Outer membrane vesicles (OMVs), nanoparticles released by Shiga toxin-producing Escherichia coli (STEC), have been identified as novel efficient virulence tools of these pathogens. STEC O157 OMVs carry a cocktail of virulence factors including Shiga toxin 2a (Stx2a), cytolethal distending toxin V (CdtV), EHEC hemolysin, flagellin, and lipopolysaccharide. OMVs are taken up by human intestinal epithelial and microvascular endothelial cells, the major targets during STEC infection, and deliver the virulence factors into host cells. There the toxins separate from OMVs and are trafficked via different pathways to their target compartments, i.e., the cytosol (Stx2a-A subunit), nucleus (CdtV-B subunit), and mitochondria (EHEC hemolysin). This leads to a toxin-specific host cell injury and ultimately apoptotic cell death. Besides their cytotoxic effects, STEC OMVs trigger an inflammatory response via their lipopolysaccharide and flagellin components. In this chapter, we describe methods for the isolation and purification of STEC OMVs, for the detection of OMV-associated virulence factors, and for the analysis of OMV interactions with host cells including OMV cellular uptake and intracellular trafficking of OMVs and OMV-delivered toxins.

Institute for Hygiene University of Münster Münster Germany

Institute for Infectiology Center for Molecular Biology of Inflammation University of Münster Münster Germany

National Reference Laboratory for E coli and Shigellae National Institute of Public Health Prague Czech Republic

Zobrazit více v PubMed

Karch H, Tarr PI, Bielaszewska M (2005) Enterohaemorrhagic Escherichia coli in human medicine. Int J Med Microbiol 295:405–418 DOI

Tarr PI, Gordon CA, Chandler WL (2005) Shiga-toxin-producing Escherichia coli and haemolytic uraemic syndrome. Lancet 365:1073–1086 PubMed

Richardson SE, Karmali MA, Becker LE et al (1988) The histopathology of the hemolytic uremic syndrome associated with verocytotoxin-producing Escherichia coli infections. Hum Pathol 19:1102–1108 DOI

Obrig TG (2010) Escherichia coli Shiga toxin mechanisms of action in renal disease. Toxins (Basel) 2:2769–2794 DOI

Zoja C, Buelli S, Morigi M (2010) Shiga toxin-associated hemolytic uremic syndrome: pathophysiology of endothelial dysfunction. Pediatr Nephrol 25:2231–2240. https://doi.org/10.1007/s00467-010-1522-1 PubMed DOI

Melton-Celsa AR (2014) Shiga toxin (Stx) classification, structure, and function. Microbiol Spectr 2(2):EHEC-0024-2013. https://doi.org/10.1128/microbiolspec.EHEC-0024-2013 PubMed DOI

Karpman D, Loos S, Tati R et al (2017) Haemolytic uraemic syndrome. J Intern Med 281:123–148. https://doi.org/10.1111/joim.12546 PubMed DOI

Janka A, Bielaszewska M, Dobrindt U et al (2003) Cytolethal distending toxin gene cluster in enterohemorrhagic Escherichia coli O157:H DOI

Schmidt H, Beutin L, Karch H (1995) Molecular analysis of the plasmid-encoded hemolysin of Escherichia coli O157:H7 strain EDL 933. Infect Immun 63:1055–1061 DOI

Bielaszewska M, Sinha B, Kuczius T et al (2005) Cytolethal distending toxin from Shiga toxin-producing Escherichia coli O157 causes irreversible G2/M arrest, inhibition of proliferation, and death of human endothelial cells. Infect Immun 73:552–562 DOI

Friedrich AW, Lu S, Bielaszewska M et al (2006) Cytolethal distending toxin in Escherichia coli O157:H7: spectrum of conservation, structure, and endothelial toxicity. J Clin Microbiol 44:1844–1846 DOI

Aldick T, Bielaszewska M, Zhang W et al (2007) Hemolysin from Shiga toxin-negative Escherichia coli O26 strains injures microvascular endothelium. Microbes Infect 9:282–290 DOI

Bielaszewska M, Rüter C, Kunsmann L et al (2013) Enterohemorrhagic Escherichia coli hemolysin employs outer membrane vesicles to target mitochondria and cause endothelial and epithelial apoptosis. PLoS Pathog 9(12):e1003797. https://doi.org/10.1371/journal.ppat.1003797 PubMed DOI PMC

Ellis TN, Kuehn MJ (2010) Virulence and immunomodulatory roles of bacterial outer membrane vesicles. Microbiol Mol Biol Rev 74:81–94. https://doi.org/10.1128/MMBR.00031-09 PubMed DOI PMC

Schwechheimer C, Kuehn MJ (2015) Outer-membrane vesicles from Gram-negative bacteria: biogenesis and functions. Nat Rev Microbiol 13:605–619. https://doi.org/10.1038/nrmicro3525 PubMed DOI PMC

