Quantifying the cytoprotective efficiency of extracellular vesicles (EVS) against Staphylococcus aureus alpha-hemolysin

The bacterial toxin α-hemolysin (Hlα) is a water soluble monomer which oligomerizes in the plasma membrane of host cells, generating a membrane pore which induces permeabilization and cell death. Through this activity, Hlα plays an important role in methicillin-resistant S. aureus (MRSA) pathogenicity. A role of animal extracellular vesicles (EVs) as part of the innate immune system, and more specifically as decoys to sequester and inactivate α-hemolysin was recently proposed. In this work we aimed to quantify the efficacy of EVs in inhibiting the toxicity of S. aureus Hlα. Cytotoxicity assays were carried out against the L-929 mouse fibroblast cell line, employing commercially available S. aureus Hlα, and LC50 values were determined. S. aureus Hlα was found to have an LC50 of 174 nM in complete medium and 72 nM in EV-depleted medium, confirming that EV depletion promoted Hlα-mediated cell death. Dramatic inhibition of Hlα permeabilizing activity by EVs was confirmed in confocal fluorescence microscopy permeabilization assays of both L-929 cells and giant unilamellar vesicles (GUVs), in the presence and absence of isolated EVs. Sequestration of Hlα by isolated extracellular vesicles was also evaluated through fluorescence correlation spectroscopy (FCS) with Alexa-488 labeled Hlα. Surprisingly, modification of lipid composition in isolated EVs was found to potentiate Hlα neutralization efficacy of these vesicles, suggesting that bacterial toxin neutralization by EVs can be further augmented for therapeutic purposes in the context of drug-resistant MRSA infections.