Characterization of Escherichia coli outer membrane vesicles and the impact of pathogenic ones on NLR signaling pathways
Abstract
Background and Objectives: The secretion of outer membrane vesicles (OMVs) is a universal event among bacteria. In this study, we characterized OMVs from pathogenic and non-pathogenic strains of Escherichia coli and assessed the effect of pathogenic OMVs on NLR signaling pathways.
Materials and Methods: OMVs were extracted by differential centrifugation and characterized by scanning electron microscopy (SEM), SDS-PAGE, Limulus amebocyte lysate (LAL) test, and nucleic acid extraction. Then, the Caco-2 cells were treated with the pathogenic OMVs to evaluate their effect on NLR signaling pathways.
Results: SEM showed that pathogenic and non-pathogenic strains produced OMVs in the range of 9-72.9 and 45-270 nm, respectively. The SDS-PAGE revealed that both OMVs had protein bands ranging from 25 to 100 kDa. The LAL test displayed that the concentration of LPS was 2.368 and 0.055 EU/ml in pathogenic and non-pathogenic OMVs, respectively. The evaluation of nucleic acid contents showed no significant difference between both types of OMVs. The assessment of pathogenic OMVs' effect on NLR genes demonstrated that the expression level was changed in some genes.
Conclusion: The characterization of OMVs showed that both strains of E. coli secrete OMVs in different sizes and contents. Besides, it was revealed that OMVs can regulate gene expression.
1. Kuehn MJ, Kesty NC. Bacterial outer membrane vesicles and the host–pathogen interaction. Genes Dev 2005; 19: 2645-2655.
2. Mashburn-Warren LM, Whiteley M. Special delivery: vesicle trafficking in prokaryotes. Mol Microbiol 2006; 61: 839-846.
3. Mohammadzadeh R, Ghazvini K, Farsiani H, Soleimanpour S. Mycobacterium tuberculosis extracellular vesicles: exploitation for vaccine technology and diagnostic methods. Crit Rev Microbiol 2021; 47: 13-33.
4. Jan AT. Outer membrane vesicles (OMVs) of gram-negative bacteria: a perspective update. Front Microbiol 2017; 8: 1053.
5. Nagakubo T, Nomura N, Toyofuku M. Cracking open bacterial membrane vesicles. Front Microbiol 2020; 10: 3026.
6. Schwechheimer C, Sullivan CJ, Kuehn MJ. Envelope control of outer membrane vesicle production in Gram-negative bacteria. Biochemistry 2013; 52: 3031-3040.
7. Moon DC, Choi CH, Lee JH, Choi C-W, Kim H-Y, Park JS, et al. Acinetobacter baumannii outer membrane protein A modulates the biogenesis of outer membrane vesicles. J Microbiol 2012; 50: 155-160.
8. Schwechheimer C, Rodriguez DL, Kuehn MJ. NlpI-mediated modulation of outer membrane vesicle production through peptidoglycan dynamics in Escherichia coli. Microbiologyopen 2015; 4: 375-389.
9. Mashburn LM, Whiteley M. Membrane vesicles traffic signals and facilitate group activities in a prokaryote. Nature 2005; 437: 422-425.
10. Schwechheimer C, Kulp A, Kuehn MJ. Modulation of bacterial outer membrane vesicle production by envelope structure and content. BMC Microbiol 2014; 14: 324.
11. Amano A, Takeuchi H, Furuta N. Outer membrane vesicles function as offensive weapons in host–parasite interactions. Microbes Infect 2010; 12: 791-798.
12. Bomberger JM, MacEachran DP, Coutermarsh BA, Ye S, O'Toole GA, Stanton BA. Long-distance delivery of bacterial virulence factors by Pseudomonas aeruginosa outer membrane vesicles. PLoS Pathog 2009; 5(4): e1000382.
13. Jurkoshek KS, Wang Y, Athman JJ, Barton MR, Wearsch PA. Interspecies communication between pathogens and immune cells via bacterial membrane vesicles. Front Cell Dev Biol 2016; 4: 125.
14. Ellis TN, Kuehn MJ. Virulence and immunomodulatory roles of bacterial outer membrane vesicles. Microbiol Mol Biol Rev 2010; 74: 81-94.
