Vacuolisation of human microvascular endothelial cells by enterohaemorrhagic Escherichia coli

Martina Bielaszewska; Andreas Bauwens; Lilo Greune; Björn Kemper; Ulrich Dobrindt; Joyce M. Geelen; Kwang S. Kim; M. Alexander Schmidt; Helge Karch

doi:10.1160/TH09-07-0499

Thrombosis and Haemostasis, Inhaltsverzeichnis

Thromb Haemost 2009; 102(06): 1080-1092
DOI: 10.1160/TH09-07-0499

Theme Issue Article

Schattauer GmbH

Vacuolisation of human microvascular endothelial cells by enterohaemorrhagic Escherichia coli

Autor*innen

Martina Bielaszewska^*

¹Institute of Hygiene, University of Münster, Münster, Germany
Andreas Bauwens^*

¹Institute of Hygiene, University of Münster, Münster, Germany
Lilo Greune

²Institute of Infectiology, Center for Molecular Biology of Inflammation (ZMBE), University of Münster, Münster, Germany
Björn Kemper

³Center for Biomedical Optics and Photonics, University of Münster, Münster, Germany
Ulrich Dobrindt

⁴Institute for Molecular Infection Biology, University of Würzburg, Würzburg, Germany
Joyce M. Geelen

⁵Department of Pediatric Nephrology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands
Kwang S. Kim

⁶Division of Pediatric Infectious Diseases, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
M. Alexander Schmidt

²Institute of Infectiology, Center for Molecular Biology of Inflammation (ZMBE), University of Münster, Münster, Germany
Helge Karch

¹Institute of Hygiene, University of Münster, Münster, Germany

Abstract

Summary

Enterohaemorrhagic Escherichia coli (EHEC) cause haemolytic uraemic syndrome (HUS),a thrombotic microangiopathy resulting from endothelial injury in the renal glomeruli and other organs. EHEC virulence factors that damage the microvascular endothelium play therefore major roles in the pathogenesis of HUS.We identified an EHEC strain that vacuolates and kills primary human glomerular microvascular endothelial cells (GMVECs) and a human brain microvascular endothelial cell (HBMEC) line. Because the vacuolating effect closely resembles those elicited on other cells by the vacuolating cytotoxin of Helicobacter pylori (VacA), we designated the factor responsible for this effect EHEC vacuolating cytotoxin (EHEC-Vac). EHEC-Vac (a secreted non-serine protease protein) binds to HBMECs rapidly and irreversibly, vacuolates within 30 min after exposure and the effect is maximally apparent at 48 h. Despite the vacuolisation, HBMECs survive for several days before they undergo necrosis. Electron and immunofluorescence microscopy demonstrate that the vacuoles induced by EHEC-Vac originate from lysosomes.Accordingly, they stain with neutral red indicating an acidic microenvironment. Similar to VacA, the EHEC-Vac-mediated vacuolisation is both prevented and reverted by the vacuolar proton pump inhibitor bafilomycin A1, suggesting a similar mechanism of vacuole formation by these toxins. Despite the similarity of phenotypes elicited by EHEC-Vac and VacA, genomic DNA from the EHEC-Vac-producing strain failed to hybridise to a vacA probe, as well as to probes derived from presently known E. coli vacuolating toxins.Through its microvascular endothelium-injuring potential combined with the ability to induce interleukin 6 release from these cells EHEC-Vac might contribute to the pathogenesis of HUS.

Keywords

Enterohaemorrhagic E. coli - haemolytic uraemic syndrome - vacuolating cytotoxin - EHEC-Vac - microvascular endothelial injury

