Thromb Haemost 2011; 105(03): 515-528
DOI: 10.1160/TH10-02-0140
Endothelium and Vascular Development
Schattauer GmbH

Differential cytotoxic actions of Shiga toxin 1 and Shiga toxin 2 on microvascular and macrovascular endothelial cells

Andreas Bauwens
1   Institute for Hygiene, University of Münster, Münster, Germany
,
Martina Bielaszewska
1   Institute for Hygiene, University of Münster, Münster, Germany
,
Björn Kemper
2   Center for Biomedical Optics and Photonics, University of Münster, Münster, Germany
,
Patrik Langehanenberg
2   Center for Biomedical Optics and Photonics, University of Münster, Münster, Germany
,
Gert von Bally
2   Center for Biomedical Optics and Photonics, University of Münster, Münster, Germany
,
Rudolf Reichelt
3   Institute for Medical Physics and Biophysics, University of Münster, Münster, Germany
,
Dennis Mulac
4   Institute for Food Chemistry, University of Münster, Münster, Germany
,
Hans-Ulrich Humpf
4   Institute for Food Chemistry, University of Münster, Münster, Germany
,
Alexander W. Friedrich
1   Institute for Hygiene, University of Münster, Münster, Germany
,
Kwang S. Kim
5   Division of Pediatric Infectious Diseases, Johns Hopkins University School of Medicine, David M. Rubenstein Child Health Building, Baltimore, Maryland, USA
,
Helge Karch
1   Institute for Hygiene, University of Münster, Münster, Germany
,
Johannes Müthing
1   Institute for Hygiene, University of Münster, Münster, Germany
› Institutsangaben
Financial support: This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG) MU 845/4–1, FR 2569/1–1, the Graduate School “Molecular Interactions of Pathogens with Biotic and Abiotic Surfaces“ (GRK 1409), and the program “Infections of the Endothelium“ SPP 1130 project KA 717/4–3, by a grant from the Interdisciplinary Center of Clinical Research (IZKF) Münster, project no. Ka2/061/04, and by a grant from the German Federal Ministry of Education and Research (BMBF), project FKZ 13N8183 within the research focus program “Biophotonics“.
Weitere Informationen

Publikationsverlauf

Received: 23. Februar 2010

Accepted after major revision: 25. November 2010

Publikationsdatum:
27. November 2017 (online)

Summary

Shiga toxin (Stx)-mediated injury to vascular endothelial cells in the kidneys, brain and other organs underlies the pathogenesis of haemolytic uraemic syndrome (HUS) caused by enterohaemorrhagic Escherichia coli (EHEC). We present a direct and comprehensive comparison of cellular injury induced by the two major Stx types, Stx1 and Stx2, in human brain microvascular endothelial cells (HBMECs) and EA.hy 926 macro-vascular endothelial cells. Scanning electron microscopy of microcarrier-based cell cultures, digital holographic microscopy of living single cells, and quantitative apoptosis/necrosis assays demonstrate that Stx1 causes both necrosis and apoptosis, whereas Stx2 induces almost exclusively apoptosis in both cell lines. Moreover, microvascular and macrovascular endothelial cells have different susceptibilities to the toxins: EA.hy 926 cells are slightly, but significantly (~ 10 times) more susceptible to Stx1, whereas HBMECs are strikingly (≥ 1,000 times) more susceptible to Stx2. These findings have implications in the pathogenesis of HUS, and suggest the existence of yet to be delineated Stx type-specific mechanisms of endothelial cell injury beyond inhibition of protein bio-synthesis.