15. Sharpe SW, Kuehn MJ, Mason KM. Elicitation of epithelial cell-derived immune effectors by outer membrane vesicles of nontypeable Haemophilus influenzae. Infect Immun 2011; 79: 4361-4369.
16. Bielaszewska M, Rüter C, Kunsmann L, Greune L, Bauwens A, Zhang W, et al. Enterohemorrhagic Escherichia coli hemolysin employs outer membrane vesicles to target mitochondria and cause endothelial and epithelial apoptosis. PLoS Pathog 2013; 9(12): e1003797.
17. Parker H, Chitcholtan K, Hampton MB, Keenan JI. Uptake of Helicobacter pylori outer membrane vesicles by gastric epithelial cells. Infect Immun 2010; 78: 5054-5061.
18. Johnston EL, Kufer TA, Kaparakis-Liaskos M (2020). Immunodetection and pathogenesis mediated by bacterial membrane vesicles. Bacterial membrane vesicles. pp.159-188. https://link.springer.com/chapter/10.1007/978-3-030-36331-4_8
19. Kaparakis-Liaskos M, Ferrero RL. Immune modulation by bacterial outer membrane vesicles. Nat Rev Immunol 2015; 15: 375-387.
20. Mukherjee T, Hovingh ES, Foerster EG, Abdel-Nour M, Philpott DJ, Girardin SE. NOD1 and NOD2 in inflammation, immunity and disease. Arch Biochem Biophys 2019; 670: 69-81.
21. Motta V, Soares F, Sun T, Philpott DJ. NOD-like receptors: versatile cytosolic sentinels. Physiol Rev 2015; 95: 149-178.
22. Ahmadi Badi S, Moshiri A, Ettehad Marvasti F, Mojtahedzadeh M, Kazemi V, Siadat SD. Extraction and evaluation of outer membrane vesicles from two important gut microbiota members, Bacteroides fragilis and Bacteroides thetaiotaomicron. Cell J 2020; 22: 344-349.
23. Behrouzi A, Vaziri F, Riazi Rad F, Amanzadeh A, Fateh A, Moshiri A, et al. Comparative study of pathogenic and non-pathogenic Escherichia coli outer membrane vesicles and prediction of host-interactions with TLR signaling pathways. BMC Res Notes 2018; 11: 539.
24. Sambuy Y, De Angelis I, Ranaldi G, Scarino ML, Stammati A, Zucco F. The Caco-2 cell line as a model of the intestinal barrier: influence of cell and culture-related factors on Caco-2 cell functional characteristics. Cell Biol Toxicol 2005; 21: 1-26.
25. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001; 25: 402-408.
26. Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 2008; 3: 1101-1108.
27. Deatherage BL, Lara JC, Bergsbaken T, Barrett SL, Lara S, Cookson BT. Biogenesis of bacterial membrane vesicles. Mol Microbiol 2009; 72: 1395-1407.
28. Johnston EL, Heras B, Kufer TA, Kaparakis-Liaskos M. Detection of bacterial membrane vesicles by NOD-like receptors. Int J Mol Sci 2021; 22: 1005.
29. Cecil JD, O’Brien-Simpson NM, Lenzo JC, Holden JA, Chen Y-Y, Singleton W, et al. Differential responses of pattern recognition receptors to outer membrane vesicles of three periodontal pathogens. PLoS One 2016; 11(4): e0151967.
30. Kaparakis M, Turnbull L, Carneiro L, Firth S, Coleman HA, Parkington HC, et al. Bacterial membrane vesicles deliver peptidoglycan to NOD1 in epithelial cells. Cell Microbiol 2010; 12: 372-385.
31. Patten DA, Hussein E, Davies SP, Humphreys PN, Collett A. Commensal-derived OMVs elicit a mild proinflammatory response in intestinal epithelial cells. Microbiology (Reading) 2017; 163: 702-711.
32. Fábrega MJ, Aguilera L, Giménez R, Varela E, Alexandra Canas M, Antolín M, et al. Activation of immune and defense responses in the intestinal mucosa by outer membrane vesicles of commensal and probiotic Escherichia coli strains. Front Microbiol 2016; 7: 705.
33. Kunsmann L, Rüter C, Bauwens A, Greune L, Glüder M, Kemper B, et al. Virulence from vesicles: Novel mechanisms of host cell injury by Escherichia coli O104: H4 outbreak strain. Sci Rep 2015; 5: 13252.
