Michio Ohba

Unique activity associated with non-insecticidal Bacillus thuringiensis parasporal inclusions : in vitro cell-killing action on human cancer cells

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.

Distribution of Bacillus thuringiensis in soils of Japan

Abstract A total of 136 soil samples collected from nonsericultural areas of 18 prefectures of Japan were examined for the distribution of Bacillus thuringiensis . Of 6910 isolates referable to the Bacillus cereus-B. thuringiensis group, 189 (2.7%) produced parasporal inclusions (PIs). Among these PI-forming isolates, only 66 (34.9%) were identified as B. thuringiensis H serotypes 3a, 4a:4c, 5a:5c, 8a:8c, 10, 11a:11c, and 17. Of the remaining 123 isolates, 84 were well-motile but untypeable by H antisera against the known B. thuringiensis serotypes 1–21, and 39 were untestable because of nonmotility or marked autoagglutination. The results suggested that B. thuringiensis flora in soils was remarkably different from that in the sericultural environment.

Insect toxicity of Bacillus thuringiensis isolated from soils of Japan

Abstract A total of 189 isolates of Bacillus thuringiensis producing parasporal inclusions (PIs), obtained from soils of Japan, were examined for their oral toxicity against larvae of the silkworm, Bombyx mori , and the mosquito, Aedes aegypti , and adults of a chrysomelid coleopteran, Pyrrhalta tibialis . Of these isolates, 68 (36.0%) were toxic to insects tested: 48 were Lepidoptera-toxic and 20 were mosquito-toxic. The Lepidoptera-specific isolates were referable to B. thuringiensis serotypes 3a (2 isolates), 5a:5c (1 solate), and 10 (44 isolates), while one isolate was untypable by the known B. thuringiensis H antisera. Unlike PIs of the known Lepidoptera-toxic B. thuringiensis strains, PIs produced by these untypable and serotype 10 isolates were spherical in shape. Of 20 mosquitotoxic isolates, 7 were referable to B. thuringiensis serotype 11a:11c, and 13 were untypable. Although 121 isolates (64%) produced PIs with various morphologies including typically bipyramidal ones, they did not demonstrate any toxicity against insects of three orders, suggesting that nontoxic PI-forming bacteria predominate in natural environments rather than toxic ones.

Parasporin, a Human Leukemic Cell-Recognizing Parasporal Protein of Bacillus thuringiensis

ABSTRACT An unusual property, human leukemic cell-recognizing activity, associated with parasporal inclusions of a noninsecticidalBacillus 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.

Isolation of a Bacillus thuringiensis strain (serotype 8a:8b) highly and selectively toxic against mosquito larvae

Abstract An isolate of Bacillus thuringiensis designated as PG-14 obtained from the Philippines was highly toxic to the mosquitoes Aedes aegypti and Culex molestus but nontoxic to the silkworm, Bombyx mori , and adults of a daphnid. The degree of toxicity to mosquito larvae was the same as that of the reference strain of Bacillus thuringiensis subsp. israelensis (serotype 14). Parasporal inclusion produced by the isolate PG-14 was spherical or irregular in shape and morphologically similar to that produced by the reference strain of subsp. israelensis . The H antigenic structure of the isolate was identical to that of the reference strain of B. thuringiensis subsp. morrisoni (serotype 8a:8b). Differences were shown in the O antigenic structures and in the production of lecithinase. Thermostable exotoxin was not produced by the isolate PG-14. The results indicate the isolation of a B. thuringiensis strain which shows the same toxicity as that of subsp. israelensis .

Serological identification of Bacillus thuringiensis and related bacteria isolated in Japan

Abstract Distribution of Bacillus thuringiensis and related sporeforming bacteria in Japan was investigated and it was found that most of the crystalliferous isolates belonged to B. thuringiensis serotypes 3a, 4a:4b, 7, and 8. Serotypes 1, 3a:3b, 4a:4c, and 11 were rarely isolated. H antigens of 189 isolates of acrystalliferous sporeformers were analyzed and 26 isolates were agglutinated by B. thuringiensis H antisera against serotypes 3a, 4a:4b, 5a:5c, 6, 7, 8, 10, 11, and 12 at high serum dilutions. Heat-stable somatic antigens of these isolates differed significantly from that of reference B. thuringiensis .

