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, 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.

[1]  Yutaka Suzuki,et al.  RAB5A and TRAPPC6B are novel targets for Shiga toxin 2a inactivation in kidney epithelial cells , 2020, Scientific Reports.

[2]  A. Mellmann,et al.  Real- time interaction analysis of Shiga toxins and membrane microdomains of primary human brain microvascular endothelial cells. , 2019, Glycobiology.

[3]  G. Wunderlich,et al.  Nano-multilamellar lipid vesicles (NMVs) enhance protective antibody responses against Shiga toxin (Stx2a) produced by enterohemorrhagic Escherichia coli strains (EHEC) , 2018, Brazilian Journal of Microbiology.

[4]  A. Mellmann,et al.  Enterohemorrhagic Escherichia coli O157 outer membrane vesicles induce interleukin 8 production in human intestinal epithelial cells by signaling via Toll-like receptors TLR4 and TLR5 and activation of the nuclear factor NF-κB. , 2018, International journal of medical microbiology : IJMM.

[5]  A. Mellmann,et al.  Combining Mass Spectrometry, Surface Acoustic Wave Interaction Analysis, and Cell Viability Assays for Characterization of Shiga Toxin Subtypes of Pathogenic Escherichia coli Bacteria. , 2018, Analytical chemistry.

[6]  H. Karch,et al.  Shiga Toxin Glycosphingolipid Receptors in Human Caco-2 and HCT-8 Colon Epithelial Cell Lines , 2017, Toxins.

[7]  S. Loos,et al.  Haemolytic uraemic syndrome , 2017, Journal of internal medicine.

[8]  A. Mellmann,et al.  Host cell interactions of outer membrane vesicle-associated virulence factors of enterohemorrhagic Escherichia coli O157: Intracellular delivery, trafficking and mechanisms of cell injury , 2017, PLoS pathogens.

[9]  Hongwei Zhang,et al.  Thin-Film Hydration Followed by Extrusion Method for Liposome Preparation. , 2017, Methods in molecular biology.

[10]  S. Antimisiaris Preparation of DRV Liposomes. , 2017, Methods in molecular biology.

[11]  B. Kemper,et al.  Shiga toxin of enterohaemorrhagic Escherichia coli directly injures developing human erythrocytes , 2016, Cellular microbiology.

[12]  M. Kuehn,et al.  Outer-membrane vesicles from Gram-negative bacteria: biogenesis and functions , 2015, Nature Reviews Microbiology.

[13]  Björn Kemper,et al.  Virulence from vesicles: Novel mechanisms of host cell injury by Escherichia coli O104:H4 outbreak strain , 2015, Scientific Reports.

[14]  A. Mellmann,et al.  Enterohemorrhagic Escherichia coli Hemolysin Employs Outer Membrane Vesicles to Target Mitochondria and Cause Endothelial and Epithelial Apoptosis , 2013, PLoS pathogens.

[15]  Hui Sun,et al.  Enterohemorrhagic Escherichia coli Specific Enterohemolysin Induced IL-1β in Human Macrophages and EHEC-Induced IL-1β Required Activation of NLRP3 Inflammasome , 2012, PloS one.

[16]  Mary K. Lewinski,et al.  Renal and neurological involvement in typical Shiga toxin-associated HUS , 2012, Nature Reviews Nephrology.

[17]  B. Kemper,et al.  Facing glycosphingolipid–Shiga toxin interaction: dire straits for endothelial cells of the human vasculature , 2012, Cellular and Molecular Life Sciences.

[18]  K. Sandvig,et al.  Shiga toxins. , 2012, Toxicon : official journal of the International Society on Toxinology.

[19]  Patrik Langehanenberg,et al.  Differential cytotoxic actions of Shiga toxin 1 and Shiga toxin 2 on microvascular and macrovascular endothelial cells , 2010, Thrombosis and Haemostasis.

[20]  A. Kane,et al.  Shiga Toxin 2 and Flagellin from Shiga-Toxigenic Escherichia coli Superinduce Interleukin-8 through Synergistic Effects on Host Stress-Activated Protein Kinase Activation , 2010, Infection and Immunity.

[21]  M. Kuehn,et al.  Virulence and Immunomodulatory Roles of Bacterial Outer Membrane Vesicles , 2010, Microbiology and Molecular Biology Reviews.

