Enterohaemorrhagic Escherichia coli outer membrane vesicles: The influence of the colonic milieu and their interaction with host cells

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

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Enterohaemorrhagic Escherichia coli (EHEC) is a major foodborne pathogen which can cause bloody diarrhoea and potentially fatal haemolytic uremic syndrome (HUS). HUS is associated with the release of Shiga toxin (Stx) by EHEC, leading to the damage of globotriaosylceramide (Gb3) expressing cells located in the vascular system, kidneys and the central nervous system. In addition to release following bacterial cell lysis, Stxs can be secreted via outer membrane vesicles (OMVs). While OMVs are constitutively produced by Gram-negative bacteria, specific growth conditions can influence vesiculation.
To successfully colonise, EHEC must survive the various hostile conditions present in the gastrointestinal tract. The production of OMVs can facilitate bacterial survival as OMVs are known to constitute a defence mechanism against harsh environmental conditions. Thus, in this investigation, the effect of various colonic environmental factors on EHEC OMV vesiculation was examined by quantifying OMV yield through different techniques. This study concluded that while simulated colonic environmental medium, which mimics various abiotic factors of the colonic milieu, and the presence of bile salts increased OMV vesiculation, no significant differences in OMV yields were detected when EHEC was grown in physiologically relevant levels of carbon dioxide and in the presence of host colonic T84 cells.
In addition to their defensive role, OMVs can also contribute to pathogenesis by transferring virulence proteins into host cells. Since EHEC is not an invasive pathogen and the colonic epithelium does not express Gb3, OMVs may provide a mechanism which allows trafficking of Stx and other virulence factors across the colonic epithelium. Therefore, the ability of the colonic epithelium to internalise and translocate EHEC OMVs was investigated. This study determined that while OMVs are internalised and subsequently trafficked to late endosomal/lysosomal compartments in non-polarised Gb3-positive colonic-derived Caco-2 cells, OMVs are transported to the basolateral membrane and subsequently released by polarised Caco-2 cells. Nevertheless, using the more physiologically relevant Gb3-negative colonic-derived T84 cell line, little OMV internalisation was exhibited. To evaluate this discrepancy in a more biologically relevant context, EHEC OMV trafficking was evaluated in human colonic organoids which demonstrated OMV translocation across the polarised cell monolayer. The trafficking of EHEC OMVs was also elucidated to be similar in renal Vero cells as that in non-polarised Caco-2 cells. Furthermore, it was elucidated that upon OMV internalisation, encapsulated Stx2 dissociates from OMVs and follows retrograde trafficking to the Golgi apparatus and the endoplasmic reticulum, eventually leading to cell death.
In summary, this PhD study has shown that different conditions of the colonic milieu can affect EHEC OMV vesiculation in different ways. Moreover, the colonic epithelium can internalise and translocate EHEC OMVs, with cytotoxic effects in target cells. This study underlines the potential role OMVs have in the development of HUS.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Medicine and Health Sciences > Norwich Medical School
Depositing User: Nicola Veasy
Date Deposited: 21 Jun 2021 10:24
Last Modified: 21 Jun 2021 10:24
URI: https://ueaeprints.uea.ac.uk/id/eprint/80303

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