Characterization of Actinobacillus actinomycetemcomitans leukotoxin pore formation in HL60 cells

Abstract

The mechanism of cell death induced by Actinobacillus actinomycetemcomitans leukotoxin (LTX) has been investigated with flow cytometry and patch electrode recording using cultured HL60 cells. The kinetics of propidium iodide (PI) positive staining of HL60 cells was measured as a function of LTX concentration at 37°C. Results showed a concentration-dependent decrease in the t(k) times. Cell kill was slow at <1 μg/ml LTX concentrations with fewer than 50% of the cells killed after 1 h; at 1 μg/ml, the t(k) times ranged from ~15 to 30 min. At higher concentrations, the t(k) times decreased rapidly. The rate of cell kill was appreciably slowed at 20°C. HL60 whole cell currents were recorded with patch electrodes. Immediately following exposure to high concentrations of LTX, large currents were recorded suggesting that the membrane potential of these cells had collapsed due to the large conductance increases. At low toxin concentrations, rapid conductance fluctuations were seen suggestive of a limited number of toxin-mediated events. Cells exposed to low concentrations of LTX exhibited these conductance fluctuations for up to 1 h, whereas toxin-insensitive cells were unaffected by long exposures to high concentrations of toxin. Our results are consistent with LTX-induced pores in susceptible cells which overwhelm the ability of the cell to maintain osmotic homeostasis causing cell death.