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

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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Flocculation of Escherichia coli Cells in Association with Enhanced Production of Outer Membrane Vesicles

Applied and Environmental Microbiology ◽

10.1128/aem.01011-15 ◽

2015 ◽

Vol 81 (17) ◽

pp. 5900-5906 ◽

Cited By ~ 9

Author(s):

Yoshihiro Ojima ◽

Minh Hong Nguyen ◽

Reiki Yajima ◽

Masahito Taya

Keyword(s):

Escherichia Coli ◽

Outer Membrane ◽

Sodium Dodecyl ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Laser Scanning Microscopy ◽

Content Type ◽

E Coli ◽

Enhanced Production ◽

K 12

ABSTRACTMicrobial flocculation is a phenomenon of aggregation of dispersed bacterial cells in the form of flocs or flakes. In this study, the mechanism of spontaneous flocculation ofEscherichia colicells by overexpression of thebcsBgene was investigated. The flocculation induced by overexpression ofbcsBwas consistent among the variousE. colistrains examined, including the K-12, B, and O strains, with flocs that resembled paper scraps in structure being about 1 to 2 mm. The distribution of green fluorescent protein-labeledE. colicells within the floc structure was investigated by three-dimensional confocal laser scanning microscopy. Flocs were sensitive to proteinase K, indicating that the main component of the flocs was proteinous. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and nano-liquid chromatography tandem mass spectrometry analyses of the flocs strongly suggested the involvement of outer membrane vesicles (OMVs) inE. coliflocculation. The involvement of OMVs in flocculation was supported by transmission electron microscopy observation of flocs. Furthermore,bcsB-inducedE. coliflocculation was greatly suppressed in strains with hypovesiculation phenotypes (ΔdsbAand ΔdsbBstrains). Thus, our results demonstrate the strong correlation between spontaneous flocculation and enhanced OMV production ofE. colicells.

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Outer Membrane Vesicle Production by Escherichia coli Is Independent of Membrane Instability

Journal of Bacteriology ◽

10.1128/jb.00498-06 ◽

2006 ◽

Vol 188 (15) ◽

pp. 5385-5392 ◽

Cited By ~ 191

Author(s):

Amanda J. McBroom ◽

Alexandra P. Johnson ◽

Sreekanth Vemulapalli ◽

Meta J. Kuehn

Keyword(s):

Escherichia Coli ◽

Outer Membrane ◽

Cell Envelope ◽

Membrane Vesicle ◽

Membrane Vesicles ◽

Fold Increase ◽

Outer Membrane Vesicles ◽

Host Cells ◽

Physiological Basis ◽

Gram Negative

ABSTRACT It has been long noted that gram-negative bacteria produce outer membrane vesicles, and recent data demonstrate that vesicles released by pathogenic strains can transmit virulence factors to host cells. However, the mechanism of vesicle release has remained undetermined. This genetic study addresses whether these structures are merely a result of membrane instability or are formed by a more directed process. To elucidate the regulatory mechanisms and physiological basis of vesiculation, we conducted a screen in Escherichia coli to identify gene disruptions that caused vesicle over- or underproduction. Only a few low-vesiculation mutants and no null mutants were recovered, suggesting that vesiculation may be a fundamental characteristic of gram-negative bacterial growth. Gene disruptions were identified that caused differences in vesicle production ranging from a 5-fold decrease to a 200-fold increase relative to wild-type levels. These disruptions included loci governing outer membrane components and peptidoglycan synthesis as well as the σE cell envelope stress response. Mutations causing vesicle overproduction did not result in upregulation of the ompC gene encoding a major outer membrane protein. Detergent sensitivity, leakiness, and growth characteristics of the novel vesiculation mutant strains did not correlate with vesiculation levels, demonstrating that vesicle production is not predictive of envelope instability.

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Outer Membrane Vesicle Induction and Isolation for Vaccine Development

Frontiers in Microbiology ◽

10.3389/fmicb.2021.629090 ◽

2021 ◽

Vol 12 ◽

Author(s):

Melanie D. Balhuizen ◽

Edwin J. A. Veldhuizen ◽

Henk P. Haagsman

Keyword(s):

Outer Membrane ◽

Vaccine Development ◽

Current Knowledge ◽

Membrane Vesicle ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Gram Negative Bacteria ◽

Bacterial Surface ◽

Advantages And Disadvantages ◽

Isolation Methods

Gram-negative bacteria release vesicular structures from their outer membrane, so called outer membrane vesicles (OMVs). OMVs have a variety of functions such as waste disposal, communication, and antigen or toxin delivery. These vesicles are the promising structures for vaccine development since OMVs carry many surface antigens that are identical to the bacterial surface. However, isolation is often difficult and results in low yields. Several methods to enhance OMV yield exist, but these do affect the resulting OMVs. In this review, our current knowledge about OMVs will be presented. Different methods to induce OMVs will be reviewed and their advantages and disadvantages will be discussed. The effects of the induction and isolation methods used in several immunological studies on OMVs will be compared. Finally, the challenges for OMV-based vaccine development will be examined and one example of a successful OMV-based vaccine will be presented.

