Interaction of C2I with PA₆₃ channels.

<p>A: Titration of PA₆₃ induced membrane conductance with His₆-C2I. The membrane was painted from 1% (w/v) diphytanoyl phosphatidylcholine dissolved in n-decane. It contained about 300 PA₆₃-channels. His₆-C2I was added at the concentrations shown at the top of the panel to the <i>cis</i>-side of the membrane. Finally, about 83% of the PA₆₃-channels were blocked. The aqueous phase contained 1 ng/ml activated PA₆₃ (added only to the <i>cis</i>-side of the membrane), 150 mM KCl, 10 mM MES-KOH pH 6. The temperature was 20°C and the applied voltage was 20 mV. B: Lineweaver-Burk (double reciprocal) plot of the inhibition of the PA₆₃-induced membrane conductance by His₆-C2I using <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046964#pone.0046964.e002" target="_blank">equation (2</a>). The fit was obtained by linear regression of the data points taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046964#pone-0046964-g001" target="_blank">Figure 1A</a> and corresponds to a stability constant <i>K</i> for His₆-C2I binding to PA₆₃ of (3.93±0.39)×10⁷ M⁻¹ (r = 0.955; half saturation constant <i>K_s</i> = 25 nM).</p

Voltage dependency of PA₆₃-channels in the presence of EDIN and His₆-EDIN.

<p>A: Current response of PA₆₃-channels in presence of EDIN. Voltage pulses between +20 and +70 mV were applied to a diphytanoyl phosphatidylcholine/n-decane membrane in the presence of PA₆₃-pores and EDIN (both added only to the cis side of the membrane). The aqueous phase contained 150 mM KCl, 10 mM MES-KOH, pH 6. The temperature was 20°C. B: Current response of PA₆₃ channels in the presence of His₆-EDIN. Voltage pulses between +10 and +90 mV were applied to a diphytanoyl phosphatidylcholine/n-decane membrane in the presence of PA₆₃-pores and His₆-EDIN (both added only to the cis side of the membrane). The aqueous phase contained 150 mM KCl, 10 mM MES-KOH, pH 6. The temperature was 20°C. Note the change of the scale (Arrow).</p

His₆-tag allows internalization of EDIN in endothelial cells through PA₆₃.

<p>A: Upper panel: SDS-PAGE of recombinant His₆-tagged EDIN before (left) and after thrombin treatment (right). Lower panel: immunoblot anti–His₆-tag on His₆-tagged EDIN before and after cleavage by thrombin. B, C: Immunoblots showing cellular levels of active RhoA (RhoA-GTP) in HUVECs determined by GST-Rhotekin RBD pulldown (labeled RhoA-GTP). Cellular content of RhoA (Total RhoA) was assessed by anti-RhoA on 2% of total protein extracts. Immunoblot anti-actin antibody exhibits equal protein loading. (B) Cells were intoxicated with different concentrations of His₆-EDIN (1, 10 and 100 µg/ml) with and without 3 µg/ml of PA₆₃, as indicated. (C) Cells were intoxicated with 10 µg/ml His₆-EDIN, 10 µg/ml EDIN, and 3 µg/ml PA₆₃ as indicated.</p

Stability constants <i>K</i> and half saturation constants <i>K_s</i> for binding of proteins with and without His₆-tags to membrane channels formed by anthrax PA₆₃ and C2II.

<p>Stability constants <i>K</i> and half saturation constants <i>K_s</i> for the binding of His₆-tagged and untagged EF, LF, C2I, gpJ or EDIN to PA₆₃- or C2II-channels in lipid bilayer membranes. The membranes were painted from 1% (w/v) diphytanoyl phosphatidylcholine dissolved in n-decane. The aqueous phase contained 150 mM KCl, buffered to pH between 5.5 and 6 using 10 mM MES-KOH; T = 20°C. Measurements were performed at a membrane potential of 20 mV. The data represent the means (± SD) of at least three individual titration experiments. <i>K_S</i> is the half saturation constant, i.e. <i>K_S</i> = 1/<i>K</i>. Stability constants given in bold were adjusted to the voltage dependent behavior of binding. (* taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046964#pone.0046964-Neumeyer1" target="_blank">[21]</a> ** taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046964#pone.0046964-Kronhardt1" target="_blank">[43]</a>).</p

Immunofluorescence studies of HUVECs treated with EDIN and His₆-EDIN and PA₆₃.

<p>A: HUVECs were intoxicated for 24 h with a combination of PA₆₃ 3 µg/ml, His₆-EDIN 10 µg/ml and LF_1–254-EDIN (LFN-EDIN) 1 µg/ml, as indicated. Cells were fixed and actin cytoskeleton was labelled using FITC-conjugated phalloidin. Bar = 10 µm. Arrows indicate transendothelial cell macroaperture tunnels (TEMs, transcellular tunnels). B: Graph shows percentage of cells with toxin-induced transendothelial cell macroaperture tunnels (TEMs, transcellular tunnels). HUVECs were intoxicated for 24 h with a combination of PA₆₃ 3 µg/ml, His₆-EDIN or EDIN 10 µg/ml and LF_1–254-EDIN (LFN-EDIN) 1 µg/ml, as indicated on the graph legend. Data correspond to means ± SEM (n = 3, 400 cells per condition).</p

Correlation of affinity constant <i>K</i> and voltage dependence of PA₆₃-channels in presence of EDIN and His₆-EDIN.

<p>The stability constants of EDIN and His6-EDIN binding to the PA₆₃-channel are given as a function of the applied membrane potential taken from experiments similar to that shown in Fig. 5 A/B. Means of three experiments are shown.</p

Estimation of the surfaces of <i>S.b</i>-B, ST and beads.

<p>Estimation of the surface of <i>S.b</i>-B, ST and beads was made by using ImageJ plugin (more details in supplemental data <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033796#pone-0033796-g001" target="_blank">Figure 1</a>).</p

Comparison between motility (panel A) and track linearity (panel B) of ST maintained alone and ST in the presence of <i>S. b</i>-B or beads added during infection.

<p>Video-microscopic acquisitions were made 30 min PI, and 100 ST were tracked in each condition. In panel A are presented the statistical comparison between the average speeds of ST alone versus the average speeds of ST maintain in the presence of <i>S. b</i>-B or beads. In panel B are presented the quantification of different type of tracks: LT, CT or RT. The percentages marked in each column correspond to: the number of specific track type/the total number of track for each condition ×100. The data derived from a sequence using MTrack J processing software.</p

Modification of ST invasion by <i>S.b</i>-B, beads or <i>S.b</i>-B-CM.

<p>T84 cells were infected 60 min with the wild type strain SL 1344 alone or in the presence of <i>S.b</i>-B, beads or the conditioned medium by the yeast (<i>S.b</i>-B-CM) added during the infection. Experiments were also performed in cell incubated overnight (ON) with <i>S.b</i>–B (<i>S.b</i>-B(ON)) prior infection. At least beads and <i>S.b</i>-B-CM were added together during infection with ST. Invasion was assessed by the gentamicin protection method (23). % of invasion was calculated as intracellular bacteria/CFU of ST added by well (10⁷ CFU/well). * Indicates statistical difference <i>vs</i> ST-alone infected cells (P<0.05) (n = 4).</p>#<p>Indicates statistical difference <i>vs</i> beads+SL1344 infected cells or <i>S.b</i>-B-CM+SL1344 (P<0.05) (n = 3).</p

Strains used in this study.

<p>Strains used in this study.</p