Scalable Reporter Assays to Analyze the Regulation of stx2 Expression in Shiga Toxin-Producing Enteropathogens

Stx2 is the major virulence factor of EHEC and is associated with an increased risk for HUS in infected patients. The conditions influencing its expression in the intestinal tract are largely unknown. For optimal management and treatment of infected patients, the identification of environmental conditions modulating Stx2 levels in the human gut is of central importance. In this study, we established a set of chromosomal stx2 reporter assays. One system is based on superfolder GFP (sfGFP) using a T7 polymerase/T7 promoter-based amplification loop. This reporter can be used to analyze stx2 expression at the single-cell level using FACSs and fluorescence microscopy. The other system is based on the cytosolic release of the Gaussia princeps luciferase (gluc). This latter reporter proves to be a highly sensitive and scalable reporter assay that can be used to quantify reporter protein in the culture supernatant. We envision that this new set of reporter tools will be highly useful to comprehensively analyze the influence of environmental and host factors, including drugs, small metabolites and the microbiota, on Stx2 release and thereby serve the identification of risk factors and new therapies in Stx-mediated pathologies.

Automated summary

This study established a set of chromosomal stx2 reporter assays to investigate the expression and regulation of Stx2 in the gut, utilizing two systems based on sfGFP and gluc for detection at single-cell level or in culture supernatant. This new reporter tools are envisioned to be highly useful in analyzing the impact of environmental and host factors on Stx2 release and identifying risk factors and new therapies in Stx-mediated pathologies.