Outer membrane vesicles (OMVs) carrying virulence factors of enterohemorrhagic Escherichia coli (EHEC) are assumed to play a role in the pathogenesis of life-threatening hemolytic uremic syndrome (HUS). However, it is unknown if and how OMVs, which are produced in the intestinal lumen, cross the intestinal epithelial barrier (IEB) to reach the renal glomerular endothelium, the major target in HUS. We investigated the ability of EHEC O157 OMVs to translocate across the IEB using a model of polarized Caco-2 cells grown on Transwell inserts and characterized important aspects of this process. Using unlabeled or fluorescently labeled OMVs, tests of the intestinal barrier integrity, inhibitors of endocytosis, cell viability assay, and microscopic techniques, we demonstrated that EHEC O157 OMVs translocated across the IEB. OMV translocation involved both paracellular and transcellular pathways and was significantly increased under simulated inflammatory conditions. In addition, translocation was not dependent on OMV-associated virulence factors and did not affect viability of intestinal epithelial cells. Importantly, translocation of EHEC O157 OMVs was confirmed in human colonoids thereby supporting physiological relevance of OMVs in the pathogenesis of HUS.

Centre for Epidemiology and Microbiology National Institute of Public Health Prague Czechia

Norwich Medical School University of East Anglia Norwich United Kingdom

Zobrazit více v PubMed

Aijaz S., Balda M. S., Matter K. (2006). Tight junctions: Molecular architecture and function. Int. Rev. Cytol. 248 261–298. 10.1016/S0074-7696(06)48005-0 PubMed DOI

Aldick T., Bielaszewska M., Uhlin B. E., Humpf H. U., Wai S. N., Karch H. (2009). Vesicular stabilization and activity augmentation of enterohaemorrhagic Escherichia coli haemolysin. Mol. Microbiol. 71 1496–1508. 10.1111/j.1365-2958.2009.06618.x PubMed DOI

Aldick T., Bielaszewska M., Zhang W., Brockmeyer J., Schmidt H., Friedrich A. W., et al. (2007). Hemolysin from Shiga toxin-negative Escherichia coli O26 strains injures microvascular endothelium. Microbes Infect. 9 282–290. 10.1016/j.micinf.2006.12.001 PubMed DOI

Bauwens A., Kunsmann L., Marejková M., Zhang W., Karch H., Bielaszewska M., et al. (2017b). Intrahost milieu modulates production of outer membrane vesicles, vesicle-associated Shiga toxin 2a and cytotoxicity in Escherichia coli O157:H7 and O104:H4. Environ. Microbiol. Rep. 9 626–634. 10.1111/1758-2229.12562 PubMed DOI

Bauwens A., Kunsmann L., Karch H., Mellmann A., Bielaszewska M. (2017a). Antibiotic-mediated modulations of outer membrane vesicles in enterohemorrhagic Escherichia coli O104:H4 and O157:H7. Antimicrob. Agents Chemother. 61 e00937-17. 10.1128/AAC.00937-17 PubMed DOI PMC

Bielaszewska M., Karch H. (2005). Consequences of enterohaemorrhagic Escherichia coli infection for the vascular endothelium. Thromb. Haemost. 94 312–318. 10.1160/TH05-04-0265 PubMed DOI

Bielaszewska M., Greune L., Bauwens A., Dersch P., Mellmann A., Rüter C. (2021). Virulence factor cargo and host cell interactions of Shiga toxin-producing Escherichia coli outer membrane vesicles. Methods Mol. Biol. 2291 177–205. 10.1007/978-1-0716-1339-9_8 PubMed DOI

Bielaszewska M., Marejková M., Bauwens A., Kunsmann-Prokscha L., Mellmann A., Karch H. (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. 10.1016/j.ijmm.2018.06.004 PubMed DOI

Bielaszewska M., Rüter C., Bauwens A., Greune L., Jarosch K. A., Steil D., 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:e1006159. 10.1371/journal.ppat.1006159 PubMed DOI PMC

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

Bielaszewska M., Sinha B., Kuczius T., Karch H. (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. 10.1128/IAI.73.1.552-562.2005 PubMed DOI PMC