Volltext

Referenzen

References
1 Karch H, Tarr PI, Bielaszewska M. Enterohaemorrhagic Escherichia coli in human medicine. Int J Med Microbiol 2005; 295: 405-418.
2 Tarr PI, Gordon CA, Chandler WL. Shiga-toxinproducing Escherichia coli and haemolytic uraemic syndrome. Lancet 2005; 365: 1073-1086.
3 Andreoli SP, Trachtman H, Acheson DWK. et al. Hemolytic uremic syndrome: epidemiology, pathophysiology, and therapy. Pediatr Nephrol 2002; 17: 293-298.
4 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.
5 Upadhyaya K, Barwick K, Fishaut M. et al. The importance of nonrenal involvement in hemolytic-uremic syndrome. Pediatrics 1980; 65: 115-120.
6 Bielaszewska M, Karch H. Consequences of enterohaemorrhagic Escherichia coli infection for the vascular endothelium. Thromb Haemost 2005; 94: 312-318.
7 Dehio C. Bartonella interactions with endothelial cells and erythrocytes. Trends Microbiol 2001; 09: 279-285.
8 Sandvig K. Shiga toxins. Toxicon 2001; 39: 1629-1635.
9 Müthing J, Schweppe CH, Karch H. et al. Shiga toxins, glycosphingolipid diversity, and endothelial cell injury. Thromb Haemost 2009; 101: 252-264.
10 Janka A, Bielaszewska M, Dobrindt U. et al. Cytolethal distending toxin gene cluster in enterohemorrhagic Escherichia coli O157:H⁻ and O157:H7: characterization and evolutionary considerations. Infect Immun 2003; 71: 3634-3638.
11 Friedrich AW, Lu S, Bielaszewska M. et al. Cytolethal distending toxin in Escherichia coli O157:H7: spectrum of conservation, structure, and endothelial toxicity. J Clin Microbiol 2006; 44: 1844-1846.
12 Bielaszewska M, Fell M, Greune L. et al. Characterization of cytolethal distending toxin genes and expression in Shiga toxin-producing Escherichia coli strains of non-O157 serogroups. Infect Immun 2004; 72: 1812-1816.
13 Schmidt H, Beutin L, Karch H. Molecular analysis of the plasmid-encoded hemolysin of Escherichia coli O157:H7 strain EDL 933. Infect Immun 1995; 63: 1055-1061.
14 Bielaszewska M, Sinha B, Kuczius T. et al. 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 2005; 73: 552-562.
15 Schmidt H, Maier E, Karch H. et al. Pore-forming properties of the plasmid-encoded hemolysin of enterohemorrhagic Escherichia coli O157:H7. Eur J Biochem 1996; 241: 594-601.
16 Aldick T, Bielaszewska M, Zhang W. et al. Hemolysin from Shiga toxin-negative Escherichia coli O26 strains injures microvascular endothelium. Microbes Infect 2007; 09: 282-290.
17 Paton AW, Srimanote P, Talbot UM. et al. A new family of potent AB5 cytotoxins produced by Shiga toxigenic Escherichia coli . J Exp Med 2004; 200: 35-46.
18 Leunk RD, Johnson PT, David BC. et al. Cytotoxic activity in broth-culture filtrates of Campylobacter pylori . J Med Microbiol 1988; 26: 93-99.
19 Zhang W, Bielaszewska M, Friedrich AW. et al. Transcriptional analysis of genes encoding Shiga toxin 2 and its variants in Escherichia coli . Appl Environ Microbiol 2005; 71: 558-561.
20 Brzuszkiewicz E, Bruggemann H, Liesegang H. et al. How to become a uropathogen: Comparative genomic analysis of extraintestinal pathogenic Escherichia coli strains. Proc Natl Acad Sci USA 2006; 103: 12879-12884.
21 Welch RA, Burland V, Plunkett III G. et al. Extensive mosaic structure revealed by the complete genome sequence of uropathogenic Escherichia coli . Natl Acad Sci USA 2002; 99: 17020-17024.
22 Stins MF, Gilles F, Kim KS. Selective expression of adhesion molecules on human brain microvascular endothelial cells. J Neuroimmunol 1997; 76: 81-90.
23 van Setten PA, van Hinsbergh VWM, van der Velden TJAN. et al. Effects of TNF on verocytotoxin cytotoxicity in purified human glomerular microvascular endothelial cells. Kidney Int 1997; 51: 1245-1256.
24 Cover TL, Reddy LY, Blaser MJ. Effects of ATPase inhibitors on the response of HeLa cells to Helicobacter pylori vacuolating toxin. Infect Immun 1993; 61: 1427-1431.
25 Coelho A, Andrade JR, Vicente AC. et al. Cytotoxic cell vacuolating activity from Vibrio cholerae hemolysin. Infect Immun 2000; 68: 1700-1705.
26 Figueroa-Arredondo P, Heuser JE, Akopyants NS. et al. Cell vacuolation caused by Vibrio cholerae hemolysin. Infect Immun 2001; 69: 1613-1624.
