Shiga toxin sub-type 2a increases the efficiency of Escherichia coli O157 transmission between animals and restricts epithelial regeneration in bovine enteroids.

Specific Escherichia coli isolates lysogenised with prophages that express Shiga toxin (Stx) can be a threat to human health, with cattle being an important natural reservoir. In many countries the most severe pathology is associated with enterohaemorrhagic E. coli (EHEC) serogroups that express Stx subtype 2a. In the United Kingdom, phage type (PT) 21/28 O157 strains have emerged as the predominant cause of life-threatening EHEC infections and this phage type commonly encodes both Stx2a and Stx2c toxin types. PT21/28 is also epidemiologically linked to super-shedding (>10(3) cfu/g of faeces) which is significant for inter-animal transmission and human infection as demonstrated using modelling studies. We demonstrate that Stx2a is the main toxin produced by stx2a(+)/stx2c(+) PT21/28 strains induced with mitomycin C and this is associated with more rapid induction of gene expression from the Stx2a-encoding prophage compared to that from the Stx2c-encoding prophage. Bacterial supernatants containing either Stx2a and/or Stx2c were demonstrated to restrict growth of bovine gastrointestinal organoids with no restriction when toxin production was not induced or prevented by mutation. Isogenic strains that differed in their capacity to produce Stx2a were selected for experimental oral colonisation of calves to assess the significance of Stx2a for both super-shedding and transmission between animals. Restoration of Stx2a expression in a PT21/28 background significantly increased animal-to-animal transmission and the number of sentinel animals that became super-shedders. We propose that while both Stx2a and Stx2c can restrict regeneration of the epithelium, it is the relatively rapid and higher levels of Stx2a induction, compared to Stx2c, that have contributed to the successful emergence of Stx2a+ E. coli isolates in cattle in the last 40 years. We propose a model in which Stx2a enhances E. coli O157 colonisation of in-contact animals by restricting regeneration and turnover of the colonised gastrointestinal epithelium. Author summary Enterohaemorrhagic E. coli (EHEC) O157 strains are found in cattle where they are asymptomatic, while human exposure can lead to severe symptoms including bloody diarrhoea and kidney damage due to the activity of Shiga toxin (Stx). The most serious symptoms in humans are associated with isolates that encode Stx subtype 2a. The advantage of these toxins in the animal reservoir is still not clear, however there is experimental evidence implicating Stx with increased bacterial adherence, immune modulation and suppression of predatory protozoa. In this study, the hypothesis that Stx2a is important for super-shedding and calf-to-calf transmission was tested by comparing excretion and transmission dynamics of E. coli O157 strains with and without Stx2a. While Stx2a did not alter excretion levels when calfs were orally challenge, it enabled colonisation of more in contact 'sentinel' animals in our transmission model. We show that Stx2a is generally induced more rapidly than Stx2c, resulting in increased levels of Stx2a expression. Both Stx2a and Stx2c were able to restrict cellular proliferation of epithelial cells in cultured bovine enteroids. Taken together, we propose that rapid production of Stx2a and its role in establishing E. coli O157 colonisation in the bovine gastrointestinal tract facilitate effective transmission and have led to its expansion in the cattle E. coli O157 population.