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

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

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

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

<i>E. coli</i> strains and plasmids used in this study.

a<p>Cb^r: carbenicillin resistant, Cm^r: chloramphenicol resistant, Km^r: kanamycin resistant, Spec^r; spectinomycin resistant.</p

Loss of outer membrane integrity in strain RN102.

<p>Bacterial samples were collected from 48-hour-cultured biofilms for TEM analysis. TEM images of the bacterial cells and the cell appendages are shown for strains: (A) BW25113, (B) RN102, (C) BW25113/pNTR-SD, (D) RN102/pNTR-SD, and (E) RN102/pNT3(<i>hldE</i>). The outer membranes are indicated by arrows. Representative electron-microphotographs of each strain are shown. A 500-nm-long bar is shown in the lower left corner of each eclectron-micrograph. (F) Western blot analysis of supernatants from BW25113 and RN102. Supernatants were harvested by centrifugation from bacterial liquid culture grown for 48 hours under static conditions. Results of Western blot using anti-Crp, anti-DsbA, anti-OmpC, and anti-OmpA antisera are shown. (G) Supernatants from bacterial liquid cultures of BW25113 or RN102 grown for 48 hours under static conditions were serially diluted with TE. The diluted samples were used as template DNA for PCR using <i>E. coli atoS</i> gene-specific primer pairs. Lanes: 1, without dilution; 2, 10⁻¹ dilution; 3, 10⁻² dilution; 4, 10⁻³ dilution; 5, 10⁻⁴ dilution; 6, 10⁻⁵ dilution; 7, 10⁻⁶ dilution.</p

Autoaggregation phenotype by LPS mutants.

<p>Each strain standardized at OD₆₀₀ = 1.0 in PBS was used for autoaggregation assay. The value at OD₆₀₀ after an18-hour incubation is shown as the mean ± SD of results from three independent experiments. Statistical analysis was performed using ANOVA. *<i>P</i><0.05, against autoaggregation level of strain BW25113.</p

Contribution of eDNA to biofilm structure formed by RN102.

<p>(A–E) CLSM images of biofilms formed by strains: (A) BW25113 (B) RN102, (C) BW25113/pNTR-SD, (D) RN102/pNTR-SD, and (D) RN102/pNT3(<i>hldE</i>). Images of biofilms stained with acrydine orange are shown as digital CLSM images. In each strain, a section which has the largest sum of signals in the defined area (127.3 μm by 127.3 μm) among all X–Y sections is shown in the upper row (X–Y). The overview of biofilms in the same area of each X–Y section is shown as 3D image in the lower row (3D). The volume of each 3D image (μm³) in the area of the X–Y planes was quantified and the mean ± SD obtained from 3 different areas chosen at random are denoted in the upper-right corners. The data shown are representative microphotographs of two independent experiments. (F) Quantification of eDNA from BW25113 and RN102 strains. The bars represent the ratio of extracellular DNA to intracellular DNA (eDNA/iDNA). Results are shown as the mean ± SD from 3 independent experiments. *<i>P</i><0.05. Stastical analysis was performed using Mann-Whitney's U-test. (G and H) Effect of DNase I on biofilm formation by the RN102 in a clear test tube (G) and as quantified in a 96-well plate (H). The RN102 was grown in presence of different concentrations of DNase I or in presence of pre-heated DNase I or without DNase I for 48 hours under static conditions at 37°C. The mean ± SD of results from 3 independent experiments are shown. Statistical analysis was performed using ANOVA. *<i>P</i><0.05, against the biofilm formation by RN102 without DNase I treatment.</p

Analysis of OMV preparations.

<p>(A) The OMVs were isolated from the supernatant of a 80-ml bacterial liquid culture incubated 14 hours under shaking conditions. Finally, each OMV preparation was resuspended with 200 μl of 20 mM Tris-Cl (pH 8.0). Five μl of each OMV sample per well was run on 12% PAGE and subjected to silver staining. Total protein amounts (mg) of OMVs from a 80-ml bacterial culture of each strain were determined by Bradford assay and are presented below each lane. Lanes; 1, BW25113; 2, RN102; 3, BW25113/pNTR-SD; 4, RN102/pNTR-SD; 5, RN102/pNT3(<i>hldE</i>). (B and C) AFM images of OMVs prepared from BW25113 and RN102 on 1 μm² surfaces were shown.</p