Tight association of α-toxin with <i>S. aureus</i> MVs.

<p>(A) Immunoblot detection of α-toxin (Hla) and protein A (SPa) in density gradient fractions of MVs from strain 8325-4. Fractions (15 µl applied on the gel) are numbered from left to right (2–10) according to increasing density. A polyclonal antiserum specific for α-toxin was used for immunoblot detection. The sizes (kDa) of the proteins in the prestained molecular weight marker (M) are indicated along the right side. (B) Relative hemolytic activity of density gradient fractions 3, 6, 8, and 15, respectively, as determined using an <i>in vitro</i> assay (20% rabbit erythrocytes). Data were normalized to the activity of fraction 3, having the highest α-toxin/protein A-ratio. Shown are the means ± SEM for three independent experiments. *<i>P</i><0.03, fraction 3 activity vs the activity of either of the other tested fractions. (C) Dissociation assays using MVs isolated from strain 8325-4. An MV preparation in PBS was treated for 60 min on ice in the presence of: PBS (buffer), urea (0.8 M and 8 M), or SDS (1%), respectively. The resulting pellets (P) and supernatants (S) after centrifugation were analyzed by immunoblotting, using a polyclonal anti-α-toxin (Hla) antiserum.</p

Detection of α-toxin in <i>S. aureus</i> MV preparations.

<p>Immunoblot detection of α-toxin (Hla; panel A), and CodY (lysis marker; panel B) in MV preparations, and in whole cell (WC) preparation samples from <i>S. aureus</i> strain 8325-4 (WT), and from the strain 8325-4 <i>hla</i> mutant, DU1090 (<i>hla</i>). Polyclonal antisera specific for <i>S. aureus</i> α-toxin, and <i>B. subtilis</i> CodY, respectively were used for immunoblot detection, and the reactive bands corresponding to these proteins are indicated with an arrowhead. The sizes (kDa) of the proteins in the prestained molecular weight marker (M) are indicated along the left sides. Protein samples equal to 10 µg were applied on the gels. Bar graphs indicate results of densitometric analysis of the immunoblots. Shown are the means ± SEM of relative band density for Hla (A) and CodY (B) from three independent experiments. Data were normalized to the whole cell lysate of the parental strain.</p

MV-associated α-toxin is biologically active.

<p>Hemolytic activity <i>in vitro</i> of MVs isolated from <i>S. aureus</i> strains 8325-4 (WT), and DU1090 (<i>hla</i>), respectively. Rabbit erythrocytes (100% in PBS) were incubated for 60 min with MVs or with MVs disrupted by sonication (2, 4, and 10 µg protein as indicated). Control treatment (C) erythrocytes incubated with PBS. Shown are the means ± SEM for three independent experiments. *<i>P</i><0.02, 8325-4 MVs vs DU1090 MVs for all tested concentrations; **<i>P</i><0.05, sonicated vs non-sonicated strain 8325-4 MVs for all tested concentrations; ***<i>P</i><0.03, 8325-4 MVs vs DU1090 MVs for all tested concentrations.</p

Detection of α-toxin in association with <i>S. aureus</i> MVs.

<p>Electron microscopy and immunogold-labeling of α-toxin, using a polyclonal antiserum specific for <i>S. aureus</i> α-toxin (A), or <i>B. subtilis</i> CodY (B). Immunoelectron micrographs of MVs isolated from strain 8325-4 (WT), DU1090 (<i>hla</i>), and WA764 (<i>spa</i>) are shown. Examples of vesicle structures are indicated by arrows, or highlighted by square boundaries and also shown in larger magnification below the corresponding micrograph. Arrows also indicate gold particles surrounding one 8325-4 (WT) vesicle structure in panel A. Gold particles associated with disrupted vesicle structures are indicated by arrowheads. Bars  = 100 nm. (C) Atomic force micrograph of strain 8325-4 (WT) cultivated on agar. Arrows indicate examples of the released MVs. Bar  = 300 nm.</p

Cholesterol-dependent fusion of <i>S. aureus</i> MVs with HeLa cells.

<p>Localization of rhodamine B-R18-labeled MVs (red fluorescence used as a readout for MV fusion with the host cell plasma membrane), and FITC-conjugated lipid raft marker CtxB (green fluorescence) in HeLa cells after 30 min of incubation with membrane-derived vesicles obtained from strain 8325-4 (MV; panel A), and with PBS (buffer; panel B). Treatment was done in the absence (−FIL) and in the presence (+FIL), respectively, of the cholesterol-sequestering agent Filipin III (final concentration 10 µg/ml). Bar graphs show quantitative analysis of red (B-R18) and green (FITC) fluorescence in treated HeLa cell samples. Values represent arbitrary units of pixel intensity for red and green fluorescence determined using ImageJ, and shown are the means ± SEM of data collected from 10 cells. *<i>P</i> = 0.0001, **<i>P</i><0.0001, and ***<i>P</i> = 0.0002, for treatment in the absence vs presence of Filipin III. The merged images show the labeling with both fluorescent dyes. The scattergrams in panel A with red (B-R18) and green (FITC) pixels plotted on graphs were used to obtain the colocalization coefficient (r_p) between MVs and CtxB in the HeLa cells treated with strain 8325-4 MVs for 30 min. (C) B-R18-labeled MVs alone. Magnification: 1000×. Bars  = 10 µm.</p