A novel method for the extraction of outer membrane vesicles (OMVs) from Bordetella pertussis Tohama strain

Background and Objectives: There are many pertussis outbreaks which is mainly due to the reduction in the immunity of acellular pertussis (aP) vaccines. Therefore, there is a crucial necessity to develop a new generation of pertussis vaccine. Pre- ceding researches have shown that Bordetella pertussis outer membrane vesicles (OMVs) have appropriate specifications, making them a suitable vaccine candidate against pertussis. Materials and Methods: The OMVs were separated by a new serial ultra centrifugation technique. Transmission electron microscopy (TEM) examination, SDS-PAGE, Western blotting and ELISA assay were used to characterize the OMVs. Results: TEM studies showed the size of the extracted OMVs at 40-200 nm. The presence of pertussis toxin, filamentous hemagglutinin, and pertactin was verified using Western blot and ELISA assay. Conclusion: The presented technique is a simple and effective way to obtain OMVs from Bordetella pertussis. So it can be utilized as an appropriate procedure in the development of an OMV-based vaccine against pertussis.

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Canonical and Non-canonical Inflammasome Activation by Outer Membrane Vesicles Derived From Bordetella pertussis

Frontiers in Immunology ◽

10.3389/fimmu.2020.01879 ◽

2020 ◽

Vol 11 ◽

Author(s):

Maia L. Elizagaray ◽

Marco Túlio R. Gomes ◽

Erika S. Guimaraes ◽

Martín Rumbo ◽

Daniela F. Hozbor ◽

...

Keyword(s):

Outer Membrane ◽

Bordetella Pertussis ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Inflammasome Activation

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A streamlined method for the extraction of outer membrane vesicles (OMVs) from Bordetella pertussis

Vaccine Research ◽

10.29252/vacres.5.2.43 ◽

2018 ◽

Vol 5 (2) ◽

pp. 43-46

Author(s):

M Sekhavati ◽

SD Siadat ◽

M Noofeli ◽

A Mohebati Mobarez ◽

◽

...

Keyword(s):

Outer Membrane ◽

Bordetella Pertussis ◽

Membrane Vesicles ◽

Outer Membrane Vesicles

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Isolation and Characterization of Outer Membrane Vesicles of Pectobacterium brasiliense 1692

Microorganisms ◽

10.3390/microorganisms9091918 ◽

2021 ◽

Vol 9 (9) ◽

pp. 1918

Author(s):

Silindile Maphosa ◽

Lucy Novungayo Moleleki

Keyword(s):

Electron Microscopy ◽

Transmission Electron Microscopy ◽

Outer Membrane ◽

Critical Role ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Broad Host Range ◽

Isolation And Characterization ◽

Transmission Electron ◽

Animal Pathogens

Pectobacterium brasiliense (Pbr) 1692 is an aggressive phytopathogen affecting a broad host range of crops and ornamental plants, including potatoes. Previous research on animal pathogens, and a few plant pathogens, revealed that Outer Membrane Vesicles (OMVs) are part of Gram-negative bacteria’s (GNB) adaptive toolkit. For this reason, OMV production and subsequent release from bacteria is a conserved process. Therefore, we hypothesized that OMVs might transport proteins that play a critical role in causing soft rot disease and in the survival and fitness of Pbr1692. Here, we show that the potato pathogen, Pbr1692, releases OMVs of various morphologies in Luria Bertani media at 31 °C. Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) confirmed the production of OMVs by Pbr1692 cells. Transmission Electron Microscopy showed that these exist as chain-, single-, and double-membrane morphologies. Mass spectrometry followed by Gene Ontology, Clusters of Orthologous Groups, Virulence Factor, CAZymes, Antibiotic Resistance Ontology, and Bastion6 T6SE annotations identified 129 OMV-associated proteins with diverse annotated roles, including antibiotic stress response, virulence, and competition. Pbr1692 OMVs contributed to virulence in potato tubers and elicited a hypersensitive response in Nicotiana benthamiana leaves. Furthermore, Pbr1692 OMVs demonstrated antibacterial activity against Dickeya dadantii.

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Acellular outer membrane vesicles of Brucella abortus strain 19 elicits both humoral and cell mediated immune response in mice

Indian Journal of Animal Research ◽

10.18805/ijar.b-3428 ◽

2018 ◽

Author(s):

Losa Rose ◽

Bablu Kumar ◽

Ankita Jain ◽

M K Singh ◽

Abhishek .

