Bacterial strains used in the study.

<p>WT, wild-type</p><p>Bacterial strains used in the study.</p

Intracellular survival of wild-type GBS in human astrocytes.

<p>A, Wild-type GBS, COH1, intracellular survival assays in SVG-A cells were carried out for up to 12 hr at MOI of 1 and 10. To assess intracellular survival in SVG-A cells over time, intracellular bacteria were enumerated after the addition of antibiotics (gentamicin (50 μg/ml) and penicillin (25 μg/ml) from 2 to 12 h time points. B, Transmission electron micrographs demonstrating GBS (COH1) intracellular survival over time in SVG-A cells at magnifications (6,000X for A-J, and 30,000X for K-O). All assays were performed in triplicate or quadruplet and repeated at least three times.</p

GBS adhesion, invasion and intracellular survival of SVG-A cells.

<p>A and B, Confluent monolayers of SVG-A cells were incubated with GBS, COH1 wild-type strain, at an MOI of 1 for various times at 37°C, 5% CO₂ and adhesion and invasion was examined as described in Materials and Methods. The data shown are a representative of 3 independent experiments performed in triplicate. Incubation at shorter and longer times resulted in a similar level of adhesion and invasion of COH1 to SVG-A cells and was not statistically significant as determined by one-way ANOVA and Tukey's multiple comparison tests. C, SVG-A cells were infected with COH1 expressing GFP at an MOI of 10 and incubated for 8 h at 37°C. Cells were fixed and processed for fluorescence microscopy and stained with Cell Mask (red) to visualize the plasma membrane or with DAPI (blue) to visualize the nucleus. The image is representative of many images from 2 independent experiments.</p

Activation of human astrocytes following GBS infection.

<p>A-D, Transcript abundance of IL-1β, IL-6, VEGF, and IL-8 in SVG-A cells was determined using quantitative RT-PCR following infection with GBS COH1 (MOI = 50). Fold change was calculated using GAPDH and then normalized to media controls as described in Materials and Methods. E, Protein production of IL-8 was determined by ELISA as described in Materials and Methods. Data is one representative experiment of at least 2 independent experiments performed in a minimum of 4 replicates. Data was analyzed by two-way ANOVA with Bonferroni’s multiple comparisons post-test. (*) = P< 0.05, (***) = P< 0.001, (****) = P< 0.0001.</p

Interaction of GBS with human SVG-A astrocytes.

<p>A, confluent monolayers of SVG-A cells were infected with GBS strain COH1 at the indicated MOI for 30 minutes for adhesion and 2 h for invasion assays and the extent of bacterial adherence and invasion were quantified as described in Materials and Methods. The data are expressed as the percentage of recovered CFU/initial inoculum. All experiments were performed in triplicate and repeated in at least three independent experiments. B, SVG-A cells were infected with COH1 at an MOI of 1 and 10 for 24 hours and percent cell viability determined by trypan blue dye exclusion and compared to an uninfected control. C, SVG-A cells were pretreated in media alone, DMSO vehicle control or cytochalasin D (Cyt D) at 1 to 5 μg/ml for 30 min at 37°C and 5% CO₂. Cells were then incubated with COH1 at an MOI of 1 for 2 h and intracellular GBS was quantified. The data shown represents the percentage of bacterial uptake after cytochalasin D treatment relative to untreated control. Significance was determined by one-way ANOVA using tukeys multiple comparison test, P < 0.0001 (****). D, Transmission electron microscopic (TEM) images of SVG-A cells that were either uninfected (control) or infected with GBS COH1 at an MOI of 10. For TEM scale bars for panels A, B, D, and E are 2um and for C and F are 500nm.</p

Meningioma cell injury induced by wild type GBS strains.