 
  • References

  • 1 Schnittler H, Preissner KT. Between microbial attack and defence: the endothelium as a vulnerable player in infectious diseases. Thromb Haemost 2009; 102: 1011-1013.
  • 2 Lemichez E, Lecuit M, Nassif X. et al. Breaking the wall: targeting of the endothelium by pathogenic bacteria. Nat Rev Microbiol 2010; 8: 93-104.
  • 3 Hippenstiel S, Suttorp N. Interaction of pathogens with the endothelium. Thromb Haemost 2003; 89: 18-24.
  • 4 Karmali MA. Infection by Shiga toxin-producing Escherichia coli: an overview. Mol Biotechnol 2004; 26: 117-122.
  • 5 Karch H, Tarr PI, Bielaszewska M. Enterohaemorrhagic Escherichia coli in human medicine. Int J Med Microbiol 2005; 295: 405-418.
  • 6 Tarr PI, Gordon CA, Chandler WL. Shiga-toxin-producing Escherichia coli and haemolytic uraemic syndrome. Lancet 2005; 365: 1073-1086.
  • 7 Proulx F, Seidman EG, Karpman D. Pathogenesis of Shiga toxin-associated hemolytic uremic syndrome. Pediatr Res 2001; 50: 163-171.
  • 8 Bitzan M. Treatment options for HUS secondary to Escherichia coli O157:H7. Kidney Int Suppl 2009; 112: S62-66.
  • 9 Bielaszewska M, Karch H. Consequences of enterohaemorrhagic Escherichia coli infection for the vascular endothelium. Thromb Haemost 2005; 94: 312-318.
  • 10 Sandvig K. Shiga toxins. Toxicon 2001; 39: 1629-1635.
  • 11 Ling H, Boodhoo A, Hazes B. et al. Structure of the Shiga-like toxin I B-pentamer complexed with an analogue of its receptor Gb3. Biochemistry 1998; 37: 1777-1788.
  • 12 Jackson MP, Neill RJ, O’Brien AD. et al. Nucleotide sequence analysis and comparison of the structural genes for Shiga-like toxin I and Shiga-like toxin II encoded by bacteriophages from Escherichia coli . FEMS Microbiol Lett 1987; 44: 109-114.
  • 13 Lingwood CA, Law H, Richardson S. et al. Glycolipid binding of purified and recombinant Escherichia coli produced verotoxin in vitro. J Biol Chem 1987; 262: 8834-8839.
  • 14 Waddell T, Head S, Petric M. et al. Globotriosyl ceramide is specifically recognized by the Escherichia coli verocytotoxin 2. Biochem Biophys Res Commun 1988; 152: 674-679.
  • 15 Müthing J, Schweppe CH, Karch H. et al. Shiga toxins, glycosphingolipid diversity, and endothelial cell injury. Thromb Haemost 2009; 101: 252-264.
  • 16 Head SC, Karmali MA, Lingwood CA. Preparation of VT1 and VT2 hybrid toxins from their purified dissociated subunits. Evidence for B subunit modulation of a subunit function. J Biol Chem 1991; 266: 3617-3621.
  • 17 Fraser ME, Cherney MM, Marcato P. et al. Binding of adenine to Stx2, the protein toxin from Escherichia coli O157:H7. Acta Crystallogr Sect F Struct Biol Cryst Commun 2006; 62: 627-630.
  • 18 Hurley BP, Thorpe CM, Acheson DW. Shiga toxin translocation across intestinal epithelial cells is enhanced by neutrophil transmigration. Infect Immun 2001; 69: 6148-6155.
  • 19 Brigotti M, Caprioli A, Tozzi AE. et al. Shiga toxins present in the gut and in the polymorphonuclear leukocytes circulating in the blood of children with hemolytic-uremic syndrome. J Clin Microbiol 2006; 44: 313-317.
  • 20 Brigotti M, Carnicelli D, Ravanelli E. et al. Interactions between Shiga toxins and human polymorphonuclear leukocytes. J Leukoc Biol 2008; 84: 1019-1027.
  • 21 Johannes L, Goud B. Facing inward from compartment shores: how many pathways were we looking for?. Traffic 2000; 1: 119-123.
  • 22 Sandvig K, van Deurs B. Transport of protein toxins into cells: pathways used by ricin, cholera toxin and Shiga toxin. FEBS Lett 2002; 529: 49-53.
  • 23 Römer W, Berland L, Chambon V. et al. Shiga toxin induces tubular membrane invaginations for its uptake into cells. Nature 2007; 450: 670-675.
  • 24 Sandvig K, Garred O, Prydz K. et al. Retrograde transport of endocytosed Shiga toxin to the endoplasmic reticulum. Nature 1992; 358: 510-512.
  • 25 Lingwood CA. Aglycone modulation of glycolipid receptor function. Glycoconj J 1996; 13: 495-503.
  • 26 Sandvig K, Spilsberg B, Lauvrak SU. et al. Pathways followed by protein toxins into cells. Int J Med Microbiol 2004; 293: 483-490.