Bittel M., Reichert P., Sarfati I., Dressel A., Leikam S., Uderhardt S., et al. (2021). Visualizing transfer of microbial biomolecules by outer membrane vesicles in microbe-host-communication in vivo. J. Extracell. Vesicles 10:e12159. 10.1002/jev2.12159 PubMed DOI PMC

Caruana J. C., Walper S. A. (2020). Bacterial membrane vesicles as mediators of microbe – microbe and microbe – host community interactions. Front. Microbiol. 11:432. 10.3389/fmicb.2020.00432 PubMed DOI PMC

Choi Y., Kwon Y., Kim D. K., Jeon J., Jang S. C., Wang T., et al. (2015). Gut microbe-derived extracellular vesicles induce insulin resistance, thereby impairing glucose metabolism in skeletal muscle. Sci. Rep. 5:15878. 10.1038/srep15878 PubMed DOI PMC

Devos S., Van Putte W., Vitse J., Van Driessche G., Stremersch S., Van Den Broek W., et al. (2017). Membrane vesicle secretion and prophage induction in multidrug-resistant Stenotrophomonas maltophilia in response to ciprofloxacin stress. Environ. Microbiol. 19 3930–3937. 10.1111/1462-2920.13793 PubMed DOI

Díaz-Garrido N., Badia J., Baldomà L. (2021). Microbiota-derived extracellular vesicles in interkingdom communication in the gut. J. Extracell. Vesicles 10:e12161. 10.1002/jev2.12161 PubMed DOI PMC

Fitzpatrick M. M., Shah V., Trompeter R. S., Dillon M. J., Barratt T. M. (1992). Interleukin-8 and polymorphoneutrophil leucocyte activation in hemolytic uremic syndrome of childhood. Kidney Int. 42 951–956. 10.1038/ki.1992.372 PubMed DOI

Friedrich A. W., Lu S., Bielaszewska M., Prager R., Bruns P., Xu J. G., et al. (2006). Cytolethal distending toxin in Escherichia coli O157:H7: Spectrum of conservation, structure, and endothelial toxicity. J. Clin. Microbiol. 44 1844–1846. 10.1128/JCM.44.5.1844-1846.2006 PubMed DOI PMC

Ghaffarian R., Muro S. (2013). Models and methods to evaluate transport of drug delivery systems across cellular barriers. J. Vis. Exp. 80:50638. 10.3791/50638 PubMed DOI PMC

Griffin P. M., Olmstead L. C., Petras R. E. (1990). Escherichia coli O157:H7-associated colitis. A clinical and histological study of 11 cases. Gastroenterology 99 142–149. 10.1016/0016-5085(90)91241-w PubMed DOI

Haas-Neill S., Forsythe P. (2020). A budding relationship: Bacterial extracellular vesicles in the microbiota-gut-brain axis. Int. J. Mol. Sci. 21:8899. 10.3390/ijms21238899 PubMed DOI PMC

Hendrix A., De Wever O. (2022). Systemically circulating bacterial extracellular vesicles: Origin, fate, and function. Trends Microbiol. 30 213–216. 10.1016/j.tim.2021.12.012 PubMed DOI

Hidalgo I. J., Raub T. J., Borchardt R. T. (1989). Characterization of the human colon carcinoma cell line (Caco-2) as a model system for intestinal epithelial permeability. Gastroenterology 96 736–749. PubMed

Hurley B. P., Pirzai W., Eaton A. D., Harper M., Roper J., Zimmermann C., et al. (2016). An experimental platform using human intestinal epithelial cell lines to differentiate between hazardous and non-hazardous proteins. Food Chem. Toxicol. 92 75–87. 10.1016/j.fct.2016.04.003 PubMed DOI

Hurley B. P., Thorpe C. M., Acheson D. W. (2001). Shiga toxin translocation across intestinal epithelial cells is enhanced by neutrophil transmigration. Infect. Immun. 69 6148–6155. 10.1128/IAI.69.10.6148-6155.2001 PubMed DOI PMC

