Novel path to apoptosis: small transmembrane pores created by staphylococcal alpha-toxin in T lymphocytes evoke internucleosomal DNA degradation

Abstract

Peripheral-blood human T lymphocytes were treated with Staphylococcus aureus alpha-toxin. Membrane permeabilization was assessed by measuring elilux of K+ and Rb+ and influx of Na+, Ca2+, and propidium iodide. Cellular ATP and [3H]thymidine incorporation following lectin stimulation were measured as parameters for cell viability. Internucleosomal cleavage characteristic of programmed cell death was assessed by agarose gel electrophoresis and by quantifying low-molecular-weight, [3H]thymidine-labeled DNA fragments. Nanomolar concentrations of alpha-toxin evoked protracted, irreversible ATP depletion in both activated and resting T lymphocytes. Toxin-damaged cells also lost their ability to incorporate [3H] thymidine upon subsequent stimulation with phytohemagglutinin. These cells carried toxin hexamers, and their plasma membranes became permeable for monovalent ions but not for Ca2+ and propidium iodide. The permeabilization event was followed by internucleosomal DNA degradation characteristic of programmed cell death. Membranes of cells treated with high toxin doses (>300 nM) became permeable to both Ca2+ and propidium iodide. In this case, ATP depletion occurred within minutes and no DNA degradation was observed. When cells were suspended in Na+-free buffer, alpha-toxin applied at low doses still bound and formed hexamers. However, these cells displayed neither DNA degradation nor loss of viability. The data indicate that formation of very small but not of large alpha-toxin pores may trigger programmed cell death in lymphocytes and that uncontrolled flux of Na+ ions may be an important event precipitating the suicide cascade. Alpha-toxin, the major cytolysin of Staphylococcus aureus (3, 7), damages cells by generating pores in the plasma membrane (7, 11, 27, 30). At low concentrations, the 34-kDa toxin monomer binds to susceptible cells by interaction with as-yetunidentified acceptor molecules on the membrane. At high concentrations (>200 nM; 6 jig/ml), binding additionally occurs via nonspecific absorption to the lipid bilayer (9, 16). In rabbit erythrocyte membranes, both modes of binding result in the formation of hexameric pores with an effective diameter of 1 to 2 nm (7, 11). Aqueous pores of similar size have been produced through the action of alpha-toxin on liposomes (17) and planar lipid membranes (2, 20) in endothelial cells (25), polymorphonuclear granulocytes (26), platelets (6), and neuroendocrine cells (1). It is therefore generally assumed that, once formed, alpha-toxin pores will display effective diameters of 1 to 2 nm, allowing passage of mono-and divalent ions and of small macromolecules, including ATP, with molecular weights of .1,000 to 2,000 (7). Recent studies in our laboratories have disclosed, however, that this original concept of homogeneous alpha-toxin pores requires revision. Thus, pores forming in keratinocytes at low toxin doses were found to permit passage of monovalent ions but not of Ca2+ or larger marker molecules such as propidium

Alpha‐toxin is produced by skin colonizing Staphylococcus aureus and induces a T helper type 1 response in atopic dermatitis

“…The induction of apoptosis in T cells by a-toxin has been described earlier. Consistent with our results, T cells underwent apoptosis after incubation with subcytolytic a-toxin doses exceeding 100 ng/ mL [20,40].…”

“…In keratinocytes, TNF-a is released simultaneously to lysis [22]. Besides this well described mechanism of lysis, a-toxin may induce apoptosis in T lymphocytes [20]. In the present study, we detected a-toxin in lesional skin of AD patients.…”

“…A distinct percentage of S. aureus strains are able to produce a toxin, a potent 33 000 Da cytolysin which does not belong to the group of enterotoxins (superantigens) [19]. A wide range of human cells has been shown to be prone to lysis by a-toxin with greatly varying susceptibility, among them keratinocytes and lymphocytes [20,21]. In keratinocytes, TNF-a is released simultaneously to lysis [22].…”

Staphylococcus aureus α-toxin induces apoptosis in endothelial cells

“…Use of K-toxin concentrations between 5 and 20 nM consistently induced apoptosis in endothelial cell monolayers; however, use of higher concentrations caused lysis of cells or necrosis. Jonas et al hypothesized that lower concentrations of K-toxin produced pores of small diameter ( 6 1^2 nm) that would allow selective monovalent ion £uxes which could trigger programmed cell death; conversely, higher K-toxin concentrations would produce larger pores allowing ATP depletion and subsequent necrotic cell death [11]. Their ¢ndings are consistent with our observations in K-toxintreated endothelial cells.…”

“…Indeed, in bovine endothelial cells, cytotoxicity as measured by loss of membrane integrity was associated with presumed intracellular expression of Ktoxin by a high K-toxin-producing strain [10]. However, another mechanism of cell death by K-toxin, apoptosis, was demonstrated in lymphocytes treated with low concentrations of K-toxin [11]. In that investigation, this poreforming toxin appeared to induce DNA fragmentation at low concentrations and necrosis at higher concentrations.…”