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Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References

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.

Bacillus thuringiensis was first isolated in Japan as a pathogen of the sotto disease of the silkworm, Bombyx mori, by Ishiwata (1901) early in this century. The organism is a Gram-positive, spore-forming bacterium that produces crystalline parasporal inclusions during sporulation. The inclusions often exhibit strong insecticidal activity against several orders of insects, and this makes B. thuringiensis a reliable agent for microbial control of insect pests of agricultural and medical importance ( Lambert & Peferoen 1992; Cannon 1996). The insecticidal parasporal inclusions contain two families of insect-toxic molecules, Cry and Cyt proteins. The Cry protein is specifically toxic to insects and is currently classified into 22 genetically different major groups, Cry1 to Cry22 ( Crickmore et al. 1998 ). It has been well established that the high specificity of Cry proteins in killing insects is attributable to specific binding of the proteins to receptors that reside on the midgut cell membranes of susceptible insects ( Cannon 1996). Another family of the toxin, the Cyt protein, has a broad cytolytic activity against invertebrate and vertebrate cells including erythrocytes, and is divided into two genetically different groups, Cyt1 and Cyt2 (formerly CytA and CytB, respectively) ( Crickmore et al. 1998 ).

Historically, it has long been believed that B. thuringiensis as a species is characterized by insecticidal activity associated with its parasporal inclusions. Earlier studies, however, have demonstrated that in natural environments, B. thuringiensis strains producing non-insecticidal parasporal inclusions are more widely distributed than insecticidal ones ( Ohba & Aizawa 1986; Hastowo et al. 1992 ; Meadows et al. 1992 ; Ohba 1996, 1997; Roh et al. 1996 ). Thus, the question arises as to whether such non-insecticidal parasporal inclusions have any biological activity as yet undiscovered ( Ohba et al. 1988 ). Although antibacterial activity has recently been demonstrated in inclusion proteins of some non-insecticidal strains of B. thuringiensis ( Yudina & Burtseva 1997), very little, if anything, is known about the other activities. It is reported here for the first time that a unique activity, cytocidal against human cancer cells, is associated with non-insecticidal parasporal inclusions of certain B. thuringiensis strains.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References

Bacterial strains and culture conditions

Most Japanese Bacillus thuringiensis isolates used in this study were from the collection of the Institute of Biological Control, Kyushu University ( Saitoh et al. 1998 ). They had been serotyped by the method described previously ( Ohba & Aizawa 1978), although H serotyping of some new isolates was done in the present study. The reference type strains of B. thuringiensis were obtained from the Institut Pasteur, Paris. To prepare sporulated cultures, the strains were grown on nutrient agar, pH 7·6, at 28 °C for 4 d. Nutrient agar consisted of meat extract (10 g), polypeptone (10 g), NaCl (2 g), agar (20 g) and distilled water (1000 ml).

Cells and culture conditions

Seven vertebrate and insect cell lines were obtained from RIKEN Cell Bank (Tsukuba, Japan): MOLT-4, human leukaemia T cell; A549, human lung cancer cell; MRC-5, normal human lung fibroblast cell; HeLa, human uterus cervix cancer cell; GF-Scale, gold fish (Carassius auratus) scale cell; BM-N, silkworm (Bombyx mori) cell; NIAS-AeAl-2, mosquito (Aedes albopictus) cell. They were cultured under the conditions recommended by the manufacturer. Normal human T cells were prepared from buffy coats obtained from Fukuoka Red Cross Blood Centre (Fukuoka, Japan). T cells were separated from lymphocyte cells using a CellectTM Human T Cell kit (Biotex Laboratories Inc., Alberta, Canada), and were cultured in RPMI1640 medium supplemented with 10% foetal bovine serum and kanamycin (30 μg ml−1) at 37 °C. Sheep erythrocytes were purchased from Nippon Bio-Test Lab., Tokyo, Japan. Human erythrocytes (blood group O) were freshly prepared from a volunteer.

