Extracellular vesicles (EVs) are produced by all domains of life. In Gram-negative bacteria, EVs are produced by the pinching off of the outer membrane; however, how EVs escape the thick cell walls of Gram-positive bacteria, mycobacteria and fungi is still unknown. Nonetheless, EVs have been described in a variety of cell-walled organisms, including Staphylococcus aureus, Mycobacterium tuberculosis and Cryptococcus neoformans. These EVs contain varied cargo, including nucleic acids, toxins, lipoproteins and enzymes, and have important roles in microbial physiology and pathogenesis. In this Review, we describe the current status of vesiculogenesis research in thick-walled microorganisms and discuss the cargo and functions associated with EVs in these species.

Summary Previously, extracellular vesicle production in Gram-positive bacteria was dismissed due to the absence of an outer membrane, where Gram-negative vesicles originate, and the difficulty in envisioning how such a process could occur through the cell wall. However, recent work has shown that Gram-positive bacteria produce extracellular vesicles and that the vesicles are biologically active. In this study, we show that Bacillus subtilis produces extracellular vesicles similar in size and morphology to other bacteria, characterized vesicles using a variety of techniques, provide evidence that these vesicles are actively produced by cells, show differences in vesicle production between strains, and identified a mechanism for such differences based on vesicle disruption. We found that in wild strains of B. subtilis, surfactin disrupted vesicles while in laboratory strains harboring a mutation in the gene sfp, vesicles accumulated in the culture supernatant. Surfactin not only lysed B. subtilis vesicles, but also vesicles from Bacillus anthracis, indicating a mechanism that crossed species boundaries. To our knowledge, this is the first time a gene and a mechanism has been identified in the active disruption of extracellular vesicles and subsequent release of vesicular cargo in Gram-positive bacteria. We also identify a new mechanism of action for surfactin.

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...

Graphical abstract

The substantia nigra pars reticulata (SNR) is a structure involved in the control of seizures, and its effects on seizures are age-dependent. GABAergic transmission in the SNR, mediated by GABAA receptors, influences seizure susceptibility. We have found that in the adult SNR there may be at least two segregated GABAA-sensitive SNR projection networks involved in regulating the SNR effects on seizures, perhaps reflecting the existence of an anticonvulsant and a proconvulsant network. The two networks with opposing effects on seizures originate from two topographically distinct regions. The anterior-medial region of the SNR is associated with anticonvulsant effects, whereas the posterior-dorsal region of the SNR is associated with proconvulsant effects. To explain ontogenic differences in the SNR effects on seizures, we hypothesize that the two functionally segregated networks which influence the expression of seizures may become segregated with maturation. The increased seizure susceptibility of rat pups may be due, in part, to delayed development of this anterior-medial SNR anticonvulsant network.