The absence of lp36 does not affect NF-κB-mediated signaling, but is required for transcription of <i>ifna1</i> and <i>ifnb1</i>.

<p>Human PBMCs (5×10⁶) were co-incubated for 12 hours with 5×10⁷ B31-A3 or A3-M9 lp36−. Equal amounts of PBMC total RNA from each of three biological replicates was pooled, converted to cDNA and used as a template for the Human NF-κB Signaling Pathway RT² Profiler PCR Array. Genes that were transcriptionally induced or repressed ≥4-fold are indicated by red or green circles, respectively. Scatter plots depict transcriptional regulation in PBMCs stimulated with B31-A3 <i>(</i><b><i>A</i></b><i>)</i> or A3-M9 lp36− (<b><i>B</i></b>) versus unstimulated PBMCs. <b><i>C</i></b><i>,</i> Comparison of transcript profiles of PBMCs stimulated with either B31-A3 or A3-M9 lp36− <i>B. burgdorferi</i>.</p

Oligonucleotides used for plasmid content analysis.

<p>Oligonucleotides used for plasmid content analysis.</p

RST1 isolates contain a region of lp36 that is lacking in RST3 isolates.

<p>RST1 and RST3 <i>B. burgdorferi</i> isolates were grown to late log phase prior to isolation of plasmid DNA. (<b><i>A</i></b>) PCR primers were designed to amplify a ∼2.5 kilobase region on the distal end of lp36. The presence of lp36 was assessed by amplification of <i>bbk</i>19, an ORF located adjacent to the plasmid partition locus. PCR products were analyzed by gel electrophoresis in 1% agarose gels prepared in 1X TBE buffer and stained with ethidium bromide. (<b>B</b>) Southern blotting of <i>Spe</i>I digests of total genomic DNAs of <i>B. burgdorferi</i> isolates was performed using a probe specific for the variable region of lp36 (upper panel). As a control, membranes were stripped and re-probed for <i>B. burgdorferi ospA</i> (BBA15), located on lp54 (lower panel).</p

lp36 contributes to the association of <i>B. burgdorferi</i> with specific populations of dendritic cells.

<p>Human PBMCs (4×10⁶) were co-incubated with 4×10⁷ GFP-tagged B31 (black bars), A3-M9 lp36− (white bars) or A3-M9 lp36−/lp36+ (cross-hatched bars) <i>B. burgdorferi</i> for 6 hours at 4°C or 37°C. The percentages of GFP⁺ mDC1s (CD19⁻CD3⁻BDCA2⁻BDCA1⁺) (<b><i>A</i></b>), pDCs (CD19⁻CD3⁻BDCA2⁺BDCA1⁻) or (<b><i>B</i></b>) mDC2s (CD19⁻CD3⁻BDCA3⁺BDCA2⁻) (<b><i>C</i></b>) were determined by multiparameter flow cytometry. Dot plots representing 500,000 collected events are provided to illustrate gating strategies (left). Column graphs represent the mean and standard deviation of three biological replicates (right). Statistical analysis was performed using a one-way ANOVA with a Tukey’s post-test for multiple comparisons.</p

The adhesion and uptake of <i>B. burgdorferi</i> is significantly reduced when lp36 is absent.

<p>Human PBMCs (4×10⁶) were co-incubated with 4×10⁷ GFP-tagged B31-A3 (black bars), A3-M9 lp36− (white bars) or A3-M9 lp36−/lp36+ (cross-hatched bars) for 6 hours at 4°C or 37°C. <b><i>A</i></b>, Flow cytometry was performed to compare the ability of the GFP-tagged <i>B. burgdorferi</i> strains to associate with PBMCs. Representative dot plots are shown on the left. Graphs (right) depict the mean percentage ± SD of PBMCs identified as FITC+, indicating either adhesion (4°C) or adhesion and uptake (37°C) of GFP-tagged <i>B. burgdorferi</i>. Data are representative of results from three blood donors assessed in triplicate. A one-way ANOVA with a Tukey’s post-test was used to determine statistical significance. <b><i>B</i></b> and <b><i>C</i></b>, Three-dimensional fluorescence deconvolution microscopy was performed to quantify spirochetes adherent to, or internalized by, PBMCs. Samples were co-incubated with GFP-tagged <i>B. burgdorferi</i> for 6 hours at 37°C. PBMC outer cell membranes were labeled with APC-conjugated anti-CD45 to label the outer cell membrane (cyan) and DAPI (blue) was used to label the cell nucleus. Single optical sections of images acquired under 100X magnification were examined for adherent (<b><i>B</i></b>, left) or internalized (<b><i>B</i></b>, right) GFP-tagged <i>B. burgdorferi</i>. <b><i>C</i></b>, Columns show the mean (± SD) number of spirochetes outside (black bars) or inside (white bars) PBMCs. Data were collected from single optical sections taken from five 100X fields from each of three independent biological replicates. An unpaired, two-tailed Student’s <i>t</i>-test was used for statistical analysis.</p

The presence of lp36 is required for IFN-α and IFN-λ induction.

<p>Human PBMCs (4×10⁶) were co-incubated for 20 hrs with 4×10⁷ wt B31-A3 (black bars), B31-4 (gray bars), A3-M9 lp36− (white bars) or A3-M9 lp36−/lp36+ (cross-hatched bar)(MOI = 10). Protein concentrations of human IFN-α (<b><i>A</i></b>), IFN-λ1 (<b><i>B</i></b>), or IFN-γ (<b><i>C</i></b>) in cell-free culture supernatants were quantitated by ELISA (<b><i>A</i></b>,<b><i>B</i></b>) or cytometric bead array (<b><i>C</i></b>). The results are shown as the mean ± SD of values from three to five blood donors assessed in two or three independent experiments. Statistics were obtained using a non-parametric Mann-Whitney U-test.</p

Pro- and anti-inflammatory cytokine production by RST1 and RST3 <i>B. burgdorferi</i> clinical isolates.

<p>*B31-4 and A3-M9 lp36− were not included in the RST1 group when calculating averages and performing statistical analysis. Statistical significance was determined using a non-parametric Mann-Whitney U-test. Data shown are from three independent donors assessed in separate experiments.</p

<i>B. burgdorferi</i> clinical isolates and strains used in this study.

a<p>Genotype was determined by PCR-RFLP analysis of 16S-23S ribosomal RNA gene spacer <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100174#pone.0100174-Liveris2" target="_blank">[9]</a>.</p

Pro- and anti-inflammatory cytokine profiles elicited by <i>B. burgdorferi</i> B31-A3 and A3-M9 lp36−.

<p>Statistical analysis was performed using a non-parametric Mann Whitney U-test. Data shown are from three to five independent donors assessed in separate experiments.</p

RST1 isolates induce expression of multiple interferons.

<p>Human PBMCs (4×10⁶) were co-incubated for 20 hrs with RST1 (black bars) or RST3 (white bars) <i>B. burgdorferi</i> clinical isolates (4×10⁷) (MOI = 10∶1). Concentrations of human IFN-α (<b><i>A</i></b>), IFN-λ1/IL29 (<b><i>B</i></b>), or IFN-γ (<b><i>C</i></b>) proteins in cell-free culture supernatants were quantitated by ELISA (<b><i>A</i></b><i>,</i><b><i>B</i></b>) or cytometric bead array (<b><i>C</i></b>). Columns represent the mean ± SD of values from three blood donors assessed in independent experiments, with the exception of (<b><i>B</i></b>) which represents results from a single donor. Statistical analysis was performed using a non-parametric, two-tailed Mann-Whitney U-test.</p