34. Lee J, Kim OY, Gho YS. Proteomic profiling of Gram-negative bacterial outer membrane vesicles: Current perspectives. Proteomics Clin Appl 2016; 10: 897-909.
35. Van Der Pol L, Stork M, van der Ley P. Outer membrane vesicles as platform vaccine technology. Biotechnol J 2015; 10: 1689-1706.
36. Raetz CR, Reynolds CM, Trent MS, Bishop RE. Lipid A modification systems in gram-negative bacteria. Annu Rev Biochem 2007; 76: 295-329.
37. Ahmadi Badi S, Bruno SP, Moshiri A, Tarashi S, Siadat SD, Masotti A. Small RNAs in outer membrane vesicles and their function in host-microbe interactions. Front Microbiol 2020; 11: 1209.
38. Dell’Annunziata F, Folliero V, Giugliano R, De Filippis A, Santarcangelo C, Izzo V, et al. Gene transfer potential of outer membrane vesicles of gram-negative bacteria. Int J Mol Sci 2021; 22: 5985.
39. Rodriguez BV, Kuehn MJ. Staphylococcus aureus secretes immunomodulatory RNA and DNA via membrane vesicles. Sci Rep 2020; 10: 18293.
40. Chatterjee D, Chaudhuri K. Vibrio cholerae O395 outer membrane vesicles modulate intestinal epithelial cells in a NOD1 protein-dependent manner and induce dendritic cell-mediated Th2/Th17 cell responses. J Biol Chem 2013; 288: 4299-4309.
41. Schertzer JW, Whiteley M. Bacterial outer membrane vesicles in trafficking, communication and the host-pathogen interaction. J Mol Microbiol Biotechnol 2013; 23: 118-130.
42. Franchi L, Warner N, Viani K, Nuñez G. Function of Nod-like receptors in microbial recognition and host defense. Immunol Rev 2009; 227: 106-128.
43. Tavano R, Franzoso S, Cecchini P, Cartocci E, Oriente F, Aricò B, et al. The membrane expression of Neisseria meningitidis adhesin A (NadA) increases the proimmune effects of MenB OMVs on human macrophages, compared with NadA–OMVs, without further stimulating their proinflammatory activity on circulating monocytes. J Leukoc Biol 2009; 86: 143-153.
44. Kim OY, Park HT, Dinh NTH, Choi SJ, Lee J, Kim JH, et al. Bacterial outer membrane vesicles suppress tumor by interferon-γ-mediated antitumor response. Nat Commun 2017; 8: 626.
2. Mashburn-Warren LM, Whiteley M. Special delivery: vesicle trafficking in prokaryotes. Mol Microbiol 2006; 61: 839-846.
3. Mohammadzadeh R, Ghazvini K, Farsiani H, Soleimanpour S. Mycobacterium tuberculosis extracellular vesicles: exploitation for vaccine technology and diagnostic methods. Crit Rev Microbiol 2021; 47: 13-33.
4. Jan AT. Outer membrane vesicles (OMVs) of gram-negative bacteria: a perspective update. Front Microbiol 2017; 8: 1053.
5. Nagakubo T, Nomura N, Toyofuku M. Cracking open bacterial membrane vesicles. Front Microbiol 2020; 10: 3026.
6. Schwechheimer C, Sullivan CJ, Kuehn MJ. Envelope control of outer membrane vesicle production in Gram-negative bacteria. Biochemistry 2013; 52: 3031-3040.
7. Moon DC, Choi CH, Lee JH, Choi C-W, Kim H-Y, Park JS, et al. Acinetobacter baumannii outer membrane protein A modulates the biogenesis of outer membrane vesicles. J Microbiol 2012; 50: 155-160.
8. Schwechheimer C, Rodriguez DL, Kuehn MJ. NlpI-mediated modulation of outer membrane vesicle production through peptidoglycan dynamics in Escherichia coli. Microbiologyopen 2015; 4: 375-389.
9. Mashburn LM, Whiteley M. Membrane vesicles traffic signals and facilitate group activities in a prokaryote. Nature 2005; 437: 422-425.