<p>A) Release of LDH: cells were infected for 9 h with different initial MOI of GBS strains and LDH release was measured. Results shown are the mean values of LDH release compared with maximum release by lysed cells and the error bars are the SEM from at least two independent experiments, each performed in triplicate. B) Confocal microscopy: cell cultures were infected with a range of initial MOI of different serotypes of wild type GBS for 9 h. Cell death was examined using the LIVE/DEAD fluorescent dye assay, where uptake of the red dye (ethidium homodimer) identifies dead cells and uptake of the green dye (calcein AM) identifies live cells. The scale bar shows 75 µm (×40 lens magnification). Images are of monolayers infected for 9 h, except for the COH-1 strain infected for 24 h, and they are representative of experiments carried out in triplicate. Similar data were obtained using both cell lines.</p

Bacterial strains used in the study.

<p>WT, wild type; − Non-haemolytic; + Weakly haemolytic; ++ Normo-haemolytic; +++ Hyper-haemolytic.</p

Invasion of meningioma cells by GBS.

<p>Monolayers were infected for 9 h with an initial MOI of 0.3 (10⁴ cfu bacteria), washed and then treated with gentamicin.</p>*<p>The percentage of invading bacteria (invasion rate) was calculated using the formula: Invasion rate (%) = (internalised bacteria/associated bacteria)×100. The mean and SEM for GBS were calculated from n = 3–6 experiments. <i>E. coli</i> IH3080 was included as a positive control and showed an invasion rate of 1.167% (SEM ± 0.283 from n = 10 experiments). Similar data were obtained using both meningioma cell lines.</p

The surfactant DPPC protects meningioma cells from GBS-induced death.

<p>(A) Effect of DPPC on cell death induced by live bacterial infection. Meningioma cells were infected with doses of GBS strains that induced the highest levels of LDH release (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042660#pone-0042660-g002" target="_blank">Figure 2A</a>) and cell death quantified at 9 h. The columns represent the mean levels of LDH released from monolayers and the arrows indicate the percentage of reduction of LDH release in the presence of DPPC in comparison with LDH in the absence of DPPC. The error bars show the SEM of at least two independent experiments. The two confocal microscopy images show monolayers infected with a cytotoxic dose (initial MOI, 30) of the hyper-haemolytic NCTC10/84 strain for 9 h in the absence and presence of DPPC. (B) The protective effect of DPPC is dependent on time of addition. Meningioma cell monolayers were infected with GBS strain NCTC10/84 and DPPC (3 mg/ml) added at 0, 3 and 6 h. LDH release was measured at 9 h. The columns represent the mean levels of LDH release and the error bars the SEM of 3 independent infection experiments. (C) Cell death induced by β-h/c toxin extracts and inhibition by DPPC. The graph shows LDH release from monolayers treated with β-h/c toxin extracts prepared from wild type NCTC and A909 strains (containing 100HU and 50HU, respectively). Equal volumes of extracts prepared from their β-h/c deficient mutant strains were also used. Results shown are mean values of LDH release and error bars are the SEM from four independent experiments using two independent batches of β-h/c toxin extracts. Arrows indicate the percentage inhibition of LDH release in the presence and absence of DPPC. The confocal microscopy images show monolayers treated with β-h/c extracts (250HU) prepared from wild type GBS strains for 9 h in the absence and presence of DPPC. An equivalent volume of extract prepared from the Δ<i>cylE</i> (β-h/c deficient) mutant strain was also used. Using the LIVE/DEAD assay, the red colour identifies dead cells, whereas the green colour corresponds to viable cells. The scale bar at bottom right of each image measures 75 µm (×40 magnification). Similar data were obtained using both cell lines.</p

GBS adherence to hBMEC is mediated by the interaction of Srr1 and fibrinogen.

<p>(A) Fibrinogen on the surface of hBMEC pretreated with or without exogenous fibrinogen (Fg; 20 µg/ml). Nuclei were stained with DAPI (blue) and fibrinogen was detected with anti-fibrinogen IgG, followed by Alexa Fluor 488 conjugated anti-rabbit IgG (red). (B) NCTC 10/84 (WT) or PS2645 (Δ<i>srr1</i>) incubated with hBMEC, with or without fibrinogen pretreatment (20 µg/ml). Unbound bacteria were washed out and bound bacteria were counted. Values represent percent (mean ± S.D.) of total GBS inoculum bound to the monolayers. * = P<0.01.</p