In J. G., Foulke-Abel J., Estes M. K., Zachos N. C., Kovbasnjuk O., Donowitz M. (2016). Human mini-guts: New insights into intestinal physiology and host-pathogen interactions. Nat. Rev. Gastroenterol. Hepatol. 13 633–642. 10.1038/nrgastro.2016.142 PubMed DOI PMC

In J., Foulke-Abel J., Zachos N. C., Hansen A.-M., Kaper J. B., Bernstein H. B., et al. (2016). Enterohemorrhagic Escherichia coli reduces mucus and intermicrovillar bridges in human stem cell-derived colonoids. Cell. Mol. Gastroenterol. Hepatol. 2 48–62. 10.1016/j.jcmgh.2015.10.001 PubMed DOI PMC

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

Jones E. J., Booth C., Fonseca S., Parker A., Cross K., Miquel-Clopés A., et al. (2020). The uptake, trafficking, and biodistribution of Bacteroides thetaiotaomicron generated outer membrane vesicles. Front. Microbiol. 11:57. 10.3389/fmicb.2020.00057 PubMed DOI PMC

Karch H., Tarr P. I., Bielaszewska M. (2005). Enterohaemorrhagic Escherichia coli in human medicine. Int. J. Med. Microbiol. 295 405–418. 10.1016/j.ijmm.2005.06.009 PubMed DOI

Keepers T. R., Psotka M. A., Gross L. K., Obrig T. G. (2006). A murine model of HUS: Shiga toxin with lipopolysaccharide mimics the renal damage and physiologic response of human disease. J. Am. Soc. Nephrol. 17 3404–3414. 10.1681/ASN.2006050419 PubMed DOI

Kelly J., Oryshak A., Wenetsek M., Grabiec J., Handy S. (1990). The colonic pathology of Escherichia coli O157:H7 infection. Am. J. Surg. Pathol. 14 87–92. 10.1097/00000478-199001000-00010 PubMed DOI

Kim S. H., Lee Y. H., Lee S. H., Lee S. R., Huh J. W., Kim S. U., et al. (2011). Mouse model for hemolytic uremic syndrome induced by outer membrane vesicles of Escherichia coli O157:H7. FEMS Immunol. Med. Microbiol. 63 427–434. 10.1111/j.1574-695X.2011.00869.x PubMed DOI

Kolling G. L., Matthews K. R. (1999). Export of virulence genes and Shiga toxin by membrane vesicles of Escherichia coli O157:H7. Appl. Environ. Microbiol. 65 1843–1848. 10.1128/AEM.65.5.1843-1848.1999 PubMed DOI PMC

Konowalchuk J., Speirs J., Stavric S. (1977). Vero response to a cytotoxin of Escherichia coli. Infect. Immun. 18 775–779. 10.1128/iai.18.3.775-779.1977 PubMed DOI PMC

Kouzel I. U., Pohlentz G., Schmitz J. S., Steil D., Humpf H. U., Karch H., et al. (2017). Shiga toxin glycosphingolipid receptors in human Caco-2 and HCT-8 colon epithelial cell lines. Toxins (Basel) 9:338. 10.3390/toxins9110338 PubMed DOI PMC

Kunsmann L., Rüter C., Bauwens A., Greune L., Glüder M., Kemper B., et al. (2015). Virulence from vesicles: Novel mechanisms of host cell injury by Escherichia coli O104:H4 outbreak strain. Sci. Rep. 5:13252. 10.1038/srep13252 PubMed DOI PMC

Macia E., Ehrlich M., Massol R., Boucrot E., Brunner C., Kirchhausen T. (2006). Dynasore, a cell- permeable inhibitor of dynamin. Dev. Cell. 10 839–850. 10.1016/j.devcel.2006.04.002 PubMed DOI

Mellmann A., Bielaszewska M., Köck R., Friedrich A. W., Fruth A., Middendorf B., et al. (2008a). Analysis of collection of hemolytic uremic syndrome-associated enterohemorrhagic Escherichia coli. Emerg. Infect. Dis. 14 1287–1290. 10.3201/eid1408.071082 PubMed DOI PMC