Inclusion protein preparation and production of antisera

Sporulated cultures were washed three times with distilled water before solubilization and used as crude parasporal inclusions. Parasporal inclusions were purified from sporulated cultures as previously described ( Saitoh et al. 1998 ). The crude or purified inclusions were suspended in 50 mmol l−1 Na2CO3 (pH 10) + 10 mmol l−1 DTT (dithiothreitol) + 1 mmol l−1 EDTA for 60 min at 37 °C. After solubilization of the inclusions, the solution was added with phenylmethylsulphonyl fluoride (Wako Pure Chemical, Tokyo, Japan) to stop the proteolytic reaction. The mixture was then centrifuged at 20 000 g for 5 min at 4 °C. Protein concentration of the supernatant fluid was determined by the method of Lowry et al. (1951) using bovine serum albumin as the standard. The supernatant fluid was then treated with proteinase K as previously described ( Ishii & Ohba 1993). After incubation, the pH of the fluid was adjusted to 7·2 and filtered through a 0·45 μm membrane filter. Antisera were raised in rabbits against solubilized whole inclusion proteins of the type strains of serovars israelensis, kyushuensis and kurstaki according to the method of Higuchi et al. (1998) .

Screening methods

A two-step method ( Saitoh et al. 1996 ) was used for mass screening of alkali-solubilized, proteinase K-treated, parasporal proteins. For primary screening 1744 strains were grouped into 349 sets, usually five isolates per set. For the sets exhibiting cytocidal activity against human leukaemia T cells (MOLT-4), the isolates were individually examined for cytotoxicity against MOLT-4 cells and haemolytic activity against sheep erythrocytes. The assay procedure in the screening involved (i) observation of the cytopathic effect (CPE) under a phase-contrast microscope and (ii) assessment of the degree of cytotoxicity by a cell proliferation assay technique.

Cytotoxicity assay and haemolysis assay

Each well of a microtest plate received 90 μl of cell suspension containing 2 × 104 cells. The plate was incubated at 37 °C for 16 h. Then, to each well was added 10 μl of the sample solution, which contained approximately 12 μg of protease-activated inclusion proteins. Each test was done in duplicate or triplicate and was repeated at least three times. The CPE was monitored under a phase-contrast microscope 1 h and 24 h post-inoculation. The degree of CPE was graded as follows on the basis of the proportion of damaged cells in about 1000 cells: –, < 5% ± , 5% to < 10% + , 10% to < 30% + + , 30% to < 60% + + + , 60% to < 90% + + + + , ≥90%.

For assessment of the level of cytotoxicity, a cell proliferation test using a MTT [3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide)] assay ( Behl et al. 1992 ; Heiss et al. 1997 ) was done 24 h post-inoculation using a Cell Titer 96TM kit (Promega Co., Madison, WI, USA) or a Premix WST-1 kit (Takara Co., Kyoto, Japan). In this assay, the average of absorbance values for a negative control (mock inoculation of parasporal protein) was used as a blank value. The degree of cytotoxicity (CT) was graded on the basis of the relative value of absorbance to the blank (1·00): + + + + , extremely high (<0·10); + + + , high (0·10 to <0·40); + + , moderate (0·40 to <0·70); + , low (0·70 to <0·90); ± , very low (0·90 to <0·95); –, non-toxic (≥ 0·95). The isolates with the graded + + to + + + + in CPE and/or CT were selected for further study as cytotoxic strains.

Haemolytic activity was tested on sheep or human erythrocytes as described previously ( Saitoh et al. 1998 ). The degree of haemolytic activity was graded as follows on the basis of their absorbence at 540 nm: + + + , high (>1·00); + + , moderate (1·00 to >0·50); + , low (0·50 to >0·20); –, non-haemolytic ( ≥0·20). The isolates with the levels + to + + + in haemolytic activity were considered as haemolytic strains.

Insecticidal activity test

For insecticidal activity tests, 11 species of five orders were used: Lepidoptera (Plutella xylostella and Bombyx mori), Diptera (Aedes aegypti, Culex pipiens molestus, Anopheles stephensi, Telmatoscopus albipunctatus and Musca domestica), Orthoptera (Gryllus bimaculatus and Atractomorpha bedeli), Dictyoptera (Blattella germanica) and Isoptera (Reticulitermes separatus separatus). The assay was done according to the method of Higuchi et al. (1998) .

