No abstract

Bacillus thuringiensis crystal proteins, well known to be toxic to certain insects but not pathogenic to mammals, are used as insecticidal proteins in agriculture and forest management. We here identified a crystal protein that is non-insecticidal and non-hemolytic but has strong cytocidal activity against various human cells with a markedly divergent target specificity, e.g. highly cytotoxic to HepG2 and Jurkat and less cytotoxic to the normal hepatocyte (HC) and HeLa. In slices of liver and colon cancer tissues, the toxin protein preferentially killed the cancer cells, leaving other cells unaffected. The cytocidal effect of the protein is non-apoptotic with swelling and fragmentation of the susceptible cells, although the apoptotic process does occur when the cell damage proceeded slowly. The amino acid sequence deduced from the nucleotide sequence of the cloned gene of the protein has little sequence homology with the insecticidal crystal proteins of B. thuringiensis. These observations raise the presence of a new group of the B. thuringiensis toxin and the possibility of new applications for the protein in the medical field.

Parasporin-2, a new crystal protein derived from noninsecticidal and nonhemolytic Bacillus thuringiensis, recognizes and kills human liver and colon cancer cells as well as some classes of human cultured cells. Here we report that a potent proteinase K-resistant parasporin-2 toxin shows specific binding to and a variety of cytocidal effects against human hepatocyte cancer cells. Cleavage of the N-terminal region of parasporin-2 was essential for the toxin activity, whereas C-terminal digestion was required for rapid cell injury. Protease-activated parasporin-2 induced remarkable morphological alterations, cell blebbing, cytoskeletal alterations, and mitochondrial and endoplasmic reticulum fragmentation. The plasma membrane permeability was increased immediately after the toxin treatment and most of the cytoplasmic proteins leaked from the cells, whereas mitochondrial and endoplasmic reticulum proteins remained in the intoxicated cells. Parasporin-2 selectively bound to cancer cells in slices of liver tumor tissues and susceptible human cultured cells and became localized in the plasma membrane until the cells were damaged. Thus, parasporin-2 acts as a cytolysin that permeabilizes the plasma membrane with target cell specificity and subsequently induces cell decay.

Parasporin-1 is a novel non-insecticidal inclusion protein fromBacillus 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-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(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 Ca 2؉ level and that the Ca 2؉ influx is the first step in the pathway that underlies parasporin-1 toxicity.Pathogenic bacteria produce a wide variety of protein toxins and toxin-like molecules. These toxins and toxin-like molecules have been studied extensively to understand the diseases caused by pathogenic bacteria and to find effective preventive treatments. It is known that bacterial toxins affect the enzymic or non-enzymic activities of specific host molecules, which are often critical for cell function, resulting in inhibition or excess activation of these targets. Although bacterial toxins differ in their modes of action, they often show strict target specificities when compared with chemically synthesized drugs. Thus, bacterial toxins can be used as powerful therapeutic agents or as tools in biological studies (1).Bacillus thuringiensis is a Gram-positive, spore-forming bacterium that produces parasporal inclusions during sporulation. The inclusions often contain one or more insecticidal proteins that are toxic to the larvae of certain insects and, in some cases, to nematodes, mites, and protozoa (2). There is a remarkable diversity of B. thuringiensis strains and inclusion proteins. Previous studies have identified a number of noninsecticidal B. thuringiensis strains in natural env...

Bacillus thuringiensis is a valuable source of protein toxins that are specifically effective against certain insects and worms but harmless to mammals. In contrast, a protein toxin obtained from B. thuringiensis strain A1547, designated parasporin-2, is not insecticidal but has a strong cytocidal activity against human cells with markedly divergent target specificity. The 37 kDa inactive protein is proteolytically activated to a 30 kDa active form. The active form of the recombinant protein toxin was crystallized in the presence of ethylene glycol and polyethylene glycol 8000 at neutral pH. The crystals belong to the hexagonal space group P6(1) or P6(5), with unit-cell parameters a = b = 134.37, c = 121.24 A. Diffraction data from a native crystal were collected to 2.75 A resolution using a synchrotron-radiation source.