title: 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.
abstract: 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 toxin-sensitive 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-1-treated cells. Parasporin-1 decreased the level of cellular protein and DNA synthesis in parasporin-1-sensitive HeLa cells. The earliest change observed in cells treated with this toxin was a rapid elevation of the intracellular free-Ca(2+) concentration; increases in the intracellular Ca(2+) 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 Ca(2+) concentration. The toxin-induced elevation of the intracellular Ca(2+) concentration was markedly decreased in low-Ca(2+) buffer and was not observed in Ca(2+)-free buffer. Accordingly, the cytotoxicity of parasporin-1 decreased in the low-Ca(2+) buffer and was restored by the addition of Ca(2+) to the extracellular medium. Suramin, which inhibits trimeric G-protein signaling, suppressed both the Ca(2+) influx and the cytotoxicity of parasporin-1. In parasporin-1-treated HeLa cells, degradation of pro-caspase-3 and poly(ADP-ribose) 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 Ca(2+) level and that the Ca(2+) influx is the first step in the pathway that underlies parasporin-1 toxicity.
authors.affiliation: Biotechnology and Food Research Institute, Fukuoka Industrial Technology Center, Aikawa, Kurume, Fukuoka 839-0861, Japan. hkatayam@fitc.pref.fukuoka.jp
authors.forename: Hideki
authors.initials: H
authors.lastname: Katayama
authors.forename: Yoshitomo
authors.initials: Y
authors.lastname: Kusaka
authors.forename: Haruo
authors.initials: H
authors.lastname: Yokota
authors.forename: Tetsuyuki
authors.initials: T
authors.lastname: Akao
authors.forename: Masayasu
authors.initials: M
authors.lastname: Kojima
authors.forename: Osamu
authors.initials: O
authors.lastname: Nakamura
authors.forename: Eisuke
authors.initials: E
authors.lastname: Mekada
authors.forename: Eiich
authors.initials: E
authors.lastname: Mizuki
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D001413:Bacillus thuringiensis / Q000472:pathogenicity*; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002463:Cell Membrane Permeability / Q000187:drug effects; D003593:Cytoplasm / Q000378:metabolism*; D004247:DNA / Q000096:biosynthesis; D004731:Endotoxins / Q000494:pharmacology*; D006367:HeLa Cells; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology; D014176:Protein Biosynthesis / Q000187:drug effects; D013498:Suramin / Q000494:pharmacology
chemical_list: D004731:Endotoxins; C412914:parasporin; D000001:Calcimycin; D015759:Ionomycin; D013498:Suramin; D004247:DNA; D002118:Calcium