27 Kügler S, Böcker K, Heusipp G. et al. Pertussis toxin transiently affects barrier integrity, organelle organization and transmigration of monocytes in a human brain microvascular endothelial cell barrier model. Cell Microbiol 2007; 09: 619-632.
28 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.
29 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.1-61910T.8.
30 Kemper B, Carl D, Schnekenburger J. et al. Investigation of living pancreas tumor cells by digital holographic microscopy. J Biomed Opt 2006; 11: 34005.
31 Tomb JF, White O, Kerlavage AR. et al. The complete genome sequence of the gastric pathogen Helicobacter pylori. . Nature 1997; 388: 539-547.
32 Parreira VR, Gyles CL. A novel pathogenicity island integrated adjacent to the thrW tRNA gene of avian pathogenic Escherichia coli encodes a vacuolating autotransporter toxin. Infect Immun 2003; 71: 5087-5096.
33 Guyer DM, Henderson IR, Nataro JP. et al. Identification of Sat, an autotransporter toxin produced by uropathogenic Escherichia coli . Mol Microbiol 2000; 38: 53-66.
34 Ewers C, Janssen T, Kiessling S. et al. Molecular epidemiology of avian pathogenic Escherichia coli (APEC) isolated from colisepticemia in poultry. Vet Microbiol 2004; 104: 91-101.
35 Cover TL, Puryear W, Perez-Perez GI. et al. Effect of urease on HeLa cell vacuolation induced by Helicobacter pylori cytotoxin. Infect Immun 1991; 59: 1264-70.
36 Bowman EJ, Siebers A, Altendorf K. Bafilomycins: a class of inhibitors of membrane ATPases from micro-organisms, animal cells, and plant cells. Proc Natl Acad Sci USA 1988; 85: 7972-7976.
37 Papini E, Bugnoli M, De Bernard M. et al. Bafilomycin A1 inhibits Helicobacter pylori-induced vacuolization of HeLa cells. Mol Microbiol 1993; 07: 323-327.
38 Szabò I, Brutsche S, Tombola F. et al. Formation of anion-selective channels in the cell plasma membrane by the toxin VacA of Helicobacter pylori is required for its biological activity. EMBO J 1999; 18: 5517-5527.
39 Cover TL, Blanke SR. Helicobacter pylori VacA, a paradigm for toxin multifunctionality. Nat Rev Microbiol 2005; 03: 320-332.
40 Holtzman E. Lysosomes. Springer Verlag; 2007.
41 Salvadori MR, Yano T, Carvalho HE. et al. Vacuolating cytotoxin produced by avian pathogenic Escherichia coli . Avian Dis 2001; 45: 43-51.
42 Guyer DM, Radulovic S, Jones FE. et al. Sat, the secreted autotransporter toxin of uropathogenic Escherichia coli, is a vacuolating cytotoxin for bladder and kidney epithelial cells. Infect Immun 2002; 70: 4539-4546.
43 Mitra R, Figueroa P, Mukhopadhyay AK. et al. Cell vacuolation a manifestation of the El Tor hemolysin of Vibrio cholerae . Infect. Immun 2000; 68: 1928-1933.
44 Hertle R, Hilger M, Weingardt-Kocher S. et al. Cytotoxic action of Serratia marcescens hemolysin on human epithelial cells. Infect Immun 1999; 67: 817-825.
45 Abrami L, Fivaz M, Glauser PE. et al. A pore-forming toxin interacts with a GPI-anchored protein and causes vacuolation of the endoplasmic reticulum. J Cell Biol 1998; 140: 525-540.
46 Morinaga N, Yahiro K, Matsuura G. et al. Two distinct cytotoxic activities of subtilase cytotoxin produced by shiga-toxigenic Escherichia coli . Infect Immun 2007; 75: 488-496.
47 Papini E, de Bernard M, Milia E. et al. Cellular vacuoles induced by Helicobacter pylori originate from late endosomal compartments. Proc Natl Acad Sci USA 1994; 91: 9720-9724.
48 Molinari M, Galli C, Norais N. et al. Vacuoles induced by Helicobacter pylori toxin contain both late endosomal and lysosomal markers. J Biol Chem 1997; 272: 25339-25344.
49 Reading NC, Torres AG, Kendall MM. et al. A novel two-component signaling system that activates transcription of an enterohemorrhagic Escherichia coli effector involved in remodeling of host actin. J Bacteriol 2007; 189: 2468-2476.
50 Guimarães MS, Andrade JR, Freitas-Almeida AC. et al. Aeromonas hydrophila vacuolating activity in the Caco-2 human enterocyte cell line as a putative virulence factor. FEMS Microbiol Lett 2002; 207: 127-131.
51 Kang G, Pulimood AB, Koshi R. et al. A monkey model for enterohemorrhagic Escherichia coli infection. J Infect Dis 2001; 184: 206-210.