Comparison of Methods for Quantifying Extracellular Vesicles of Gram-Negative Bacteria
Abstract
:1. Introduction
2. Results
2.1. Impact of Lysis Buffers on Protein and Lipid Quantification
2.2. Impact of Diluent on Vesicle Quantification
2.3. Determining Correlation between Protein and Particle Measurement Assays
3. Discussion
4. Materials and Methods
4.1. Bacterial Strain and Growth Conditions
4.2. Generation and Isolation of Bacterial Membrane Vesicles
4.3. Lysis Buffers and Diluents
4.4. Protein Quantification Assays
4.5. Lipid Quantification Assays
4.6. Nanoparticle Tracking Analysis (NTA)
4.7. Effect of OMV Dilution on Protein and NTA Quantification
4.8. Data Analysis and Statistics
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- Toyofuku, M.; Nomura, N.; Eberl, L. Types and origins of bacterial membrane vesicles. Nat. Rev. Microbiol. 2019, 17, 13–24. [Google Scholar] [CrossRef] [PubMed]
- Bhar, S.; Edelmann, M.J.; Jones, M.K. Characterization and proteomic analysis of outer membrane vesicles from a commensal microbe, Enterobacter cloacae. J. Proteom. 2021, 231, 103994. [Google Scholar] [CrossRef] [PubMed]
- Mosby, C.A.; Edelmann, M.J.; Jones, M.K. Murine Norovirus Interaction with Enterobacter cloacae Leads to Changes in Membrane Stability and Packaging of Lipid and Metabolite Vesicle Content. Microbiol. Spectr. 2023, 11, e0469122. [Google Scholar] [CrossRef] [PubMed]
- Sartorio, M.G.; Valguarnera, E.; Hsu, F.F.; Feldman, M.F. Lipidomics Analysis of Outer Membrane Vesicles and Elucidation of the Inositol Phosphoceramide Biosynthetic Pathway in Bacteroides thetaiotaomicron. Microbiol. Spectr. 2022, 10, e0063421. [Google Scholar] [CrossRef] [PubMed]
- Gerritzen, M.J.H.; Martens, D.E.; Wijffels, R.H.; Stork, M. High throughput nanoparticle tracking analysis for monitoring outer membrane vesicle production. J. Extracell. Vesicles 2017, 6, 1333883. [Google Scholar] [CrossRef] [PubMed]
- Reimer, S.L.; Beniac, D.R.; Hiebert, S.L.; Booth, T.F.; Chong, P.M.; Westmacott, G.R.; Zhanel, G.G.; Bay, D.C. Comparative Analysis of Outer Membrane Vesicle Isolation Methods with an Escherichia coli tolA Mutant Reveals a Hypervesiculating Phenotype with Outer-Inner Membrane Vesicle Content. Front. Microbiol. 2021, 12, 628801. [Google Scholar] [CrossRef]
- Mosby, C.A.; Bhar, S.; Phillips, M.B.; Edelmann, M.J.; Jones, M.K. Interaction with mammalian enteric viruses alters outer membrane vesicle production and content by commensal bacteria. J. Extracell. Vesicles 2022, 11, e12172. [Google Scholar] [CrossRef] [PubMed]
- Théry, C.; Witwer, K.W.; Aikawa, E.; Alcaraz, M.J.; Anderson, J.D.; Andriantsitohaina, R.; Antoniou, A.; Arab, T.; Archer, F.; Atkin-Smith, G.K.; et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): A position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J. Extracell. Vesicles 2018, 7, 1535750. [Google Scholar] [CrossRef] [PubMed]
- De Sousa, K.P.; Rossi, I.; Abdullahi, M.; Ramirez, M.I.; Stratton, D.; Inal, J.M. Isolation and characterization of extracellular vesicles and future directions in diagnosis and therapy. Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 2023, 15, e1835. [Google Scholar] [CrossRef] [PubMed]
