Elucidation of the mode of action of the pore-forming toxin aerolysin on T lymphomas

Date

2018-07-11

Authors

Nelson, Kimberlea Lynne

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Abstract

Aerolysin is a channel-forming protein toxin secreted by virulent Aeromonas species. The toxin binds to receptors on cells, is proteolytically activated, and then assembles into a heptameric oligomer, which inserts into the plasma membrane forming a functional channel, resulting in cell death. To further characterize these steps receptor identification, the effect of membrane domains on channel formation and the mode of cell death were investigated on T lymphomas. Screening of cell lysates for proaerolysin-binding proteins N-glycosidase and phosphatidylinositol specific phospholipase C treatment and/or purification of these proteins resulted in the identification of a group of glycosylphosphatidylinositol (GPI)-anchored proteins, which included contactin, Thy-1, and placental alkaline phosphatase. Liposomes were used to show that these proteins were receptors for aerolysin as those containing Thy1 or placental alkaline phosphatase in their membranes were at least 100-fold more sensitive to aerolysin than those without protein. Similarly, cells expressing GPI-anchored proteins were 10⁴-fold more sensitive to aerolysin than cells lacking them. This is likely the result of these proteins concentrating aerolysin on the cell surface and thus promoting oligomerization. The fact that these proteins can be localized to membrane domains known as rafts, which are enriched in sphingomyelin and cholesterol has the potential to affect oligomerization. To investigate this possibility erythrocytes and T lymphomas were treated with methyl-β-cyclodextrin, which destroys rafts by sequestering cholesterol. Raft disruption did not decrease the sensitivity of these cells to aerolysin. Similarly, aerolysin was no more active against liposomes containing placental alkaline phosphatase in raft domains than those in which the receptor was in non-raft domains. Thus raft domains do not promote channel formation by aerolysin. The mechanism of cell death was next investigated. At high toxin concentrations cell death was shown to proceed by necrosis, whereas at subnanomolar concentrations aerolysin triggers apoptosis. Using inactive aerolysin variants it was determined that apoptosis was not a result of binding to GPI-anchored proteins nor was it triggered by receptor clustering induced by oligomerization. Instead the formation of a small number of channels was shown to trigger apoptosis. Taken together these studies have helped to clarify the mode of action of aerolysin.

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Keywords

Aerolysin, Bacterial toxins, Cell death

Citation