Loaded delta-hemolysin shapes the properties of Staphylococcus aureus membrane vesicles

Abstract

BackgroundMembrane vesicles (MVs) are nanoscale vesicular structures produced by bacteria during their growth in vitro and in vivo. Some bacterial components can be loaded in bacterial MVs, but the roles of the loaded MV molecules are unclear.MethodsMVs of Staphylococcus aureus RN4220 and its derivatives were prepared. Dynamic light scattering analysis was used to evaluate the size distribution, and 4D-label-free liquid chromatography–tandem mass spectrometry analysis was performed to detect protein composition in the MVs. The site-mutation S. aureus RN4220-Δhld and agrA deletion mutant RN4220-ΔagrA were generated via allelic replacement strategies. A hemolysis assay was performed with rabbit red blood cells. CCK-8 and lactate dehydrogenase release assays were used to determine the cytotoxicity of S. aureus MVs against RAW264.7 macrophages. The serum levels of inflammatory factors such as IL-6, IL-1β, and TNFα in mice treated with S. aureus MVs were detected with an enzyme-linked immunosorbent assay kit.ResultsDelta-hemolysin (Hld) was identified as a major loaded factor in S. aureus MVs. Further study showed that Hld could promote the production of staphylococcal MVs with smaller sizes. Loaded Hld affected the diversity of loaded proteins in MVs of S. aureus RN4220. Hld resulted in decreased protein diversity in MVs of S. aureus. Site-mutation (RN4220-Δhld) and agrA deletion (RN4220-ΔagrA) mutants produced MVs (ΔhldMVs and ΔagrAMVs) with a greater number of bacterial proteins than those derived from wild-type RN4220 (wtMVs). Moreover, Hld contributed to the hemolytic activity of wtMVs. Hld-loaded wtMVs were cytotoxic to macrophage RAW264.7 cells and could stimulate the production of inflammatory factor IL-6 in vivo.ConclusionThis study presented that Hld was a major loaded factor in S. aureus MVs, and the loaded Hld played vital roles in the MV-property modification.

Staphylococcus Aureus Membrane Vesicles Kill Tumor Cells Through a Caspase-1-Dependent Pyroptosis Pathway

“…However, the body weights of the MV-treated C57BL/6 mice on day 11 notably decreased ( Figure 1I ), but not in BALB/c mice ( Supplementary Figure 1D ), which may be caused by the mouse sensibility to uncertain virulence factors packaged in the MVs. 28 Collectively, these results suggest that MVs prepared from S. aureus USA300 have antitumor activity in vivo.…”

“…Some toxic components can be enriched in MVs during formation and are the chief contributors to MV toxicity. 23 , 28 , 37 However, the identification of the actual factors that play vital roles in USA300 MV toxicity is important, and we will investigate them in the future.…”

“… 14 S. aureus MVs can package an array of prototypic pore-forming toxins, including alpha-hemolysin, delta-hemolysin, and leukocidins. 28 , 36 However, the selective cytotoxicity of S. aureus MVs against tumor cells is unclear. One possible explanation is that the expression levels of toxin receptors vary between tumorous and non-carcinomatous cells; however, this explanation requires further investigation.…”

“…However, the survival rates of the MV-challenged mice decreased with increasing MV dose, and 20 μg of USA300 MVs killed all tested mice within 24 h post-injection (Figure 1D). Some toxic components can be enriched in MVs during formation and are the chief 23,28,37 However, the identification of the actual factors that play vital roles in USA300 MV toxicity is important, and we will investigate them in the future.…”

“…Experiments were conducted with protocols as previously described. 18 , 28 As for CCK-8 assay, B16F10, CT26, and bEnd.3 cells were cultivated in 96-well plates until the cell density reached 70%–80%. Then, the medium was replaced with fresh medium (100 μL per well) supplemented with 10 μL of S. aureus MVs at final concentrations of 5, 20, and 100 μg/mL.…”