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Scalable Reporter Assays to Analyze the Regulation of stx2 Expression in Shiga Toxin-Producing Enteropathogens.
- Source :
-
Toxins [Toxins (Basel)] 2021 Jul 29; Vol. 13 (8). Date of Electronic Publication: 2021 Jul 29. - Publication Year :
- 2021
-
Abstract
- 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.
- Subjects :
- Animals
Chlorocebus aethiops
Citrobacter rodentium genetics
Citrobacter rodentium metabolism
Escherichia coli genetics
Escherichia coli metabolism
Gene Expression Regulation, Bacterial
Genes, Reporter
Green Fluorescent Proteins genetics
Green Fluorescent Proteins metabolism
Luciferases genetics
Luciferases metabolism
Vero Cells
Biological Assay
Shiga Toxin 2 genetics
Subjects
Details
- Language :
- English
- ISSN :
- 2072-6651
- Volume :
- 13
- Issue :
- 8
- Database :
- MEDLINE
- Journal :
- Toxins
- Publication Type :
- Academic Journal
- Accession number :
- 34437405
- Full Text :
- https://doi.org/10.3390/toxins13080534