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Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy
Published on: October 1, 2012
Bimodal Response to Shiga Toxin 2 Subtypes Results from Relatively Weak Binding to the Target Cell
Patrick Cherubin1, Dennis Fidler1, Beatriz Quiñones2
1Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, Florida, USA.
Abstract:
There are two major antigenic forms of Shiga toxin (Stx), Stx1 and Stx2, which bind the same receptor and act on the same target but nonetheless differ in potency. Stx1a is more toxic to cultured cells, but Stx2 subtypes are more potent in animal models. To understand this phenomenon in cultured cells, we used a system that combines flow cytometry with a fluorescent reporter to monitor the Stx-induced inhibition of protein synthesis in single cells. We observed that Vero cells intoxicated with Stx1a behave differently than those intoxicated with Stx2 subtypes: cells challenged with Stx1a exhibited a population-wide loss of protein synthesis, while cells exposed to Stx2a or Stx2c exhibited a dose-dependent bimodal response in which one subpopulation of cells was unaffected (i.e., no loss of protein synthesis). Cells challenged with a hybrid toxin containing the catalytic subunit of Stx1a and the cell-binding subunit of Stx2a also exhibited a bimodal response to intoxication, while cells challenged with a hybrid toxin containing the catalytic subunit of Stx2a and the cell-binding subunit of Stx1a exhibited a population-wide loss of protein synthesis. Other experiments further supported a primary role for the subtype of the B subunit in the outcome of host-Stx interactions. Our collective observations indicate that the bimodal response to Stx2 subtypes is due to relatively weak binding between Stx2 and the host cell that reduces the total functional pool of Stx2 in comparison to that of Stx1a. This explains, in part, the molecular basis for the differential cellular toxicity between Stx1a and Stx2 subtypes.
Insights
Shiga toxin (Stx) subtypes Stx1a and Stx2 exhibit differential cellular toxicity due to variations in their B subunits. Stx2
Area of Science:
- Microbiology
- Toxicology
- Cell Biology
Background:
- Shiga toxins (Stx) exist in two major antigenic forms, Stx1 and Stx2, with differing potencies in cell cultures versus animal models.
- Stx1a is more toxic to cultured cells, whereas Stx2 subtypes demonstrate higher potency in animal models, suggesting a differential mechanism of action.
- Understanding the molecular basis for this differential toxicity is crucial for comprehending host-pathogen interactions and developing targeted interventions.
Purpose of the Study:
- To investigate the differential cellular toxicity between Shiga toxin (Stx) subtypes Stx1a and Stx2 in cultured cells.
- To elucidate the role of toxin subunits in mediating the observed differences in protein synthesis inhibition and cellular response.
- To explain the molecular basis for the bimodal response observed with Stx2 subtypes compared to the population-wide response of Stx1a.
Main Methods:
- Utilized a combined flow cytometry and fluorescent reporter system to monitor Shiga toxin-induced inhibition of protein synthesis in single cells.
- Assessed cellular responses to Stx1a, Stx2a, Stx2c, and hybrid toxins with swapped catalytic and binding subunits.
- Analyzed dose-dependent responses and population-wide versus bimodal inhibition patterns.
Main Results:
- Stx1a induced a population-wide loss of protein synthesis in Vero cells.
- Stx2a and Stx2c exhibited a dose-dependent bimodal response, with one subpopulation of cells remaining unaffected.
- Hybrid toxin experiments indicated a primary role for the B subunit subtype in determining the host-Stx interaction outcome and cellular response.
Conclusions:
- The bimodal response to Stx2 subtypes is attributed to weaker binding between Stx2 and the host cell, reducing the functional toxin pool.
- Differential binding affinity of the B subunit is a key factor explaining the varying cellular toxicity between Stx1a and Stx2 subtypes.
- These findings provide insight into the molecular mechanisms underlying Shiga toxin's differential effects on host cells.
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