- Cocozza, F.; Grisard, E.; Martin-Jaular, L.; Mathieu, M.; Théry, C. SnapShot: Extracellular Vesicles. Cell 2020, 182, 262–262.e261. [Google Scholar] [CrossRef]
- Bachurski, D.; Schuldner, M.; Nguyen, P.H.; Malz, A.; Reiners, K.S.; Grenzi, P.C.; Babatz, F.; Schauss, A.C.; Hansen, H.P.; Hallek, M.; et al. Extracellular vesicle measurements with nanoparticle tracking analysis—An accuracy and repeatability comparison between NanoSight NS300 and ZetaView. J. Extracell. Vesicles 2019, 8, 1596016. [Google Scholar] [CrossRef] [PubMed]
- Smith, P.K.; Krohn, R.I.; Hermanson, G.T.; Mallia, A.K.; Gartner, F.H.; Provenzano, M.D.; Fujimoto, E.K.; Goeke, N.M.; Olson, B.J.; Klenk, D.C. Measurement of protein using bicinchoninic acid. Anal. Biochem. 1985, 150, 76–85. [Google Scholar] [CrossRef] [PubMed]
- Aschtgen, M.-S.; Wetzel, K.; Goldman, W.; McFall-Ngai, M.; Ruby, E. Vibrio fischeri-derived outer membrane vesicles trigger host development. Cell. Microbiol. 2016, 18, 488–499. [Google Scholar] [CrossRef] [PubMed]
- Zavan, L.; Bitto, N.J.; Johnston, E.L.; Greening, D.W.; Kaparakis-Liaskos, M. Helicobacter pylori Growth Stage Determines the Size, Protein Composition, and Preferential Cargo Packaging of Outer Membrane Vesicles. Proteomics 2019, 19, e1800209. [Google Scholar] [CrossRef] [PubMed]
- Jones, L.J.; Haugland, R.P.; Singer, V.L. Development and Characterization of the NanoOrange® Protein Quantitation Assay: A Fluorescence-Based Assay of Proteins in Solution. BioTechniques 2003, 34, 850–861. [Google Scholar] [CrossRef] [PubMed]
- Gerritzen, M.J.H.; Maas, R.H.W.; van den Ijssel, J.; van Keulen, L.; Martens, D.E.; Wijffels, R.H.; Stork, M. High dissolved oxygen tension triggers outer membrane vesicle formation by Neisseria meningitidis. Microb. Cell Factories 2018, 17, 157. [Google Scholar] [CrossRef] [PubMed]
- Sjöström, A.E.; Sandblad, L.; Uhlin, B.E.; Wai, S.N. Membrane vesicle-mediated release of bacterial RNA. Sci. Rep. 2015, 5, 15329. [Google Scholar] [CrossRef] [PubMed]
- Gasperini, G.; Biagini, M.; Arato, V.; Gianfaldoni, C.; Vadi, A.; Norais, N.; Bensi, G.; Delany, I.; Pizza, M.; Aricò, B.; et al. Outer Membrane Vesicles (OMV)-based and Proteomics-driven Antigen Selection Identifies Novel Factors Contributing to Bordetella pertussis Adhesion to Epithelial Cells. Mol. Cell. Proteom. 2018, 17, 205–215. [Google Scholar] [CrossRef] [PubMed]
- Chiang, M.H.; Chang, F.J.; Kesavan, D.K.; Vasudevan, A.; Xu, H.; Lan, K.L.; Huang, S.W.; Shang, H.S.; Chuang, Y.P.; Yang, Y.S.; et al. Proteomic Network of Antibiotic-Induced Outer Membrane Vesicles Released by Extensively Drug-Resistant Elizabethkingia anophelis. Microbiol. Spectr. 2022, 10, e0026222. [Google Scholar] [CrossRef] [PubMed]
- Bhar, S.; Zhao, G.; Bartel, J.D.; Sterchele, H.; Del Mazo, A.; Emerson, L.E.; Edelmann, M.J.; Jones, M.K. Bacterial extracellular vesicles control murine norovirus infection through modulation of antiviral immune responses. Front. Immunol. 2022, 13, 909949. [Google Scholar] [CrossRef] [PubMed]