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Reciprocal Packaging of the Main Structural Proteins of Type 1 Fimbriae and Flagella in the Outer Membrane Vesicles of “Wild Type” Escherichia coli Strains

Frontiers in Microbiology ◽

10.3389/fmicb.2021.557455 ◽

2021 ◽

Vol 12 ◽

Author(s):

Sarah A. Blackburn ◽

Mark Shepherd ◽

Gary K. Robinson

Keyword(s):

Escherichia Coli ◽

Outer Membrane ◽

Membrane Vesicle ◽

Outer Membrane Vesicles ◽

Wild Type ◽

Protein Fusion ◽

E Coli ◽

Type 1 Fimbriae ◽

K 12

Fundamental aspects of outer membrane vesicle (OMV) biogenesis and the engineering of producer strains have been major research foci for many in recent years. The focus of this study was OMV production in a variety of Escherichia coli strains including wild type (WT) (K12 and BW25113), mutants (from the Keio collection) and proprietary [BL21 and BL21 (DE3)] strains. The present study investigated the proteome and prospective mechanism that underpinned the key finding that the dominant protein present in E. coli K-12 WT OMVs was fimbrial protein monomer (FimA) (a polymerizable protein which is the key structural monomer from which Type 1 fimbriae are made). However, mutations in genes involved in fimbriae biosynthesis (ΔfimA, B, C, and F) resulted in the packaging of flagella protein monomer (FliC) (the major structural protein of flagella) into OMVs instead of FimA. Other mutations (ΔfimE, G, H, I, and ΔlrhA–a transcriptional regulator of fimbriation and flagella biosynthesis) lead to the packaging of both FimA and Flagellin into the OMVs. In the majority of instances shown within this research, the production of OMVs is considered in K-12 WT strains where structural appendages including fimbriae or flagella are temporally co-expressed throughout the growth curve as shown previously in the literature. The hypothesis, proposed and supported within the present paper, is that the vesicular packaging of the major FimA is reciprocally regulated with the major FliC in E. coli K-12 OMVs but this is abrogated in a range of mutated, non-WT E. coli strains. We also demonstrate, that a protein of interest (GFP) can be targeted to OMVs in an E. coli K-12 strain by protein fusion with FimA and that this causes normal packaging to be disrupted. The findings and underlying implications for host interactions and use in biotechnology are discussed.

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Active Cytotoxic Necrotizing Factor 1 Associated with Outer Membrane Vesicles from Uropathogenic Escherichia coli

Infection and Immunity ◽

10.1128/iai.74.4.2022-2030.2006 ◽

2006 ◽

Vol 74 (4) ◽

pp. 2022-2030 ◽

Cited By ~ 64

Author(s):

J. Clavin Kouokam ◽

Sun Nyunt Wai ◽

Maria Fällman ◽

Ulrich Dobrindt ◽

Jörg Hacker ◽

...

Keyword(s):

Escherichia Coli ◽

Outer Membrane ◽

Membrane Vesicle ◽

Active Form ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Upec Strain ◽

Cytotoxic Necrotizing Factor ◽

Factor Type ◽

Bacterial Cytoplasm

ABSTRACT Cytotoxic necrotizing factor type 1 (CNF1) is one of the virulence factors produced by uropathogenic Escherichia coli (UPEC). How this toxin is translocated from the bacterial cytoplasm to the surrounding environment is not well understood. Our data suggest that CNF1 may be regarded as a secreted protein, since it could be detected in culture supernatants. Furthermore, we found that CNF1 was tightly associated to outer membrane vesicles, suggesting that such vesicles play a role in the secretion of this protein. Interestingly, vesicle samples containing CNF1 could exert the effects known for this protein on HeLa cell cultures, showing that CNF1 is transported by vesicles in its active form. Taken together, our results strongly suggest that outer membrane vesicles could be a means for the bacteria to deliver CNF1 to the environment and to the infected tissue. In addition, our results indicate that the histone-like nucleoid structuring protein H-NS has a role in the downregulation of CNF1 production and that it affects the outer membrane vesicle release in UPEC strain J96.

