Journal: Frontiers in Microbiology
Article Title: Staphylococcus aureus Membrane-Derived Vesicles Promote Bacterial Virulence and Confer Protective Immunity in Murine Infection Models
Figure Lengend Snippet: Staphylococcus aureus MVs promote bacterial survival in human whole blood and in the presence of neutrophils ex vivo and in vivo . (A) Survival of S. aureus MSSA476 in blood is increased in the presence of 20 μg MV (0.1 μg/μl) isolated from MSSA476 grown in LB and BHI [marked as LB (MVs) or BHI (MVs) in the figure] compared to absence of MVs (marked as control in the figure). The number of inoculated bacteria at time point 0 was set to 100% and the number of surviving bacteria after 3 h is represented as the percentage of inoculation. (B) S. aureus MSSA476 survival in blood is increased in the presence of MVs, in a dose-dependent manner (5–20 μg of total MVs, i.e., 0.025–0.1 μg/μl). The number of surviving bacteria after 3 h in the absence of MVs was arbitrary set as 1, and the number of surviving bacteria in the presence of MVs is represented as the fold change compared to bacteria in the absence of MV. (C) Survival of USA300 MRSA in human blood is increased in the presence of MVs isolated from USA300 (MV-Hla) and USA300ΔHla (MV-ΔHla) grown in BHI. The percentage of survival was calculated as described in (A) . (D) Sonication of purified MVs followed by proteinase K (PK) treatment abolished the effect of MVs on bacterial survival in human whole blood. The fold change of survival was calculated as described in (B) . (E) Survival of opsonized S. aureus MSSA476 in the presence of neutrophils is enhanced by supplementation of MVs isolated from bacteria growing in LB and BHI. Percentage of survival was calculated as described in (A) . (F) S. aureus MSSA476 were labeled with FITC and incubated with human whole blood in the absence or presence of MVs isolated from MSSA476 grown in LB and BHI. Data represents geometric mean of the fluorescence intensity (GMFI). (G) Bacterial loads in the blood (CFU/ml) of 8-week-old C57BL/6 mice were counted 24 h after the mice were intravenously infected with S. aureus MSSA476 supplemented with PBS or an exogenous source of MVs isolated from MSSA476 grown in BHI. (H) HaCaT (100 μg of total MVs, i.e., 0.1 μg/μl) and freshly purified neutrophils were treated with MVs (5–20 μg of total MVs, i.e., 0.025–0.1 μg/μl) isolated from S. aureus MSSA476 grown in LB or BHI at the time points indicated. Percentage of cytotoxicity was calculated by measuring the amount of LDH released from the cytosol of damaged cells into the supernatant after exposure to MVs. (I) Viability staining of neutrophils in the presence (20 μg of total MVs, i.e., 0.1 μg/μl) or absence of MVs were performed using propidium iodide (PI). Live imaging was performed after 0 and 0.45 or 1.5 h using fluorescence microscopy. Scale bar is shown. The data represent as the mean ± SEM of at least three independent experiments except for (D) , which the data are expressed as the mean ± SEM of two independent experiments performed in triplicate. Mice study corresponds to one experiment performed with 10 mice/group. The significance is indicated by asterisks ( ∗ ): ∗ P
Article Snippet: In order to perform proteomic analysis, fractionation of MVs from S. aureus MSSA476 grown in LB and BHI broth was carried out by density gradient centrifugation using Optiprep (Sigma–Aldrich, Germany) as previously described , with minor modifications.
Techniques: Ex Vivo, In Vivo, Isolation, Sonication, Purification, Labeling, Incubation, Fluorescence, Mouse Assay, Infection, Staining, Imaging, Microscopy