A Bacillus thuringiensis Crystal Protein with Selective Cytocidal Action to Human Cells

Abstract

Bacillus thuringiensis crystal proteins, well known to be toxic to certain insects but not pathogenic to mammals, are used as insecticidal proteins in agriculture and forest management. We here identified a crystal protein that is non-insecticidal and non-hemolytic but has strong cytocidal activity against various human cells with a markedly divergent target specificity, e.g. highly cytotoxic to HepG2 and Jurkat and less cytotoxic to the normal hepatocyte (HC) and HeLa. In slices of liver and colon cancer tissues, the toxin protein preferentially killed the cancer cells, leaving other cells unaffected. The cytocidal effect of the protein is non-apoptotic with swelling and fragmentation of the susceptible cells, although the apoptotic process does occur when the cell damage proceeded slowly. The amino acid sequence deduced from the nucleotide sequence of the cloned gene of the protein has little sequence homology with the insecticidal crystal proteins of B. thuringiensis. These observations raise the presence of a new group of the B. thuringiensis toxin and the possibility of new applications for the protein in the medical field. Bacillus thuringiensis crystal proteins, well known to be toxic to certain insects but not pathogenic to mammals, are used as insecticidal proteins in agriculture and forest management. We here identified a crystal protein that is non-insecticidal and non-hemolytic but has strong cytocidal activity against various human cells with a markedly divergent target specificity, e.g. highly cytotoxic to HepG2 and Jurkat and less cytotoxic to the normal hepatocyte (HC) and HeLa. In slices of liver and colon cancer tissues, the toxin protein preferentially killed the cancer cells, leaving other cells unaffected. The cytocidal effect of the protein is non-apoptotic with swelling and fragmentation of the susceptible cells, although the apoptotic process does occur when the cell damage proceeded slowly. The amino acid sequence deduced from the nucleotide sequence of the cloned gene of the protein has little sequence homology with the insecticidal crystal proteins of B. thuringiensis. These observations raise the presence of a new group of the B. thuringiensis toxin and the possibility of new applications for the protein in the medical field. Bacillus thuringiensis is a Gram-positive, spore-forming bacterium that forms a parasporal crystal during the stationary phase of its growth cycle. The crystal proteins studied so far are toxic to the larvae of certain insects of several orders, and even to nematodes, mites, and protozoa, but not pathogenic to mammals, birds, amphibians, or reptiles (1Schnepf E. Crickmore N. Van Rie J. Lereclus D. Baum J. Feitelson J. Zeigler D.R. Dean D.H. Microbiol. Mol. Biol. Rev. 1998; 62: 775-806Crossref PubMed Google Scholar, 2de Maagd R.A. Bravo A. Crickmore N. Trends Genet. 2001; 17: 193-199Abstract Full Text Full Text PDF PubMed Scopus (473) Google Scholar). Thus, this bacterium is a valuable source of insecticidal proteins for use in conventional sprayable formulations and in transgenic crops and may even be the most promising alternative to synthetic chemical pesticides used in commercial agriculture and forest management as these proteins are beneficial and friendly to the environment relative to chemical pesticides (1Schnepf E. Crickmore N. Van Rie J. Lereclus D. Baum J. Feitelson J. Zeigler D.R. Dean D.H. Microbiol. Mol. Biol. Rev. 1998; 62: 775-806Crossref PubMed Google Scholar, 2de Maagd R.A. Bravo A. Crickmore N. Trends Genet. 2001; 17: 193-199Abstract Full Text Full Text PDF PubMed Scopus (473) Google Scholar). Genes of the crystal proteins of B. thuringiensis appear to reside on plasmids, often as parts of composite structures that include a variety of transportable elements (2de Maagd R.A. Bravo A. Crickmore N. Trends Genet. 