Meningococcal <i>pilC1</i> mediates calcium efflux from intracellular stores of host endothelial cells.

<p>HBMEC were pre-treated with 50 µM of 2-APB before meningococcal infection. 2-APB inhibits IP₃ receptors and blocks release of calcium from store operated channels. Fluorescence microscopy was performed at the indicated time points. Uninfected cells (control) and cells infected with <i>L. lactis</i> (<i>L. lactis</i>), respectively, were used as controls. Representative images from an individual experiment repeated five times in triplicate are shown. Scale bars represent 50 µm in all panels. (B) Quantification of changes in calcium concentrations in host endothelial cells pre-treated with 2-APB during infection with different meningococcal strains are shown at the indicated time points. Uninfected cells (control) and cells infected with <i>L. lactis</i> (<i>L. lactis</i>), respectively, were used as controls. Data are expressed as the mean ± S.D of fifteen independent readings. *<i>P</i><0.05, **<i>P</i><0.01, versus values from control samples by Student’s <i>t</i>-test.</p

Meningococci mediate calcium mobilization in host cells via <i>pilC1.</i>

<p>(A) Changes of host cells intracellular calcium concentrations during infection with the meningococcal strains WT, <i>ΔpilC1</i> and <i>ΔpilC2</i> for the indicated times were monitored by performing immunofluorescence microscopy of <i>Fluo</i>-8 labeled HBMEC. Uninfected cells (control) and cells infected with <i>L. lactis</i> (<i>L. lactis</i>), respectively, were used as controls. Representative images from an individual experiment repeated five times in triplicate are shown. Scale bars represent 50 µm in all panels. (B) Quantification of changes in calcium concentrations in host endothelial cells during infection with different meningococcal strains are shown at the indicated time points. Uninfected cells (control) and cells infected with <i>L. lactis</i> (<i>L. lactis</i>), respectively, were used as controls. Data are expressed as the mean ± S.D of fifteen independent readings. *<i>P</i><0.05, **<i>P</i><0.01, versus values from control samples by Student’s <i>t</i>-test.</p

Meningococcal <i>pilC1-</i>mediated adherence to and invasion into host cells require PLC.

<p>Host cells were grown in wells of 24 well plates, pre-treated with different concentrations of U73122 (A and B), U73343 (C and D) or left untreated (control). Infection assays with three meningococcal strains (WT, <i>ΔpilC1</i> and <i>ΔpilC2</i>) were carried out for 4 h at 37°C and 5% CO₂. (A and C) The total number of bacteria associated with host cells was determined after removing unbound bacteria by washing the cells grown in wells of 24 well plates with infection medium. Subsequently, host cells were lysed with saponin and serial dilutions of the lysates were plated on chocolate agar and incubated overnight at 37°C. Control of WT was taken as 100% and other values are presented as relative to this control. (B and D) After removing unbound bacteria by washing, the number of internalized meningococci was determined by the antibiotic protection assay after 4 h of infection in presence of 2-APB. Data are expressed as the mean ± S.D of five independent experiments performed in duplicate. *<i>P</i><0.05, **<i>P</i><0.01 versus values from control samples by Student’s <i>t</i>-test.</p

Intracellular release of calcium ions facilitates meningococcal internalization.

