Bimodal response to Shiga toxin 2 subtypes results from relatively weak binding to the target cell

Authors: CHERUBIN, PATRICK - University Of Central Florida; FIDDLER, DENNIS - University Of Central Florida; Quinones, Beatriz; TETER, KEN - University Of Central Florida

Submitted to: Infection and Immunity
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/7/2019
Publication Date: 11/18/2019
Citation: Cherubin, P., Fiddler, D., Quinones, B., Teter, K. 2019. Bimodal response to Shiga toxin 2 subtypes results from relatively weak binding to the target cell. Infection and Immunity. 87:e00428-19. https://doi.org/10.1128/IAI.00428-19.
DOI: https://doi.org/10.1128/IAI.00428-19

Interpretive Summary: Shiga toxin-producing Escherichia coli (STEC) strains are a major public health concern worldwide, and STEC serotype O157:H7 is frequently associated with human gastroenteritis outbreaks. STEC infections can range from mild to life-threatening conditions such as the hemolytic-uremic syndrome. Shiga toxins are a key virulence factor contributing to the development of severe disease symptoms in humans. To examine the effects of Shiga toxins, most quantitative assays monitor the Shiga toxin-induced inhibition of protein synthesis by averaging the results from a population of human cells. Using these methods, it is not possible to differentiate between intoxicated and unintoxicated human cells within the population. As an alternative approach, our study used flow cytometry in conjunction with a fluorescent mammalian cell line (Vero-d2EGFP) that expresses a variant of the enhanced green fluorescent protein (EGFP) with a short half-life. This cell line allowed us to quantify the Shiga toxin-induced inhibition of protein synthesis and resulting loss of EGFP fluorescence from individual cells within the population of toxin-challenged cells. The use of a cytofluorometry-based intoxication assay allowed us, for the first time, to document the bimodal population response to Shiga toxin 2 subtypes that appears to result from the relatively low affinity of toxin binding to the human cell surface. These findings provide a new basis to better understand the differential toxicity between Shiga toxin subtypes and establish an innovative conceptual foundation for the development of post-exposure toxin therapeutics that function by lowering the amount of toxin that reaches the internal (intracellular) compartments in human cells.

Technical 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. Stx1 is more toxic to cultured cells, but Stx2 subtypes are more potent in animal models. To understand this phenomenon in cultured mammalian 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 Stx1 behave differently than those intoxicated with Stx2: cells challenged with Stx1 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 and resulted in no loss of protein synthesis. Cells challenged with a hybrid toxin containing the catalytic subunit of Stx1 and the cell receptor-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 receptor-binding subunit of Stx1 exhibited a population-wide loss of protein synthesis. Other experiments further supported a primary role for the original subtype of the B subunit in the outcome of host cell-Stx interactions. Our collective observations indicate that the bimodal cellular response to Stx2 subtypes is due to relatively weak affinity between Stx2 and the host cell receptor that reduces the total cell-associated pool of Stx2 in comparison to Stx1. These findings provide the compelling molecular evidence for the differential cellular toxicity between Stx1 and Stx2 subtypes in cultured mammalian cells.