10. Schwechheimer C, Kulp A, Kuehn MJ. Modulation of bacterial outer membrane vesicle production by envelope structure and content. BMC Microbiol 2014; 14: 324.
11. Amano A, Takeuchi H, Furuta N. Outer membrane vesicles function as offensive weapons in host–parasite interactions. Microbes Infect 2010; 12: 791-798.
12. Bomberger JM, MacEachran DP, Coutermarsh BA, Ye S, O'Toole GA, Stanton BA. Long-distance delivery of bacterial virulence factors by Pseudomonas aeruginosa outer membrane vesicles. PLoS Pathog 2009; 5(4): e1000382.
13. Jurkoshek KS, Wang Y, Athman JJ, Barton MR, Wearsch PA. Interspecies communication between pathogens and immune cells via bacterial membrane vesicles. Front Cell Dev Biol 2016; 4: 125.
14. Ellis TN, Kuehn MJ. Virulence and immunomodulatory roles of bacterial outer membrane vesicles. Microbiol Mol Biol Rev 2010; 74: 81-94.
15. Sharpe SW, Kuehn MJ, Mason KM. Elicitation of epithelial cell-derived immune effectors by outer membrane vesicles of nontypeable Haemophilus influenzae. Infect Immun 2011; 79: 4361-4369.
16. Bielaszewska M, Rüter C, Kunsmann L, Greune L, Bauwens A, Zhang W, et al. Enterohemorrhagic Escherichia coli hemolysin employs outer membrane vesicles to target mitochondria and cause endothelial and epithelial apoptosis. PLoS Pathog 2013; 9(12): e1003797.
17. Parker H, Chitcholtan K, Hampton MB, Keenan JI. Uptake of Helicobacter pylori outer membrane vesicles by gastric epithelial cells. Infect Immun 2010; 78: 5054-5061.
18. Johnston EL, Kufer TA, Kaparakis-Liaskos M (2020). Immunodetection and pathogenesis mediated by bacterial membrane vesicles. Bacterial membrane vesicles. pp.159-188. https://link.springer.com/chapter/10.1007/978-3-030-36331-4_8
19. Kaparakis-Liaskos M, Ferrero RL. Immune modulation by bacterial outer membrane vesicles. Nat Rev Immunol 2015; 15: 375-387.
20. Mukherjee T, Hovingh ES, Foerster EG, Abdel-Nour M, Philpott DJ, Girardin SE. NOD1 and NOD2 in inflammation, immunity and disease. Arch Biochem Biophys 2019; 670: 69-81.
21. Motta V, Soares F, Sun T, Philpott DJ. NOD-like receptors: versatile cytosolic sentinels. Physiol Rev 2015; 95: 149-178.
22. Ahmadi Badi S, Moshiri A, Ettehad Marvasti F, Mojtahedzadeh M, Kazemi V, Siadat SD. Extraction and evaluation of outer membrane vesicles from two important gut microbiota members, Bacteroides fragilis and Bacteroides thetaiotaomicron. Cell J 2020; 22: 344-349.
23. Behrouzi A, Vaziri F, Riazi Rad F, Amanzadeh A, Fateh A, Moshiri A, et al. Comparative study of pathogenic and non-pathogenic Escherichia coli outer membrane vesicles and prediction of host-interactions with TLR signaling pathways. BMC Res Notes 2018; 11: 539.
24. Sambuy Y, De Angelis I, Ranaldi G, Scarino ML, Stammati A, Zucco F. The Caco-2 cell line as a model of the intestinal barrier: influence of cell and culture-related factors on Caco-2 cell functional characteristics. Cell Biol Toxicol 2005; 21: 1-26.
25. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001; 25: 402-408.
26. Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 2008; 3: 1101-1108.
27. Deatherage BL, Lara JC, Bergsbaken T, Barrett SL, Lara S, Cookson BT. Biogenesis of bacterial membrane vesicles. Mol Microbiol 2009; 72: 1395-1407.
28. Johnston EL, Heras B, Kufer TA, Kaparakis-Liaskos M. Detection of bacterial membrane vesicles by NOD-like receptors. Int J Mol Sci 2021; 22: 1005.
29. Cecil JD, O’Brien-Simpson NM, Lenzo JC, Holden JA, Chen Y-Y, Singleton W, et al. Differential responses of pattern recognition receptors to outer membrane vesicles of three periodontal pathogens. PLoS One 2016; 11(4): e0151967.
