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 C.
AU - Lauvau, Gregoire
AU - Nakayasu, Ernesto S.
AU - Brady, Nathan R.
AU - Hamacher-Brady, Anne
AU - Coppens, Isabelle
AU - Casadevall, Arturo
N1 - Funding Information:
4 Supported by National Institutes of Health Training Grant GMS GM08752. 6 Supported by National Institutes of Health Grants 5R01HL059842, 5R01AI033774, 5R37AI033142, and 5R01AI052733.
Funding Information:
Acknowledgments—We thank Geoffrey Perumal, Benjamin Clark, and Leslie Gunther at the Analytical Imaging Facility, Albert Einstein College of Medicine, for assistance with the transmission EM. Negative staining was performed at the Johns Hopkins Microscopy Facility, and we particularly acknowledge Barbara McClintock. We also thank the technical competence of Kimberley Zichichi from the Electron Microscopy Facility at Yale University for the immuno-EM. We thank Howard Goldfine for the kind gift of PI-PLC antiserum. The MPLEx experiment was performed in the Environmental Molecular Science Laboratory, a United States Department of Energy (DOE) national scientific user facility at the Pacific Northwest National Laboratory (PNNL) in Richland, WA. Battelle operates PNNL for the DOE under contract DE-AC05-76RLO01830.
Funding Information:
The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This article contains Movie S1, Tables S1–S4, and Figs. S1–S4. Multiomics data were deposited in the MassIVE repository under accession num-bers MSV000081402, MSV000081403, and MSV000081404. 1 Both authors contributed equally and should be considered co-first authors. 2To whom correspondence may be addressed: Hopkins Bloomberg School of Public Health, 615 North Wolfe St., Baltimore, MD 21205. E-mail: carolina.coelho@abdn.ac.uk. 3The data were from a thesis submitted in partial fulfillment of the require-ments for the degree of Doctor of Philosophy in the Sue Golding Graduate Division of Medical Science, Albert Einstein College of Medicine, Yeshiva University, Bronx, NY. 4 Supported by National Institutes of Health Training Grant GMS GM08752. 5 These authors contributed equally to this work. 6Supported by National Institutes of Health Grants 5R01HL059842, 5R01AI033774, 5R37AI033142, and 5R01AI052733. To whom correspon-dence may be addressed: Johns Hopkins Bloomberg School of Public Health, 615 North Wolfe St., Baltimore, MD 21205. E-mail: acasade1@ jhu.edu.
Publisher Copyright:
© 2019 Coelho et al. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc.
PY - 2019/1/25
Y1 - 2019/1/25
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.
UR - http://www.scopus.com/inward/record.url?scp=85060570094&partnerID=8YFLogxK
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 -