SDS-PAGE and immunoblotting

SDS-PAGE of parasporal inclusion proteins was performed as described by Laemmli (1970) using 10% separating and 4% stacking gels. The gels were stained with 0·1% (w/v) Coomassie blue R250 (Sigma). The molecular masses of proteins were determined using protein standards (Sigma). For immunoblot analysis, the resolved parasporal inclusion proteins were transferred to a PVDF membrane using a Transblot SD semi-dry electrophoretic blotting cell (Bio-Rad, Hercules, CA, USA). The membrane was then probed with an appropriate dilution of antiserum. Bound antibodies were detected using alkaline phosphatase-conjugated anti-rabbit IgG (TAGO Inc. Burlinegame, CA, USA). Visualization was performed with nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl toluidinate (Wako Pure Chemical) as the colour substrate.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References

Screening of leukaemia cell-toxic but non-haemolytic inclusion proteins

A total of 1744 B. thuringiensis strains, comprising 1700 Japanese isolates and 44 reference type strains of existing H serovars ( Table 1), were examined first for cytocidal activity of inclusion proteins against human leukaemia T cells and for cytolytic activity on sheep erythrocytes.

Table 1.  Cytotoxic activities of Bacillus thuringiensis parasporal inclusion proteins against human leukaemia T cells and sheep erythrocytes
 Number of isolates mono-toxic to 
H antigenH serovarNumber of isolates tested‡Leukaemia T cellSheep erythrocyteNumber of isolates toxic to both cellsNumber of non-toxic isolates
  • *

    Sub-factor, not identified.

  • Seroreactive for two reference antisera.

  • Bold figure includes the type strain.

  • UTE, Untestable due to no flagellation and/or autoagglutination.

  • UTY, Untypable by reference antisera.

  • NE, Not examined.

1thuringiensis2020
2finitimus 11
3a*33
3acalesti257257
3abckurstaki8989
3acd11
3adsumiyoshiensis 88
3adefukuokaensis2727
4absotto120120
4ackenyae188188
5*11
5abgalleriae 88
5ab/21†11
5accanadensis 66
5ac/21†33
6*22
6abentomocidus 11
6acoyamensis 99
7aizawai107107
8abmorrisoni6060
8acostriniae 55
8bdnigeriensis 11
9tolworthi 44
10abdamstadiensis90189
11*211
11abtoumanoffii 11
11ackyushuensis21849
12thompsoni 11
13pakistani2323
13/29†11
14israelensis 514
14/19†22
15dakota11812
15/21†110
16indiana1616
17tohokuensis211119
17/21†11
17/27†1616
18abkumamotoensis 55
19tochigiensis2828
20*22
20abyunnanensis 88
20acpondicheriensis 11
21colmeri 44
22shandongiensis164111
23japonensis 44
24*66
24abneoleonensis 312
25coreanensis 211
26silo 11
27mexicanensis1414
28abmonterrey 11
29amagiensis234217
31toguchini 22
32cameroun 11
33leesis 22
34konkukian 633
35seoulensis11
37andalousiensis22
43oswaldocruzi55
44higo1414
UTE1632032138
UTY183791166
NE113 13496
Total 17444244161642

Of the strains tested, 60 showed haemolytic activity against sheep erythrocytes. Among these haemolytic strains, 16, including the type strains of serovar israelensis and serovar kyushuensis, exhibited significant cytotoxicity against leukaemia cells. However, the 44 other haemolytic strains did not have leukaemia cell-killing activity. Of the 1684 B. thuringiensis strains with no haemolytic activity, 42 showed cytocidal activity against leukaemia cells ( Table 1). These strains, non-haemolytic but leukaemia cell-killing, consisted of 41 Japanese isolates derived from soils and phylloplanes of nine geographically different localities, and the type strain of serovar neoleonensis from Mexico ( Rodriguez-Padilla et al. 1990 ) ( Table 2). The 41 Japanese strains belonged to H serovars dakota, shandongiensis, coreanensis and other unidentified serogroup(s). None of the predominant serovars in our B. thuringiensis library, such as alesti, kenyae, sotto, aizawai, kurstaki and morrisoni, were toxic to leukaemia cells, although these serovars contained many strains highly toxic to insects of the order Lepidoptera. Interestingly, the 42 leukaemia cell-toxic strains showed no significant insecticidal activity against 11 species from five orders (data not shown), except that one strain exhibited only low larvicidal activity against the mosquito, C. pipiens molestus. As shown in Table 2, the shape and size of parasporal inclusions varied markedly depending on the strains.

Table 2. Bacillus thuringiensis strains whose parasporal inclusion proteins are non-insecticidal and non-haemolytic but cytocidal against human leukaemia cells
H antigenH serovarNumber of isolates Locality (Prefecture)SourceInclusion morphology
  • *

    Seroreactive for two reference antisera.

  • †Type strain ( Rodriguez-Padilla et al. 1990 ).

  • Low toxicity to Culex pipiens molestus (Diptera).

