Parasporal inclusion proteins from a total of 1744 Bacillus thuringiensis strains, consisting of 1700 Japanese isolates and 44 reference type strains of existing H serovars, were screened for cytocidal activity against human leukaemia T cells and haemolytic activity against sheep erythrocytes. Of 1684 B. thuringiensis strains having no haemolytic activity, 42 exhibited in vitro cytotoxicity against leukaemia T cells. These non‐haemolytic but leukaemia cell‐toxic strains belonged to several H‐serovars including dakota, neoleonensis, shandongiensis, coreanensis and other unidentified serogroups. Purified parasporal inclusions of the three selected strains, designated 84‐HS‐1‐11, 89‐T‐26‐17 and 90‐F‐45‐14, exhibited no haemolytic activity and no insecticidal activity against dipteran and lepidopteran insects, but were highly cytocidal against leukaemia T cells and other human cancer cells, showing different toxicity spectra and varied activity levels. Furthermore, the proteins from 84‐HS‐1‐11 and 89‐T‐26‐17 were able to discriminate between leukaemia and normal T cells, specifically killing the former cells. These findings may lead to the use of B. thuringiensis inclusion proteins for medical purposes.

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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.

An unusual property, human leukemic cell-recognizing activity, associated with parasporal inclusions of a noninsecticidal Bacillus thuringiensis soil isolate was investigated, and a protein (named parasporin in this study) responsible for the activity was cloned. The parasporin, encoded by a gene 2,169 bp long, was a polypeptide of 723 amino acid residues with a predicted molecular weight of 81,045. The sequence of parasporin contained the five conserved blocks commonly found in B. thuringiensis Cry proteins; however, only very low homologies (<25%) between parasporin and the existing classes of Cry and Cyt proteins were detected. Parasporin exhibited cytocidal activity only when degraded by proteases into smaller molecules of 40 to 60 kDa. Trypsin and proteinase K activated parasporin, while chymotrypsin did not. The activated parasporin showed strong cytocidal activity against human leukemic T cells (MOLT-4) and human uterus cervix cancer cells (HeLa) but not against normal T cells.