC−MS/MS) analyses of the purified peptides identified a total of 102 and 198 individual proteins from GG and Lc705, respectively. Comparison of both data sets suggested that the Msp-type antigens (Msp1, Msp2) and the serine protease HtrA were uniquely exposed at the cell surface of GG, whereas the Lc705-specific proteins included lactocepin and a wider range of different moonlighting proteins. ImmunoEM analyses with the GG and Lc705 antibodies suggested that the whole-cell immunization yielded antibodies toward surface-bound proteins and proteins that were secreted or released from the cell-surface. One of the detected antigens was a pilus-like structure on the surface of GG cells, which was not detected with Lc705 antibodies. Further 2-DE immunoblotting analysis of GG proteins with both <i>L. rhamnosus</i> antisera revealed that majority of the detected antigens were moonlighting proteins with potential roles in adhesion, pathogen exclusion or immune stimulation. The present study provides the first catalog of surface-exposed proteins from lactobacilli and highlights the importance of the specifically exposed moonlighting proteins for adaptation and probiotic functions of <i>L. rhamnosus</i>

    Induction of cytokine production in human moDCs by recombinant SpaCBA-piliated lactococcal cells.

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    <p>Stimulation of TNF-α, IL-12, IL-10, and IL-6 cytokine production (A, B, C, and D, respectively) in human moDCs by normalized cultures of recombinant SpaCBA-piliated lactococcal cells (GRS1185, GRS1195, GRS1211, and GRS1223) was carried out according to the method described in Materials and Methods. Cells from GRS71 and GRS1052 <i>L. lactis</i> were included as negative controls. RPMI cell culture media and <i>E. coli</i> lipopolysaccharide (LPS; 1 ”g/ml) were also included as controls. Measurements were carried out in quadruplicate and each experiment was done three times with moDCs from four different donors, with the data used being the most representative of the experiments. Error bars indicate standard error of mean (SEM).</p

    Effect of heat-treated WT SpaCBA-piliated lactococcal cells on TLR2-dependent immune activity.

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    <p>Stimulation of TLR2-dependent NF-ÎșB activation (A) and IL-8 cytokine production (B) in the HEK-TLR2 cell line by either live (-) or heat-treated (100°C for 10 minutes) (+) WT SpaCBA-piliated lactococcal cells (GRS1185) was performed as described in Materials and Methods. Live and heat-treated GRS1052 lactococcal cells were also tested. DMEM cell culture media, Pam3CSK4 (1 ng/ml), and <i>E. coli</i> lipopolysaccharide (LPS; 1 ng/ml) were included as controls. Quadruplicate and duplicate measurements for NF-ÎșB activation and IL-8 production, respectively, were performed. Error bars indicate standard error of mean (SEM). Differences between GRS1185 and GRS1052 data are deemed extremely significant (<i>P</i><0.001).</p

    Characterization of recombinant-expressed <i>L. rhamnosus</i> GG SpaCBA pili in <i>L. lactis</i>.

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    <p>(A) The coding region of the <i>L. rhamnosus</i> GG <i>spaCBA</i> pilus operon and a summary of the nisin promoter-regulated (P<i>nisA</i>) WT and pilin-deleted SpaBCA-piliated recombinant constructs in <i>L. lactis</i> are depicted schematically. Genes for the pilin subunits (<i>spaA</i>, <i>spaB</i>, and <i>spaC</i>) and pilin-specific sortase (<i>srtC1</i>), including those genes that are deleted (×) in the recombinant constructs, are indicated. Identities for the various SpaCBA-piliated lactococcal constructs (GRS1185, GRS1195, GRS1211, and GRS1223), including the <i>spaCBA</i> pilus operon-containing plasmids they each propagate (pKTH5391, pKTH5497, pKTH5413, and pKTH5440, respectively), are indicated. (B) Immunoblot analysis of cell wall-extracted proteins recovered from <i>L. rhamnosus</i> GG (lane 2), the empty vector (pKTH5080)-carrying GRS1052 <i>L. lactis</i> construct (lane 3), and the nisin-induced WT SpaCBA-piliated GRS1085 <i>L. lactis</i> construct (lane 4) probed with the various pilin-specific antisera (anti-SpaA, anti-SpaB, or anti-SpaC, as indicated). Asterisks on the right of each immunoblot indicate the presumed location of monomeric SpaA, SpaB, and SpaC pilin proteins. The compressed and laddered region of high-molecular-weight (HMW) protein bands representing various lengths of pili is indicated on the left. Molecular weight markers (lane 1) and their positions are indicated on the left. (C) Immunogold double-labeling electron microscopy of WT SpaCBA-piliated lactococcal cells (GRS1185) using SpaA antiserum with protein A-10-nm gold particles as well as SpaC antiserum with protein A-5-nm gold particles is shown. The location of 5-nm (white arrow) and 10-nm (black arrow) gold particles on the pilus structure are indicated. (D) Immunoblot analysis of cell wall-extracted proteins recovered from <i>L. rhamnosus</i> GG (lane 1) and the empty vector (pKTH5080)-carrying GRS1052 <i>L. lactis</i> construct (lane 2), as well as the nisin-induced WT (GRS1085) (lane 3), SpaB-deleted (GRS1195) (lane 4), SpaC-deleted (GRS1211) (lane 5), and SpaB- and SpaC-deleted (GRS1223) (lane 6) piliated <i>L. lactis</i> constructs probed with anti-SpaA, anti-SpaB, or anti-SpaC sera, each of which is indicated on the left of the respective immunoblot. Positions of monomeric SpaA (∌31 kDa), SpaB (∌21 kDa), and SpaC (∌91 kDa) protein bands are marked by an asterisk on the right of corresponding immunoblots.</p

