Oxacillin promotes membrane vesicle secretion in <i>Staphylococcus aureus via</i> a SarA–Sle1 regulatory cascade

Membrane vesicles (MVs) are nanoscale particles secreted by living bacteria <i>in vitro</i> and <i>in vivo</i>. Bacterial MVs encapsulate various proteins, making them promising candidates for developing vaccines, drug carriers, and cancer immunotherapy agents. However, the mechanisms underlying MV secretion in Gram-positive bacteria remain unclear. Here, we showed that the subinhibitory concentration of oxacillin (OXA) stimulated MV production in <i>Staphylococcus aureus</i> with diverse genetic backgrounds. OXA treatment remarkably increased the expression of <i>sle1</i>, which encodes a main peptidoglycan hydrolase for adjusting peptidoglycan cross-linking. Deletion of <i>sle1</i> decreased the OXA-mediated MV yield, whereas overexpression of <i>sle1</i> considerably increased MV production. The accessory regulator SarA increased in response to OXA treatment, and SarA inactivation substantially attenuated OXA-stimulated MV production. We also demonstrated that SarA controlled <i>sle1</i> expression by directly binding to its promoter region. Thus, the SarA-Sle1 regulatory axis was formed to mediate OXA-induced MV production in <i>S. aureus</i>. MVs derived from OXA-treated <i>S. aureus</i> RN4220 (MVs/OXA) exhibited a smaller particle size compared with those purified from wild-type RN4220; however, proteomic analysis revealed a comparable protein profile between MVs and MVs/OXA. Overall, our research reveals a mechanism underlying OXA-promoted <i>S. aureus</i> MV secretion and highlights the potential application of OXA-induced MVs.