Shiga toxin translocation across human intestinal epithelium in a microaerobic infection model

Lead Research Organisation: University of East Anglia
Department Name: Norwich Medical School

Abstract

Shiga toxin-producing E. coli bacteria (STEC) are a leading cause of foodborne illness worldwide. In humans, STEC infection often causes severe bloody diarrhoea and abdominal cramps. In some persons, particularly children under five years of age and the elderly, the infection can also cause a complication called haemolytic uraemic syndrome (HUS), in which red blood cells are destroyed and small blot clots develop which can lead to kidney damage and failure. The HUS fatality rate is around 5%, and up to 40% of HUS patients suffer from long-term renal dysfunction. So far, there is no specific medication available, and treatment is merely supportive involving dialysis and blood transfusion.

After ingestion via contaminated food or water, STEC bacteria adhere to the intestinal lining and produce a toxin (Shiga toxin), which moves from the gut to the kidneys to cause HUS. It is not known how this happens as there are no appropriate experimental model systems available to study this process.
We have recently developed a human gut cell culture system which enables us to study STEC infections at reduced oxygen levels. Usually, such kind of infection experiments are performed in air (which contains 20% oxygen) to keep the host cells alive. However, the environment in the gut only contains around 1-5% oxygen which (amongst other factors) has been shown to provide a signal to the bacteria that they have reached their final destination. Results from our previous study have confirmed this for STEC bacteria and have shown that low oxygen levels increase the production of bacterial factors which promote colonisation of the gut wall. In addition, reduced oxygen levels also lead to the production of bacterial components which increase toxin movement across the gut wall.

In this application, we propose to continue these studies to determine (1) how the toxin crosses the gut barrier, (2) how the bacteria facilitate this process and which bacterial factors are switched on by low oxygen levels, and (3) whether STEC bacteria which produce high amounts of toxin and/or are good at moving toxin across the gut wall, cause more serious disease in humans.

The information obtained by the proposed study will help understand the early events that occur in the human gut after STEC infection and might contribute to the development of treatment strategies against HUS and STEC vaccine development.

Technical Summary

Shiga toxin-producing E. coli (STEC) are foodborne pathogens that cause bloody diarrhoea and severe systemic complications such as haemolytic uraemic syndrome (HUS). HUS is the leading cause of acute renal failure in children and associated with production of bacterial Shiga toxins (Stx), which are highly cytotoxic to renal microvascular endothelium. After ingestion via contaminated food or water, STEC colonise the intestinal epithelium but do not invade. Therefore, Stx has to cross the intestinal epithelial barrier to get access into the bloodstream. The mode of Stx translocation across human intestinal epithelium, which lacks the Stx receptor (Gb3) and is resistant to Stx cytotoxicity, is unknown. Progress has been hampered by the absence of a suitable model for experimental study.

We have recently established an experimental system based on polarised monolayers of Gb3-negative human colon carcinoma cells in a vertical diffusion chamber (VDC) which allows to perform infections under microaerobic (MA) conditions. Most cell culture infection models use atmospheric oxygen levels to maintain cell viabilty, but this is unlike the intestinal milieu where oxygen is restricted. Using the VDC system, we have shown that microaerobiosis enhances STEC type III secretion and colonisation and also promotes Stx2 translocation during STEC infection. As this latter effect was only observed in the context of infection and not with purified Stx2 alone, these results lead to the hypothesis that MA conditions induce STEC factors which promote Stx2 translocation across human intestinal epithelium.

In this application, we propose to continue the MA VDC studies to (1) characterise the Stx transport pathway during MA infection, (2) to identify STEC factors which are induced during MA infection and test their involvement in Stx2 translocation, and (3) to examine whether Stx2 production and translocation during infection are linked to human virulence.