2001; 17: 193-199Abstract Full Text Full Text PDF PubMed Scopus (473) Google Scholar, 3Mahillon J. Rezsohavy R. Hallet B. Delcour J. Genetica (Dordrecht). 1994; 93: 13Crossref PubMed Scopus (85) Google Scholar). This high degree of genetic plasticity results in the remarkable diversity of B. thuringiensis strains and crystal proteins, and a growing number of the strains and toxin proteins are being isolated and cloned (1Schnepf E. Crickmore N. Van Rie J. Lereclus D. Baum J. Feitelson J. Zeigler D.R. Dean D.H. Microbiol. Mol. Biol. Rev. 1998; 62: 775-806Crossref PubMed Google Scholar, 4Crickmore N. Zeigler D.R. Feitelson J. Schnepf E. Van Rie J. Lereclus D. Baum J. Dean D.H. Microbiol. Mol. Biol. Rev. 1998; 62: 807-813Crossref PubMed Google Scholar). Earlier studies have shown that non-insecticidal B. thuringiensis strains are ubiquitous in natural environments and are even more widely distributed than insecticidal strains (5Ohba M. Aizawa K.J. J. Invertebr. Pathol. 1986; 47: 12-20Crossref Scopus (119) Google Scholar, 6Meadows M.P. Ellis D.J. Butt J. Jarrett P. Burges H.D. Appl. Environ. Microbiol. 1992; 58: 1344-1350Crossref PubMed Google Scholar). Among the non-insecticidal B. thuringiensis isolates, we found isolates with a novel property, non-hemolytic but highly cytotoxic to a wide range of mammalian cells, including human cancer cells (7Mizuki E. Ohba M. Akao T. Yamashita S. Saitoh H. Park Y.S. J. Appl. Microbiol. 1999; 86: 477-486Crossref PubMed Scopus (136) Google Scholar, 8Mizuki E. Park Y.S. Saitoh H. Yamashita S. Akao T. Higuchi K. Ohba M. Clin. Diagn. Lab. Immunol. 2000; 7: 625-634Crossref PubMed Scopus (126) Google Scholar, 9Kim H.S. Yamashita S. Akao T. Saitoh H. Higuchi K. Park Y.S. Mizuki E. Ohba M. J. Appl. Microbiol. 2000; 89: 16-23Crossref PubMed Scopus (27) Google Scholar, 10Lee D.W. Akao T. Yamashita S. Katayama H. Maeda M. Saitoh H. Mizuki E. Ohba M. Biochem. Biophys. Res. Commun. 2000; 272: 218-223Crossref PubMed Scopus (38) Google Scholar, 11Lee D.W. Katayama H. Akao T. Maeda M. Tanaka R. Yamashita S. Saitoh H. Mizuki E. Ohba M. Biochim. Biophys. Acta. 2001; 1547: 57-63Crossref PubMed Scopus (27) Google Scholar). The cytotoxic proteins were heterogeneous in cytotoxicity spectra, and some were active on a wide range of human cells, whereas the others killed a few specific cells. These proteins are not allied to the class of Cyt proteins, which also exhibit cytotoxicity against mammalian cells but do have hemolytic and insecticidal activities (1Schnepf E. Crickmore N. Van Rie J. Lereclus D. Baum J. Feitelson J. Zeigler D.R. Dean D.H. Microbiol. Mol. Biol. Rev. 1998; 62: 775-806Crossref PubMed Google Scholar). The B. thuringiensis A1547 (designated previously as strain 94-F-45-14), belonging to serovar dakota, is one of the strains with parasporal crystal proteins that are non-hemolytic but are cytocidal against human leukemic T cells, MOLT-4 (7Mizuki E. Ohba M. Akao T. Yamashita S. Saitoh H. Park Y.S. J. Appl. Microbiol. 1999; 86: 477-486Crossref PubMed Scopus (136) Google Scholar, 8Mizuki E. Park Y.S. Saitoh H. Yamashita S. Akao T. Higuchi K. Ohba M. Clin. Diagn. Lab. Immunol. 2000; 7: 625-634Crossref PubMed Scopus (126) Google Scholar, 9Kim H.S. Yamashita S. Akao T. Saitoh H. Higuchi K. Park Y.S. Mizuki E. Ohba M. J. Appl. Microbiol. 2000; 89: 16-23Crossref PubMed Scopus (27) Google Scholar). In the present study, we obtained the gene for the purified toxin protein, examined the cytotoxic activity of the expressed recombinant toxin protein against the cultured human cells and the cancer tissues, and found that the toxin protein has strong cytocidal activity against various human cells with markedly divergent target specificity and preferentially kills the liver and colon cancer cells, leaving the normal cells in the tumor tissue slice unaffected. Bacterial Strains and Culture Media—The organisms used in this study was the soil isolate B. thuringiensis A1547, belonging to serovar dakota (7Mizuki E. Ohba M. Akao T. Yamashita S. Saitoh H. Park Y.S. J. Appl. Microbiol. 