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

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

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

Specificity of HUVECs intoxication by C2I using PA₆₃ in comparison to C2II.

<p>HUVECs (5×10⁵ cells/100 mm well) were intoxicated with the indicated concentration of polypeptides during 48 (A) or 24 hours (B). <b>A</b>: Cells were intoxicated as indicated and levels of cellular ADP-ribosylated actin (ADPr-actin) were determined by <i>in vitro</i> ADP-ribosylation of cell lysates with C2I and radiolabeled [³²P]-NAD. Under these conditions ADP-ribosylated actin formed during the intoxication process is no longer labeled by <i>in vitro</i> ADP-ribosylation, which is indicated by decrease of radioactivity. Immunoblotting anti-beta-actin was performed in parallel on cell lysates to show actin protein levels engaged in the ADP-ribosylation experiments. ADP-ribosylation signals were normalized to actin immunoblot signals. <b>B</b>: Efficiency of cell intoxication. Cells were intoxicated and the number of round cells was directly assessed by counting floating cells. The columns show mean values of 5 independent counting for the individual conditions ± SEM (ns: non significant; * p<0.05 versus control).</p

Interaction of C2I with PA₆₃ channels.

<p><b>A</b>: Titration of PA₆₃ induced membrane conductance with C2I. The membrane was formed from diphytanoyl phosphatidylcholine/n-decane, containing about 5,500 channels. C2I was added at the concentrations shown at the top of the panel. Finally, about 40% of the PA₆₃ channels were blocked. The aqueous phase contained 1 ng/ml activated PA₆₃ protein (added only to the <i>cis</i>-side of the membrane), 150 mM KCl, 10 mM MES pH 6. The temperature was 20°C and the applied voltage was 20 mV. Note that C2I only blocks PA₆₃ channels when it is added to the <i>cis</i>-side of the membrane (data not shown). <b>B</b>: Lineweaver-Burke plot of the inhibition of the PA₆₃-induced membrane conductance by C2I. 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.0023133#pone-0023133-g001" target="_blank">Figure 1A</a> (r² = 0.996654) and corresponds to a stability constant <i>K</i> for C2I binding to PA₆₃ of (3.98±0.063) ×10⁶ M⁻¹ for C2I binding to PA₆₃ (half saturation constant K_S = 251 nM).</p

Stability constants <i>K</i> and half saturation constants <i>K_S</i> for the cross-reaction of anthrax and C2 toxin.

<p>bility constants <i>K</i> for the binding of C2I, EF or LF to PA₆₃ or C2II channels reconstituted in lipid bilayer membranes. The membranes were formed from diphytanoyl phosphatidylcholine/n-decane. The aqueous phase contained 150 mM KCl, buffered to pH 5.5 to 6 using 10 mM MES-KOH; T = 20°C. Measurements were performed at a membrane potential of 20 mV. The data represent the means of at least three individual titration experiments. <i>K_S</i> is the half saturation constant, i.e. 1/<i>K</i>. Some of the wild-type toxin combinations (given in bold) were taken from reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023133#pone.0023133-Neumeyer1" target="_blank">[20]</a>.</p