Fusion of azurophilic granules with phagosomes containing <i>S. pyogenes</i> bacteria.

<p>A. Western blot of isolated phagosomes and cell lysate. Differentiated HL-60 cells were allowed to phagocytose IgG-opsonized, heat-killed and magnetically labeled <i>S. pyogenes</i> bacteria for 20 min at a bacteria/cell ratio of 5∶1. Following washes, the cells were lysed by nitrogen cavitation and phagosomes were retrieved by magnetic selection. Equal amounts of phagosomes were loaded and probed with antibodies against cathepsin D (azurophilic granule content marker) and GM130 (Golgi marker) with anti-<i>S. pyogenes</i> as loading control. Increasing amounts of cell lysate (relative protein content.05×, 0.5× and 1.0×) was used as a control. B. Azurophilic granule–phagosome fusion. Differentiated HL-60 cells were allowed to phagocytose opsonized Oregon Green-labeled <i>S. pyogenes</i> bacteria, either the BMJ71 (i-iii) or the AP1 (iv–v) strains, at a bacteria/cell ratio of 10∶1. After a synchronized presentation, the samples were incubated at 37°C for 5 min, fixed and incubated with antibodies directed against CD63, subsequently detected using Alexa 594 F(ab')₂ fragments. Single deconvolved focal planes from serial z-stacks were taken from the mid-part of the HL-60 cells. Oregon Green staining shows the localization of bacteria (ii, v). Magenta staining shows the localization of the azurophilic granule membrane marker CD63 (i, iv). The images are also presented as merged (iii, vi). Size bar: 5 µm.</p

Average single-channel conductance, <i>G</i>, of HlyA_Δ71–110 and HlyA_Δ264–286 in different salt solutions.^a.

a<p>The membranes were formed from 1% (mass/volume) asolectin dissolved in n-decane. The aqueous solutions were unbuffered and had a pH of 6. The applied voltage was 20 mV, and the temperature was 20°C. The average single-channel conductance, <i>G</i> (i.e. current divided by voltage), was calculated from at least 80 single events. The standard deviation of the single-channel conductance was generally below ±15%. <i>c</i> is the concentration of the aqueous salt solutions. The single-channel conductance of wildtype HlyA of <i>E. coli</i> is given for comparison <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112248#pone.0112248-Benz3" target="_blank">[28]</a>. The values denoted with an asterix were measured during this study with purified HlyA. n.m. means not measured.</p><p>Average single-channel conductance, <i>G</i>, of HlyA_Δ71–110 and HlyA_Δ264–286 in different salt solutions.^a.</p

Extracellular secretion and hemolytic activity of <i>E. coli</i> HlyA and of HlyA mutants.

<p>(A) SDS-PAGE of extracellular proteins from <i>E. coli</i> 5K containing different plasmids. Lane 1, molecular mass markers given in kDa; lane 2, <i>E. coli</i> 5K/pACYC184 (vector control); lane 3, <i>E. coli</i> 5K/pANN202–312* overproducing HlyA; lane 4, isogenic strain overproducing HlyA_Δ71–110; lane 5, isogenic strain overproducing HlyA_Δ158–167; lane 6, isogenic strain overproducing HlyA_Δ180–203; lane 7, isogenic strain overproducing HlyA_Δ264–286. The proteins in cell-free culture supernatants (harvested in the late log phase) were precipitated by addition of ice-cold trichloroacetic acid (final concentration, 10%), pelleted by centrifugation at 12,000×g, washed with acetone, dried under vacuum, and dissolved in sample buffer <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112248#pone.0112248-Ludwig2" target="_blank">[11]</a>. Proteins from 100 µl culture supernatant were separated on the gel and visualized by silver staining. (B) Hemolytic phenotype of <i>E. coli</i> 5K/pANN202–312* overproducing HlyA and of isogenic strains overproducing the HlyA mutants with the indicated deletions. Bacteria from individual colonies were picked onto a sheep blood/Cm agar plate that was subsequently incubated for 24 hours at 37°C.</p

Results of osmotic protection experiments with HlyA, HlyA_Δ71–110, and HlyA_Δ264–286.

<p>Sheep erythrocytes were incubated with the toxins at 37°C for 60 min in saline solution (control) or in saline solution supplemented with 30 mM of PEGs of different molecular masses (PEG 400, 600, 1000, 2000, 3000, and 4000, with diameters of 1.07, 1.32, 1.72, 2.47, 3.05, and 3.54 nm, respectively). The concentration of HlyA was 0.5 µg/ml and that of HlyA_Δ71–110 and HlyA_Δ264–286 2.5 µg/ml. The degree of hemolysis is shown as a function of the molecular mass of the PEGs.</p

Calculation of the channel diameters of HlyA and HlyA_Δ71–110 from the single-channel conductance.