  • 27 Bonifacino JS, Rojas R. Retrograde transport from endosomes to the trans-Golgi network. Nat Rev Mol Cell Biol 2006; 7: 568-579.
  • 28 Yu M, Haslam DB. Shiga toxin is transported from the endoplasmic reticulum following interaction with the luminal chaperone HEDJ/ERdj3. Infect Immun 2005; 73: 2524-2532.
  • 29 Garred O, Dubinina E, Holm PK. et al. Role of processing and intracellular trans-port for optimal toxicity of Shiga toxin and toxin mutants. Exp Cell Res 1995; 218: 39-49.
  • 30 O’Brien AD, Tesh VL, Donohue-Rolfe A. et al. Shiga toxin: biochemistry, genetics, mode of action, and role in pathogenesis. Curr Top Microbiol Immunol 1992; 180: 65-94.
  • 31 Barbieri L, Valbonesi P, Brigotti M. et al. Shiga-like toxin I is a polynucleotide:adenosine glycosidase. Mol Microbiol 1998; 29: 661-662.
  • 32 Endo Y, Tsurugi K, Yutsudo T. et al. Site of action of a Vero toxin (VT2) from Escherichia coli O157:H7 and of Shiga toxin on eukaryotic ribosomes. RNA N-glycosidase activity of the toxins. Eur J Biochem. 1988; 171: 45-50.
  • 33 Katagiri YU, Mori T, Nakajima H. et al. Activation of Src family kinase yes induced by Shiga toxin binding to globotriaosyl ceramide (Gb3/CD77) in low density, detergent-insoluble microdomains. J Biol Chem 1999; 274: 35278-35282.
  • 34 Takenouchi H, Kiyokawa N, Taguchi T. et al. Shiga toxin binding to globotriaosyl ceramide induces intracellular signals that mediate cytoskeleton remodeling in human renal carcinoma-derived cells. J Cell Sci 2004; 117: 3911-3922.
  • 35 Fujii J, Wood K, Matsuda F. et al. Shiga toxin 2 causes apoptosis in human brain microvascular endothelial cells via C/EBP homologous protein. Infect Immun 2008; 76: 3679-3689.
  • 36 Brigotti M, Accorsi P, Carnicelli D. et al. Shiga toxin 1: damage to DNA in vitro . Toxicon 2001; 39: 341-348.
  • 37 Brigotti M, Carnicelli D, Vara AG. Shiga toxin 1 acting on DNA in vitro is a heat-stable enzyme not requiring proteolytic activation. Biochimie 2004; 86: 305-309.
  • 38 Sestili P, Alfieri R, Carnicelli D. et al. Shiga toxin 1 and ricin inhibit the repair of H2O2-induced DNA single strand breaks in cultured mammalian cells. DNA Repair 2005; 4: 271-277.
  • 39 Sandvig K, Torgersen ML, Raa HA. et al. Clathrin-independent endocytosis: from nonexisting to an extreme degree of complexity. Histochem Cell Biol 2008; 129: 267-276.
  • 40 Obrig TG, Del Vecchio PJ, Brown JE. et al. Direct cytotoxic action of Shiga toxin on human vascular endothelial cells. Infect Immun 1988; 56: 2373-2378.
  • 41 Louise CB, Obrig TG. Specific interaction of Escherichia coli O157:H7-derived Shiga-like toxin II with human renal endothelial cells. J Infect Dis 1995; 172: 1397-1401.
  • 42 Ohmi K, Kiyokawa N, Takeda T. et al. Human microvascular endothelial cells are strongly sensitive to Shiga toxins. Biochem Biophys Res Commun 1998; 251: 137-141.
  • 43 Jacewicz MS, Acheson DW, Binion DG. et al. Responses of human intestinal microvascular endothelial cells to Shiga toxins 1 and 2 and pathogenesis of hemorrhagic colitis. Infect Immun 1999; 67: 1439-1444.
  • 44 Ramegowda B, Samuel JE, Tesh VL. Interaction of Shiga toxins with human brain microvascular endothelial cells: cytokines as sensitizing agents. J Infect Dis 1999; 180: 1205-1213.
  • 45 Stins MF, Gilles F, Kim KS. Selective expression of adhesion molecules on human brain microvascular endothelial cells. J Neuroimmunol 1997; 76: 81-90.
  • 46 Edgell CJ, McDonald CC, Graham JB. Permanent cell line expressing human factor VIII-related antigen established by hybridization. Proc Natl Acad Sci USA 1983; 80: 3734-3737.
  • 47 Smith HW, Linggood MA. The transmissible nature of enterotoxin production in human enteropathogenic strain of Escherichia coli . J Med Microbiol 1971; 4: 301-305.
  • 48 Strockbine NA, Marques LR, Newland JW. et al. Two toxin-converting phages from Escherichia coli O157:H7 strain 933 encode antigenically distinct toxins with similar biologic activities. Infect Immun 1986; 53: 135-140.
