Staphylococcus aureus produces membrane-derived vesicles that induce host cell death

Abstract

Gram-negative bacteria produce outer membrane vesicles that play a role in the delivery of virulence factors to host cells. However, little is known about the membrane-derived vesicles (MVs) produced by gram-positive bacteria. The present study examined the production of MVs from Staphylococcus aureus and investigated the delivery of MVs to host cells and subsequent cytotoxicity. Four S. aureus strains tested, two type strains and two clinical isolates, produced spherical nanovesicles during in vitro culture. MVs were also produced during in vivo infection of a clinical S. aureus isolate in a mouse pneumonia model. Proteomic analysis showed that 143 different proteins were identified in the S. aureus-derived MVs. S. aureus MVs were interacted with the plasma membrane of host cells via a cholesterol-rich membrane microdomain and then delivered their component protein A to host cells within 30 min. Intact S. aureus MVs induced apoptosis of HEp-2 cells in a dose-dependent manner, whereas lysed MVs neither delivered their component into the cytosol of host cells nor induced cytotoxicity. In conclusion, this study is the first report that S. aureus MVs are an important vehicle for delivery of bacterial effector molecules to host cells.

Figure 1. Production of MVs from S. aureus.

(A to D) Transmission electron micrograph of MVs prepared from S. aureus ATCC 25923 (A), ATCC 700699 (B), 103D (C), and 06ST1048 (D) cultured in LB broth. (E and F) Production and secretion of MVs from S. aureus 06ST1048 during in vivo infection. Neutropenic mice were infected with 1×10⁷ CFU of bacteria intratracheally. The S. aureus-infected mice were sacrificed 18 h after bacterial injection. Arrows indicate the spherical nanovesicles from S. aureus.

Figure 2. Proteins identified in the S. aureus-derived MVs.

SDS-PAGE of proteins packaged in the MVs from S. aureus 06ST1048 (A) and its Western blot analysis (B). The samples were separated on 12% SDS-PAGE and immunoblotted with anti-protein A and anti-β-lactamases antibodies. Lanes M, molecular weight maker; CL, bacterial cell lysate fraction; S, supernatant fraction; and MVs, membrane-derived vesicle fraction.

Figure 3. Delivery of protein A packaged in the S. aureus MVs to host cells.

(A) Delivery of protein A to host cells via the MVs. HEp-2 cells were treated with S. aureus MVs (20 µg/ml of protein concentrations) for the indicated times. Cell lysates were separated on 12% SDS-PAGE, transferred to membranes, and immunoblotted with anti-protein A and β-actin antibodies. Both full-length and degraded forms of protein A were appeared. (B) HEp-2 cells were treated with intact or lysed MVs (20 µg/ml of protein concentrations) for 24 h. HEp-2 cells were pretreated with 10 mM MβCD for 45 min at 37°C. The cells were fixed, permeabilized, and stained with a rabbit anti-protein A antibody, followed by Alexa Fluor® 488-conjugated rabbit IgG (green). DAPI was used to stain the nuclei (blue). The analytical sectioning was performed from the top to the bottom of the cells. The figure represents all projection of sections in one picture.

Figure 4. Host cell death induced by the S. aureus MVs.

(A) HEp-2 cells were treated with 50 µg/ml of MVs for 24 h and stained with DAPI. Upper and lower panels are untreated control cells and S. aureus MV-treated cells, respectively. S. aureus MVs induced host cell pathology, such as condensation of nuclei, nuclear fragmentation and cellular shrinkage. DIC, differential interference contrast microscopy. (B) Flow cytometric analysis of cell death induced by the S. aureus MVs. Upper panel, control cells without MVs and with PBS for 24 h. Lower panel, HEp-2 cells were treated with various concentrations of S. aureus MVs for 24 h. Representative data from three independent experiments are shown. In the graph, cells in right upper and lower parts represent apoptotic cells, and cells in left upper part represent necrotic cells. (C) HEp-2 cells were treated with various concentrations of intact MVs or MVs lysed with EDTA for 24 h. Cells were stained with Annexin V and PI, and then 10⁴ cells were analyzed by flow cytometry. All dead cell population, including Annexin V⁺/PI⁺, Annexin V⁺/PI⁻ and Annexin V⁻/PI⁺ fractions, were calculated. Data are presented as mean ± standard error in three independent experiments. *Statistically significant at p<0.05 using a Student's t-test.