Considerations for the Analysis of Bacterial Membrane Vesicles: Methods of Vesicle Production and Quantification Can Influence Biological and Experimental Outcomes

Bitto, Natalie J.; Zavan, Lauren; Johnston, Ella L.; Stinear, Timothy P.; Hill, Andrew F.; Kaparakis‐Liaskos, Maria

doi:10.1128/spectrum.01273-21

Cited by 41 publications

(64 citation statements)

References 68 publications

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“…In addition, we did not observe TLR9 activation in response to stimulation with B. fragilis OMVs at any MOI examined in this study, which suggests that the amount of DNA delivered by B. fragilis OMVs may not have been sufficient to mediate TLR9 activation (35). We have previously shown that BMVs produced by different strains of a bacterial species vary in their amount of DNA and RNA cargo and therefore differ in their ability to activate their respective TLRs (35). Therefore, although we did not see activation of TLR8 and TLR9 by B. fragilis OMVs in our study, we cannot exclude the possibility that stimulation of these cells with an increased amount of OMVs, or with OMVs produced by a different B. fragilis strain that harbor a greater concentration of DNA and RNA, may activate these TLRs.…”

Section: Discussionmentioning

confidence: 53%

“…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%

“…TEM sample preparation was performed as previously described ( 5 , 35 ). Briefly, OMVs were coated onto carbon‐coated 400 mesh copper grids (ProSciTech, Australia) for 10 min, fixed in 1% (w/v) glutaraldehyde (Sigma-Aldrich, USA) and negatively-stained with 2% (w/v) uranyl-acetate (ProSciTech, Australia).…”

Section: Methodsmentioning

confidence: 99%

“…B. fragilis OMVs were isolated using our established methods of OMV isolation (5,6,12,(34)(35)(36). Briefly, BHI broth was inoculated using an overnight B. fragilis culture at a starting optical density (O.D.…”

Section: Isolation Of B Fragilis Omvsmentioning

confidence: 99%

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

et al. 2022

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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.

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Section: Discussionmentioning

confidence: 53%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Isolation Of B Fragilis Omvsmentioning

confidence: 99%

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

et al. 2022

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“…Interestingly, we found that the averaged diameter of 103 + -EVs (130 nm) is slightly bigger than 103 − -EVs (105 nm) ( Figure 1 ). The size of EVs in Gram-positive bacterial varies between different bacterial strains [ 34 , 35 , 36 , 37 ], throughout various stages of bacterial growth [ 38 ], and environmental conditions [ 27 , 39 , 40 ]. As the reports that the size distribution of EVs of the invasive Streptococcus pyogenes strains SSI-1 were 1.31 times bigger than the non-invasive Streptococcus pyogenes strains JRS4 [ 37 ].…”

Section: Discussionmentioning

confidence: 99%

Rhodococcus equi-Derived Extracellular Vesicles Promoting Inflammatory Response in Macrophage through TLR2-NF-κB/MAPK Pathways

Hao

et al. 2022

IJMS

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Rhodococcus equi (R. equi) is a Gram-positive coccobacillus that causes pneumonia in foals of less than 3 months, which have the ability of replication in macrophages. The ability of R. equi persist in macrophages is dependent on the virulence plasmid pVAPA. Gram-positive extracellular vesicles (EVs) carry a variety of virulence factors and play an important role in pathogenic infection. There are few studies on R. equi-derived EVs (R. equi-EVs), and little knowledge regarding the mechanisms of how R. equi-EVs communicate with the host cell. In this study, we examine the properties of EVs produced by the virulence strain R. equi 103+ (103+-EVs) and avirulenct strain R. equi 103− (103−-EVs). We observed that 103+-EVs and 103−-EVs are similar to other Gram-positive extracellular vesicles, which range from 40 to 260 nm in diameter. The 103+-EVs or 103−-EVs could be taken up by mouse macrophage J774A.1 and cause macrophage cytotoxicity. Incubation of 103+-EVs or 103−-EVs with J774A.1 cells would result in increased expression levels of IL-1β, IL-6, and TNF-α. Moreover, the expression of TLR2, p-NF-κB, p-p38, and p-ERK were significantly increased in J774A.1 cells stimulated with R. equi-EVs. In addition, we presented that the level of inflammatory factors and expression of TLR2, p-NF-κB, p-p38, and p-ERK in J774A.1 cells showed a significant decreased when incubation with proteinase K pretreated-R. equi-EVs. Overall, our data indicate that R. equi-derived EVs are capable of mediating inflammatory responses in macrophages via TLR2-NF-κB/MAPK pathways, and R. equi-EVs proteins were responsible for TLR2-NF-κB/MAPK mediated inflammatory responses in macrophage. Our study is the first to reveal potential roles for R. equi-EVs in immune response in R. equi-host interactions and to compare the differences in macrophage inflammatory responses mediated by EVs derived from virulent strain R. equi and avirulent strain R. equi. The results of this study have improved our knowledge of the pathogenicity of R. equi.

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Production and purification of bacterial membrane vesicles for biotechnology applications: Challenges and opportunities

2022

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Bacterial membrane vesicles (BMVs) are bi‐layered nanostructures derived from Gram‐negative and Gram‐positive bacteria. Among other pathophysiological roles, BMVs are critical messengers in intercellular communication. As a result, BMVs are emerging as a promising technology for the development of numerous therapeutic applications. Despite the remarkable progress in unveiling BMV biology and functions in recent years, their successful isolation and purification have been limited. Several challenges related to vesicle purity, yield, and scalability severely hamper the further development of BMVs for biotechnology and clinical applications. This review focuses on the current technologies and methodologies used in BMV production and purification, such as ultracentrifugation, density‐gradient centrifugation, size‐exclusion chromatography, ultrafiltration, and precipitation. We also discuss the current challenges related to BMV isolation, large‐scale production, storage, and stability that limit their application. More importantly, the present work explains the most recent strategies proposed for overcoming those challenges. Finally, we summarize the ongoing applications of BMVs in the biotechnological field.

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Considerations for the Analysis of Bacterial Membrane Vesicles: Methods of Vesicle Production and Quantification Can Influence Biological and Experimental Outcomes

Abstract: Recent years have seen a surge in interest in the roles of BMVs in host-microbe interactions and interbacterial communication. As a result of such rapid growth in the field, there is a lack of uniformity in BMV enumeration.

Cited by 41 publications

References 68 publications

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

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

Rhodococcus equi-Derived Extracellular Vesicles Promoting Inflammatory Response in Macrophage through TLR2-NF-κB/MAPK Pathways

Production and purification of bacterial membrane vesicles for biotechnology applications: Challenges and opportunities

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