1999; 86: 477-486Crossref PubMed Scopus (136) Google Scholar, 8Mizuki E. Park Y.S. Saitoh H. Yamashita S. Akao T. Higuchi K. Ohba M. Clin. Diagn. Lab. Immunol. 2000; 7: 625-634Crossref PubMed Scopus (126) Google Scholar, 9Kim H.S. Yamashita S. Akao T. Saitoh H. Higuchi K. Park Y.S. Mizuki E. Ohba M. J. Appl. Microbiol. 2000; 89: 16-23Crossref PubMed Scopus (27) Google Scholar). The bacteria were grown on nutrient medium, containing 0.1% meat extract, 0.1% polypeptone, and 0.2% NaCl, pH 7.6, at 27 °C for 8 days. Human Cells and Culture of the Cells—Human cells used, MOLT-4, Jurkat, HL60, Sawano, HepG2, HeLa, TCS, HC, A549, MRC-5, and Caco-2 cells, were obtained from Riken Cell Bank (Tsukuba, Japan). All cells were maintained under conditions recommended by the manufacturer. Normal T-cells were prepared from buffy coats obtained from the Fukuoka Red Cross Blood Center (Fukuoka, Japan) and were separated from lymphocytes and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 30 μg/ml kanamycin at 37 °C. Isolation of Toxin Protein from Parasporal Crystals—Sporulated cells of B. thuringiensis strain A1547 were harvested and washed three times and disrupted in distilled water. After purification of the parasporal crystals using a biphasic separation technique (12Goodman N.S. Gottfried R.J. Rogoff M.H. J. Bacteriol. 1967; 94: 485Crossref PubMed Google Scholar), the crystals were solubilized with 50 mm Na2CO3 (pH 10.0) at 37 °C for 1 h in the presence of 10 mm dithiothreitol and 1 mm EDTA and were treated with proteinase K (final concentration, 10 μg/ml) at 37 °C for 30 min followed by the addition of 1 mm phenylmethylsulfonyl fluoride to stop the proteolysis. The protease-treated proteins were applied to DE-52 ion-exchange column, and the toxin protein was eluted with 50 mm NaCl in 20 mm Tris-HCl buffer, pH 8.0. The active fraction was then subjected to gel filtration with Sephacryl S-300. Assay of Cytocidal Activity—The cytocidal activity was measured by the 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-dipheny-2H tetrazolium bromide (MTT) 1The abbreviations used are: MTT, 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-dipheny-2H tetrazolium bromide; Z, benzyloxycarbonyl; fmk, fluoromethyl ketone. method (13Behl C. Davis J. Cole G.M. Schubert D. Biochem. Biophys. Res. Commun. 1992; 186: 944-950Crossref PubMed Scopus (586) Google Scholar, 14Heiss P. Bernatz S. Bruchelt G. Senekowitsch-Schmidtke R. Anticancer Res. 1997; 17: 3177-3178PubMed Google Scholar), using a non-radioactive cell proliferation assay system (Promega). Briefly, each well of a microtest plate contained 90 μl of cell suspension containing 2 × 104 cells. After preincubation for 16 h at 37 °C, 10 μl of the proteinase K-activated sample was added to the well, and the cell proliferation was measured 24 h after the administration. Determination of N-terminal Amino Acid Sequence—The purified protein was transferred to a polyvinylidene difluoride membrane (Millipore), and the N-terminal sequence was determined using an automatic sequencer Model 473A (Applied Biosystems). Cloning of the Toxin Protein—The library for plasmid DNA of the bacteria was prepared from a mixture of size-selected (2-8-kb) fragments of the plasmid integrated into Lambda ZAP II vector. Selection of positive plaques was done using a Gene Images CDP-star detection module (Amersham Biosciences). The degenerate primer, designed from the N-terminal amino acid sequence of the 30-kDa toxin protein, was labeled using Gene Images 3′-oligolabeling module then used for