<p>HBMEC were grown on (A) cover slips or (B and C) directly in wells of 24 well plates. Host cells were treated with or without 2-APB and infected with meningococcal strains WT, <i>ΔpilC1</i> and <i>ΔpilC2</i> for 4 h at 37°C. (A) After removing unbound bacteria by washing, adherent meningococci were incubated with chicken anti-meningococcal primary IgG and Alexa-Fluor-594 (red) coupled anti-chicken secondary antibodies. Cells were permeabilized by Triton X-100 and extra- and intracellular meningococci were further incubated with chicken anti-meningococcal IgG and Alexa-Fluor-488 (green) coupled anti-chicken antibodies. DAPI was used to stain the host cell nuclei. Fluorescence microscopy was performed using a Zeiss Axiobserver Z1 microscope equipped with an Apotome. Extracellular bacteria appear yellow (red and green) and intracellular bacteria appear red. Representative images from an individual experiment repeated five times in triplicate are shown. Scale bars represent 50 µm in all panels. (B) The total number of bacteria associated with host cells was determined after removing unbound extracellular bacteria by washing the cells grown in wells of 24 well plates with infection medium. Host cells were lysed with saponin followed by plating of the serially diluted cells on chocolate agar plates overnight at 37°C. Control of WT was taken as 100% and other values are presented as relative to this control. (C) After removing unbound bacteria by repeated washing steps, the number of internalized meningococci was determined by the antibiotic protection assay after 4 h of infection in presence of 2-APB. Data are expressed as the mean ± S.D of five independent experiments performed in duplicate. *<i>P</i><0.05, ***<i>P</i><0.001, versus values from control samples by Student’s <i>t</i>-test.</p

Chelating intracellular calcium inhibits meningococcal invasion into host endothelial cells.

<p>BAPTA-AM (50 µM) pre-treated host cells were grown on (A) glass cover slips for microscopy or directly in (B and C) wells of 24 well plates to quantify the adhered and internalized bacteria. HBMEC were infected with meningococci (MOI50) for 4 h at 37°C and 5% CO₂. After infection, host cells were washed thoroughly to remove the unbound meningococci. (A) Cells were fixed with paraformaldehyde, incubated with anti-meningococcal IGg, followed by an Alexa-Fluor-594 labelled secondary antibody. Host cells were permeabilized to stain intracellular bacteria with Alexa-Fluor-488 and nuclei with DAPI. Scale bars represent 50 µm. (B) The number of adhered meningococci to host cells grown in wells of 24 well plates was determined after removing unbound extracellular bacteria and plating the recovered bacteria on blood agar plates. (C) Effect of pre-treating host endothelial cells with BAPTA-AM on internalization meningococci after 4 h of infection of HBMEC as determined by the antibiotic protection assay. Adherence and internalization of WT meningococci by host cells in the absence of BAPTA-AM was set to 100% and other values are presented as relative percentage relative to the WT control. Data represent means ± S.D (n = 5) (*<i>P</i><0.05).</p

Phospholipase C is required to induce calcium signaling by meningococci.

<p>Host endothelial cells were treated with <i>Fluo</i>-8 calcium dye for 1 h prior to infection with meningococcal strains. (A, B and C) Cells were either treated with the PLC inhibitor U73122 or its negative control U73343. Uninfected cells (control) and cells infected with <i>L. lactis</i> (<i>L. lactis</i>), respectively, were used as controls to monitor the calcium fluxes by fluorescence microscope. (A) Representative images from an individual experiment repeated five times in triplicate are shown. U73122 significantly blocked the calcium efflux mediated by meningococci (<i>pilC1⁺</i> strains) compared to non-treated or control-treated (U73343) cells. Scale bars represent 50 µm in all panels. (B and C) Data are expressed as the mean ± S.D of five independent experiments performed in duplicate. *<i>P</i><0.05, ***<i>P</i><0.001, versus values from control samples by Student’s <i>t</i>-test.</p

Extracellular Ca²⁺ concentration has no effect on meningococcal adherence to and subsequent invasion into HBMEC.

