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

Hideki Katayama,Yoshitomo Kusaka,Haruo Yokota,Tetsuyuki Akao,Masayasu Kojima,Osamu Nakamura,Eisuke Mekada,Eiich Mizuki

doi:10.1074/jbc.m611382200

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.

Highlights

Pathogenic bacteria produce a wide variety of protein toxins and toxin-like molecules
Among the non-insecticidal B. thuringiensis strains, we found strains that produced a novel class of inclusion proteins
Effects of Parasporin-1 on HeLa Cell Membrane Permeability— Some bacterial toxins, including B. thuringiensis Cry toxin, form pores in the plasma membranes of target cells, thereby increasing membrane permeability, Kϩ efflux, exerts its toxic effects quickly by a process that does not require the entry of parasporin-1 into the cellular cytoplasm

Summary

Introduction

Pathogenic bacteria produce a wide variety of protein toxins and toxin-like molecules. The cytotoxicity of parasporin-1 was attenuated by low extracellular concentrations of Ca2ϩ, suggesting that parasporin-1 induces excess Ca2ϩ influx, resulting in the apoptotic death of the target cell. Parasporin-1 Induces Ca2ϩ Influx and a Sustained Elevation of Intracellular Ca2ϩ Concentration in Toxin-sensitive Cells—The nature of parasporin-1 cytotoxicity, i.e. rapid action without a lag time and partial inhibition of DNA and protein synthesis, implied that parasporin-1 may affect the cells by enhancing or suppressing the levels of intracellular second messengers through the binding of this toxin to the cell surface.

Results

Conclusion