- Ellis, T.N.; Kuehn, M.J. Virulence and immunomodulatory roles of bacterial outer membrane vesicles. Microbiol. Mol. Biol. Rev. 2010, 74, 81–94. [Google Scholar] [CrossRef] [PubMed]
- Gurung, M.; Moon, D.C.; Choi, C.W.; Lee, J.H.; Bae, Y.C.; Kim, J.; Lee, Y.C.; Seol, S.Y.; Cho, D.T.; Kim, S.I.; et al. Staphylococcus aureus produces membrane-derived vesicles that induce host cell death. PLoS ONE 2011, 6, e27958. [Google Scholar] [CrossRef] [PubMed]
- Dong, X.H.; Ho, M.H.; Liu, B.; Hildreth, J.; Dash, C.; Goodwin, J.S.; Balasubramaniam, M.; Chen, C.H.; Xie, H. Role of Porphyromonas gingivalis outer membrane vesicles in oral mucosal transmission of HIV. Sci. Rep. 2018, 8, 8812. [Google Scholar] [CrossRef]
- van der Ley, P.A.; Zariri, A.; van Riet, E.; Oosterhoff, D.; Kruiswijk, C.P. An Intranasal OMV-Based Vaccine Induces High Mucosal and Systemic Protecting Immunity Against a SARS-CoV-2 Infection. Front. Immunol. 2021, 12, 781280. [Google Scholar] [CrossRef] [PubMed]
- Bae, E.H.; Seo, S.H.; Kim, C.U.; Jang, M.S.; Song, M.S.; Lee, T.Y.; Jeong, Y.J.; Lee, M.S.; Park, J.H.; Lee, P.; et al. Bacterial Outer Membrane Vesicles Provide Broad-Spectrum Protection against Influenza Virus Infection via Recruitment and Activation of Macrophages. J. Innate Immun. 2019, 11, 316–329. [Google Scholar] [CrossRef]
- Zhao, Z.; Wang, L.; Miao, J.; Zhang, Z.; Ruan, J.; Xu, L.; Guo, H.; Zhang, M.; Qiao, W. Regulation of the formation and structure of biofilms by quorum sensing signal molecules packaged in outer membrane vesicles. Sci. Total Environ. 2022, 806, 151403. [Google Scholar] [CrossRef]
- Manning, A.J.; Kuehn, M.J. Contribution of bacterial outer membrane vesicles to innate bacterial defense. BMC Microbiol. 2011, 11, 258. [Google Scholar] [CrossRef] [PubMed]
- Wai, S.N.; Lindmark, B.; Soderblom, T.; Takade, A.; Westermark, M.; Oscarsson, J.; Jass, J.; Richter-Dahlfors, A.; Mizunoe, Y.; Uhlin, B.E. Vesicle-mediated export and assembly of pore-forming oligomers of the enterobacterial ClyA cytotoxin. Cell 2003, 115, 25–35. [Google Scholar] [CrossRef]
- Zhao, G.; Jones, M.K. Role of Bacterial Extracellular Vesicles in Manipulating Infection. Infect. Immun. 2023, 91, e0043922. [Google Scholar] [CrossRef]
- Stentz, R.; Carvalho, A.L.; Jones, E.J.; Carding, S.R. Fantastic voyage: The journey of intestinal microbiota-derived microvesicles through the body. Biochem. Soc. Trans. 2018, 46, 1021–1027. [Google Scholar] [CrossRef]
- Jones, E.J.; Booth, C.; Fonseca, S.; Parker, A.; Cross, K.; Miquel-Clopés, A.; Hautefort, I.; Mayer, U.; Wileman, T.; Stentz, R.; et al. The Uptake, Trafficking, and Biodistribution of Bacteroides thetaiotaomicron Generated Outer Membrane Vesicles. Front. Microbiol. 2020, 11, 57. [Google Scholar] [CrossRef] [PubMed]
- Matthias, K.A.; Connolly, K.L.; Begum, A.A.; Jerse, A.E.; Macintyre, A.N.; Sempowski, G.D.; Bash, M.C. Meningococcal Detoxified Outer Membrane Vesicle Vaccines Enhance Gonococcal Clearance in a Murine Infection Model. J. Infect. Dis. 2022, 225, 650–660. [Google Scholar] [CrossRef]
- Huynh, D.T.; Jong, W.S.P.; Koningstein, G.M.; van Ulsen, P.; Luirink, J. Overexpression of the Bam Complex Improves the Production of Chlamydia trachomatis MOMP in the E. coli Outer Membrane. Int. J. Mol. Sci. 2022, 23, 7393. [Google Scholar] [CrossRef] [PubMed]