Mellmann A., Lu S., Karch H., Xu J., Harmsen D., Schmidt M. A., et al. (2008b). Recycling of Shiga toxin 2 genes in sorbitol-fermenting enterohemorrhagic Escherichia coli O157:NM. Appl. Environ. Microbiol. 74 67–72. 10.1128/AEM.01906-07 PubMed DOI PMC

Mosmann T. (1983). Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J. Immunol. Methods 65 55–63. 10.1016/0022-759(83)90303-4 PubMed DOI

Obrig T. G. (2010). Escherichia coli Shiga toxin mechanisms of action in renal disease. Toxins (Basel) 2 2769–2794. 10.3390/toxins2122769 PubMed DOI PMC

Orench-Rivera N., Kuehn M. J. (2016). Environmentally controlled bacterial vesicle-mediated export. Cell. Microbiol. 18 1525–1536. 10.1111/cmi.12676 PubMed DOI PMC

Orlandi P. A., Fishman P. H. (1998). Filipin-dependent inhibition of cholera toxin: Evidence for toxin internalization and activation through caveolae-like domains. J. Cell. Biol. 141 905–915. 10.1083/jcb.141.4.905 PubMed DOI PMC

Park J. Y., Choi J., Lee Y., Lee J. E., Lee E. H., Kwon H. J., et al. (2017). Metagenome analysis of bodily microbiota in a mouse model of Alzheimer disease using bacteria-derived membrane vesicles in blood. Exp. Neurobiol. 26 369–379. 10.5607/en.2017.26.6.369 PubMed DOI PMC

Poritz L. S., Garver K. I., Green C., Fitzpatrick L., Ruggiero F., Koltun W. A. (2007). Loss of the tight junction protein ZO-1 in dextran sulfate sodium induced colitis. J. Surg. Res. 140 12–19. 10.1016/j.jss.2006.07.050 PubMed DOI

Richardson S. E., Karmali M. A., Becker L. E., Smith C. R. (1988). The histopathology of the hemolytic uremic syndrome associated with verocytotoxin-producing Escherichia coli infections. Hum. Pathol. 19 1102–1108. 10.1016/s0046-8177(88)80093-5 PubMed DOI

Rosales A., Hofer J., Zimmerhackl L. B., Jungraithmayr T. C., Riedl M., Giner T., et al. (2012). Need for long-term follow-up in enterohemorrhagic Escherichia coli-associated hemolytic uremic syndrome due to late-emerging sequelae. Clin. Infect. Dis. 54 1413–1421. 10.1093/cid/cis196 PubMed DOI

Roxas J. L., Koutsouris A., Bellmeyer A., Tesfay S., Royan S., Falzari K., et al. (2010). Enterohemorrhagic E. coli alters murine intestinal epithelial tight junction protein expression and barrier function in a Shiga toxin independent manner. Lab. Invest. 90 1152–1168. 10.1038/labinvest.2010.91 PubMed DOI PMC

Rueter C., Bielaszewska M. (2020). Secretion and delivery of intestinal pathogenic Escherichia coli virulence factors via outer membrane vesicles. Front. Cell. Infect. Microbiol. 10:91. 10.3389/fcimb.2020.00091 PubMed DOI PMC

Schüller S. (2011). Shiga toxin interaction with human intestinal epithelium. Toxins (Basel) 3 626–639. 10.3390/toxins3060626 PubMed DOI PMC

Schüller S., Frankel G., Phillips A. D. (2004). Interaction of Shiga toxin from Escherichia coli with human intestinal epithelial cell lines and explants: Stx2 induces epithelial damage in organ culture. Cell. Microbiol. 6 289–301. 10.1046/j.1462-5822.2004.00370.x PubMed DOI

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

Siegler R., Oakes R. (2005). Hemolytic uremic syndrome; pathogenesis, treatment, and outcome. Curr. Opin. Pediatr. 17 200–204. 10.1097/01.mop.0000152997.66070.e9 PubMed DOI

Stentz R., Carvalho A. L., Jones E. J., Carding S. R. (2018). Fantastic voyage: The journey of intestinal microbiota-derived microvesicles through the body. Biochem. Soc. Trans. 46 1021–1027. 10.1042/BST20180114 PubMed DOI PMC