<p>Host cells pre-treated with extracellular calcium chelators BAPTA and EGTA or CaCl₂ were infected with three different strains of meningococci with MOI of 50 for 4 h at 37°C and 5% CO₂. (A, C and E) The number of meningococci adhered to host endothelial cells was determined in the absence (control) or presence of BAPTA, EGTA or CaCl₂ by lysing the host cells and plating of the lysates on agar plates for overnight at 37°C and 5% CO₂. (B, D and F) The number of meningococci invaded into HBMEC was determined by using the antibiotic protection assay. Adherence and invasion of WT meningococci in the absence of inhibitors was set to 100%. Meningococcal adherence to and subsequent invasion into host cells was not altered by manipulation of the extracellular calcium. Data represent means ± S.D (n = 5) (*<i>P</i><0.01).</p

MOESM1 of The choroid plexus may be an underestimated site of tumor invasion to the brain: an in vitro study using neuroblastoma cell lines

Additional file 1: Figure 1. Effect of a pretreatment of the HIBCPP layer with the actin microfilament-disrupting agent Cytochalasin D on the transmigration rate of IMR32 and SH-SY5Y neuroblastoma cell lines (a) and barrier integrity, as assessed by TEER (b) and dextran flux (c) measurements. Transmigration experiments were performed and transmigration rates were determined as described in Materials and Methods section (a). Before transmigration experiments, filters with HIBCPPs were incubated for 75 min with 1 µg.ml−1 Cytochalasin D (Sigma) diluted in serum-free medium containing 0.5 % BSA (‘+ cytochalasin D’ condition). In parallel, control filters were incubated with serum-free medium containing 0.5 % BSA (‘- cytochalasin D’ condition). The TEER was measured before the treatment and after the treatment to confirm break-down of the barrier properties (b, ‘before’ and ‘cyto D’ conditions). All filters were then placed in new wells containing medium without Cytochalasin D, the transmigration experiment was launched. 5 µl Dextran-TexasRed (MW: 3000 Da, Life Technologies) were added to the upper compartment of the inserts together with IMR32 or SH-SY5Y cells, in order to monitor permeability of HIBCPPs treated with and without cytochalasin D during the experiment (c). After 4 h of transmigration, the TEER was measured again (b, condition ‘after’). TEER values increased again and the experiment was stopped. The fluid in the lower compartments was collected for determination of the amount of Dextran having crossed the barrier during the experiment by fluorescence measurement using a Tecan 200 M Infinite Multiwell reader (c). All results were expressed as mean ± SD from two independent experiments, each performed in triplicates. Statistical significance was assessed by unpaired t-tests. A p-value < 0.05 was considered as significant. Statistical analyses were performed using GraphPad Prism 5.0 for Windows (GraphPad Software, San Diego, California, USA)

Polar invasion of bacteria into HIBCPP.

<p>Invasion of <i>S. suis</i> and <i>N. meningitidis</i> strains was analysed by double immunofluorescence. Standard (white bars) and inverted (black bars) transwell filter cultures were infected with the indicated strains for 4 h before immunofluorescence staining and quantification of invasion as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030069#s4" target="_blank">Experimental procedures</a>. Data shown are the mean+SD of a minimum of four independent experiments each performed at least in duplicates. ** (highly significant; p<0.01), *** (extremely significant; p<0.001); when invasion in the inverted filter system compared to invasion in the standard filter system. # (significant; p<0.05), ### (extremely significant; p<0.001); when invasion of the unencapsulated strain compared to invasion of the capsulated strain in the standard or inverted filter system, respectively. §§ (highly significant; p<0.01); when invasion of the Opc complemented strain compared to the respective not complemented strain.</p

Electron microscopic analysis of HIBCPP TJ structure.

<p>HIBCPP were grown on transwell filter supports in the standard (<b>A</b>, <b>C</b>) and the inverted (<b>B</b>, <b>D</b>) culture system, respectively. Transmission electron microscopy studies (<b>A</b>, <b>B</b>) show that in both culture systems the cells are connected by TJs (arrows), which are located close to the apical side as indicated by the presence of microvilli. Examination of HIBCPP by freeze fracture electron microscopy (<b>C</b>, <b>D</b>) revealed a broad band of closely meshed TJ strands. The diameter of meshes were in the magnitude of 0.2 to 0.4 µm.</p