  • UTE, Untestable due to no flagellation and/or autoagglutination.

  • UTY, Untypable by reference antisera.

15dakota8FukuokaSoilIrregularly pointed
15/21 *dakota/colmeri1FukuokaPhylloplaneIrregularly pointed
22shandongiensis3TokyoSoilIrregularly pointed
  1TokushimaSoilBipyramidal
24abneoleonensis 1 Guanajuato, MexicoSoilTriangular
25coreanensis1KyotoSoilIrregularly pointed
UTE 19HiroshimaSoilSpherical
  1FukuokaPhylloplaneSpherical
UTY 6TokyoSoilBipyramidal
   1 KumamotoSoilBipyramidal
Total 42   

Cancer cell-toxicity of purified parasporal inclusions

The purified parasporal inclusions of the three selected Japanese isolates (84-HS-1-11, 89-T-26-17 and 90-F-45-14), and type strains of the three H serovars (israelensis, kyushuensis and kurstaki), were examined for their in vitro cytotoxic activity against vertebrate and insect cells. Inclusion proteins of the three isolates showed neither insecticidal activity against lepidopteran and dipteran insects (data not shown), nor haemolytic activity against human erythrocytes. In contrast, they exhibited marked cytotoxic activities, with different spectra and varied activity levels, against human, fish and insect cells ( Table 3, Fig. 1). The proteins of the two strains, 84-HS-1-11 and 89-T-26-17, had cytocidal activity against leukaemia T cells ( Fig. 1a,b) but not against normal T cells ( Fig. 1e,f). The strain 84-HS-1-11 also showed strong cytocidal activity against HeLa cells ( Fig. 1i). The serovar dakota strain 90-F-45-14 was highly cytotoxic to all the human and insect cells tested ( Fig. 1c,g,k) but not active on human erythrocytes and fish cells ( Table 3). The activities of the three isolates were heat-(100 °C, 10 min)labile and dose-dependent; the minimum protein concentrations causing detectable cytopathy varied from 1 to 100 μg ml−1, depending on the cell species (data not shown). In general, the cytopathy occurred in 1 h, except for strain 84-HS-1-11 in which CPE became visible after several hours.

Table 3. In vitro cytocidal and haemolytic activities of parasporal inclusion proteins in the three selected Japanese isolates and the type strains of the three H serovars of Bacillus thuringiensis
Cytocidal activity* against
 Human T cellHuman lung cell Insect cell
StrainH antigen (serovar)Haemolysis on human erythrocyte LeukaemiaNormalCancerNormalHuman uterus cervix cancer cellGoldfish (Carassius auratus) scale cell Bombyx moriAedes albopictus
84-HS-1-11UTE++ (++) − (−) + (±) ++ (+) +++ (+++) + (+) − (+) − (+)
89-T-26-17UTY++ (++) − (−) ± (±) − (−) ++ (++) − (+) − (+) + (+)
90-F-45-1415 (dakota) +++ (+++) +++ (++) +++ (+++) +++ (+++) +++ (++++) − (+) ++ (+++) +++ (++)
israelensis type strain14 (israelensis) ++++++ (+++) ++ (+) ++++ (+++) ++++ (+++) ++++ (++++) +++ (+++) +++ (+++) ++++ (+++)
kyushuensis type strain11ac (kyushuensis) +++++ (++) ± (±) ++++ (+++) ++++ (+++) + (++) NT+++ (+++) ++++ (+++)
HD-73‡3abc (kurstaki) − (−) − (−) − (−) − (−) − (+) NT− (−) − (+)
*Alkali-solubilized, protease-treated inclusion proteins (124 μg ml−1) were examined for cytotoxic activities on: MOLT-4 (human leukaemia T cell), normal human T cell (primary culture), A549 (human lung cancer cell), MRC-5 (normal human lung fibroblast cell), HeLa (human uterus cervix cancer cell), GF-Scale (Carassius auratus goldfish scale cell), BM-N (Bombyx mori silkworm cell), and NIAS-AeAl-2 (Aedes albopictus mosquito cell). The results with microscopic observations are shown in upper rows, and the levels of cytotoxicity, assessed by cell proliferation assay, are presented in parentheses. The degree of cell damage was graded as follows: extremely high (++++), high (+++), moderate (++), low (+), very low (±), and no damage (−). †The type strain of serovar kurstaki. UTE, Untestable due to no flagellation and/or autoagglutination. UTY, Untypable by reference antisera. NT, Not tested.
image

Figure 1. Cytopathic effect of parasporal inclusion proteins of the three non-insecticidal Bacillus thuringiensis strains. Phase-contrast microscopy at 24 h post-inoculation. Bar shown in l represents 50 μm for a–h, 100 μm for i–l

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Indiscriminative cytotoxicities to human cells, fish cells and insect cells were associated with parasporal inclusions of the two mosquitocidal reference strains, israelensis and kyushuensis. The strain HD-73 (serovar kurstaki), a Lepidoptera-toxic strain, was virtually non-toxic to the vertebrate and invertebrate cells used here.