selection of positive plaques. DNA sequencing of the positive clone was done with the Thermo sequenase per mixed cycle sequencing kit (Amersham Biosciences) and an automated SQ-5500 DNA sequencer (Hitachi). Expression and Purification of Recombinant Toxin Proteins—Plasmids, pET-37k and pET-30k, were constructed from pET-23a. pET-37k contains a gene of the full-length toxin protein, and pET-30k contains a gene for the proteinase K-activated protein with the initiation methionine codon at the 5′-terminus. A hexahistidine tag was introduced into the C termini of both proteins. Escherichia coli BL21 (DE3) cells transformed with each plasmid were cultured in LB medium containing 0.1 mm isopropyl β-d-thiogalactopyranoside for 24 h at 25 °C. After harvesting the cells by centrifugation for 10 min at 1,000 × g, the cells were suspended in 10 mm Tris-HCl buffer, pH 7.5, containing 10 mm MgSO4, 50 mm KCl, and 10% glycerol, and then lysed by sonication followed by solubilization with 50 mm Na2CO3 (pH 10.0) at 37 °C for 1 h in the presence of 10 mm dithiothreitol and 1 mm EDTA. The soluble extract was recovered by centrifugation for 20 min at 15,000 × g. The extract was loaded on a nickel-chelating Sepharose column (Amersham Biosciences; His-trap) and eluted with 500 mm imidazole. The purity was confirmed by SDS-PAGE. Culture of Human Liver Tissue Slice—The cancer tissue was isolated immediately after surgical resected. The cancer tissue was cut into about 3-mm-diameter pieces. After washing with cold phosphate-buffered saline, the fragments were cultured in Dulbecco's modified Eagle's medium, supplemented with 10% fetal calf serum and an antibiotic mixture containing 100 ng/ml streptomycin, 100 units/ml penicillin, and 5 mg/ml fungizone, at 37 °C. The toxin protein was added to the culture medium (final concentration, 0.3 μg/ml), and the tissue fragments were further incubated for 8 h. The tissues were then fixed with 20% formaldehyde and embedded in paraffin. After being stained with hematoxylin and eosin, the tissue was observed under a light microscope. Assay of Caspase Activity—Cells were incubated with the toxin protein (0.1 or 10 μg/ml), staurosporine (10 μg/ml), Triton X-100 (0.1%) at 37 °C for the indicated times. After lysis of the cells, the fluorogenic peptide substrate, z-DEVD-rhodamine 110, was added and incubated at the room temperature. The caspase activation (fold) was determined on the basis of the relative intensity of fluorescence to the mock-incubated cells. For inhibition experiments, cells were pretreated with or without caspase inhibitor, Z-VAD-fmk (100 μm), at 37 °C for 1 h and then incubated with the toxin protein (0.1 or 10 μg/ml), staurosporine (10 μg/ml) at 37 °C for 20 h. A half-portion of each sample was subjected to the caspase assay, and for another half, the cell proliferation was measured as described above. Cloning of a Cytocidal Protein of B. thuringiensis A1547 and Its Expression in E. coli—After treatment of the parasporal inclusions of B. thuringiensis A1547 strain with proteinase K, the toxin protein was purified through two column chromatographies, measuring their cytotoxic effects on MOLT-4 cells using the MTT assay system. The cytocidal activity was associated with a protein with a molecular mass of 30 kDa. Without protease digestion, the inclusion proteins, like the Cry toxins, exhibited no cytocidal activity (15Lecadet M.M. Dedonder R. Bull. Soc. Chim. Biol. 1966; 48: 631-660PubMed Google Scholar, 16Tojo A. Aizawa K. Appl. Environ. Microbiol. 