Bacterial strains and plasmids used in this study.

<p>Bacterial strains and plasmids used in this study.</p

Fluorescence microscopy of NleH-GFP.

<p>pAHE8 (NleH1-GFP) and pAHE22 (NleH2-GFP) were transformed in ZAP193, ZAP193Δler and ZAP193ΔgrlA and at OD₆₀₀ = 0.8, dried onto a microscope slide in 4% PFA and stained for EspA filaments. Volocity quantification software was used to determine the average GFP fluorescence per voxel of 100 individual bacteria for NleH1 (A) and NleH2 (B). Each point represents the average GFP fluorescence from a composite from 16 z-slice images thus reducing planar effects. Error bars represent the standard deviation.</p

Quantitative PCR of NleH transcripts in LEE regulator knockouts.

<p>RNA was collected from ZAP193 strains WT, Δler and ΔgrlA grown to OD₆₀₀ = 1.2 in MEM and cDNA prepared. NleH1, NleH2, GapA, Tir and 16S RNA transcript was then quantified by q-PCR, NleH values normalised to that of 16S RNA, and the fold change calculated comparing mutant to wild-type. Bars represent the average of three biological samples. Error bars represent the standard error of the mean.</p

Expression of NleH-GFP upon <i>E. coli</i> O157:H7 ZAP193 contact with EBL cells.

<p>ZAP193 transformed with plasmids expressing GFP constitutively (pAJR145; <i>rpsm</i>::<i>gfp</i>) or translational fusions of <i>nleH</i> or <i>tir</i> to <i>gfp</i> under the control of their native promoter (pAHE8; NleH1-GFP, pAHE22; NleH2-GFP, pAJR75; Tir-GFP) were added to EBL cells and incubated for 0, 5, 30, 60 or 180 minutes at 37°C, 5% CO₂ before the removal of supernatant and fixation of cells with 4% paraformaldehyde. The panel of images is representative of all time points tested, apart from Tir-GFP, that showed strong early expression during cell contact but was markedly reduced at 180 minutes.</p

Expression of NleH-GFP and Tir-GFP in <i>E. coli</i> O157:H7 defined LEE regulator mutants.

<p><i>E. coli</i> O157:H7 ZAP193, ZAP193Δ<i>ler</i> and ZAP193Δ<i>grlA</i> were transformed with constructs expressing NleH1-GFP (pAHE8; A), NleH2-GFP (pAHE22; B) and Tir-GFP (pAJR132; C). GFP expression was monitored during growth of the transformants in MEM media, with a promoterless GFP construct (pAJR70) as a background control. Fluorescence values were corrected for background and lines represent the average of three biological repeats.</p

Expression of NleH-GFP constructs in <i>E. coli</i> O157:H7 grown in defined media.

<p>Constructs consisting of 120 bp (pAHE18), 283 bp (pAHE19) or 531 bp (pAHE8) of the NleH1 5′ UTR and 113 bp (pAHE20), 291 bp (pAHE21) or 655 bp (pAHE22) of the NleH2 5′ UTR cloned upstream of <i>gfp</i> were transformed into ZAP193, grown in MEM-HEPES (A) or DMEM (B) and GFP fluorescence measured during growth. All values were corrected for background from a promoter-less GFP (pAJR70) control measured at the same optical density. Graphs represent the average of three experimental repeats.</p

NF-κB activity in the presence of NleH variants.

<p>HEK293T cells were co-transfected with a luciferase reporter plasmid under the control of consensus κB sites, a β-galactosidase plasmid and a control (pCMV), NleH or OspG vector. After 40 hours, cells were stimulated by the addition of TNF-α (25 ng/ml; 24 hours). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033408#s2" target="_blank">Results</a> represent three biological replicates, where variants were tested in triplicate and assayed in duplicate. Statistical analysis with one-way ANOVA shows no significant difference compared with the pCMV control. Error bars represent the standard error of the mean.</p

AIM assay detects tetanus-specific CD4⁺ T cells.

<p>(A) Representative flow cytometry plots of CD25⁺OX40⁺ upregulation by CD4⁺ T cells in naïve (CD45RA⁺CCR7⁺; lower panel) and antigen-experienced memory (excluding naïve cells; upper panel) cells after stimulation with TT-megapool [TT (MP)], Tetanus Toxoid [TT (ag)], Dengue virus-megapool [DV (MP)], or PHA as a positive control. (B) Median CD25⁺OX40⁺ expression by CD4⁺ memory T cells after 24 hours. Each dot represents one donor originally primed with DTwP vaccine and not recently boosted. Kruskal-Wallis multiple comparison test, *, <i>p</i><0.05. (C and D) Percentage IFNγ- (C) or IL-4-producing (D) CD4⁺ memory T cells in response to TT megapool, or PMA/Ion.</p

Differential polarization of T cell responses as a function of the original vaccine type used for childhood vaccination.

<p>IFNγ and IL-5 responses were measured by dual color ELISPOT assays. Each data point represents the ratio of IFNγ/IL-5 responses of each positive individual peptide from all the reactive donors. Median ± interquartile range for donors originally primed with DTwP or DTaP vaccine is represented. Two-tailed Mann-Whitney test.</p

Bioinformatics predictions efficiently identify the preponderance of the tetanus toxin response.

<p>(A) Total SFC detected against known (black bar) and novel (grey bar) epitopes. (B) Percentage of the total response captured by the indicated percentile score from peptide binding predictions.</p