Cellular recovery from exposure to sub-optimal concentrations of AB toxins that inhibit protein synthesis

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

Submitted to: Scientific Reports
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/25/2018
Publication Date: 2/6/2018
Citation: Cherubin, P., Quinones, B., Teter, K. 2018. Cellular recovery from exposure to sub-optimal concentrations of AB toxins that inhibit protein synthesis. Scientific Reports. https://doi.org/10.1038/s41598-018-20861-9.
DOI: https://doi.org/10.1038/s41598-018-20861-9

Interpretive Summary: The AB-type protein toxins, diphtheria toxin, ricin, Shiga toxin 1 and exotoxin A, are released into the extracellular environment but attack targets within the host cytoplasm, resulting in inhibition of protein synthesis. These AB toxins initially enter the cell through receptor-mediated endocytosis and reach the cytosol via retrograde transport thru membrane-bound organelles between 30 min and 5 h after internalization from the plasma membrane. Based on extrapolations from in vitro studies with toxin serial dilutions or kinetic analyses of intoxication, it is believed that the inhibition of protein synthesis and resulting cell death can result from the delivery of a single toxin molecule to the cytosol. Dose response curves generated with AB toxins would thus reflect the probability of intoxication in a population of cells, and by this model, the half-maximal effective dose of a toxin represents an all-or-nothing condition in which half the exposed cells contain no cytosolic toxin and are therefore unaffected while the other half exhibit the full effects of intoxication. An alternative interpretation for toxin effective dose values is based on proportionality rather than probability. At the effective dose for protein synthesis inhibition, it is possible all cells in the exposed population contain an amount of cytosolic toxin that only reduces protein synthesis by 50%. According to this proportionality model, a limiting but not eliminating quantity of cytosolic toxin could protect an eukaryotic cell from the lethal outcome of intoxication. This issue has important implications for inhibitor development, as the potentially lethal effect resulting from a single molecule of cytosolic toxin would greatly limit treatment regimes that are not 100% effective or target the cell-associated toxin after first contact.

Technical Abstract: Shiga toxin 1, exotoxin A, diphtheria toxin and ricin are all AB-type protein toxins that act within the host cytosol to kill the host cell through a pathway involving the inhibition of protein synthesis. It is thought that a single molecule of cytosolic toxin is sufficient to kill the host cell. Intoxication is therefore viewed as an irreversible process. Using flow cytometry and a fluorescent reporter system to monitor protein synthesis, the present study provided evidence that a single molecule of cytosolic AB-toxin is not sufficient for complete inhibition of protein synthesis or cell death. After testing toxin concentrations at the half-maximal effective dose by flow cytometry, a population-wide downshift was observed instead of a bimodal cell population during a 50% loss of protein synthesis. Furthermore, cells exposed to 0.01 ng/mL of ricin or 0.001 ng/mL of Stx1 had higher levels of protein synthesis after a chase in toxin-free medium for 36 h when compared to 18 h of continual intoxication. These findings indicated that cells can recover from intoxication since cells with a partial loss of protein synthesis will, upon removal of the toxin, increase the level of protein production and survive the toxin challenge. Thus, in contrast to the prevailing model, ongoing toxin delivery to the cytosol appears to be required for the death of cells exposed to sub-optimal toxin concentrations. This issue has important implications for inhibitor development, as the potentially lethal effect resulting from a single molecule of cytosolic toxin would greatly limit intervention strategies that are not 100% effective.