30. Kaparakis M, Turnbull L, Carneiro L, Firth S, Coleman HA, Parkington HC, et al. Bacterial membrane vesicles deliver peptidoglycan to NOD1 in epithelial cells. Cell Microbiol 2010; 12: 372-385.
31. Patten DA, Hussein E, Davies SP, Humphreys PN, Collett A. Commensal-derived OMVs elicit a mild proinflammatory response in intestinal epithelial cells. Microbiology (Reading) 2017; 163: 702-711.
32. Fábrega MJ, Aguilera L, Giménez R, Varela E, Alexandra Canas M, Antolín M, et al. Activation of immune and defense responses in the intestinal mucosa by outer membrane vesicles of commensal and probiotic Escherichia coli strains. Front Microbiol 2016; 7: 705.
33. Kunsmann L, Rüter C, Bauwens A, Greune L, Glüder M, Kemper B, et al. Virulence from vesicles: Novel mechanisms of host cell injury by Escherichia coli O104: H4 outbreak strain. Sci Rep 2015; 5: 13252.
34. Lee J, Kim OY, Gho YS. Proteomic profiling of Gram-negative bacterial outer membrane vesicles: Current perspectives. Proteomics Clin Appl 2016; 10: 897-909.
35. Van Der Pol L, Stork M, van der Ley P. Outer membrane vesicles as platform vaccine technology. Biotechnol J 2015; 10: 1689-1706.
36. Raetz CR, Reynolds CM, Trent MS, Bishop RE. Lipid A modification systems in gram-negative bacteria. Annu Rev Biochem 2007; 76: 295-329.
37. Ahmadi Badi S, Bruno SP, Moshiri A, Tarashi S, Siadat SD, Masotti A. Small RNAs in outer membrane vesicles and their function in host-microbe interactions. Front Microbiol 2020; 11: 1209.
38. Dell’Annunziata F, Folliero V, Giugliano R, De Filippis A, Santarcangelo C, Izzo V, et al. Gene transfer potential of outer membrane vesicles of gram-negative bacteria. Int J Mol Sci 2021; 22: 5985.
39. Rodriguez BV, Kuehn MJ. Staphylococcus aureus secretes immunomodulatory RNA and DNA via membrane vesicles. Sci Rep 2020; 10: 18293.
40. Chatterjee D, Chaudhuri K. Vibrio cholerae O395 outer membrane vesicles modulate intestinal epithelial cells in a NOD1 protein-dependent manner and induce dendritic cell-mediated Th2/Th17 cell responses. J Biol Chem 2013; 288: 4299-4309.
41. Schertzer JW, Whiteley M. Bacterial outer membrane vesicles in trafficking, communication and the host-pathogen interaction. J Mol Microbiol Biotechnol 2013; 23: 118-130.
42. Franchi L, Warner N, Viani K, Nuñez G. Function of Nod-like receptors in microbial recognition and host defense. Immunol Rev 2009; 227: 106-128.
43. Tavano R, Franzoso S, Cecchini P, Cartocci E, Oriente F, Aricò B, et al. The membrane expression of Neisseria meningitidis adhesin A (NadA) increases the proimmune effects of MenB OMVs on human macrophages, compared with NadA–OMVs, without further stimulating their proinflammatory activity on circulating monocytes. J Leukoc Biol 2009; 86: 143-153.
44. Kim OY, Park HT, Dinh NTH, Choi SJ, Lee J, Kim JH, et al. Bacterial outer membrane vesicles suppress tumor by interferon-γ-mediated antitumor response. Nat Commun 2017; 8: 626.
| Files | ||
| Issue | Vol 17 No 1 (2025) | |
| Section | Original Article(s) | |
| DOI | https://doi.org/10.18502/ijm.v17i1.17801 | |
| Keywords | ||
| Escherichia coli; Outer membrane vesicles; NOD-like receptors; Signaling pathways | ||
| Rights and permissions | |
|
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. |
How to Cite
1.
Mohammadzadeh R, Behrouzi A, Vaziri F, Siadat SD. Characterization of Escherichia coli outer membrane vesicles and the impact of pathogenic ones on NLR signaling pathways. Iran J Microbiol. 2025;17(1):51-58.
