Investigating Mechanisms Underlying Staphylococcus aureus Extracellular Membrane Vesicle Biogenesis

Abstract

The release of extracellular vesicles (EV) 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 in Gram-positive EV biogenesis and release is limited, partly due to labor-intensive vesicle isolation and assessment methods. Here, we design a high-throughput approach to analyze the Nebraska Transposon Mutant Library to identify genetic determinants of EV production in S. aureus. We demonstrate that our high throughput method can successfully identify mutants with vesiculation phenotypes by verification using the traditional ultracentrifugation method of EV isolation. Our screen revealed CodY as a regulator EV biogenesis. Additionally, we demonstrate that S. aureus EV biogenesis is largely orchestrated though the agr quorum sensing system, and is mainly facilitated through RNAIII. On the other hand, we show that α-PSMs inhibit EV production. We establish a link between nutrient availability and EV production, in that EV biogenesis is upregulated in response to nutrient limitation. Lastly, our results suggest that EV biogenesis may be governed by crosstalk between several cellular processes including the quorum sensing system and the stringent response, and supports a conserved communication strategy that allows metabolic state to influence EV production.