RT Journal Article SR Electronic T1 Listeria monocytogenes virulence factors are secreted in biologically active Extracellular Vesicles JF bioRxiv FD Cold Spring Harbor Laboratory SP 210906 DO 10.1101/210906 A1 Carolina Coelho A1 Lisa Brown A1 Maria Maryam A1 Meagan C. Burnet A1 Jennifer E. Kyle A1 Heino M. Heyman A1 Rafael Prados-Rosales A1 Gregoire Lauvau A1 Ernesto S. Nakayasu A1 Nathan Ryan Brady A1 Anne Hamacher-Brady A1 Isabelle Coppens A1 Arturo Casadevall YR 2017 UL http://biorxiv.org/content/early/2017/10/29/210906.abstract AB Outer membrane vesicles produced by Gram-negative bacteria have been studied for half a century but the possibility that Gram-positive bacteria secreted extracellular vesicles (EVs) was not pursued due to the assumption that the thick peptidoglycan cell wall would prevent their release to the environment. However, following discovery in fungi, which also have cell walls, EVs have now been described for a variety of Gram-positive bacteria. EVs purified from Gram-positive bacteria are implicated in virulence, toxin release and transference to host cells, eliciting immune responses, and spread of antibiotic resistance. Listeria monocytogenes is a Gram-positive bacterium that is the etiological agent of listeriosis. Here we report that L. monocytogenes produces EVs with diameter ranging from 20-200 nm, containing the pore-forming toxin listeriolysin O (LLO) and phosphatidylinositol-specific phospholipase C (PI-PLC). Using simultaneous metabolite, protein, and lipid extraction (MPLEx) multi-omics we characterized protein, lipid and metabolite composition of bacterial cells and secreted EVs and found that EVs carry the majority of listerial virulence proteins. Cell-free EV preparations were toxic to the murine macrophage cell line J774.16, in a LLO-dependent manner, evidencing EV biological activity. The deletion of plcA increased EV toxicity, suggesting PI-PLC can restrain LLO activity. Using immunogold electron microscopy we detect LLO localization at several organelles within infected human epithelial cells and with high-resolution fluorescence imaging we show that dynamic lipid structures are released from L. monocytogenes that colocalize with LLO during infection. Our findings demonstrate that L. monocytogenes utilize EVs for toxin release and implicate these structures in mammalian cytotoxicity.