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Microbiology Society logo Microbiology

Volume 171, Issue 4

Co-purification of the GroEL chaperone during outer membrane vesicle purification: insights from subsp. Open Access

Abstract

Outer membrane vesicles (OMVs) are naturally produced by Gram-negative bacteria and originate from their outer membrane. They can be extracted using ultracentrifugation or ultrafiltration using concentration columns, followed by purification with a density gradient. However, these methods may co-purify contaminants with similar physical properties. Several studies have identified GroEL, a chaperonin, as a major protein in OMV preparations. Using subsp. as a model, we detected GroEL by mass spectrometry and observed it in transmission electron microscopy images as separate from OMVs. As a cytoplasmic protein complex, GroEL is more likely a contaminant resulting from bacterial lysis during growth rather than an intrinsic OMV component. The model subsp. proved valuable in reaching this conclusion because it produces high levels of extracellular GroEL and low amounts of OMVs. This study emphasizes the need for caution when interpreting the presence of GroEL in OMV preparations and highlights the importance of rigorous purification methods to ensure OMV purity.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): GroEL , Aeromonas salmonicida subsp. salmonicida , Amicon® column , electron microscopy , optiprep gradient , outer membrane vesicles (OMVs) and purification
Funding
This study was supported by the:
  • Université Laval
    • Principal Award Recipient: MaudeF Paquet
  • Natural Sciences and Engineering Research Council of Canada (Award RGPIN-2019-04444)
    • Principal Award Recipient: SteveJ Charette
  • Natural Sciences and Engineering Research Council of Canada
    • Principal Award Recipient: MaudeF Paquet
  • Fonds de recherche du Québec
    • Principal Award Recipient: MaudeF Paquet
© 2025 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License.
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2025-04-28
2026-04-15