Ubiquity of Bacillus thuringiensis on phylloplanes of arboreous and herbaceous plants in Japan

A total of 120 Bacillus thuringiensis strains was isolated from phylloplanes of 35 species of arboreous and herbaceous plants in an area of northern Kyushu, Japan. The isolates belonged to at least 17 serotypes and the group of H serotype 3 was predominant. Twenty strains were untypable by the existing reference H antisera and 47 were untestable due to autoagglutination or poor motility. Of the 120 isolates, 25 produced bipyramidal parasporal inclusions and the others, spherical or irregular‐shaped. Insecticidal activity against mosquitoes (Culex pipiens molestus and Anopheles stephensi) and/or diamondback moth, Plutella xylostella, was associated with 28 isolates (23·3%). Overall results revealed that: B. thuringiensis is ubiquitous on a variety of plants; bacterial flora on phylloplanes consists of highly heterogeneous H serogroups; and there is little correlation between plant species and phenotypes of B. thuringiensis isolates.

The isolates of Bacillus thuringiensis serotype 10 with a highly preferential toxicity to mosquito larvae.

Abstract Comparative bacteriological and serological studies of three isolates and the reference strain of Bacillus thuringiensis subsp. darmstadiensis (serotype 10) were conducted. No difference was shown in the flagellar antigenic structure between the three isolates and the reference strain. Differences were observed in the O antigenic structures and in the following biochemical properties: lecithinase production, DNase production, arginine decarboxylase production, acid production from inulin, and malonate utilization. β-Exotoxin production was not detected in these three isolates. The reference strain produced parasporal inclusions toxic to the lepidopterous larvae but nontoxic to mosquito larvae. On the contrary, two among the three isolates, which produced spherical parasporal inclusions, were not toxic to the lepidopterous larvae but highly toxic to larvae of the mosquitoes, Culex tritaenlorhynchus, Culex molestus , and Aedes aegypti . Another isolate produced large irregular-shaped inclusions nontoxic to the insects of both orders. Accordingly, B. thuringiensis serotype 10 was divided into three groups from the viewpoint of toxicity against lepidopterous and mosquito larvae.

Crystal Structure of the Parasporin-2 Bacillus thuringiensis Toxin That Recognizes Cancer Cells

Parasporin-2 is a protein toxin that is isolated from parasporal inclusions of the Gram-positive bacterium Bacillus thuringiensis. Although B. thuringiensis is generally known as a valuable source of insecticidal toxins, parasporin-2 is not insecticidal, but has a strong cytocidal activity in liver and colon cancer cells. The 37-kDa inactive nascent protein is proteolytically cleaved to the 30-kDa active form that loses both the N-terminal and the C-terminal segments. Accumulated cytological and biochemical observations on parasporin-2 imply that the protein is a pore-forming toxin. To confirm the hypothesis, we have determined the crystal structure of its active form at a resolution of 2.38 A. The protein is unusually elongated and mainly comprises long beta-strands aligned with its long axis. It is similar to aerolysin-type beta-pore-forming toxins, which strongly reinforce the pore-forming hypothesis. The molecule can be divided into three domains. Domain 1, comprising a small beta-sheet sandwiched by short alpha-helices, is probably the target-binding module. Two other domains are both beta-sandwiches and thought to be involved in oligomerization and pore formation. Domain 2 has a putative channel-forming beta-hairpin characteristic of aerolysin-type toxins. The surface of the protein has an extensive track of exposed side chains of serine and threonine residues. The track might orient the molecule on the cell membrane when domain 1 binds to the target until oligomerization and pore formation are initiated. The beta-hairpin has such a tight structure that it seems unlikely to reform as postulated in a recent model of pore formation developed for aerolysin-type toxins. A safety lock model is proposed as an inactivation mechanism by the N-terminal inhibitory segment.

A unique isolate of Bacillus thuringiensis serovar japonensis with a high larvicidal activity specific for scarabaeid beetles

A spore‐forming bacterium isolated from the soil of Japan was assigned to Bacillus thuringiensis serovar japonensis (flagellar antigen 23). Parasporal inclusions of this isolate were spherical to ovoid in shape and exhibited high larvicidal activity against coleopterous scarabaeid beetles, the cupreous chafer. Anomala cuprea, the soybean beetle, Anomala rufocuprea, and the Japanese beetle. Popillia japonica. No toxicity was shown by this isolate against larvae of Lepidoptera, Diptera, and Orthoptera, and adults of a chrysomelid coleopteran.