Keyword(s):

Immune Response ◽

Outer Membrane ◽

Brucella Abortus ◽

Outer Membrane Proteins ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Biologically Active ◽

Cell Mediated Immunity ◽

Humoral Immune ◽

Sds Page

Outer membrane vesicles (OMVs) contain biologically active proteins, lipoolysaccharide (LPS), periplasmic and membrane-bound proteins and are known to perform diverse biological functions. OMVs from Brucella abortus S19 were isolated and characterized by transmission electron microscopy (TEM), SDS-PAGE and immunoreactivity was investigated by western blotting. On TEM, bilayered spherical structures of 50-200 nm were observed. SDS-PAGE of OMVs revealed approximate bands size of 82 kDa, 68 kDa, 38 kDa, 32 kDa, 29 kDa and 18 kDa. Western blot analysis of OMVs revealed a dominant immunoreactive band of 38 kDa that correspond to some major outer membrane proteins. Humoral immune response was measured by indirect ELISA which showed that OMV specific antibodies were detected from 7th day post immunization (DPI) onwards and showed a rising trend up to 35th DPI. Cell mediated immune (CMI) response against OMVs as evidenced by the proliferation of splenocytes have also been observed. Thus OMVs were found to possess immunogenic proteins which had potential to induce both humoral as well as cell mediated immunity. After correlating this immune response with protection it has been concluded that OMV can be used as one of the vaccine candidate against brucellosis.

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Outer membrane vesicles derived from Bordetella parapertussis as an acellular vaccine against Bordetella parapertussis and Bordetella pertussis infection

Vaccine ◽

10.1016/j.vaccine.2013.08.059 ◽

2013 ◽

Vol 31 (45) ◽

pp. 5262-5268 ◽

Cited By ~ 19

Author(s):

D. Bottero ◽

M.E. Gaillard ◽

A. Errea ◽

G. Moreno ◽

E. Zurita ◽

...

Keyword(s):

Outer Membrane ◽

Bordetella Pertussis ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Pertussis Infection ◽

Bordetella Parapertussis ◽

Acellular Vaccine

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Outer membrane vesicles obtained from Bordetella pertussis Tohama expressing the lipid A deacylase PagL as a novel acellular vaccine candidate

Vaccine ◽

10.1016/j.vaccine.2010.12.068 ◽

2011 ◽

Vol 29 (8) ◽

pp. 1649-1656 ◽

Cited By ~ 52

Author(s):

Cristian J.A. Asensio ◽

María Emilia Gaillard ◽

Griselda Moreno ◽

Daniela Bottero ◽

Eugenia Zurita ◽

...

Keyword(s):

Outer Membrane ◽

Bordetella Pertussis ◽

Lipid A ◽

Vaccine Candidate ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Acellular Vaccine

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Comparative Analysis of Outer Membrane Vesicle Isolation Methods With an Escherichia coli tolA Mutant Reveals a Hypervesiculating Phenotype With Outer-Inner Membrane Vesicle Content

Frontiers in Microbiology ◽

10.3389/fmicb.2021.628801 ◽

2021 ◽

Vol 12 ◽

Author(s):

Shelby L. Reimer ◽

Daniel R. Beniac ◽

Shannon L. Hiebert ◽

Timothy F. Booth ◽

Patrick M. Chong ◽

...

Keyword(s):

Escherichia Coli ◽

Outer Membrane ◽

Membrane Vesicle ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Size Distributions ◽

Transmission Electron ◽

Isolation Methods ◽

Morphological Differences ◽

K 12

Outer membrane vesicles (OMVs) produced by Gram-negative bacteria are mediators of cell survival and pathogenesis by facilitating virulence factor dissemination and resistance to antimicrobials. Studies of OMV properties often focus on hypervesiculating Escherichia coli mutants that have increased OMV production when compared to their corresponding wild-type (WT) strains. Currently, two conventional techniques, ultracentrifugation (UC) and ultradiafiltration (UF), are used interchangeably to isolate OMVs, however, there is concern that each technique may inadvertently alter the properties of isolated OMVs during study. To address this concern, we compared two OMV isolation methods, UC and UF, with respect to final OMV quantities, size distributions, and morphologies using a hypervesiculating Escherichia coli K-12 ΔtolA mutant. Nanoparticle tracking analysis (NTA) indicated that UC techniques result in lower vesicle yields compared to UF. However, UF permitted isolation of OMVs with smaller average sizes than UC, highlighting a potential OMV isolation size bias by each technique. Cryo-transmission electron microscopy (cryo-TEM) visualization of isolated OMVs revealed distinct morphological differences between WT and ΔtolA OMVs, where ΔtolA OMVs isolated by either UC or UF method possessed a greater proportion of OMVs with two or more membranes. Proteomic OMV analysis of WT and ΔtolA OMVs confirmed that ΔtolA enhances inner plasma membrane carryover in multi-lamellar OMVs. This study demonstrates that UC and UF are useful techniques for OMV isolation, where UF may be preferable due to faster isolation, higher OMV yields and enrichment of smaller sized vesicles.

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