Extracellular Vesicle Production in Gram-positive Bacteria

Abstract

Extracellular vesicles (EVs) are small, lipid bilayered spheres ranging in size from 20500 nm in diameter that are produced by all domains of life. Outer membrane vesicles (OMVs), produced by the bulging and pinching off of the outer membrane of Gram-negative bacteria, have been studied for decades. However, the production of EVs by Gram-positive bacteria was neglected due to the erroneous impression that vesicular structures could not escape the thick peptidoglycan cell wall. The production of EVs has now been described in a handful of Gram-positive bacteria, and while the field is expanding, not much is understood about the various biological roles of EVs in bacterial survival and pathogenesis. We have utilized various techniques to investigate the biological functions of EV production from the Gram-positive bacteria Bacillus subtilis and Listeria monocytogenes.;The genetically tractable bacterium, B. subtilis, has long been utilized for the study of Gram-positive bacterial physiology. We characterized EVs purified from B. subtilis cultures and describe a novel function for the B. subtilis lipopeptide, surfactin. Surfactin disrupts EVs and subsequently releases EV cargo into the extracellular space. Many lab strains of B. subtilis are unable to synthesize surfactin and therefore large quantities of EVs are recoverable from these cultures. The high yield of EVs combined with the genetic tractability of this bacterium makes it an efficient model for the study of vesiculogenesis in Gram-positive bacteria.;EVs produced by the intracellular pathogen, L. monocytogenes , are associated with the virulence factor, listeriolysin O (LLO), and we therefore investigated the role that EVs may play in the pathogenesis of this bacterium. We found that EVs purified from L. monocytogenes can deliver LLO to macrophages and that intact but not sonicated EVs were cytotoxic to macrophages.;The data herein describes EV production in nonpathogenic and pathogenic species of Gram-positive bacteria. This work expands the knowledge and applications of this emerging field by characterizing EVs and highlighting the various functional roles that EVs can play in Gram-positive bacterial physiology.