Tarr P. I., Gordon C. A., Chandler W. L. (2005). Shiga-toxin-producing Escherichia coli and haemolytic uraemic syndrome. Lancet 365 1073–1086. 10.1016/S0140-6736(05)71144-2 PubMed DOI

Tulkens J., Vergauwen G., Van Deun J., Geeurickx E., Dhondt B., Lippens L., et al. (2020). Increased levels of systemic LPS-positive bacterial extracellular vesicles in patients with intestinal barrier dysfunction. Gut 69 191–193. 10.1136/gutjnl-2018-317726 PubMed DOI PMC

Tzipori S., Karch H., Wachsmuth K. I., Robins-Browne R. M., O’Brien A. D., Lior H., et al. (1987). Role of a 60-megadalton plasmid and Shiga-like toxins in the pathogenesis of infection caused by enterohemorrhagic Escherichia coli O157:H7 in gnotobiotic piglets. Infect. Immun. 55 3117–3125. 10.1128/iai.55.12.3117-3125 PubMed DOI PMC

Ugalde-Silva P., Gonzalez-Lugo O., Navarro-Garcia F. (2016). Tight junction disruption induced by type 3 secretion system effectors injected by enteropathogenic and enterohemorrhagic Escherichia coli. Front. Cell. Infect. Microbiol. 6:87. 10.3389/fcimb.2016.00087 PubMed DOI PMC

Valilis E., Ramsey A., Sidiq S., DuPont H. L. (2018). Non-O157 Shiga toxin-producing Escherichia coli-A poorly appreciated enteric pathogen: Systematic review. Int. J. Infect. Dis. 76 82–87. 10.1016/j.ijid.2018.09.002 PubMed DOI

Villageliu D. N., Samuelson D. R. (2022). The role of bacterial membrane vesicles in human health and disease. Front. Microbiol. 13:828704. 10.3389/fmicb.2022.828704 PubMed DOI PMC

Wadia J. S., Stan R. V., Dowdy S. F. (2004). Transducible TAT-HA fusogenic peptide enhances escape of TAT fusion proteins after lipid raft macropinocytosis. Nat. Med. 10 310–315. 10.1038/nm996 PubMed DOI

Wang J., Zhang C., Guo C., Li X. (2019). Chitosan ameliorates DSS-induced ulcerative colitis mice by enhancingi intestinal barrier function and improving microflora. Int. J. Mol. Sci. 20:5751. 10.3390/ijms20225751 PubMed DOI PMC

Wang L. H., Rothberg K. G., Anderson R. G. (1993). Mis-assembly of clathrin lattices on endosomes reveals a regulatory switch for coated pit formation. J. Cell. Biol. 123 1107–1117. 10.1083/jcb.123.5.1107 PubMed DOI PMC

Wang X., Wang N., Yuan L., Li N., Wang J., Yang X. (2016). Exploring tight junction alteration using double fluorescent probe combination of lanthanide complex with gold nanoclusters. Sci. Rep. 6:32218. 10.1038/srep32218 PubMed DOI PMC

Xie J., Cools L., Van Imschoot G., Van Wonterghem E., Pauwels M. J., Vlaeminck I., et al. (2023). Helicobacter pylori-derived outer membrane vesicles contribute to Alzheimer’s disease pathogenesis via C3-C3aR signalling. J. Extracell. Vesicles 12:e12306. 10.1002/jev2.12306 PubMed DOI PMC

Yara D. (2020). Enterohaemorrhagic Escherichia coli outer membrane vesicles: The influence of the colonic milieu and their interaction with host cells. Doctoral thesis, Norwich Medical School, University of East Anglia. Norwich: University of East Anglia.

Yokoyama K., Horiim T., Yamashino T., Hashikawa S., Barua S., Hasegawa 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. 10.1111/j.1574-6968.2000.tb09372.x PubMed DOI

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

Growth phase matters: Boosting immunity via Lacticasebacillus-derived membrane vesicles and their interactions with TLR2 pathways