Planned Impact

This proposal falls within the MRC priority area of emerging pathogens and mainly addresses the "Improvement to health and life quality" remit of the MRC. STEC have only been linked to haemorrhagic colitis and HUS since 1983, and epidemiological studies show a considerable increase in HUS rates associated with more recent outbreaks (from 5 to more than 33% as observed in the current STEC outbreak in northern Germany) indicating the emergence of highly virulent strains. HUS usually requires hospitalisation for two weeks with dialysis and blood transfusion, and 40% of HUS patients suffer from chronic, irreversible renal dysfunction. Therefore, STEC infections are not only associated with considerable economic costs but also lead to long-term reduction of life quality.
Despite the severity of HUS, there is still no specific treatment available, and events during STEC infection of the gut which lead to systemic disease remain poorly understood. By addressing this knowledge gap, results from this work are likely to benefit HUS patients and individuals infected with STEC or at risk of STEC infection in the long term by contributing information towards treatment strategies, vaccine development, and diagnosis.

(1) Characterisation of cellular transport mechanisms involved in Stx uptake and translocation could lead to the identification of drug targets for blocking toxin access into the bloodstream and thereby provide early therapeutic intervention strategies for HUS.

(2) Identification of STEC virulence factors involved in intestinal colonisation and/or Stx transport could identify candidate proteins for vaccine design and, as outlined in (1), lead to HUS-specific therapies.

(3) Comparison of infection and Stx production/transport characteristics of STEC strains with different virulence potential for humans could lead to the identification of select virulence markers for highly pathogenic strains which will be useful in predicting virulence potential of new isolates.

As this project is basic research, most of the outlined possibilities for impact are on a long-term scale and will need further research beyond the duration of the project. However, part 1 which will be carried out in the first year of the project, has the potential to lead to the identification of drug targets which could then be tested in vitro during the lifetime of the project (see pathways to impact).

Publications

10 25 50
 
Description Doctoral Training Partnership (DTP)
Amount £92,000 (GBP)
Funding ID SCHÜLLER_F16FDTP2 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2016 
End 09/2020
 
Description Doctoral Training Partnership (DTP)
Amount £92,000 (GBP)
Funding ID SCHULLER_F14DTP 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2014 
End 09/2018
 
Title Isolation of EHEC mRNA from infected cells for RNA sequencing 
Description In this project, we have established protocols and methods for isolation of high quality EHEC mRNA from infected intestinal epithelial cells for transcriptomic analysis by RNA sequencing. This included enrichment and stabilisation of bacterial RNA by differential lysis, isolation of high-quality bacterial RNA, and depletion of ribosomal RNA. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact Enables use of RNA-seq to determine EHEC gene expression during intestinal infection. RNA-seq provides a greater dynamic range and sensitivity of transcriptomic profile compared with microarray analysis. 
 
Title Isolation of EHEC outer membrane vesicles 
Description Establishment of protocol to isolate outer membrane vesicles (OMVs) from EHEC during human intestinal infection 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact Development of this method has led to pilot data resulting in a successful DTP studentship application (Bacterial bombs or stress response - how does the environment influence production and function of bacterial outer membrane vesicles?, 2016-2020) and an MRC project application (Cross-talk between outer membrane vesicles from enterohaemorrhagic E. coli and human colonic epithelium, shortlisted for IIB meeting but not funded). 
 
Description Association of Shiga toxin with outer membrane vesicles (OMVs) 
Organisation Quadram Institute Bioscience
Country United Kingdom 
Sector Academic/University 
PI Contribution Development of labelling techniques for OMVs, cell culture experiments
Collaborator Contribution Training of PDRA and PhD student in OMV purification and quantification methods
Impact Oral presentation at VTEC meeting in Boston, 2015 BBSRC DTP studentship on influence of the intestinal environment on EHEC OMV production (2016-2020)
Start Year 2014
 
