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Modeling Native EHEC Outer Membrane Vesicles by Creating Synthetic Surrogates

Enterohemorrhagic Escherichia coli (EHEC) is a zoonotic pathogen responsible for life-threating diseases such as hemolytic uremic syndrome. While its major virulence factor, the Shiga toxin (Stx), is known to exert its cytotoxic effect on various endothelial and epithelial cells when in its free, so...

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Autores principales: Kehl, Alexander, Kuhn, Ronja, Detzner, Johanna, Steil, Daniel, Müthing, Johannes, Karch, Helge, Mellmann, Alexander
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
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Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7284840/
https://www.ncbi.nlm.nih.gov/pubmed/32384757
http://dx.doi.org/10.3390/microorganisms8050673
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author Kehl, Alexander
Kuhn, Ronja
Detzner, Johanna
Steil, Daniel
Müthing, Johannes
Karch, Helge
Mellmann, Alexander
author_facet Kehl, Alexander
Kuhn, Ronja
Detzner, Johanna
Steil, Daniel
Müthing, Johannes
Karch, Helge
Mellmann, Alexander
author_sort Kehl, Alexander
collection PubMed
description Enterohemorrhagic Escherichia coli (EHEC) is a zoonotic pathogen responsible for life-threating diseases such as hemolytic uremic syndrome. While its major virulence factor, the Shiga toxin (Stx), is known to exert its cytotoxic effect on various endothelial and epithelial cells when in its free, soluble form, Stx was also recently found to be associated with EHEC outer membrane vesicles (OMVs). However, depending on the strain background, other toxins can also be associated with native OMVs (nOMVs), and nOMVs are also made up of immunomodulatory agents such as lipopolysaccharides and flagellin. Thus, it is difficult to determine to which extent a single virulence factor in nOMVs, such as Stx, contributes to the molecular pathogenesis of EHEC. To reduce this complexity, we successfully developed a protocol for the preparation of synthetic OMVs (sOMVs) with a defined lipid composition resembling the E. coli outer membrane and loaded with specific proteins, i.e., bovine serum albumin (BSA) as a proxy for functional Stx2a. Using BSA for parameter evaluation, we found that (1) functional sOMVs can be prepared at room temperature instead of potentially detrimental higher temperatures (e.g., 45 °C), (2) a 1:10 ratio of protein to lipid, i.e., 100 µg protein with 1 mg of lipid mixture, yields homogenously sized sOMVs, and (3) long-term storage for up to one year at 4 °C is possible without losing structural integrity. Accordingly, we reproducibly generated Stx2a-loaded sOMVs with an average diameter of 132.4 ± 9.6 nm that preserve Stx2a’s injuring activity, as determined by cytotoxicity assays with Vero cells. Overall, we successfully created sOMVs and loaded them with an EHEC toxin, which opens the door for future studies on the degree of virulence associated with individual toxins from EHEC and other bacterial pathogens.
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spelling pubmed-72848402020-06-15 Modeling Native EHEC Outer Membrane Vesicles by Creating Synthetic Surrogates Kehl, Alexander Kuhn, Ronja Detzner, Johanna Steil, Daniel Müthing, Johannes Karch, Helge Mellmann, Alexander Microorganisms Communication Enterohemorrhagic Escherichia coli (EHEC) is a zoonotic pathogen responsible for life-threating diseases such as hemolytic uremic syndrome. While its major virulence factor, the Shiga toxin (Stx), is known to exert its cytotoxic effect on various endothelial and epithelial cells when in its free, soluble form, Stx was also recently found to be associated with EHEC outer membrane vesicles (OMVs). However, depending on the strain background, other toxins can also be associated with native OMVs (nOMVs), and nOMVs are also made up of immunomodulatory agents such as lipopolysaccharides and flagellin. Thus, it is difficult to determine to which extent a single virulence factor in nOMVs, such as Stx, contributes to the molecular pathogenesis of EHEC. To reduce this complexity, we successfully developed a protocol for the preparation of synthetic OMVs (sOMVs) with a defined lipid composition resembling the E. coli outer membrane and loaded with specific proteins, i.e., bovine serum albumin (BSA) as a proxy for functional Stx2a. Using BSA for parameter evaluation, we found that (1) functional sOMVs can be prepared at room temperature instead of potentially detrimental higher temperatures (e.g., 45 °C), (2) a 1:10 ratio of protein to lipid, i.e., 100 µg protein with 1 mg of lipid mixture, yields homogenously sized sOMVs, and (3) long-term storage for up to one year at 4 °C is possible without losing structural integrity. Accordingly, we reproducibly generated Stx2a-loaded sOMVs with an average diameter of 132.4 ± 9.6 nm that preserve Stx2a’s injuring activity, as determined by cytotoxicity assays with Vero cells. Overall, we successfully created sOMVs and loaded them with an EHEC toxin, which opens the door for future studies on the degree of virulence associated with individual toxins from EHEC and other bacterial pathogens. MDPI 2020-05-06 /pmc/articles/PMC7284840/ /pubmed/32384757 http://dx.doi.org/10.3390/microorganisms8050673 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Communication
Kehl, Alexander
Kuhn, Ronja
Detzner, Johanna
Steil, Daniel
Müthing, Johannes
Karch, Helge
Mellmann, Alexander
Modeling Native EHEC Outer Membrane Vesicles by Creating Synthetic Surrogates
title Modeling Native EHEC Outer Membrane Vesicles by Creating Synthetic Surrogates
title_full Modeling Native EHEC Outer Membrane Vesicles by Creating Synthetic Surrogates
title_fullStr Modeling Native EHEC Outer Membrane Vesicles by Creating Synthetic Surrogates
title_full_unstemmed Modeling Native EHEC Outer Membrane Vesicles by Creating Synthetic Surrogates
title_short Modeling Native EHEC Outer Membrane Vesicles by Creating Synthetic Surrogates
title_sort modeling native ehec outer membrane vesicles by creating synthetic surrogates
topic Communication
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7284840/
https://www.ncbi.nlm.nih.gov/pubmed/32384757
http://dx.doi.org/10.3390/microorganisms8050673
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