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References

  1. Ellis TN, Kuehn MJ. Virulence and immunomodulatory roles of bacterial outer membrane vesicles. Microbiol Mol Biol Rev 2010; 74:81–94 [View Article] [PubMed]
     [Google Scholar]
  2. Pasqua M, Zennaro A, Trirocco R, Fanelli G, Micheli G et al. Modulation of OMV production by the lysis module of the DLP12 defective prophage of Escherichia coli K12. Microorganisms 2021; 9:369 [View Article] [PubMed]
     [Google Scholar]
  3. Sartorio MG, Pardue EJ, Feldman MF, Haurat MF. Bacterial outer membrane vesicles: from discovery to applications. Annu Rev Microbiol 2021; 75:609–630 [View Article] [PubMed]
     [Google Scholar]
  4. Toyofuku M, Schild S, Kaparakis-Liaskos M, Eberl L. Composition and functions of bacterial membrane vesicles. Nat Rev Microbiol 2023; 21:415–430 [View Article] [PubMed]
     [Google Scholar]
  5. Kulp A, Kuehn MJ. Biological functions and biogenesis of secreted bacterial outer membrane vesicles. Annu Rev Microbiol 2010; 64:163–184 [View Article] [PubMed]
     [Google Scholar]
  6. Nevot M, Deroncelé V, Messner P, Guinea J, Mercadé E. Characterization of outer membrane vesicles released by the psychrotolerant bacterium Pseudoalteromonas antarctica NF3. Environ Microbiol 2006; 8:1523–1533 [View Article] [PubMed]
     [Google Scholar]
  7. Chen Z, Liu Y, Jiang L, Zhang C, Qian X et al. Bacterial outer membrane vesicles increase polymyxin resistance in Pseudomonas aeruginosa while inhibiting its quorum sensing. J Hazard Mater 2024; 478:135588 [View Article]
     [Google Scholar]
  8. Horstman AL, Kuehn MJ. Enterotoxigenic Escherichia coli secretes active heat-labile enterotoxin via outer membrane vesicles. J Biol Chem 2000; 275:12489–12496 [View Article] [PubMed]
     [Google Scholar]
  9. McBroom AJ, Kuehn MJ. Release of outer membrane vesicles by gram-negative bacteria is a novel envelope stress response. Mol Microbiol 2007; 63:545–558 [View Article] [PubMed]
     [Google Scholar]
  10. Reyes-Robles T, Dillard RS, Cairns LS, Silva-Valenzuela CA, Housman M et al. Vibrio cholerae outer membrane vesicles inhibit bacteriophage infection. J Bacteriol 2018; 200:e00792-17 [View Article] [PubMed]
     [Google Scholar]
  11. Erfanmanesh A, Mohajerfar T, Nikaein D, Mokhtari A, Beikzadeh B. Comparative protection of two antigens (whole-cell and outer membrane vesicle) of yersinia ruckeri in rainbow trout (oncorhynchus mykiss). Iran J Fish Sci 2022; 21:187–201
     [Google Scholar]
  12. Peeters CC, Rümke HC, Sundermann LC, Rouppe van der Voort EM, Meulenbelt J et al. Phase I clinical trial with a hexavalent PorA containing meningococcal outer membrane vesicle vaccine. Vaccine 1996; 14:1009–1015 [View Article] [PubMed]
     [Google Scholar]
  13. Seike S, Kobayashi H, Ueda M, Takahashi E, Okamoto K et al. Outer membrane vesicles released from Aeromonas strains are involved in the biofilm formation. Front Microbiol 2020; 11:613650 [View Article] [PubMed]
     [Google Scholar]
  14. Simpson RJ. Staining Proteins in Gels with Silver Nitrate Cold Spring Harbor Protocols; 2007
     [Google Scholar]
  15. Marcoux P-É, Attéré SA, Paquet VE, Paquet MF, Girard SB et al. Host dependent-transposon for a plasmid found in Aeromonas salmonicida subsp. salmonicida that bears a catB3 gene for chloramphenicol resistance. Antibiotics 2023; 12:257 [View Article]
     [Google Scholar]
  16. Leduc GR, Paquet VE, Vincent AT, Charette SJ. Characterization of bacteriophage T7-Ah reveals its lytic activity against a subset of both mesophilic and psychrophilic Aeromonas salmonicida strains. Arch Virol 2021; 166:521–533 [View Article] [PubMed]
     [Google Scholar]
  17. Paquet VE, Vincent AT, Moineau S, Charette SJ. Beyond the A-layer: adsorption of lipopolysaccharides and characterization of bacteriophage-insensitive mutants of Aeromonas salmonicida subsp. salmonicida. Mol Microbiol 2019; 112:667–677 [View Article] [PubMed]
     [Google Scholar]
  18. Ishii N. GroEL and the GroEL-GroES complex. In Marles-Wright J, Harris JR. eds In Macromolecular Protein Complexes: Structure and Function Cham: Springer International Publishing; 2017 pp 483–504
     [Google Scholar]
  19. Berlanda Scorza F, Colucci AM, Maggiore L, Sanzone S, Rossi O et al. High yield production process for Shigella outer membrane particles. PLoS One 2012; 7:e35616 [View Article] [PubMed]
     [Google Scholar]
  20. Hong J, Dauros-Singorenko P, Whitcombe A, Payne L, Blenkiron C et al. Analysis of the Escherichia coli extracellular vesicle proteome identifies markers of purity and culture conditions. J Extracell Vesicles 2019; 8:1632099 [View Article] [PubMed]
     [Google Scholar]
  21. Lindmark B, Rompikuntal PK, Vaitkevicius K, Song T, Mizunoe Y et al. Outer membrane vesicle-mediated release of cytolethal distending toxin (CDT) from Campylobacter jejuni. BMC Microbiol 2009; 9:220 [View Article] [PubMed]
