Parasporin-1, a novel cytotoxic protein from Bacillus thuringiensis, induces Ca2+ influx and a sustained elevation of the cytoplasmic Ca2+ concentration in toxin-sensitive cells

Parasporin-1 is a novel non-insecticidal inclusion protein from Bacillus thuringiensis that is cytotoxic to specific mammalian cells. In this study, we investigated the effects of parasporin-1 on toxinsensitive cell lines to elucidate the cytotoxic mechanism of parasporin-1. Parasporin-1 is not a membrane pore-forming toxin as evidenced by measurements of lactate dehydrogenase release, propidium iodide penetration, and membrane potential in parasporin-l-treated cells. Parasporin-1 decreased the level of cellular protein and DNA synthesis in parasporin-l-sensitive HeLa cells. The earliest change observed in cells treated with this toxin was a rapid elevation of the intracellular free-Ca2+ concentration; increases in the intracellular Ca2+ levels were observed 1-3 min following parasporin-1 treatment. Using four different cell lines, we found that the degree of cellular sensitivity to parasporin-1 was positively correlated with the size of the increase in the intracellular Ca2+ concentration. The toxin-induced elevation of the intracellular Ca2+ concentration was markedly decreased in low-Ca2+ buffer and was not observed in Ca2+-free buffer. Accordingly, the cytotoxicity of parasporin-1 decreased in the low-Ca2+ buffer and was restored by the addition of Ca2+ to the extracellular medium. Suramin, which inhibits trimeric G-protein signaling, suppressed both the Ca 21 influx and the cytotoxicity of parasporin-1. In parasporin-1-treated HeLa cells, degradation of pro-caspase-3 and poly(ADPribose) polymerase was observed. Furthermore, synthetic caspase inhibitors blocked the cytotoxic activity of parasporin-1. These results indicate that parasporin-1 activates apoptotic signaling in these cells as a result of the increased Ca2+ level and that the Ca2+ influx is the first step in the pathway that underlies parasporin-1 toxicity.