1983; 45: 576-580Crossref PubMed Google Scholar). The N-terminal amino acid sequence of the proteinase K-activated 30-kDa protein was determined to be Asp-Val-Ile-Arg-Glu-Tyr-leu-Met-Phe-Asn. The gene for the cytocidal 30-kDa protein was cloned from a phage library prepared from a plasmid DNA of the bacteria, using as the probes degenerated oligonucleotides designed from the N-terminal amino acid sequence. The amino acid sequence deduced from the nucleotide sequence of the cloned gene is shown in Fig. 1. The gene was 1,014 bp long and encoded a polypeptide of 338 amino acid residues with a predicted molecular mass of 37,446. The N-terminal amino acid sequence of the proteinase K-activated protein appears in the deduced sequence. Proteinase K would cleave between threonine 51 and aspartic acid 52, and the molecular weight of the N-terminal truncated protein was calculated to be 31,703. The block sequences conserved in the Cry proteins (1Schnepf E. Crickmore N. Van Rie J. Lereclus D. Baum J. Feitelson J. Zeigler D.R. Dean D.H. Microbiol. Mol. Biol. Rev. 1998; 62: 775-806Crossref PubMed Google Scholar, 3Mahillon J. Rezsohavy R. Hallet B. Delcour J. Genetica (Dordrecht). 1994; 93: 13Crossref PubMed Scopus (85) Google Scholar) were not detectable in the sequence of the 37-kDa precursor protein. The protein exhibits a weak sequence homology only with Cry 15Aa (17Brown K.L. Whiteley H.R. J. Bacteriol. 1992; 174: 457-549Crossref Google Scholar) among the established Cry and Cyt proteins (3Mahillon J. Rezsohavy R. Hallet B. Delcour J. Genetica (Dordrecht). 1994; 93: 13Crossref PubMed Scopus (85) Google Scholar). Recombinant toxin proteins of the full-length and N-terminal truncated proteins, tagged with a at the C were expressed in coli strain BL21 and purified on a nickel-chelating The full-length protein no cytocidal and the was for activation of the toxic protein. the other the N-terminal truncated protein a molecular mass of as by and the cytocidal activity against MOLT-4 cells as that of the 30-kDa protein from the parasporal inclusion of B. thuringiensis A1547 Cytocidal of the Recombinant Toxin Protein against Human of the recombinant toxin protein against various cultured human cells were using the MTT assay and the 24 h after the are shown in Fig. 2 and The from cells to cells. Among the cells the toxin protein was highly cytotoxic to MOLT-4, Jurkat, Sawano, and HepG2 cells, and the against cells were as as the other some cells, including HeLa, TCS, HC, A549, and MRC-5, were to the toxin protein. we found no for specificity of the cells or no for or cells, some cells from the tumor tissues more to the toxin protein than from the normal tissues, HepG2 activity of B. thuringiensis parasporal toxin protein to various cultured human T T T T in a new to the cancer cells was shown when we applied the toxin protein to cancer tissues isolated from and well II and The tissue were treated with various of the toxin protein for 8 h at 37 °C, and observed under a light In the cancer which has and with and forms treatment with the toxin protein μg/ml) markedly of the cells, and the and cell of of the cells as with the cancer cells to liver cells and cells, and in in by active was even after treatment with the toxin protein. the observations were obtained with colon cancer tissues In the cancer tissue to the toxin protein A and are markedly and of the cancer cells, and the number of cells was than the number of cells without toxin cancer cells and in a was observed in cells of the cells, and cells in the of the tissue shown in Fig. even after treatment with the toxin protein These results that the toxin protein kills preferentially liver and colon cancer cells, leaving other cells in tissues effect of parasporal toxin protein on colon cancer cells. The cancer tissue was isolated from a of in the of the colon immediately after surgical The tissue were treated with the toxin protein (final concentration, μg/ml) for 8 h at 37 °C in Dulbecco's modified Eagle's medium containing 10% fetal calf A and the cancer tissue to the toxin protein. the cancer tissue without toxin the of the cancer tissue the toxin protein. In A and the