Hiroyuki Saitoh

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.

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.

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.

Occurrence of Bacillus thuringiensis in Fresh Waters of Japan

Abstract.Bacillus thuringiensis was recovered at a relatively high frequency from both running and still fresh waters in natural environments of Kyushu, Japan. Of 107 water samples examined, 53 (49.5%) contained this organism. The frequency of B. thuringiensis colonies was 4.4% among 4414 colonies of the Bacillus cereus/B. thuringiensis group. The density of this bacterium in fresh waters averaged 0.45 cfu/ml. Serologically, B. thuringiensis isolates were assigned to 26 H serotypes. Of these, H14/36 (H serovar israelensis/malaysiensis) was the predominant, followed by the serotypes H3abc (kurstaki), H27 (mexicanensis), H3ad (sumiyoshiensis), and H35 (seoulensis). Of 195 isolates, 52 (26.7%) exhibited larvicidal activity against aquatic Diptera; 21 killed Culex pipiens molestus (Culicidae) only, and 31 were active on both the culicine mosquito and the moth-fly, Clogmia albipunctata (Psychodidae). The Diptera-toxic isolates produced spherical or irregularly pointed parasporal inclusions.

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

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 28 kDa protein of the Bacillus thuringiensis serovar shandongiensis isolate 89-T-34-22 induces a human leukemic cell-specific cytotoxicity

A 28 kDa protein that exhibits cytocidal activity specific for human leukemic T (MOLT-4) cells was purified from proteinase K-digested parasporal inclusion of a Bacillus thuringiensis serovar shandongiensis isolate. The N-terminal sequence of the protein was identical with that of the 32 kDa protein, regarded as a protoxin, of the inclusion proteins. The median effective concentration of this protein was 0.23 microg/ml against MOLT-4 cells and its specific activity was 7.9 times greater than that of the whole inclusion proteins. The 28 kDa protein induced necrosis-like cytotoxicity against MOLT-4 cells and the cytopathic effect with the passage of time was characterized by cell swelling, nuclear membrane isolation and chromatin condensation.

Larvicidal toxicity of Japanese Bacillus thuringiensis against the mosquito Anopheles stephensi

Japanese isolates of Bacillus thuringiensis were screened for larvicidal activity against the mosquito Anopheles stephensi, the urban malaria vector of the Indian subcontinent. Among more than 30 strains identified, larvicidal activity causing >80% mortality in 72 h was demonstrated for 41/1449 (2.8%) isolates. The majority of strains and isolates (97.2%) exhibited little or no larvicidal activity. Anopheles‐active strains belonged to more than 12 H serotypes, especially H3ade (serovar fukuokaensis) and H44 (serovar higo). SDS‐PAGE profiles of inclusion proteins showed 4 distinct types among 6 active strains examined. The most active Japanese isolates were H20 strain 89‐T‐34‐14 (LC50 4.4 μg/ml) and H44 serovar higo strain 74‐E‐45‐24 (LC50 7.6 μg/ml), respectively, 13‐fold and 23‐fold less active than the international standard H14 serovar israelensis (LC50 0.33 μg/ml).

Larvicidal activity of Bacillus thuringiensis natural isolates, indigenous to Japan, against two nematoceran insect pests occurring in urban sewage environments

A total of 1449 Bacillus thuringiensis strains, indigenous to Japan, were screened for larvicidal activity against two nematoceran insect pests, the mosquito, Culex pipiens molestus (Culicidae), and the moth-fly, Telmatoscopus albipunctatus (Psychodidae). Mosquito specific strains were abundant in H serotypes 3abc (serovar kurstaki), 3ade (fukuokaensis), 4ac (kenyae), 7 (aizawai), 11ac (kyushuensis) and 29 (amagiensis), while moth-fly specific strains were predominantly found in H serotype 17 (tohokuensis). Strains toxic to both insects were most frequently detected in H serotypes 10 (darmstadiensis) and 17/27. Seven selected B. thuringiensis strains were highly toxic to Culex and/or Telmatoscopus. There was a diversity in SDS-PAGE profiles of inclusion proteins of these strains.

Mode of action of parasporin-4, a cytocidal protein from Bacillus thuringiensis.

Parasporin-4 (PS4) is a cytotoxic protein produced by Bacillus thuringiensis strain A1470. It exhibits specific cytotoxicity against human cancer cell lines, CACO-2, Sawano, and MOLT-4 cells, in particular. When cells were administrated with PS4, cell swelling and nuclear shrinkage were induced, and, the ballooned cells burst within 24 h. PSI-BLAST search showed that the protein shared homology not only with B. thuringiensis Cry toxins but also with aerolysin-type β-pore-forming toxins. Circular dichroism measurements suggested that PS4 was a β-sheet-rich protein. PS4 aggregated into oligomers on the plasma membrane of PS4-susceptible CACO-2 cells, but not on that of PS4-resistant HeLa cells. Leakage of lactate dehydrogenase and influx of extracellular FITC-dextrans were observed only in susceptible cells. The activation of effectors caspase 3 and/or 7 was not observed in PS4-treated CACO-2 cells. It was shown that cytotoxicity of the PS4 against CACO-2 cells was exhibited when treated by cyclodextrin which induces cholesterol depletion. These results suggest that PS4 is a unique β-pore-forming toxin with a cholesterol-independent activity.

A Novel Isolate of Bacillus thuringiensis serovar leesis that Specifically Exhibits Larvicidal Activity against the Moth-Fly, Telmatoscopus albipunctatus

A soil isolate designated 88-KO-14-45, belonging to Bacillus thuringiensis serovar leesis (H33), exhibited larvicidal activity against the moth-fly, Telmatoscopus albipunctatus (Diptera: Psychodidae), but not for larvae of the culicine and aedine mosquitoes and Lepidoptera. Purified parasporal inclusions had an LC50 value of 5.78 micrograms/ml for the larval moth-fly, but gave no mortality against larvae of Culex pipiens molestus (Diptera: Culicidae) at protein concentrations up to 10 mg/ml. Electron microscopic observations revealed that the parasporal inclusions are homogeneous round-shaped bodies enclosed with thick, electron dense envelopes. Haemolytic activity against sheep erythrocytes was not detected in the solubilized inclusions. SDS-PAGE showed that the inclusions are composed of 72, 68, 56 and 30 kDa proteins. Immunologically, these proteins were unrelated to the inclusion proteins of B. thuringiensis serovar israelensis, while a 70 kDa protein of the strain 73-E-10-2 (B. thuringiensis serovar darmstadiensis) was seroactive to antibodies against proteins of 88-KO-14-45.