Related Experiment Video
Updated: Aug 7, 2026
Detection of Toxin Translocation into the Host Cytosol by Surface Plasmon Resonance
Published on: January 3, 2012
Probing the surface of eukaryotic cells using combinatorial toxin libraries
M R Bray1, S Bisland, S Perampalam
1Ontario Cancer Institute, Princess Margaret Hospital Rm. 7-117, 610 University Avenue, Ontario, M5G 2M9, Toronto, Canada.
Abstract:
The success of proteomics hinges in part on the development of approaches able to map receptors on the surface of cells. One strategy to probe a cell surface for the presence of internalized markers is to make use of Shiga-like toxin 1 (SLT-1), a ribosome-inactivating protein that kills eukaryotic cells [1, 2]. SLT-1 binds to the glycolipid globotriaosylceramide [3, 4], which acts as a shuttle, allowing the toxin to be imported and routed near ribosomes. We investigated the use of SLT-1 as a structural template to create combinatorial libraries of toxin variants with altered receptor specificity. Since all SLT-1 variants retain their toxic function, this property served as a search engine enabling us to identify mutants from these libraries able to kill target cells expressing internalizable receptors. Random mutations were introduced in two discontinuous loop regions of the SLT-1 receptor binding subunit. Minimal searches from screening 600 bacterial colonies randomly picked from an SLT-1 library identified toxin mutants able to kill cell lines resistant to the wild-type toxin. One such mutant toxin was shown to bind to a new receptor on these cell lines by flow cytometry. Toxin libraries provide a strategy to delineate the spectrum of receptors on eukaryotic cells.
Insights
Researchers engineered Shiga-like toxin 1 (SLT-1) variants to identify new cell surface receptors. This toxin library approach successfully discovered mutants targeting previously unknown receptors on resistant cells.
Area of Science:
- Proteomics
- Cell Biology
- Biochemistry
Background:
- Mapping cell surface receptors is crucial for proteomics.
- Shiga-like toxin 1 (SLT-1) is a ribosome-inactivating protein that binds globotriaosylceramide and internalizes into cells.
- SLT-1's toxic function can be leveraged to identify cells expressing specific receptors.
Purpose of the Study:
- To utilize SLT-1 as a template for creating combinatorial toxin libraries with modified receptor specificities.
- To develop a method for identifying toxin variants capable of binding to novel cell surface receptors.
Main Methods:
- Introduction of random mutations into two discontinuous loop regions of the SLT-1 receptor binding subunit.
- Creation of combinatorial libraries of SLT-1 variants.
- Screening of bacterial colonies from the SLT-1 library to identify mutants with altered receptor specificity.
- Utilizing the inherent toxicity of SLT-1 variants as a selection mechanism.
- Flow cytometry to confirm binding of mutant toxins to new receptors.
Main Results:
- Identification of SLT-1 toxin mutants capable of killing cell lines resistant to the wild-type toxin.
- One identified mutant toxin demonstrated binding to a novel receptor on resistant cell lines.
- The study successfully generated toxin libraries to probe for cell surface receptors.
Conclusions:
- Combinatorial toxin libraries based on SLT-1 provide an effective strategy for discovering and characterizing cell surface receptors.
- This approach enables the delineation of the spectrum of receptors present on eukaryotic cells.
- The methodology can be applied to identify receptors with altered specificities, expanding our understanding of cell-surface interactions.