is of and in and the is of In the containing the cells is with in and in the cancer cells. in and of of the have examined the of cytotoxic of the toxin protein on the human cells Fig. the of effects of the toxin protein on HepG2 cells, one of the most cells, measured using the MTT 10 μg/ml of the toxin protein, the cells and of the cells were 8 h. with a (0.1 μg/ml), the cells and the number at h. This is to that of an used at the of 10 The toxin protein the cells to and Fig. in the of HepG2 cells to the toxin protein (0.1 The cells and to from the h after to the toxin and then and to 20 h. were more observed with cells, in which the cells at 2 h and h was observed in the cells not observations that the cytotoxic effect of the toxin protein is this in more we studied DNA fragmentation and caspase The was observed at the of the toxin protein, whereas at the high no was even at the most of the cells were killed This is also the with caspase of caspase and activities was observed with a of the toxin protein, at than 8 h after the whereas at the activation of activities was when the caspase activities at the of the toxin protein were by the caspase inhibitor, to 10% of the cells without the inhibitor, the of the cells was whereas in the cell with of the caspase activity was by a to cell These results that the cytocidal effect of the parasporal toxin protein is although the apoptotic process occur when the cell damage was as effects of the a high of the toxin protein, the cells even the apoptotic process We that the toxin protein of parasporal crystal from B. thuringiensis strain A1547 has strong cytocidal activity against various human cells with markedly divergent target specificity and that this toxin preferentially kills the liver and colon cancer cells, leaving the normal cells in the tumor tissue slice unaffected. with the mammalian parasporal proteins previously (7Mizuki E. Ohba M. Akao T. Yamashita S. Saitoh H. Park Y.S. J. Appl. Microbiol. 1999; 86: 477-486Crossref PubMed Scopus (136) Google Scholar, 8Mizuki E. Park Y.S. Saitoh H. Yamashita S. Akao T. Higuchi K. Ohba M. Clin. Diagn. Lab. Immunol. 2000; 7: 625-634Crossref PubMed Scopus (126) Google Scholar, 9Kim H.S. Yamashita S. Akao T. Saitoh H. Higuchi K. Park Y.S. Mizuki E. Ohba M. J. Appl. Microbiol. 2000; 89: 16-23Crossref PubMed Scopus (27) Google Scholar, 10Lee D.W. Akao T. Yamashita S. Katayama H. Maeda M. Saitoh H. Mizuki E. Ohba M. Biochem. Biophys. Res. Commun. 2000; 272: 218-223Crossref PubMed Scopus (38) Google Scholar, 11Lee D.W. Katayama H. Akao T. Maeda M. Tanaka R. Yamashita S. Saitoh H. Mizuki E. Ohba M. Biochim. Biophys. Acta. 2001; 1547: 57-63Crossref PubMed Scopus (27) Google Scholar), the presence of a new of B. thuringiensis that are highly cytotoxic to a wide range of mammalian cells but are non-hemolytic and in addition to the established insecticidal Cry and Cyt protein (3Mahillon J. Rezsohavy R. Hallet B. Delcour J. Genetica (Dordrecht). 1994; 93: 13Crossref PubMed Scopus (85) Google Scholar). toxin protein has its specific and target against mammalian cells, and some proteins are specific to certain human cancer cells, as the toxin protein in this This the possibility that we the strains toxin cytocidal to the specific cancer cell and use proteins in the medical and specificity on target cells the presence of protein at the of the cells, as in the of the insecticidal Cry cytocidal with swelling and fragmentation of the susceptible cells that the protein of the cells, also as the Cry (1Schnepf E. Crickmore N. Van Rie J. Lereclus D. Baum J. Feitelson J. Zeigler D.R. Dean D.H. Microbiol. Mol. Biol. Rev. 1998; 62: 775-806Crossref PubMed Google Scholar, D.J. Biochim. Biophys. Acta. PubMed Scopus Google Scholar). of the cell is to some into the of target specificity and of the new of B. thuringiensis toxin protein.