Description Collaboration with Public Health England (PHE), Claire Jenkins and Marie Chattaway 
Organisation Public Health England
Country United Kingdom 
Sector Public 
PI Contribution We are participating in a current study of EHEC O157:H7 investigating the effect of multiple copies of the Shiga toxin gene (stx) on bacterial adherence, Stx production and translocation across the intestinal epithelial cells.
Collaborator Contribution PHE is determining the Stx profile of several human and bovine EHEC isolates and providing us with strains of a certain seropathotype and Stx profile for part 3 of our study.
Impact Oral presentation at VTEC conference, Florence, May 2018 Manuscript under revisions (Shiga toxin 2 translocation across intestinal epithelium is linked to virulence of Shiga toxin-producing Escherichia coli in humans; Tran, Jenkins, Livrelli & Schüller) BBSRC DTP studentship (2014-2018), Adaptation of EAEC to the human gut, in collaboration with Dr Marie Chattaway and Prof John Wain
Start Year 2012
 
Description Collaboration with University of Auvergne 
Organisation University of Verona
Country Italy 
Sector Academic/University 
PI Contribution Characterisation of bovine STEC isolates regarding Shiga toxin production, translocation and colonisation in a microaerobic environment
Collaborator Contribution Selection and provision of Stx2a-positive bovine STEC isolates
Impact Oral presentation at VTEC conference, Florence, May 2018 Manuscript under revision (Shiga toxin 2 translocation across intestinal epithelium is linked to virulence of Shiga toxin-producing Escherichia coli in humans; Tran, Jenkins, Livrelli & Schüller) Partner in Horizon 2020 ITN application, submission Jan 2016 (one PhD studentship, £250,000) Partner in ERA-NET (Infect-ERA) application, Mar 2016
Start Year 2015
 
Description Influence of EHEC infection on intestinal mucus production 
Organisation Quadram Institute Bioscience
Department Gut Health and Food Safety
Country United Kingdom 
Sector Academic/University 
PI Contribution Supervision of Year-in-Industry student, generation of experimental data and preparation of manuscript
Collaborator Contribution Funding for Year-in-Industry student, provision of cell line and experimental support
Impact Publication - Hews CL, Tran SL, Wegmann U, Brett B, Walsham AD, Kavanaugh D, Ward NJ, Juge N, Schüller S. Cell Microbiol. 2017 19(6). doi: 10.1111/cmi.12717
Start Year 2014
 
Description Meera Unnikrishnan, Wellcome Seed Award project 
Organisation University of Warwick
Department Warwick Medical School
Country United Kingdom 
Sector Academic/University 
PI Contribution Collaborator on Wellcome Seed Award "Developing an in vitro co-culture system for investigating Clostridium difficile-host interactions". Help with establishing a microaerobic vertical diffusion chamber system. Review of manuscript draft.
Collaborator Contribution Collection of experimental data and preparation of manuscript (submitted to mBio in Feb 2018)
Impact No outputs yet.
Start Year 2015
 
Description Art display at Norfolk and Norwich University Hospital 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Exhibition of images of foodborne bacterial pathogens including E. coli and information about current research projects at the Norwich Research Park - prompted interest in transmission of foodborne pathogens and safe preparation of foods

Audience asked for more information on safe preparation of foods.
Year(s) Of Engagement Activity 2014
 
Description Norwich Science Festival Oct 2017 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Science stand at the Norwich Forum with activities for children & parents to 1) raise awareness about foodborne and gut bacteria and 2) encourage children to become microbiologists. Positive feedback from Norwich Science Festival organizing committee.
Year(s) Of Engagement Activity 2017
URL http://norwichsciencefestival.co.uk/events/my-first-day-microbiologist/
 
Description Work experience placement (Spalding Grammar School, Lincolnshire) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Hosting of work experience students in the lab for one week which prompted questions about research into E. coli and interest in pursuing a career in science.

Spalding Grammar School has asked for more visits next year and also circulated details to other schools in the area.
Year(s) Of Engagement Activity 2013,2014