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...

Epigenetic modifications such as DNA methylation contribute to progression of hepatitis C virus (HCV) infection to life-threatening hepatocellular carcinoma (HCC) by promoting the silencing of tumor suppressor genes through DNA hypermethylation and by causing genomic instability through global hypomethylation. However few studies have addressed the promoter region hypomethylation status of the oncogenes involved in HCV derived HCC. In this study, we analyzed the promoter region methylation pattern of RAS oncogenes (HRAS, KRAS, and NRAS) using methylation-specific PCR for 50 chronic HCV patients infected with genotype 3a (27 HCC patients and 23 control non-HCC patients). Methylation-specific polymerase chain reaction analysis revealed that the NRAS oncogene promoter (P = .0025) was significantly hypomethylated in HCC patients compared to the non-HCC patients suggesting its contribution to the progression of HCV towards HCC. To identify the agent for alteration in the RAS oncogene expression, 7 HCV genes were expressed in the Huh-7 cell line followed by measurement of the NRAS expression level in Huh-7 by a quantitative real-time polymerase chain reaction. An increase in the messenger RNA level of the NRAS gene was detected when Huh-7 were transfected with Core, NS5a, and NS2 genes. Our findings suggest the involvement of NRAS oncogene in the pathogenesis of HCV3a derived HCC in Pakistani population and also identifies the HCV genes responsible for its enhanced expression. Our study raises the hypothesis that a single HCV gene may increase the chances of malignancy. Therefore, our study may have identified a useful epigenetic biomarker of HCC progression in HCV patients and may help to develop novel diagnostic tools.

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