Parasporin, a new anticancer protein group from Bacillus thuringiensis.

Parasporin (PS) is a collection of genealogically heterogeneous Cry proteins synthesized in Bacillus thuringiensis. A prominent feature commonly associated with PS proteins is the strong cytocidal activity preferential for human cancer cells of various origins. The proteins exhibit cytocidal activities only when digested with proteases. Currently, this protein group is classified into four families: PS1, PS2, PS3 and PS4. Marked differences are evident in cytotoxicity spectra and activity levels between the four PS families. Neither hemolytic activity nor insect toxicity is associated with PS proteins. One of the most striking aspects in the events induced by PS1Aa1 is the early and rapid increase of the intracellular Ca2+ concentration, with no change in plasma membrane permeability. There is strong evidence that PS1Aa1 kills cancer cells through apoptosis. Unlike PS1Aa1, PS2Aa1 increases plasma membrane permeability of cancer cells. The initial step in cytocidal action of PS2Aa1 is the specific binding of this cytotoxin to a putative receptor located in the lipid rafts, followed by its oligomerization and pore formation in plasma membrane.

Occurrence of Bacillus thuringiensis in canopies of a natural lucidophyllous forest in Japan.

A total of 39 Bacillus thuringiensis isolates were recovered from 38 leaves collected from 5- to 10-m-high canopies of 8 micro-/meso-phanerophyte species in a lucidophyllous forest of Japan. B. thuringiensis-positive leaves accounted for 1.4% of a total of 2805 leaves from 15 tree species. The frequency of the organism was 0.8% among the Bacillus cereus/B. thuringiensis group. Of 39 isolates obtained, 27 (69.2%) were allocated to 11 H serovars, and 12 isolates remained unidentified: 11 were motile but lacked reactivity to the 55 reference antisera, and 1 isolate was not flagellated. Two H serovars, kurstaki (H3abc) and tohokuensis (H17), occurred predominantly on canopy phylloplanes. Larvicidal activities against Bombyx mori and/or Aedes aegypti were associated with 49% of the canopy isolates. Strong hemolysis was induced by parasporal inclusion proteins of the two isolates of serovar israelensis (H14). Hemagglutinating (lectin) activity was associated with parasporal proteins of nine isolates. There was little correlation between insecticidal activity and lectin activity.

Parasporin-1Ab, a novel Bacillus thuringiensis cytotoxin preferentially active on human cancer cells in vitro.

The non-insecticidal Bacillus thuringiensis strain B0195 produces parasporin (PS) whose cytocidal activity is preferential for human cancer cells. This study identified two ps genes, ps1Aa3 and ps1Ab1, from the strain B0195. The former gene was 2,169-bp long, encoding an 81 kDa protein (PSLAa3) whose aminoacid sequence was 100% identical to that of the reference protein PS1Aa1. The latter gene was 2,178-bp long, encoding a novel protein of 82 kDa, PS1Ab1, whose sequence was 86.4% identical to that of PS1Aa1. The recombinant protein of PS1Ab1, synthesized in transformed B. thuringiensis cells, induced marked cytopathy in HeLa cells (human uterus cervix cancer cells) upon proteolytic activation. The cytopathy was characterized by cell-ballooning, followed by gradual cell-shrinking. Unlike HeLa cells, non-cancer UtSMC cells (human uterine smooth muscle-cells) were not susceptible to PS1Ab1.

Identification of parasporin genes in Vietnamese isolates of Bacillus thuringiensis.

Four genes encoding parasporins, cytotoxins preferentially killing human cancer cells in vitro, were isolated from four Vietnamese strains of Bacillus thuringiensis. Nucleotide sequence analysis revealed that: (1) three genes fall into the two known classes, ps1Aa and ps1Ab, and (2) another one belongs to ps1Ac, a novel gene class established in this study. Upon proteolytic activation, parasporal protein of the organism with ps1Ac exhibited strong cytocidal activity against human cancer cells, HeLa and Hep G2, but not to non-cancer normal cells, UtSMC and HC.

Ubiquity of parasporin-1 producers in Bacillus thuringiensis natural populations of Japan.

Parasporin, a Bacillus thuringiensis parasporal protein, is unique in having a strong cytocidal activity preferential for human cancer cells. In this study, we characterized parasporin activities associated with three novel geographical isolates of B. thuringiensis. Parasporal inclusion proteins of the three isolates were highly toxic to human uterus cervix cancer cells (HeLa), but not to non-cancer uterine smooth muscle cells (UtSMC). Inclusions of the isolates lacked insect toxicity and hemolytic activity against sheep erythrocytes. Ouchterlony immunodiffusion tests revealed that the proteins of the three isolates are immunologically closely related to parasporin-1 (Cry31A), but dissimilar to the three other existing parasporin groups. Our results provide evidence that the parasporin-1-producing organism is a common member in B. thuringiensis populations occurring in natural environments of Japan.

Occurrence of parasporin-producing Bacillus thuringiensis in Vietnam.

A total of 63 Bacillus thuringiensis isolates were recovered from urban soils of Hanoi, Vietnam. Of these, 34 were identified to 12 H serogroups. None of the isolates showed larvicidal activities against three lepidopterous insects. Three isolates belonging to the two serovars, colmeri (H21) and konkukian (H34), were highly toxic to larvae of the mosquito Aedes aegypti. Parasporal inclusion proteins of four isolates exhibited cytocidal activities against HeLa cells. Immunologically, proteins of four isolates were closely related to parasporin-1 (Cry31Aa), a parasporal protein that preferentially kills human cancer cells. Haemolytic activities were associated with parasporal proteins of the three mosquitocidal isolates but not with those of the four cancer-cell-killing isolates. PCR experiments and nucleotide sequence analysis revealed that the genes of four anti-cancer isolates are closely related to the gene parasporin-1 (cry31Aa) but are dissimilar to those of the three other existing parasporins. Our results suggest that the soil of northern Vietnam is a good reservoir of parasporin-producing B. thuringiensis.

Cloning and characterization of two novel genes, cry24B and s1orf2, from a mosquitocidal strain of Bacillus thuringiensis serovar sotto.

Two new crystal protein genes, cry24B and s1orf2, were cloned from a mosquitocidal Bacillus thuringiensis serovar sotto strain. The cry24B and s1orf2 genes encoded a 76-kDa and 62-kDa protein, respectively. The Cry24B protein retained five conserved regions commonly found in the existing Cry proteins. The amino acid sequence of the S1ORF2 had a high homology to that of the ORF2 protein of B. thuringiensis serovar jegathesan. Southern hybridization experiments with a cry24B gene-specific probe revealed that these genes are located on two large plasmids of > 100 kb. When the two genes, cry24B and s1orf2, were expressed in an acrystalliferous B. thuringiensis host, the proteins were synthesized and accumulated as inclusions. These inclusions exhibited no larvicidal activities against three mosquito species: Aedes aegypti, Anopheles stephensi, and Culex pipiens molestus. Likewise, the inclusions contained no cytocidal activity against HeLa cells.