Guerrero-Mandujano A, Hernández-Cortez C, Ibarra JA et al (2017) The outer membrane vesicles: secretion system type zero. Traffic 18:425–432. https://doi.org/10.1111/tra.12488 PubMed DOI

Jan AT (2017) Outer membrane vesicles (OMVs) of Gram-negative bacteria: a perspective update. Front Microbiol 8:1053. https://doi.org/10.3389/fmicb.2017.01053 PubMed DOI PMC

Kunsmann L, Rüter C, Bauwens A et al (2015) Virulence from vesicles: novel mechanisms of host cell injury by Escherichia coli O104:H4 outbreak strain. Sci Rep 5:13252. https://doi.org/10.1038/srep13252 PubMed DOI PMC

Bielaszewska M, Rüter C, Bauwens A et al (2017) Host cell interactions of outer membrane vesicle-associated virulence factors of enterohemorrhagic Escherichia coli O157: intracellular delivery, trafficking and mechanisms of cell injury. PLoS Pathog 13(2):e1006159. https://doi.org/10.1371/journal.ppat.1006159 PubMed DOI PMC

Kolling GL, Matthews KR (1999) Export of virulence genes and Shiga toxin by membrane vesicles of Escherichia coli O157:H7. Appl Environ Microbiol 65:1843–1848 DOI

Yokoyama K, Horii T, Yamashino T et al (2000) Production of Shiga toxin by Escherichia coli measured with reference to the membrane vesicle-associated toxins. FEMS Microbiol Lett 192:139–144 DOI

Aldick T, Bielaszewska M, Uhlin BE et al (2009) Vesicular stabilization and activity augmentation of enterohaemorrhagic Escherichia coli haemolysin. Mol Microbiol 71:1496–1508. https://doi.org/10.1111/j.1365-2958.2009.06618.x PubMed DOI

Bielaszewska M, Marejková M, Bauwens A et al (2018) 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. Int J Med Microbiol 308:882–889. https://doi.org/10.1016/j.ijmm.2018.06.004 PubMed DOI

Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254 DOI

Cascales E, Bernadac A, Gavioli M et al (2001) Pal lipoprotein of Escherichia coli plays a major role in outer membrane integrity. J Bacteriol 184:754–759 DOI

He X, Patfield S, Hnasko R et al (2013) A polyclonal antibody based immunoassay detects seven subtypes of Shiga toxin 2 produced by Escherichia coli in human and environmental samples. PLoS One 8(10):e76368. https://doi.org/10.1371/journal.pone.0076368 PubMed DOI PMC

Schmidt H, Geitz C, Tarr PI et al (1999) Non-O157:H7 pathogenic Shiga toxin-producing Escherichia coli: phenotypic and genetic profiling of virulence traits and evidence for clonality. J Infect Dis 179:115–123 DOI

Stins MF, Gilles F, Kim KS (1997) Selective expression of adhesion molecules on human brain microvascular endothelial cells. J Neuroimmunol 76:81–90 DOI

Kankaanpää P, Paavolainen L, Tiitta S et al (2012) BioImageXD: an open, general-purpose and high-throughput image-processing platform. Nat Methods 9:683–689. https://doi.org/10.1038/nmeth.2047 PubMed DOI

Tokuyasu KT (1980) Immunochemistry on ultrathin frozen sections. Histochem J 12:381–403 DOI

Humbel BM, Stierhof YD (2009) Cryosectioning according to Tokuyasu. In: Cavalier A, Spehner D, Humbel BM (eds) Handbook of cryo-preparation methods for electron microscopy. CRC Press, Boca Raton, FL

Tokuyasu KT (1973) A technique for ultracryotomy of cell suspension and tissues. J Cell Biol 57:551–565 DOI

Liou W, Geuze HJ, Slot JW (1996) Improving structural integrity of cryosections for immunogold labelling. Histochem Cell Biol 106:41–58 DOI

Slot JW, Geuze HJ (1985) A new method of preparing gold probes for multiple-labelling cytochemistry. Eur J Cell Biol 38:87–93 PubMed

Griffiths G (1993) 7.2.3 Antibody concentrations. In: Griffiths G (ed) Fine structure immunocytochemistry. Springer, Berlin, pp 245–248 DOI

Kankaanpää P (2010) BioImageXD - getting started. https://de.scribd.com/document/86133854/BioImageXD-Gettingstarted

Hed J, Hallden G, Johansson SG et al (1987) The use of fluorescence quenching in flow cytofluorometry to measure the attachment and ingestion phases in phagocytosis in peripheral blood without prior cell separation. J Immunol Methods 101:119–125 DOI

Translocation of outer membrane vesicles from enterohemorrhagic Escherichia coli O157 across the intestinal epithelial barrier

In Vivo Secretion of β-Lactamase-Carrying Outer Membrane Vesicles as a Mechanism of β-Lactam Therapy Failure