The biogenesis of extracellular vesicles fromStaphylococcus aureusand their application as a novel vaccine platform

Abstract

31Gram-positive bacteria secrete extracellular vesicles (EVs) that package diverse bacterial 32 antigens and play key roles in bacterial pathogenesis. However, the mechanisms underlying EV 33 production in Gram-positive bacteria are poorly understood. We purified and characterized EVs 34 from a community-associated methicillin-resistant Staphylococcus aureus isolate (USA300) and 35 investigated mechanisms underlying EV production. Native EVs contained 165 proteins, including 36 cytosolic, surface, and secreted proteins, autolysins, and numerous cytolysins. Staphylococcal 37 alpha-type phenol-soluble modulins (surfactant-like peptides) promoted EV biogenesis, 38presumably by acting at the cytoplasmic membrane, whereas peptidoglycan crosslinking and 39 autolysin activity were found to increase EV production by altering the permeability of the 40 staphylococcal cell wall. To address the immunogenicity of EVs, we created engineered EVs 41 (eng-EVs) by expressing detoxified proteins HlaH35L and LukE in EVs generated from a nontoxic 42 S. aureus ∆agr∆spa mutant. Eng-EVs exhibited no cytotoxicity in vitro, and mice immunized with 43 the eng-EVs produced toxin-neutralizing antibodies and showed reduced lethality in a mouse 44 sepsis model. Our study reveals novel mechanisms underlying S. aureus EV production and 45 highlights the usefulness of EVs as a novel S. aureus vaccine platform. 46 47 peer-reviewed)