PNNL

Abstract

Outer membrane vesicles (OMVs) produced by Gram-negative bacteria have been studied for half a century but the possibility that Gram-positive bacteria made extracellular vesicles (EVs) was not pursued due to the assumption that the thick peptidoglycan cell wall would prevent their release to the extracellular environment. However, following discovery in fungi, which also have cell walls, EVs have now been described in a variety of Gram-positive bacteria. EVs purified from Gram-positive bacteria have been implicated in virulence, toxin release, and delivery of molecules to host cells, eliciting immune responses, and transfer of antibiotic resistance. Listeria monocytogenes is a Gram-positive bacterium that is the etiological agent of listeriosis. Here we report that L. monocytogenes produces extracellular EVs with similar size and morphology to EVs from other microorganisms, with average diameter of approximately 20-200 nm. EV composition analyzed using a simultaneous metabolite, protein, and lipid extraction (MPLEx) multi-omics approach. Listeriolysin-O (LLO) was recently reported to be associated with EVs; we confirm this finding and report that EVs contain phosphatidylinositol-specific phospholipase C (PI-PLC). Cell free EV preparations were toxic to mammalian cells demonstrating biological activity of the EV and a complex EV-LLO-PI-PLC association. High-resolution fluorescence imaging revealed L. monocytogenes derived lipid structures colocalizing with LLO in infected human epithelial cells, a finding consistent with intracellular EV-mediated secretion of LLO. These results demonstrate that L. monocytogenes, like other toxin-producing Gram-positive bacteria, utilize EVs for toxin release and implicate these structures in mammalian cytotoxicity.