(I) Assessment of RFP expression by <i>P. multocida</i> B:2 JRMT12 pSRG during growth.

<p><i>P. multocida</i> B:2 JRMT12 without plasmid acted as a control for background fluorescence. Fluorescence was plotted against OD_600nm using Microsoft Excel software and data were corrected for background fluorescence. Experiments were performed in triplicate and the error bars indicate standard deviations of the means. (<b>II</b>) <b>Visualization of </b><b><i>P. multocida</i></b><b> B:2 JRMT12 pSRG via fluorescence microscopy.</b> Images of <i>P. multocida</i> B:2 JRMT12 pSRG were taken of the same field under three different light paths; <b>A</b>: DIC (white) light, <b>B</b>: filtered fluorescent light (Exc. = 649 nm, Ems. = 670 nm) (blue) to visualize the counterstain, Cy5 Dye (Amersham Bioscience), <b>C</b>: two filtered lights; filtered fluorescent light (Exc. = 557 nm, Ems. = 585 nm) (red) to visualize bacteria expressing RFP from plasmid pSRG and filtered fluorescent light (Exc. = 649 nm, Ems. = 670 nm) (blue) to visualize the counterstain, Cy5 Dye in order to show that RFP expression was within the bacteria, <b>D</b>: 3D imaging of the 2D image from C using Volocity software.</p

Bactofection pathway for DNA vaccine delivery.

<p>Yellow arrows indicate a schematic pathway for the bacterial plasmid delivery process in EBL cells. After internalization of the bacteria and phagolysosome fusion, the escape of the plasmid DNA (pSRG) from the vacuole into the cytosol after bacterial degradation results in the transfer of the pSRG to the intracellular environment of the EBL cell. Plasmid DNA then localises in the nucleus (blue circle) to allow expression of the GFP gene by the eukaryotic promoter, which is followed by GFP formation in the cytosol (green colouration in cytoplasm). Blue arrows point to microscopic images taken at different stages of the invasion assay; (A) <i>P. multocida</i> B:2 JRMT12 pSRG expressing RFP. Bacteria counterstained with Cy5 Dye (blue). (B) EBL cell at 3h post-invasion showing intracellular <i>P. multocida</i> B:2 JRMT12 pSRG expressing RFP. The EBL plasma membrane is counterstained blue. (C) Optical sectioning of the EBL cell in B at 0.3 µm intervals from top to bottom, confirmed the intracellular location of the RFP-expressing bacteria. (D) EBL cell in B at 3h post-invasion showing intracellular <i>P. multocida</i> B:2 JRMT12 pSRG expressing RFP. (E) The same EBL cell in D expressing GFP from intracellular plasmid DNA. (F) EBL cell expressing GFP at 5h post-invasion with <i>P. multocida</i> B:2 JRMT12 pSRG. Magnification x100; counterstaining with Cy5 Dye (blue) for bacteria and CellMask (blue) for EBL cells.</p

Localization of prokaryotic and eukaryotic protein expression.

<p>The images show EBL cells after invasion with <i>P. multocida</i> B:2 JRMT12 pSRG. Images were captured at 3 h (<b>A</b> and <b>B</b>) and 5 h (<b>C</b> and <b>D</b>) post-invasion. In <b>A</b>, an EBL cell was viewed (<b>i</b>) under DIC (white) light, (<b>ii</b>) by counterstaining with CellMask™ plasma membrane stain (GE Healthcare) (blue), (iii) merged 2D images of (ii) and image captured under filtered fluorescent light (Exc. = 557 nm, Ems. = 585 nm) (red) to visualize internalized RFP-expressing bacteria and (iv) a 3D image of (iii). In B and C, an EBL cell was viewed (i) under DIC (white) light, (ii) by counterstaining with CellMask™ plasma membrane stain (GE Healthcare) (blue), (<b>iii</b>) merged 2D images of (ii) and image captured under filtered fluorescent light (Exc. = 557 nm, Ems. = 585 nm) (red), (<b>iv</b>) merged 2D images of (ii) and image captured under filtered fluorescent light (Exc. = 488 nm, Ems. = 507 nm) (green) to visualize expression of GFP by the mammalian cells, (v) a 3D image of (iii) and (<b>vi</b>) a 3D image of (iv). In <b>D</b>, an EBL cell was viewed (<b>i</b>) under DIC (white) light, (<b>ii</b>) by counterstaining with CellMask™ plasma membrane stain (GE Healthcare) (blue), (<b>iii</b>) merged 2D images of (ii) and image captured under filtered fluorescent light (Exc. = 488 nm, Ems. = 507 nm) (green) and (<b>iv</b>) a 3D image of (iii). The merged 3D images were included to demonstrate the localization of either RFP or GFP expression. Fluorescence emitted by EBL cells infected with JRMT12 strain harbouring pMK-RED (<b>E</b>) with the same microscope settings, was used as a negative control. Each cell was viewed (i) under DIC (white) light, (<b>ii</b>) by counterstaining with CellMask™ plasma membrane stain (GE Healthcare) (blue), (<b>iii</b>) merged 2D images of (ii) and image captured under filtered fluorescent light (Exc. = 557 nm, Ems. = 585 nm) (red) to visualize internalized RFP-expressing bacteria and (<b>iv</b>) merged 2D images of (ii) and image captured under filtered fluorescent light (Exc. = 488 nm, Ems. = 507 nm) (green).</p

Bacterial strains and plasmids used in this study.

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

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

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 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