Listeria monocytogenes virulence factors, including listeriolysin O, are secreted in biologically active extracellular vesicles

Abstract

Outer membrane vesicles produced by Gram-negative bacteria have been studied for half a century but the possibility that Gram-positive bacteria secrete extracellular vesicles (EVs) was not pursued until recently due to the assumption that the thick peptidoglycan cell wall would prevent their release to the environment. However, following their 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 causes listeriosis. Here we report that L. monocytogenes produces EVs with diameters ranging from 20 to 200 nm, containing the pore-forming toxin listeriolysin O (LLO) and phosphatidylinositol-specific phospholipase C (PI-PLC). Cell-free EV preparations were toxic to mammalian cells, 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 reduced LLO activity. Using simultaneous metabolite, protein, and lipid extraction (MPLEx) multiomics we characterized protein, lipid, and metabolite composition of bacterial cells and secreted EVs and found that EVs carry the majority of listerial virulence proteins. Using immunogold EM we detected LLO 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 during infection. Our findings demonstrate that L. monocytogenes uses EVs for toxin release and implicate these structures in mammalian cytotoxicity. The pathogenic Gram-positive bacterium Listeria monocytogenes is the etiological agent of listeriosis, a disease with serious consequences for pregnant women, newborns, and immunocompromised persons. Healthy individuals who have ingested large amounts of L. monocytogenes can suffer from gastroenteritis when the bacterium passes through the gastrointestinal barrier (1-4). L. monocytogenes can cause spontaneous abortions in pregnant women and meningoencephalitis by crossing the placental and blood-brain barriers, respectively (5). To invade cells, cross these barriers, and evade the immune system, L. monocytogenes has a sophisticated intracellular life cycle and pathogenic strategy (6, 7). Initially, L. monocytogenes invades various cell types, including nonphagocytic cells, by utilizing two internalins, internalin A (InlA) and internalin B (InlB), with a minor contribution by the pore-forming toxin listeriolysin O (LLO), 7 to induce uptake of the bacterium (1, 5, 8-11). Once internalized in the host vacuole, L. monocytogenes employs LLO, phosphatidylcholinespecific phospholipase (PC-PLC), and phosphatidylinositolspecific phospholipase C (PI-PLC) to disrupt the single vacuolar membrane, releasing the bacterium into the cytoplasm The authors declare that they have...

Lipidomic and Ultrastructural Characterization of Cell Envelope ofStaphylococcus aureusGrown in the Presence of Human Serum

“…Extracellular vesicles, which are delimited by a lipid bilayer and cannot replicate, are naturally released from the cells by many different organisms (50), including S. aureus . Coelho et al found that the composition of extracellular vesicles from the gram-positive bacterial pathogen Listeria monocytogenes , grown in Brain Heart Infusion broth supplemented with 10% bovine fetal serum, were enriched in PE, sphingolipids and triacylglycerols (51). Although it is possible that serum lipids can cross the cell wall in the other direction and insert into the membrane, the fact that Triton X-100 can effectively remove these lipids make this hypothesis less likely.…”

Answers to naysayers regarding microbial extracellular vesicles

“…Performing mock extractions with killed organisms did not isolate EVs [30]. Lipid composition of EVs is different from the lipid bilayer it originates from, observed when comparing EVs with the plasma membrane of whole cells in Gram-positive bacteria Listeria monocytogenes or Streptococcus [30][31][32] as well as when comparing EVs with the outer membrane of Gram-negative bacteria [33]. The same is found for RNA cargo, where RNA cargo does not reflect the RNA content of intact cells, which is highly suggestive of selective enrichment [31].…”

Knockout of MMP3 Weakens Solid Tumor Organoids and Cancer Extracellular Vesicles

“…These populations of EVs are often distinguished based on their biogenesis, size, content, and function [1,8,9]. In addition to these EV types, other vesicles have been reported including; oncosomes, large oncosomes (1-10 µm) [10,11], matrix vesicles [12][13][14], migrasomes (50 nm to 3 µm) [15,16], exopheres (~4 µm), exomeres (~35 nm), and bacterial outer membrane vesicles (OMV) [4,[17][18][19]. Since EVs are heterogeneous populations, there is no unanimous consensus on the nomenclature of them.…”