<p>The single-channel conductance data of HlyA and HlyA_Δ71–110 were fitted by using the Renkin correction factor multiplied by the aqueous diffusion coefficients of the different cations. The single-channel conductance for the different cations taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112248#pone-0112248-t002" target="_blank">Table<u> 2</u></a> was normalized to that observed for Rb⁺ (hydrated ion radius  = 0.105 nm), which was set to 1.0, and plotted versus the hydrated ion radii taken from Table 3 of Maier <i>et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112248#pone.0112248-Maier1" target="_blank">[60]</a>. The points correspond to the single-channel conductance observed with Li⁺, Na⁺, K⁺, Cs⁺, N(CH₃)₄⁺, N(C₂H₅)₄⁺, and Tris⁺, which were all used for the pore diameter estimation (see Discussion). (A) The fit (solid lines) is shown for wildtype HlyA channels with <i>r</i> = 1.6 nm (upper line) and <i>r</i> = 1.0 nm (lower line). The best fit was achieved with <i>r</i> = 1.3 nm (diameter = 2.6 nm), which corresponds to the broken line. (B) The fit (solid lines) is shown for the HlyA_Δ71–110 channels with <i>r</i> = 1.1 nm (upper line) and <i>r</i> = 0.7 nm (lower line). The best fit of all data was achieved with <i>r</i> = 0.9 nm (diameter = 1.8 nm), which corresponds to the broken line.</p

Results of osmotic protection experiments with aerolysin of <i>A. sobria</i>.

<p>Sheep erythrocytes in saline solution (control) or in saline solution supplemented with 30 mM of different carbohydrates (arabinose, cellobiose, and melezitose, with diameters of 0.62, 0.92, and 1.14 nm, respectively) were incubated with the toxin at 37°C for 30 min (A) and 90 min (B). Erythrocyte lysis was determined as a function of increasing aerolysin concentrations.</p

Single-channel recordings with HlyA_Δ158–167 and HlyA_Δ180–203.

<p>Single-channel recordings of asolectin membranes were performed in the presence of 100 ng/ml HlyA_Δ158–167 (upper trace) and 150 ng/ml HlyA_Δ180–203 (lower trace). The aqueous phase contained 150 mM KCl (pH 6). The applied membrane potential was 20 mV; T = 20°C. The transient conductance steps in the upper trace (HlyA_Δ158–167) had a conductance of about 600 to 700 pS. The mutant HlyA_Δ180–203 produced under the given conditions only current noise (fuzzy channels) and no defined conductance states.</p

Intracellular survival of <i>S. pyogenes</i> bacteria.

<p>A. Interaction. Differentiated HL-60 cells were allowed to interact at 37°C with Oregon Green-labeled IgG-opsonized (1 mg/ml) AP1 and BMJ71, at a bacteria/cell ratio of 10∶1. After a synchronized presentation, the samples were incubated at 37°C as indicated. Analysis was by flow cytometry. An interaction ratio was calculated by dividing the number of cells interacting with Oregon Green-labeled bacteria with the total number of cells. Error bars show SEM, based on a total of three experiments. B. Internalization. After phagocytosis as in A, the internalization of bacteria was determined by immunofluorescence microscopy using non-permeabilized and permeabilized conditions and anti-<i>S. pyogenes</i> antibodies. For each condition, at least 100 cells were counted. A representative experiment is shown. C. Intracellular survival. Differentiated HL-60 cells were allowed to phagocytose AP1 and BMJ71 bacteria at 37°C, bacteria/cell ratio 10∶1. After a synchronized presentation, the samples were incubated at 37°C as indicated, before killing of extracellular bacteria by PlyC. Intracellular survival of bacteria was determined by diluting HL-60 lysates and counting the number of colonies formed after overnight growth at 37°C. Data shown are expressed as the CFU ability relative to the values at 1 min. Error bars show SEM, based on a total of three experiments.</p

Single-channel recordings with HlyA_Δ158–167 and HlyA_Δ180–203.

<p>Single-channel recordings of asolectin membranes were performed in the presence of 100 ng/ml HlyA_Δ158–167 (upper trace) and 150 ng/ml HlyA_Δ180–203 (lower trace). The aqueous phase contained 150 mM KCl (pH 6). The applied membrane potential was 20 mV; T = 20°C. The transient conductance steps in the upper trace (HlyA_Δ158–167) had a conductance of about 600 to 700 pS. The mutant HlyA_Δ180–203 produced under the given conditions only current noise (fuzzy channels) and no defined conductance states.</p

Single-channel recordings with <i>E. coli</i> HlyA, HlyA_Δ71–110, and HlyA_Δ264–286.

<p>Single-channel recordings of asolectin membranes were performed in the presence of 50 ng/ml HlyA (left side, upper trace), 50 ng/ml HlyA_Δ71–110 (left side, lower trace), and 50 ng/ml HlyA_Δ264–286 (right side). The aqueous phase contained 150 mM KCl (pH 6). The applied membrane potential was 20 mV; T = 20°C. The average single-channel conductance was 520 pS for HlyA, 150 pS for HlyA_Δ71–110, and 320 pS for HlyA_Δ264–286.</p