SDS-PAGE of inclusion proteins and immunoblot analysis

When analysed by SDS-PAGE ( Fig. 2), the purified inclusions of 84-HS-1-11 contained a single major protein of 81 kDa. In contrast, at least five major proteins were evident in purified inclusions of both 89-T-26-17 and 90-F-45-14. Protease treatment generated major bands of 61 and 28 kDa in 89-T-26-17 and 90-F-45-14, respectively, while the major protein of the strain 84-HS-1-11 was degraded rapidly into smaller molecules of < 20 kDa.

image

Figure 2. SDS-PAGE and immunoblot analysis of the cancer cell-killing parasporal proteins of the three non-insecticidal Bacillus thuringiensis strains. Immunoblotting was done with antisera raised against inclusion proteins of the type strains of (a) serovar israelensis, (b) kyushuensis, and (c) kurstaki. Each lane contained 5 μg of inclusion proteins. Molecular markers are indicated on the left

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When examined by immunoblotting, three reference polyclonal antisera against the type strains of serovars israelensis, kyushuensis and kurstaki showed little cross-reactivity to inclusion proteins of 84-HS-1-11, 89-T-26-17 and 90-F-45-14 ( Fig. 2), with the exception that the 81 kDa protein of the strain 84-HS-1-11 gave a very weak reaction for kurstaki antibodies.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References

It is reported here for the first time that certain non-insecticidal B. thuringiensis inclusion proteins have a novel, pronounced biological activity, a cytocidal toxicity to human cancer cells. A large-scale screening clearly showed the existence of B. thuringiensis strains whose parasporal inclusion proteins are neither insecticidal nor haemolytic but highly cytocidal in vitro for human cells, including certain cancer cells. Earlier, two interesting observations have been made by Indian ( Prasad & Shethna 1974) and Japanese ( Seki et al. 1978 ) workers who reported that Lepidoptera-toxic B. thuringiensis crystal proteins exhibited anti-tumour activities against mouse ascite sarcoma cells. In the present study, however, we could not find cytocidal activity against human cancer cells among Lepidoptera-toxic parasporal proteins. The two mosquitocidal strains, israelensis and kyushuensis, showed strong anti-cancer cell activity. It is very likely, however, that the activity in these strains is attributable to the broad-spectrum cytolysins, Cyt1 and Cyt2 proteins ( Koni & Ellar 1994). Immunologically, it is clear that the proteins of the three strains (84-HS-1-11, 89-T-26-17 and 90-F-45-14) are not allied to the Cyt proteins and the other established classes of the insecticidal proteins, Cry1 and Cry4.

It also appeared from the results that there is a marked variation among proteins in the cytotoxic spectrum as well as in the level of cell-killing activity. Of particular interest is the fact that the proteins from 84-HS-1-11 and 89-T-26-17 are able to discriminate between human leukaemia T cells and normal T cells, killing the former cells specifically. Recent studies have demonstrated the occurrence of B. thuringiensis strains that have very narrow insecticidal spectra. The examples include: (i) a strain of the serovar japonensis that produces the Cry8C protein specifically active on only a few genera of the family Scarabaeidae (Coleoptera) ( Ohba et al. 1992 ; Sato et al. 1994 ), and (ii) a strain of the serovar leesis whose parasporal proteins exhibit larvicidal activity highly specific for the moth-fly, Telmatoscopus albipunctatus (Diptera: Psychodidae) ( Higuchi et al. 1998 ). The findings, coupled with our present observations, strongly suggest the possible occurrence, in non-insecticidal, naturally-occurring B. thuringiensis populations, of strains with proteins highly selective for given types of cancer cells. This may lead to the use of B. thuringiensis for medical purposes.

Acknowledgement

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References

The authors thank Dr T. Kawarabata, Kyushu University, for the critical reading of the manuscript.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References
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