In vitro cytotoxicity of non-Cyt inclusion proteins of a Bacillus thuringiensis isolate against human cells, including cancer cells

Abstract

A soil isolate designated 90‐F‐45‐14, belonging to Bacillus thuringiensis serovar dakota (H15), was examined for characterization of in vitro cytotoxicity, associated with parasporal inclusion proteins, against human cells. When activated with proteolytic processing, inclusion proteins of the isolate 90‐F‐45‐14 exhibited a moderate cytotoxicity against the human uterus cervix cancer cells (HeLa) with an EC50 value of 60·8 µg ml−1, while showing extremely high activities on the human leukaemic T cells (MOLT‐4) and the normal T cells with EC50 values of 0·27 and 0·20 µg ml−1, respectively. Anti‐leukaemic cell activity of the 90‐F‐45‐14 proteins was eight to nine times greater than that of the B. thuringiensis serovar israelensis proteins containing the Cyt1 protein, a broad‐spectrum cytolysin. The cytopathy by the 90‐F‐45‐14 proteins was characterized by marked cell‐ballooning, while the israelensis proteins induced early breakdown of the cells due to cytolysis. Inclusions of the isolate consisted of five major polypeptides of 170, 103, 73, 40 and 32 kDa. A 100% homology was observed in the sequence of 15 N‐terminal amino acids between the proteins of 170 and 103 kDa. There was no N‐terminal sequence homology between 90‐F‐45‐14 proteins and the existing Cry/Cyt proteins of B. thuringiensis. Proteolytic processing by proteinase K yielded several proteins with molecular masses ranging from 40 to 28 kDa.

A Bacillus thuringiensis Crystal Protein with Selective Cytocidal Action to Human Cells

“…This, taken together with the mammalian cellrecognizing parasporal proteins reported previously (7)(8)(9)(10)(11), means the presence of a new distinct family of B. thuringiensis ␦-endotoxins that are highly cytotoxic to a wide range of mammalian cells but are non-hemolytic and non-insecticidal, in addition to the established insecticidal Cry and Cyt protein families (3). Each toxin protein has its specific and distinct target spectrum against mammalian cells, and some proteins are strictly specific to certain human cancer cells, as the toxin protein reported in this report.…”

“…Bacterial Strains and Culture Media-The organisms used in this study was the soil isolate B. thuringiensis A1547, belonging to serovar dakota (7)(8)(9). The bacteria were grown on nutrient medium, containing 0.1% meat extract, 0.1% polypeptone, and 0.2% NaCl, pH 7.6, at 27°C for 8 days.…”

“…Among the non-insecticidal B. thuringiensis isolates, we found isolates with a novel property, non-hemolytic but highly cytotoxic to a wide range of mammalian cells, including human cancer cells (7)(8)(9)(10)(11). The cytotoxic proteins were heterogeneous in cytotoxicity spectra, and some were active on a wide range of human cells, whereas the others killed a few specific cells.…”

Cytocidal Actions of Parasporin-2, an Anti-tumor Crystal Toxin from Bacillus thuringiensis

“…Proparasporin-2 is produced as parasporal protein inclusion bodies in B. thuringiensis strain A1547 (13,15). Similar to its characteristic production in the native state, the recombinant protein used in the present study is also formed as insoluble protein inclusion bodies or aggregates in a host bacterium, E. coli, that had been transformed with a parasporin-2 gene.…”

“…A potent toxin was discovered in the noninsecticidal and nonhemolytic B. thuringiensis strain A1547, which produces agglutinative Cry proteins with cytocidal activity against MOLT-4 human leukemic T cells (13). In a previous study, we obtained the gene encoding the purified new toxin protein, which was named parasporin-2 (or Cry31Aa, as designated by the B. thuringiensis ␦-endotoxin nomenclature committee) (14), and we examined the cytotoxic activities of a recombinant parasporin-2 against a variety of cultured human cells.…”