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A Suppressor of Cell Death Caused by the Loss of σE Downregulates Extracytoplasmic Stress Responses and Outer Membrane Vesicle Production in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.01534-06 ◽

2006 ◽

Vol 189 (5) ◽

pp. 1523-1530 ◽

Cited By ~ 49

Author(s):

Julie E. Button ◽

Thomas J. Silhavy ◽

Natividad Ruiz

Keyword(s):

Escherichia Coli ◽

Cell Death ◽

Outer Membrane ◽

Stress Responses ◽

Membrane Vesicle ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Loss Of Function ◽

Gene Encoding ◽

Σ Factor

ABSTRACT When envelope biogenesis is compromised or damage to envelope components occurs, bacteria trigger signaling cascades, which lead to the production of proteins that combat such extracytoplasmic stresses. In Escherichia coli, there are three pathways known to deal with envelope stresses: the Bae, Cpx, and σE responses. Although the effectors of the Bae and Cpx responses are not essential in E. coli, the effector of the σE response, the sigma factor RpoE (σE), is essential for viability. However, mutations that suppress the lethality of an rpoE-null allele can be easily obtained, and here we describe how we have isolated at least four classes of these suppressors. We present the first description of one such suppressor class, loss-of-function mutations in ydcQ, a gene encoding a putative DNA-binding protein. In wild-type rpoE + strains, ydcQ mutants have two distinct phenotypes: extracytoplasmic stress responses are significantly downregulated, and the production of outer membrane vesicles is severely reduced. We present a model in which σE is not essential per se but, rather, we propose that rpoE mutant cells die, possibly because they overreact to the absence of this σ factor by triggering a cell death signal.

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Outer membrane vesicles mediated horizontal transfer of an aerobic denitrification gene between Escherichia coli

Biodegradation ◽

10.1007/s10532-021-09945-y ◽

2021 ◽

Author(s):

Weichuan Qiao ◽

Lianjie Wang ◽

Yang Luo ◽

Jiahui Miao

Keyword(s):

Escherichia Coli ◽

Outer Membrane ◽

Horizontal Transfer ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Aerobic Denitrification ◽

Denitrification Gene

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Proteome‐minimized outer membrane vesicles from Escherichia coli as a generalized vaccine platform

Journal of Extracellular Vesicles ◽

10.1002/jev2.12066 ◽

2021 ◽

Vol 10 (4) ◽

Author(s):

Ilaria Zanella ◽

Enrico König ◽

Michele Tomasi ◽

Assunta Gagliardi ◽

Luca Frattini ◽

...

Keyword(s):

Escherichia Coli ◽

Outer Membrane ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Vaccine Platform

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Combining Augmented Radiotherapy and Immunotherapy through a Nano-Gold and Bacterial Outer-Membrane Vesicle Complex for the Treatment of Glioblastoma

Nanomaterials ◽

10.3390/nano11071661 ◽

2021 ◽

Vol 11 (7) ◽

pp. 1661

Author(s):

Mei-Hsiu Chen ◽

Tse-Ying Liu ◽

Yu-Chiao Chen ◽

Ming-Hong Chen

Keyword(s):

Outer Membrane ◽

Membrane Vesicle ◽

Glioma Cells ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

E Coli ◽

Tumor Bearing ◽

Nano Gold ◽

Gl261 Cell

Glioblastoma, formerly known as glioblastoma multiforme (GBM), is refractory to existing adjuvant chemotherapy and radiotherapy. We successfully synthesized a complex, Au–OMV, with two specific nanoparticles: gold nanoparticles (AuNPs) and outer-membrane vesicles (OMVs) from E. coli. Au–OMV, when combined with radiotherapy, produced radiosensitizing and immuno-modulatory effects that successfully suppressed tumor growth in both subcutaneous G261 tumor-bearing and in situ (brain) tumor-bearing C57BL/6 mice. Longer survival was also noted with in situ tumor-bearing mice treated with Au–OMV and radiotherapy. The mechanisms for the successful treatment were evaluated. Intracellular reactive oxygen species (ROS) greatly increased in response to Au–OMV in combination with radiotherapy in G261 glioma cells. Furthermore, with a co-culture of G261 glioma cells and RAW 264.7 macrophages, we found that GL261 cell viability was related to chemotaxis of macrophages and TNF-α production.

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