    Influence of cell-to-cell interactions on TLR2-dependent immune responses induced by WT SpaCBA-piliated lactococcal cells.

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    <p>Stimulation of human TLR2-dependent NF-ÎșB activation (A) and IL-8 cytokine production (B) in the HEK-TLR2 cell line by WT SpaCBA-piliated lactococcal cells (GRS1185; MOI 625), either non-partitioned (-) or partitioned (+) with Transwell membranes (0.4-”m pore size), was carried out as outlined in Materials and Methods. GRS1052 lactococcal cells (MOI 625) and DMEM cell culture media were included as negative controls. Measurements were conducted in quadruplicate and each experiment was carried out three times. Error bars reflect standard error of mean (SEM). Differences between GRS1185 and GRS1052 data are considered significant (<i>P</i><0.05).</p

    Binding of recombinant SpaCBA-piliated lactococcal cells to human intestinal mucus.

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    <p>The adhesion of metabolically labeled (<sup>3</sup>H) cells from normalized cultures of recombinant SpaCBA-piliated lactococcal constructs (GRS1185, GRS1195, GRS1211, and GRS1223), along with vectorless (GRS71) and empty vector (GRS1052) <i>L. lactis</i> NZ900 as negative controls and <i>L. rhamnosus</i> GG (GG) as a positive control, to human intestinal mucus was performed according to the method outlined in Materials and Methods. Measurements were done in quadruplicate and the experiment was performed three times. Error bars represent standard error of mean (SEM). Differences between data (GRS1185, GRS1195, or GRS1211) from pairwise comparisons against GRS71 data are regarded as extremely significant (<i>P</i><0.001), with the exception of data from GRS1223, which was deemed not significant (<i>P</i>>0.05).</p

    Amuc_1100 is located on the outer membrane of <i>A</i>. <i>muciniphila</i>.

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    <p>Immunofluorescence staining of <i>A</i>. <i>muciniphila</i> cells with whole cell antiserum (A) or anti-Amuc_1100 (B) and Alexa-488-conjugated secondary IgG. Phase-contrast images of the same microscopic fields are shown below. Pictures were cropped from the original image.</p

    Effect of <i>A</i>. <i>muciniphila</i>, <i>F</i>. <i>prausnitzii</i> and <i>L</i>. <i>plantarum</i> on cytokine production of human PBMCs.

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    <p>IL-10 (A), TNF-α (B), IL-8 (C) and IL-6 (D) responses of human PBMCs (n = 3 donors) stimulated with <i>A</i>. <i>muciniphila</i>, <i>F</i>. <i>prausnitzii</i> and <i>L</i>. <i>plantarum</i> live cells. IL-10 (E), TNF-α (F), IL-8 (G) and IL-6 (H) responses of human PBMCs (n = 3 donors) stimulated with <i>A</i>. <i>muciniphila</i>, <i>F</i>. <i>prausnitzii</i> and <i>L</i>. <i>plantarum</i> supernatant. *, P<0.05. Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Tukey’s HSD if homogeneity of variance was met or Games-Howell if variance was unequal.</p
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