Orchestration of S. aureus EV biogenesis by nutrient availability through quorum sensing

The release of extracellular vesicles (EVs) is a universally conserved process. Bacterial EVs package diverse cargo, including proteins and nucleic acids, and influence bacterial adaptation and survival, as well as host-pathogen interactions. Currently, our understanding of the mechanisms underlying global principles of EV biogenesis and release in Staphylococcus aureus and in gram-positive species, in general, is limited. Here, we describe a moderately high-throughput approach to analyze the Nebraska Transposon Mutant Library to broadly identify determinants of agr -dependent EV production in S. aureus . We show that the agr quorum-sensing system dictates EV production in response to nutrient availability, likely through communication with the adaptive stress response. This study demonstrates the contribution of nutritional stress to EV biogenesis and supports a conserved communication strategy that allows metabolic state to influence EV production. IMPORTANCE Studies have demonstrated a role for gram-positive extracellular vesicles (EVs) in virulence and adaptation; however, mechanistic knowledge of EV biogenesis remains limited. Here, using a screening approach, we demonstrate how the downstream targets of the agr -quorum-sensing system in Staphylococcus aureus , α-PSMs and RNAIII, differentially contribute to and alter EV biogenesis. We show a likely conserved communication strategy that allows nutrient availability to influence EV production. Additionally, we identified numerous candidate genes potentially involved in EV biogenesis and its regulation, providing a valuable resource for future mechanistic studies.