Shiga toxin production and translocation during microaerobic human colonic infection with Shiga toxin-producing E. coli O157:H7 and O104:H4.
Bottom Line: Haemolytic uraemic syndrome caused by Shiga toxin-producing E. coli (STEC) is dependent on release of Shiga toxins (Stxs) during intestinal infection and subsequent absorption into the bloodstream.An understanding of Stx-related events in the human gut is limited due to lack of suitable experimental models.Whereas microaerobiosis significantly reduced bacterial growth as well as Stx production and release into the medium, Stx translocation across the epithelial monolayer was enhanced under MA versus aerobic conditions.
Affiliation: Norwich Medical School, University of East Anglia, Norwich Research Park, Norwich, UK; Gut Health and Food Safety Programme, Institute of Food Research, Norwich Research Park, Norwich, UK.Show MeSH
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Mentions: While the VCCA represents the most sensitive assay to determine Stx levels according to toxin activity (cytotoxicity on Vero cells) it cannot distinguish between Stx1 and Stx2. This is important as EDL933 produces both toxin types. In addition, Stx activity and actual protein levels might not be directly correlated, and decreased Stx cytotoxicity in MA supernatants might be due to loss of activity rather than diminished toxin release. To quantify Stx protein levels in apical supernatants, Stx1- and Stx2-specific sandwich ELISAs were developed and applied. As before, Stx concentrations in supernatants were adjusted to OD 0.1 to account for different bacterial densities in apical media. As shown in Fig. 4A and B, both Stx1 and Stx2 release were significantly reduced under MA versus AE conditions. In addition, Stx2 was the predominant toxin type released into the medium under both AE and MA conditions (98% and 96% of total Stx respectively). This was corroborated by VCCA neutralization assays where addition of anti-Stx2 abrogated more than 95% of cytotoxicity from apical supernatants whereas addition of anti-Stx1 did not show any detectable effect (Fig. S1).
Affiliation: Norwich Medical School, University of East Anglia, Norwich Research Park, Norwich, UK; Gut Health and Food Safety Programme, Institute of Food Research, Norwich Research Park, Norwich, UK.