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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 1202-1217 |
| Number of pages | 16 |
| Journal | Journal of Biological Chemistry |
| Volume | 294 |
| Issue number | 4 |
| DOIs | |
| State | Published - Jan 1 2019 |
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ASJC Scopus subject areas
- Biochemistry
- Molecular Biology
- Cell Biology
Cite this
Listeria monocytogenes virulence factors, including listeriolysin O, are secreted in biologically active extracellular vesicles. / Coelho, Carolina; Brown, Lisa; Maryam, Maria; Vij, Raghav; Smith, Daniel F.Q.; Burnet, Meagan C.; Kyle, Jennifer E.; Heyman, Heino M.; Ramirez, Jasmine; Prados-Rosales, Rafael; Lauvau, Gregoire; Nakayasu, Ernesto S.; Brady, Nathan R.; Hamacher-Brady, Anne; Coppens, Isabelle; Casadevall, Arturo.
In: Journal of Biological Chemistry, Vol. 294, No. 4, 01.01.2019, p. 1202-1217.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Listeria monocytogenes virulence factors, including listeriolysin O, are secreted in biologically active extracellular vesicles
AU - Coelho, Carolina
AU - Brown, Lisa
AU - Maryam, Maria
AU - Vij, Raghav
AU - Smith, Daniel F.Q.
AU - Burnet, Meagan C.
AU - Kyle, Jennifer E.
AU - Heyman, Heino M.
AU - Ramirez, Jasmine
AU - Prados-Rosales, Rafael
AU - Lauvau, Gregoire
AU - Nakayasu, Ernesto S.
AU - Brady, Nathan R.
AU - Hamacher-Brady, Anne
AU - Coppens, Isabelle
AU - Casadevall, Arturo
PY - 2019/1/1
Y1 - 2019/1/1
N2 - 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.
AB - 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.
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UR - http://www.scopus.com/inward/citedby.url?scp=85060570094&partnerID=8YFLogxK
U2 - 10.1074/jbc.RA118.006472
DO - 10.1074/jbc.RA118.006472
M3 - Article
C2 - 30504226
AN - SCOPUS:85060570094
VL - 294
SP - 1202
EP - 1217
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 4
ER -