- Comfort, N.; Cai, K.; Bloomquist, T.R.; Strait, M.D.; Ferrante, A.W., Jr.; Baccarelli, A.A. Nanoparticle Tracking Analysis for the Quantification and Size Determination of Extracellular Vesicles. J. Vis. Exp. 2021, 169, e62447. [Google Scholar] [CrossRef]
- Kulp, A.; Kuehn, M.J. Biological functions and biogenesis of secreted bacterial outer membrane vesicles. Annu. Rev. Microbiol. 2010, 64, 163–184. [Google Scholar] [CrossRef] [PubMed]
- Nagakubo, T.; Nomura, N.; Toyofuku, M. Cracking Open Bacterial Membrane Vesicles. Front. Microbiol. 2020, 10, 3026. [Google Scholar] [CrossRef] [PubMed]
- Schwechheimer, C.; Kuehn, M.J. Outer-membrane vesicles from Gram-negative bacteria: Biogenesis and functions. Nat. Rev. Microbiol. 2015, 13, 605–619. [Google Scholar] [CrossRef] [PubMed]
- Boccara, M.; Fedala, Y.; Bryan, C.V.; Bailly-Bechet, M.; Bowler, C.; Boccara, A.C. Full-field interferometry for counting and differentiating aquatic biotic nanoparticles: From laboratory to Tara Oceans. Biomed. Opt. Express 2016, 7, 3736–3746. [Google Scholar] [CrossRef] [PubMed]
- Roose-Amsaleg, C.; Fedala, Y.; Vénien-Bryan, C.; Garnier, J.; Boccara, A.C.; Boccara, M. Utilization of interferometric light microscopy for the rapid analysis of virus abundance in a river. Res. Microbiol. 2017, 168, 413–418. [Google Scholar] [CrossRef] [PubMed]
- Romolo, A.; Jan, Z.; Bedina Zavec, A.; Kisovec, M.; Arrigler, V.; Spasovski, V.; Podobnik, M.; Iglič, A.; Pocsfalvi, G.; Kogej, K.; et al. Assessment of Small Cellular Particles from Four Different Natural Sources and Liposomes by Interferometric Light Microscopy. Int. J. Mol. Sci. 2022, 23, 15801. [Google Scholar] [CrossRef] [PubMed]
- Sausset, R.; Krupova, Z.; Guédon, E.; Peron, S.; Grangier, A.; Petit, M.-A.; De Sordi, L.; De Paepe, M. Comparison of interferometric light microscopy with nanoparticle tracking analysis for the study of extracellular vesicles and bacteriophages. J. Extracell. Biol. 2023, 2, e75. [Google Scholar] [CrossRef]
- Team, R.C. R: A Language and Environment for Statistical Computing, version 4.0.2; R Foundation for Statistical Computing: Vienna, Austria, 2015. [Google Scholar]
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. |
© 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Mosby, C.A.; Perez Devia, N.; Jones, M.K. Comparison of Methods for Quantifying Extracellular Vesicles of Gram-Negative Bacteria. Int. J. Mol. Sci. 2023, 24, 15096. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms242015096
Mosby CA, Perez Devia N, Jones MK. Comparison of Methods for Quantifying Extracellular Vesicles of Gram-Negative Bacteria. International Journal of Molecular Sciences. 2023; 24(20):15096. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms242015096
Chicago/Turabian StyleMosby, Chanel A., Natalia Perez Devia, and Melissa K. Jones. 2023. "Comparison of Methods for Quantifying Extracellular Vesicles of Gram-Negative Bacteria" International Journal of Molecular Sciences 24, no. 20: 15096. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms242015096