[22]  L. Johannes,et al.  Shiga toxins — from cell biology to biomedical applications , 2010, Nature Reviews Microbiology.

[23]  L. Johannes,et al.  Intracellular trafficking of Shiga‐toxin‐B‐subunit‐functionalized spherulites , 2008, Biology of the cell.

[24]  B. Delord,et al.  Specific adsorption of functionalized colloids at the surface of living cells: a quantitative kinetic analysis of the receptor-mediated binding. , 2008, Biochimica et biophysica acta.

[25]  A. Mellmann,et al.  Analysis of Collection of Hemolytic Uremic Syndrome–associated Enterohemorrhagic Escherichia coli , 2008, Emerging infectious diseases.

[26]  P. Bassereau,et al.  Key role of receptor density in colloid/cell specific interaction: a quantitative biomimetic study on giant vesicles , 2008, The European physical journal. E, Soft matter.

[27]  R. Polanowska-Grabowska,et al.  Shiga Toxin 2 and Lipopolysaccharide Induce Human Microvascular Endothelial Cells To Release Chemokines and Factors That Stimulate Platelet Function , 2005, Infection and Immunity.

[28]  H. Karch,et al.  Enterohaemorrhagic Escherichia coli in human medicine. , 2005, International journal of medical microbiology : IJMM.

[29]  P. Tarr,et al.  Shiga-toxin-producing Escherichia coli and haemolytic uraemic syndrome , 2005, The Lancet.

[30]  Tana,et al.  Induction of intestinal IgA and IgG antibodies preventing adhesion of verotoxin‐producing Escherichia coli to Caco‐2 cells by oral immunization with liposomes , 2003, Letters in applied microbiology.

[31]  N. Nagata,et al.  Protection of Monkeys against Shiga Toxin Induced by Shiga Toxin-Liposome Conjugates , 2002, International Archives of Allergy and Immunology.

[32]  L. Barbieri,et al.  Damage to nuclear DNA induced by Shiga toxin 1 and ricin in human endothelial cells 1 , 2002, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[33]  A. Friedrich,et al.  Escherichia coli harboring Shiga toxin 2 gene variants: frequency and association with clinical symptoms. , 2002, The Journal of infectious diseases.

[34]  S. Ichikawa,et al.  Enzymes inside lipid vesicles: preparation, reactivity and applications. , 2001, Biomolecular engineering.

[35]  K. Yokoyama,et al.  Production of shiga toxin by Escherichia coli measured with reference to the membrane vesicle-associated toxins. , 2000, FEMS microbiology letters.

[36]  Glynis L. Kolling,et al.  Export of Virulence Genes and Shiga Toxin by Membrane Vesicles of Escherichia coli O157:H7 , 1999, Applied and Environmental Microbiology.

[37]  N. Nagata,et al.  Induction of Protection against Oral Infection with Cytotoxin–Producing Escherichia coli O157:H7 in Mice by Shiga–like Toxin–Liposome Conjugate , 1998, International Archives of Allergy and Immunology.

[38]  T. Uchida,et al.  Protection against verocytotoxin in mice induced by liposome-coupled verocytotoxin. , 1997, International archives of allergy and immunology.

[39]  R. Macdonald,et al.  Small-volume extrusion apparatus for preparation of large, unilamellar vesicles. , 1991, Biochimica et biophysica acta.

[40]  P. K. Smith,et al.  Measurement of protein using bicinchoninic acid. , 1985, Analytical biochemistry.

[41]  H. Lior,et al.  The association between idiopathic hemolytic uremic syndrome and infection by verotoxin-producing Escherichia coli. , 1985, The Journal of infectious diseases.

[42]  J Konowalchuk,et al.  Vero response to a cytotoxin of Escherichia coli , 1977, Infection and immunity.

[43]  R. Peters,et al.  Distribution of lipids in cytoplasmic and outer membranes of Escherichia coli K12. , 1976, Biochimica et biophysica acta.

[44]  W. Lennarz,et al.  Distribution of Lipids in the Wall and Cytoplasmic Membrane Subfractions of the Cell Envelope of Escherichia coli , 1972, Journal of bacteriology.