Protecting enzymatic function through directed packaging into bacterial outer membrane vesicles

Alves, Nathan J.; Turner, Kendrick B.; Medintz, Igor L.; Walper, Scott A.

doi:10.1038/srep24866

Cited by 92 publications

(106 citation statements)

References 41 publications

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“…The bacterial cellular machinery can be genetically engineered to produce and package heterologous enzymes in OMV nanoparticles. (Alves et al, ; Alves, Turner, Medintz, & Walper, ). In our previous study, outer membrane protein W (OmpW) efficiently packaged green fluorescent protein (GFP) into OMVs of E. coli (WT strain and hypervesiculating mutants Δ degP and Δ nlpI ) by overexpression of an OmpW–GFP fused protein (Ojima, Yamaguchi, & Taya, ).…”

Section: Introductionmentioning

confidence: 99%

Construction of hypervesiculation Escherichia coli strains and application for secretory protein production

Ojima

Sawabe

Konami

et al. 2019

Biotech & Bioengineering

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Outer membrane vesicles (OMVs) are extracellular vesicles released from the surface of Gram-negative bacteria, including Escherichia coli. Several gene-deficient mutants relating to envelope stress (nlpI and degP) and phospholipid accumulation in the outer leaflet of the outer membrane (mlaA and mlaE) increase OMV production. This study examined the combinatorial deletion of these genes in E. coli and its effect on OMV production. The nlpI and mlaE double-gene-knockout mutant (ΔmlaEΔnlpI) showed the highest OMV production. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis-based quantitative analysis showed that OMV production by strain ΔmlaEΔnlpI was~30 times that by the wild-type (WT). In addition, to evaluate the protein secretion capacity of OMVs, a green fluorescent protein (GFP) fused with outer membrane protein W (OmpW) was expressed in OMVs. Western blot analysis showed that GFP secretion through OMVs reached 3.3 mg/L in the culture medium of strain ΔmlaEΔnlpI/gfp, 500 times that for the WT. Our approach using OMVs for extracellular protein secretion in E. coli is an entirely new concept compared with existing secretion systems.

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Section: Introductionmentioning

confidence: 99%

Construction of hypervesiculation Escherichia coli strains and application for secretory protein production

Ojima

Sawabe

Konami

et al. 2019

Biotech & Bioengineering

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“…Bacterial cellular machinery can be genetically engineered to produce and package heterologous enzymes into OMV nanoparticles. Specially engineered cells of Escherichia coli can simultaneously produce, package, and secrete an active enzyme into their OMVs . According to these studies, phosphotriesterase packaged into OMVs is much less susceptible to inactivation by freezing or lyophilization than free enzyme.…”

Section: Introductionmentioning

confidence: 99%

Quantitative Evaluation of Recombinant Protein Packaged into Outer Membrane Vesicles of Escherichia coli Cells

Ojima

Yamaguchi

Taya

2017

Biotechnology Progress

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Outer membrane vesicles (OMVs) are spherical bilayered proteolipids released from the cell surfaces of bacteria, which have gained traction in the biotechnology fields. Bacterial cellular machinery can be genetically engineered to produce and package heterologous enzymes into OMVs, producing nanocarriers and nanoparticle catalysts. However, the productivity or efficiency of packaging the target protein into OMVs has not been quantitatively evaluated. In this study, we packaged green fluorescence protein (GFP) into the OMVs of Escherichia coli through N-terminal fused expression to outer membrane protein W (OmpW). The OMV productivity and amount of OmpW-GFP packaged in the OMVs were quantitatively compared between two hypervesiculating mutant strains ΔnlpI and ΔdegP. Both strains increased the OMV production, but the ΔnlpI strain additionally enhanced the packaging of OmpW-GFP into OMVs. It was further confirmed that Spr, a peptidoglycan endopeptidase, plays an important role in the enhanced packaging of OmpW-GFP into OMVs through the increased OmpW-GFP expression on the ΔnlpI cells. Finally, the amount of OmpW-GFP released in the OMV fraction of both mutants was determined in terms of the OMV productivity and the packaging efficiency of OmpW-GFP into OMVs. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:51-57, 2018.

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“…These engineered OMV vaccine formulations elicited specific immune and antibody responses both in the serum and at mucosal surfaces, including the generation of antibodies able to kill plague bacteria. Finally, OMVs are stable for ultra-long periods in liquid and lyophilized form [49], and for several weeks in solution form across A. L. Carvalho et al a wide range of temperatures including 40°C. First, OMV vaccine delivery via oral or nasal administration allows for needle-free, multi-dose delivery that would enable mass vaccination programmes in challenging environments and at relatively low cost.…”

Section: Discussionmentioning

confidence: 99%

Use of bioengineered human commensal gut bacteria-derived microvesicles for mucosal plague vaccine delivery and immunization

Carvalho

Miquel-Clopés

Wegmann

et al. 2019

Clinical &Amp; Experimental Immunology

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Summary Plague caused by the Gram‐negative bacterium, Yersinia pestis, is still endemic in parts of the world today. Protection against pneumonic plague is essential to prevent the development and spread of epidemics. Despite this, there are currently no licensed plague vaccines in the western world. Here we describe the means of delivering biologically active plague vaccine antigens directly to mucosal sites of plague infection using highly stable microvesicles (outer membrane vesicles; OMVs) that are naturally produced by the abundant and harmless human commensal gut bacterium Bacteroides thetaiotaomicron (Bt). Bt was engineered to express major plague protective antigens in its OMVs, specifically Fraction 1 (F1) in the outer membrane and LcrV (V antigen) in the lumen, for targeted delivery to the gastrointestinal (GI) and respiratory tracts in a non‐human primate (NHP) host. Our key findings were that Bt OMVs stably expresses F1 and V plague antigens, particularly the V antigen, in the correct, immunogenic form. When delivered intranasally V‐OMVs elicited substantive and specific immune and antibody responses, both in the serum [immunoglobulin (Ig)G] and in the upper and lower respiratory tract (IgA); this included the generation of serum antibodies able to kill plague bacteria. Our results also showed that Bt OMV‐based vaccines had many desirable characteristics, including: biosafety and an absence of any adverse effects, pathology or gross alteration of resident microbial communities (microbiotas); high stability and thermo‐tolerance; needle‐free delivery; intrinsic adjuvanticity; the ability to stimulate both humoral and cell‐mediated immune responses; and targeting of primary sites of plague infection.

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Protecting enzymatic function through directed packaging into bacterial outer membrane vesicles

Cited by 92 publications

References 41 publications

Construction of hypervesiculation Escherichia coli strains and application for secretory protein production

Construction of hypervesiculation Escherichia coli strains and application for secretory protein production

Quantitative Evaluation of Recombinant Protein Packaged into Outer Membrane Vesicles of Escherichia coli Cells

Use of bioengineered human commensal gut bacteria-derived microvesicles for mucosal plague vaccine delivery and immunization

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