Methods of Bacterial Membrane Vesicle Production, Purification, Quantification, and Examination of Their Immunogenic Functions

Bitto, Natalie J.; Kaparakis‐Liaskos, Maria

doi:10.1007/978-1-0716-2449-4_4

Cited by 9 publications

(14 citation statements)

References 36 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“… P. aeruginosa OMVs were produced and isolated using our established techniques ( 39 ). In brief, NB was inoculated using a 1:100 (vol/vol) dilution of an overnight P. aeruginosa culture and incubated at 37°C with shaking at 200 rpm for 16 h until bacteria had reached the stationary phase of growth.…”

Section: Methodsmentioning

confidence: 99%

Planktonic and Biofilm-Derived Pseudomonas aeruginosa Outer Membrane Vesicles Facilitate Horizontal Gene Transfer of Plasmid DNA

et al. 2023

Self Cite

View full text Add to dashboard Cite

show abstract

Section: Methodsmentioning

confidence: 99%

Planktonic and Biofilm-Derived Pseudomonas aeruginosa Outer Membrane Vesicles Facilitate Horizontal Gene Transfer of Plasmid DNA

et al. 2023

Self Cite

View full text Add to dashboard Cite

show abstract

“…P. aeruginosa OMVs were further purified to remove contaminants using an iodixanol (OptiPrep; Sigma‐Aldrich, USA) density gradient [20, 34, 36]. Briefly, OMVs were resuspended in 45% (v/v) OptiPrep and underlaid in a discontinuous OptiPrep density gradient, consisting of 20%, 25%, 30%, 35%, and 40% of OptiPrep solution.…”

Section: Methodsmentioning

confidence: 99%

“…OMVs were isolated from broth cultures using established techniques [19,20,34,36]. Briefly, 2 L of overnight cultures of P. aeruginosa PAO1, PAO1Δlys, and PAO1Δlys pJN105lys were used to prepare OMVs.…”

Section: Omv Isolation and Purificationmentioning

confidence: 99%

The mechanism of Pseudomonas aeruginosa outer membrane vesicle biogenesis determines their protein composition

et al. 2023

View full text Add to dashboard Cite

Gram‐negative bacteria produce outer membrane vesicles (OMVs) and contain bacterial cargo including nucleic acids and proteins. The proteome of OMVs can be altered by various factors including bacterial growth stage, growth conditions, and environmental factors. However, it is currently unknown if the mechanism of OMV biogenesis can determine their proteome. In this study, we examined whether the mechanisms of OMV biogenesis influenced the production and protein composition of Pseudomonas aeruginosa OMVs. OMVs were isolated from three P. aeruginosa strains that produced OMVs either by budding alone, by explosive cell lysis, or by both budding and explosive cell lysis. We identified that the mechanism of OMV biogenesis dictated OMV quantity. Furthermore, a global proteomic analysis comparing the proteome of OMVs to their parent bacteria showed significant differences in the identification of proteins in bacteria and OMVs. Finally, we determined that the mechanism of OMV biogenesis influenced the protein composition of OMVs, as OMVs released by distinct mechanisms of biogenesis differed significantly from one another in their proteome and functional enrichment analysis. Overall, our findings reveal that the mechanism of OMV biogenesis is a main factor that determines the OMV proteome which may affect their subsequent biological functions.

show abstract

“…B. fragilis OMVs were isolated using our established methods of OMV isolation ( 5 , 6 , 12 , 34 – 36 ). Briefly, BHI broth was inoculated using an overnight B. fragilis culture at a starting optical density (O.D.…”

Section: Methodsmentioning

confidence: 99%

“…B. fragilis OMVs were purified by OptiPrep (60% iodixanol(v/v); Sigma-Aldrich, USA) density gradient ultracentrifugation as previously described ( 6 , 12 , 35 , 36 ). In brief, OMV samples were adjusted to 45% (v/v) OptiPrep in 2ml DPBS and were then overlaid with a discontinuous OptiPrep gradient containing 2ml each of 40%, 35%, 30%, 25% and 20% OptiPrep (v/v) in DPBS.…”

Section: Methodsmentioning

confidence: 99%

Bacteroides fragilis outer membrane vesicles preferentially activate innate immune receptors compared to their parent bacteria

et al. 2022

Self Cite

View full text Add to dashboard Cite

The release of bacterial membrane vesicles (BMVs) has become recognized as a key mechanism used by both pathogenic and commensal bacteria to activate innate immune responses in the host and mediate immunity. Outer membrane vesicles (OMVs) produced by Gram-negative bacteria can harbor various immunogenic cargo that includes proteins, nucleic acids and peptidoglycan, and the composition of OMVs strongly influences their ability to activate host innate immune receptors. Although various Gram-negative pathogens can produce OMVs that are enriched in immunogenic cargo compared to their parent bacteria, the ability of OMVs produced by commensal organisms to be enriched with immunostimulatory contents is only recently becoming known. In this study, we investigated the cargo associated with OMVs produced by the intestinal commensal Bacteroides fragilis and determined their ability to activate host innate immune receptors. Analysis of B. fragilis OMVs revealed that they packaged various biological cargo including proteins, DNA, RNA, lipopolysaccharides (LPS) and peptidoglycan, and that this cargo could be enriched in OMVs compared to their parent bacteria. We visualized the entry of B. fragilis OMVs into intestinal epithelial cells, in addition to the ability of B. fragilis OMVs to transport bacterial RNA and peptidoglycan cargo into Caco-2 epithelial cells. Using HEK-Blue reporter cell lines, we identified that B. fragilis OMVs could activate host Toll-like receptors (TLR)-2, TLR4, TLR7 and nucleotide-binding oligomerization domain-containing protein 1 (NOD1), whereas B. fragilis bacteria could only induce the activation of TLR2. Overall, our data demonstrates that B. fragilis OMVs activate a broader range of host innate immune receptors compared to their parent bacteria due to their enrichment of biological cargo and their ability to transport this cargo directly into host epithelial cells. These findings indicate that the secretion of OMVs by B. fragilis may facilitate immune crosstalk with host epithelial cells at the gastrointestinal surface and suggests that OMVs produced by commensal bacteria may preferentially activate host innate immune receptors at the mucosal gastrointestinal tract.

show abstract

Methods of Bacterial Membrane Vesicle Production, Purification, Quantification, and Examination of Their Immunogenic Functions

Cited by 9 publications

References 36 publications

Planktonic and Biofilm-Derived Pseudomonas aeruginosa Outer Membrane Vesicles Facilitate Horizontal Gene Transfer of Plasmid DNA

Planktonic and Biofilm-Derived Pseudomonas aeruginosa Outer Membrane Vesicles Facilitate Horizontal Gene Transfer of Plasmid DNA

The mechanism of Pseudomonas aeruginosa outer membrane vesicle biogenesis determines their protein composition

Bacteroides fragilis outer membrane vesicles preferentially activate innate immune receptors compared to their parent bacteria

Contact Info

Product

Resources

About