  • 49 Petric M, Karmali MA, Arbus GS. et al. Effects of cycloheximide and puromycin on cytotoxic activity of Escherichia coli verocytotoxin (Shiga-like toxin). J Clin Microbiol 1987; 25: 1265-1268.
  • 50 Head SC, Karmali MA, Roscoe ME. et al. Serological differences between verocytotoxin 2 and Shiga-like toxin II. Lancet 1988; 2: 751.
  • 51 Karmali MA, Petric M, Lim C. et al. The association between idiopathic hemolytic uremic syndrome and infection by verotoxin-producing Escherichia coli . J Infect Dis 1985; 151: 775-782.
  • 52 Reichelt R. Scanning Electron Microscopy. In: Science of Microscopy. Springer; 2007. pp. 133-272.
  • 53 Kemper B, Carl D, Höink A. et al. Modular digital holographic microscopy system for marker free quantitative phase contrast imaging of living cells. Proc SPIE 2006; 6191: 61910T.
  • 54 Carl D, Kemper B, Wernicke G. et al. Parameter-optimized digital holographic microscope for high-resolution living-cell analysis. Appl Opt 2004; 43: 6536-6544.
  • 55 Kemper B, Carl D, Schnekenburger J. et al. Investigation of living pancreas tumor cells by digital holographic microscopy. J Biomed Opt 2006; 11: 34005.
  • 56 Kemper B, Bauwens A, Vollmer A. et al. Label-free quantitative cell division monitoring of endothelial cells by digital holographic microscopy. J Biomed Opt 2010; 15: 036009.
  • 57 Nicoletti I, Migliorati G, Pagliacci MC. et al. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide and flow cytometry. J Immunol Methods 1991; 139: 271-279.
  • 58 Johannes L, Römer W. Shiga toxins – from cell biology to biomedical applications. Nat Rev Microbiol 2010; 8: 105-116.
  • 59 Ergonul Z, Hughes AK, Kohan DE. Induction of apoptosis of human brain micro-vascular endothelial cells by Shiga toxin 1. J Infect Dis 2003; 187: 154-158.
  • 60 Schweppe CH, Bielaszewska M, Pohlentz G. et al. Glycosphingolipids in vascular endothelial cells: relationship of heterogeneity in Gb3Cer/CD77 receptor expression with differential Shiga toxin 1 cytotoxicity. Glycoconj J 2008; 25: 291-304.
  • 61 Falguières T, Römer W, Amessou M. et al. Functionally different pools of Shiga toxin receptor, globotriaosyl ceramide, in HeLa cells. FEBS J 2006; 273: 5205-5218.
  • 62 Kiarash A, Boyd B, Lingwood CA. Glycosphingolipid receptor function is modified by fatty acid content. Verotoxin 1 and verotoxin 2c preferentially recognize different globotriaosyl ceramide fatty acid homologues. J Biol Chem 1994; 269: 11138-11146.
  • 63 Arab S, Lingwood CA. Influence of phospholipid chain length on verotoxin/globotriaosyl ceramide binding in model membranes: comparison of a supported bilayer film and liposomes. Glycoconj J 1996; 13: 159-166.
  • 64 Nakajima H, Kiyokawa N, Katagiri YU. et al. Kinetic analysis of binding between Shiga toxin and receptor glycolipid Gb3Cer by surface plasmon resonance. J Biol Chem 2001; 276: 42915-42922.
  • 65 Binnington B, Lingwood D, Nutikka A. et al. Effect of globotriaosyl ceramide fatty acid α-hydroxylation on the binding by verotoxin 1 and verotoxin 2. Neurochem Res 2002; 27: 807-813.
  • 66 Lord JM, Roberts LM. Toxin entry: retrograde transport through the secretory pathway. J Cell Biol 1998; 140: 733-736.
  • 67 Tam P, Mahfoud R, Nutikka A. et al. Differential intracellular transport and binding of verotoxin 1 and verotoxin 2 to globotriaosylceramide-containing lipid assemblies. J Cell Physiol 2008; 216: 750-763.
  • 68 Richardson SE, Karmali MA, Becker LE. et al. The histopathology of the hemolytic uremic syndrome associated with verocytotoxin-producing Escherichia coli infections. Hum Pathol 1988; 19: 1102-1108.
  • 69 Ostroff SM, Tarr PI, Neill MA. et al. Toxin genotypes and plasmid profiles as determinants of systemic sequelae in Escherichia coli O157:H7 infections. J Infect Dis 1989; 160: 994-998.
  • 70 Friedrich AW, Bielaszewska M, Zhang W. et al. Escherichia coli harboring Shiga toxin 2 gene variants: frequency and association with clinical symptoms. J Infect Dis 2002; 185: 74-84.
  • 71 Siegler RL, Obrig TG, Pysher TJ. et al. Response to Shiga toxin 1 and 2 in a baboon model of hemolytic uremic syndrome. Pediatr Nephrol 2003; 18: 92-96.