Eiichi Mizuki

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.

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.

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.

Recovery of Bacillus thuringiensis from marine sediments of Japan

Abstract. Marine sediments from a Japanese bay were examined for the occurrence of Bacillus thuringiensis. Of 1313 colonies belonging to the Bacillus cereus/B. thuringiensis group, 22 (1.7%) were allocated to B. thuringiensis. Marine isolates of B. thuringiensis consisted of heterogeneous multiple H serogroups; 10 isolates were assigned to the eight serovars (kurstaki, sumiyoshiensis, sotto, aizawai, darmstadiensis, thompsoni, neoleonensis, and higo); two motile isolates failed to react with the reference antisera; and the others were serologically untestable. Insecticidal activities were associated with two kurstaki isolates (toxic to both Lepidoptera and Diptera) and a higo isolate (Diptera-specific). None of the parasporal inclusion proteins of the 22 isolates exhibited in vitro cytotoxic activity against two vertebrate cells, sheep erythrocytes and HeLa cells. All B. thuringiensis isolates had no halophilism, although seawater-based medium supported their growth, sporulation, and formation of parasporal inclusions.

Cytocidal actions of parasporin-2, an anti-tumor crystal toxin from Bacillus thuringiensis

Parasporin-2, a new crystal protein derived from noninsecticidal and nonhemolytic Bacillus thuringiensis, recognizes and kills human liver and colon cancer cells as well as some classes of human cultured cells. Here we report that a potent proteinase K-resistant parasporin-2 toxin shows specific binding to and a variety of cytocidal effects against human hepatocyte cancer cells. Cleavage of the N-terminal region of parasporin-2 was essential for the toxin activity, whereas C-terminal digestion was required for rapid cell injury. Protease-activated parasporin-2 induced remarkable morphological alterations, cell blebbing, cytoskeletal alterations, and mitochondrial and endoplasmic reticulum fragmentation. The plasma membrane permeability was increased immediately after the toxin treatment and most of the cytoplasmic proteins leaked from the cells, whereas mitochondrial and endoplasmic reticulum proteins remained in the intoxicated cells. Parasporin-2 selectively bound to cancer cells in slices of liver tumor tissues and susceptible human cultured cells and became localized in the plasma membrane until the cells were damaged. Thus, parasporin-2 acts as a cytolysin that permeabilizes the plasma membrane with target cell specificity and subsequently induces cell decay.

Bacillus thuringiensis soil populations naturally occurring in the Ryukyus, a subtropic region of Japan.

Of 809 soil samples collected from the seven islands of the Ryukyus, Japan, 107 samples (13.2%) contained Bacillus thuringiensis. The frequency of B. thuringiensis among the B. cereus group was 1.1% (235/21842) on the average. The B. thuringiensis soil populations of the Ryukyus consisted of more than 22 H serogroups. The predominant H serotype was the H5ac/21 (serovar canadensis/colmeri), followed by the H3ad (serovar sumiyoshiensis) and H16 (serovar indiana). Geographically, most widely distributed H serogroups were the H16 and H10ac (serovar londrina); the former was recovered from five islands and the latter from three islands. Parasporal inclusions of the isolates were morphologically heterogeneous, roughly grouped into four categories: bipyramidal/cuboidal, spherical/ovoid, irregularly-pointed, and irregular-shaped. About 53% of the isolates formed spherical to ovoid parasporal inclusions. None of the isolates exhibited larvicidal activity against the silkworm, Bombyx mori. Only four isolates belonging to four different serotypes killed larvae of the mosquito, Aedes aegypti. These mosquito-specific isolates all produced spherical parasporal inclusions.

Three Cry toxins in two types from Bacillus thuringiensis strain M019 preferentially kill human hepatocyte cancer and uterus cervix cancer cells.

Bacillus thuringiensis strain M019, non-pathogenic to lepidopteran and dipteran insects, produces a parasporal inclusion that consists of three 84-kDa Cry proteins (CPs). CP78A and CP78B, which exhibit 83.5% amino acid identity, were new variants of the previously reported HeLa cell-killing protein (parasporin-1). CP84 was a novel CP showing low-level homology, of 21.9% (56.4% similarity), with the insecticidal Cry2 toxin. In vitro solubilization with dithiothreitol at pH 10.2 and limited hydrolysis with trypsin resulted in the removal of N-terminal portions of the CPs and their activation. The 70-kDa proteins (15- and 55-kDa fragments) from CP78A and CP78B and the 73-kDa protein (14- and 59-kDa fragments) from CP84 exhibited varying degrees of cytocidal activity preferentially toward human hepatocyte cancer HepG2 cells and uterus cervix cancer HeLa cells causing cell swelling or the formation of vacuoles in the cytoplasm. These toxins appeared to attack an identical target on human cells.

Parasporin-1, a Novel Cytotoxic Protein from Bacillus thuringiensis, Induces Ca2+ Influx and a Sustained Elevation of the Cytoplasmic Ca2+ Concentration in Toxin-sensitive Cells

Parasporin-1 is a novel non-insecticidal inclusion protein from Bacillus thuringiensis that is cytotoxic to specific mammalian cells. In this study, we investigated the effects of parasporin-1 on toxin-sensitive cell lines to elucidate the cytotoxic mechanism of parasporin-1. Parasporin-1 is not a membrane pore-forming toxin as evidenced by measurements of lactate dehydrogenase release, propidium iodide penetration, and membrane potential in parasporin-1-treated cells. Parasporin-1 decreased the level of cellular protein and DNA synthesis in parasporin-1-sensitive HeLa cells. The earliest change observed in cells treated with this toxin was a rapid elevation of the intracellular free-Ca2+ concentration; increases in the intracellular Ca2+ levels were observed 1-3 min following parasporin-1 treatment. Using four different cell lines, we found that the degree of cellular sensitivity to parasporin-1 was positively correlated with the size of the increase in the intracellular Ca2+ concentration. The toxin-induced elevation of the intracellular Ca2+ concentration was markedly decreased in low-Ca2+ buffer and was not observed in Ca2+-free buffer. Accordingly, the cytotoxicity of parasporin-1 decreased in the low-Ca2+ buffer and was restored by the addition of Ca2+ to the extracellular medium. Suramin, which inhibits trimeric G-protein signaling, suppressed both the Ca2+ influx and the cytotoxicity of parasporin-1. In parasporin-1-treated HeLa cells, degradation of pro-caspase-3 and poly(ADP-ribose) polymerase was observed. Furthermore, synthetic caspase inhibitors blocked the cytotoxic activity of parasporin-1. These results indicate that parasporin-1 activates apoptotic signaling in these cells as a result of the increased Ca2+ level and that the Ca2+ influx is the first step in the pathway that underlies parasporin-1 toxicity.