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

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.

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.

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).