AUTHOR=Chen Juan , Lv Yuhuan , Shang Weilong , Yang Yi , Wang Yuting , Hu Zhen , Huang Xiaonan , Zhang Rong , Yuan Jizhen , Huang Jingbin , Rao Xiancai 

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

JOURNAL=Frontiers in Microbiology

VOLUME=14

YEAR=2023

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2023.1254367

DOI=10.3389/fmicb.2023.1254367

ISSN=1664-302X

ABSTRACT=<sec><title>Background</title><p>Membrane vesicles (MVs) are nanoscale vesicular structures produced by bacteria during their growth <italic>in vitro</italic> and <italic>in vivo</italic>. Some bacterial components can be loaded in bacterial MVs, but the roles of the loaded MV molecules are unclear.</p></sec><sec><title>Methods</title><p>MVs of <italic>Staphylococcus aureus</italic> 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 <italic>S. aureus</italic> RN4220-Δhld and <italic>agrA</italic> 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 <italic>S. aureus</italic> MVs against RAW264.7 macrophages. The serum levels of inflammatory factors such as IL-6, IL-1β, and TNFα in mice treated with <italic>S. aureus</italic> MVs were detected with an enzyme-linked immunosorbent assay kit.</p></sec><sec><title>Results</title><p>Delta-hemolysin (Hld) was identified as a major loaded factor in <italic>S. aureus</italic> 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 <italic>S. aureus</italic> RN4220. Hld resulted in decreased protein diversity in MVs of <italic>S. aureus</italic>. Site-mutation (RN4220-Δhld) and <italic>agrA</italic> deletion (RN4220-ΔagrA) mutants produced MVs (<sup>Δ<italic>hld</italic></sup>MVs and <sup>Δ<italic>agrA</italic></sup>MVs) with a greater number of bacterial proteins than those derived from wild-type RN4220 (<sup>wt</sup>MVs). Moreover, Hld contributed to the hemolytic activity of <sup>wt</sup>MVs. Hld-loaded <sup>wt</sup>MVs were cytotoxic to macrophage RAW264.7 cells and could stimulate the production of inflammatory factor IL-6 <italic>in vivo</italic>.</p></sec><sec><title>Conclusion</title><p>This study presented that Hld was a major loaded factor in <italic>S. aureus</italic> MVs, and the loaded Hld played vital roles in the MV-property modification.</p></sec>