     [Google Scholar]
  22. Daleke-Schermerhorn MH, Felix T, Soprova Z, Ten Hagen-Jongman CM, Vikström D et al. Decoration of outer membrane vesicles with multiple antigens by using an autotransporter approach. Appl Environ Microbiol 2014; 80:5854–5865 [View Article] [PubMed]
     [Google Scholar]
  23. Kothary MH, Gopinath GR, Gangiredla J, Rallabhandi PV, Harrison LM et al. Analysis and characterization of proteins associated with outer membrane vesicles secreted by Cronobacter spp. Front Microbiol 2017; 8:134 [View Article] [PubMed]
     [Google Scholar]
  24. Pierson T, Matrakas D, Taylor YU, Manyam G, Morozov VN et al. Proteomic characterization and functional analysis of outer membrane vesicles of Francisella novicida suggests possible role in virulence and use as a vaccine. J Proteome Res 2011; 10:954–967 [View Article] [PubMed]
     [Google Scholar]
  25. Kang OJ, Vézinz L-P, Laberge S, Simard RE. Some factors influencing the autolysis of Lactobacillus bulgaricus and Lactobacillus casei. J Dairy Sci 1998; 81:639–646 [View Article]
     [Google Scholar]
  26. Ishihama Y, Schmidt T, Rappsilber J, Mann M, Hartl FU et al. Protein abundance profiling of the Escherichia coli cytosol. BMC Genomics 2008; 9:102 [View Article] [PubMed]
     [Google Scholar]
  27. Richter FM, Sander B, Golas MM, Stark H, Urlaub H. Merging molecular electron microscopy and mass spectrometry by carbon film-assisted endoproteinase digestion. Mol Cell Proteomics 2010; 9:1729–1741 [View Article] [PubMed]
     [Google Scholar]
  28. Leung C, Palmer RE. Adsorption of a model protein, the GroEL chaperonin, on surfaces. J Phys Condens Matter 2008; 20:353001 [View Article]
     [Google Scholar]
  29. Bergonzelli GE, Granato D, Pridmore RD, Marvin-Guy LF, Donnicola D et al. GroEL of Lactobacillus johnsonii La1 (NCC 533) is cell surface associated: potential role in interactions with the host and the gastric pathogen Helicobacter pylori. Infect Immun 2006; 74:425–434 [View Article] [PubMed]
     [Google Scholar]
  30. Frisk A, Ison CA, Lagergård T. GroEL heat shock protein of Haemophilus ducreyi: association with cell surface and capacity to bind to eukaryotic cells. Infect Immun 1998; 66:1252–1257 [View Article] [PubMed]
     [Google Scholar]
  31. Marques MA, Chitale S, Brennan PJ, Pessolani MC. Mapping and identification of the major cell wall-associated components of Mycobacterium leprae. Infect Immun 1998; 66:2625–2631 [View Article] [PubMed]
     [Google Scholar]
  32. Wilson DN, Nierhaus KH. The ribosome through the looking glass. Angew Chem Int Ed 2003; 42:3464–3486 [View Article]
     [Google Scholar]
  33. Yu T, Jiang J, Yu Q, Li X, Zeng F. Structural insights into the distortion of the ribosomal small subunit at different magnesium concentrations. Biomolecules 2023; 13:566 [View Article] [PubMed]
     [Google Scholar]
  34. Kuechler E, Bauer K, Rich A. Protein synthesis with ribonuclease digested ribosomes. Biochim Biophys Acta 1972; 277:615–627 [View Article] [PubMed]
     [Google Scholar]
  35. Erxleben DA, Rivas F, Smith I, Poddar S, DeAngelis PL et al. High-fidelity and iterative affinity extraction of hyaluronan. Proteoglycan Res 2024; 2:e70008 [View Article] [PubMed]
     [Google Scholar]
  36. Kwon SO, Gho YS, Lee JC, Kim SI. Proteome analysis of outer membrane vesicles from a clinical Acinetobacter baumannii isolate. FEMS Microbiol Lett 2009; 297:150–156 [View Article] [PubMed]
     [Google Scholar]
  37. Palacios E, Lobos-González L, Guerrero S, Kogan MJ, Shao B et al. Helicobacter pylori outer membrane vesicles induce astrocyte reactivity through nuclear factor-κappa B activation and cause neuronal damage in vivo in a murine model. J Neuroinflammation 2023; 20:66 [View Article] [PubMed]
     [Google Scholar]
  38. Lagos L, Tandberg JI, Repnik U, Boysen P, Ropstad E et al. Characterization and vaccine potential of membrane vesicles produced by Francisella noatunensis subsp. orientalis in an adult zebrafish model. Clin Vaccine Immunol 2017; 24:e00557-16 [View Article] [PubMed]
     [Google Scholar]
  39. Kieselbach T, Zijnge V, Granström E, Oscarsson J. Proteomics of Aggregatibacter actinomycetemcomitans outer membrane vesicles. PLoS One 2015; 10:e0138591 [View Article] [PubMed]
     [Google Scholar]
  40. Berleman JE, Allen S, Danielewicz MA, Remis JP, Gorur A et al. The lethal cargo of Myxococcus xanthus outer membrane vesicles. Front Microbiol 2014; 5:474 [View Article] [PubMed]
     [Google Scholar]
/content/journal/micro/10.1099/mic.0.001558
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Co-purification of the GroEL chaperone during outer membrane vesicle purification: insights from Aeromonas salmonicida subsp. salmonicida
Microbiology 171, 001558 (2025); https://doi.org/10.1099/mic.0.001558
/content/journal/micro/10.1099/mic.0.001558
/content/journal/micro/10.1099/mic.0.001558
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