The crystal proteins produced by Bacillus thuringiensis subsp, israelensis (Bti) and subsp. coreanensis A1519 strain were examined for the cytotoxicity against MOLT-4 and HeLa cells by MTT assay and LDH assay, The A1519 crystal proteins processed by proteinase K exhibited the specific cell-killing activity toward MOLT-4 with little damage to the cell membrane, On the other hand, the Bti crystal proteins processed by proteinase K caused the substantial damage to the cell membrane of both MOLT-4 and HeLa, leading to the cell lysis. The non-digested crystal proteins of both strains exhibited no cytotoxicity, These data suggested that while the Bti crystal proteins caused the colloid-osmotic swelling and cell lysis of MOLT-4 and HeLa, the proteinase K-digested A1519 crystal proteins induced the specific cell death of MOLT-4 through a mechanism other than that of Bti

Akao, Tetsuyuki

Mizuki, Eiichi

Namba, Akitoshi

Ohba, Michio

Sakai, Hiroshi

Yamagiwa, Masashi

English

Okayama University Digital Information Repository

Cytotoxicity of the Bacillus thuringiensis Crystal Protein against Mammalian Cells

Okayama University Scientific Achievement Repository

Memoirs of the Faculty of Engineering, Okayama University, VoL36, No.2, pp.61-66, March 2002Cytotoxicity of the Bacillus thuringiensis Crystal Proteinagainst Mammalian CellsMasashi Yamagiwa, Akitoshi Namba, Tetsuyuki, Akao", Eiichi Mizuki", MichioOhba"', and Hiroshi Sakai§Department of Bioscience and Biotechnology, Okayama University, Okayama 700-8530, Japan"Biotechnology and Food Research Institute, Fukuoka Industrial Technology Center, Fukuoka 803-0814, Japan""Graduate School of Agriculture, Kyusyu University, Fukuoka, 812-8581, JapanThe crystal proteins produced by Bacillus thuringiensis subsp, israelensis (Bti) and subsp,coreanensis Al519 strain were examined for the cytotoxicity against MOLT-4 and HeLa cellsby MTT assay and LDH assay, The AI519 crystal proteins processed by proteinase Kexhibited the specific cell-killing activity toward MOLT-4 with little damage to the cellmembrane, On the other hand, the Bti crystal proteins processed by proteinase K caused thesubstantial damage to the cell membrane of both MOLT-4 and HeLa, leading to the cell lysis,The non-digested crystal proteins of both strains exhibited no cytotoxicity, These datasuggested that while the Bti crystal proteins caused the colloid-osmotic swelling and cell lysisof MOLT-4 and HeLa, the proteinase K-digested Al519 crystal proteins induced the specificcell death of MOLT-4 through a mechanism other than that of Bti.1. INTRODUCTIONBacillus thuringiensis produces crystalline inclusions consIstIng of highly specific insecticidal proteinscalled O-endotoxins during sporulation, which are toxic to the larvae of lepidopteran, dipteran, and coleopteraninsects, and are currently classified into two families, Cry and Cyt proteins [7], However it was also reportedthat non-insecticidal B. thuringiensis occurs in natural environments more widely than insecticidal ones [11,14].In 1999 a novel activity, the cytotoxicity against leukemia T cells and other human cancer cells, was found inparasporal inclusions of non-insecticidal and non-haemolytic B. thuringiensis isolates [12]. B. thuringiensissubsp. coreanensis A1519 (90-K-14-20) strain is one of the strains whose parasporal crystal proteins are non-insecticidal and non-haemolytic but cytocidal against human leukemic cells [12].Among o-endotoxins, Cyt proteins also exhibit the cytotoxicity against mammalian cells and haemolyticactivity in addition to the insecticidal activity against dipteran larvae. Therefore, the comparative study of thenovel mammalian cell-recognizing crystal protein and the Cyt proteins for the cytotoxicity will give a lot ofinformation about its mode of action. In this study the crystal proteins produced by B. thuringiensis subsp.coreanensis A1519 strain were examined for the functional properties regarding the cytotoxicity toward MOLT-4,the leukemic T cell, in comparison with those of B. thuringiensis subsp. israelellsis.2. MATERIALS AND METHODS§E-mail sakahrsh@biotech.okayama-u.ac.jp6162 I lasashi YAM GIWA et al. MEM.F AC.ENGOKA. NL VoL36. 022.1 Bacterial strain and mammalian cellsThe B. thuringiensis subsp. roreanensis A 1519 strain (90-K-14-20, serotype H25) used in this study wasisolated from the soil in Kyoto, Japan. The human cell lines used in this study were MOLT-4. a leukemic T cell,and HeLa, a uterus cervix cancer cell.2.2 Preparation, solubilization, and digestion of crystal proteinsPurification of crystal proteins was done as described previously [13]. The crystal was solubilized in 100mM Na2COJ (pHI0.5)/ 10 mM DIT at 37°C for I hr. The soluhilized crystal proteins were digested byproteinase K (Roche) or trypsin (Roche) at 37°C for I hr. At the end of incubation, PMSF, phenylmethylsulfonylfluoride (Sigma) was added at a concentration of I mM.2.3 Cytotoxicity assayThe cytotoxicity was estimated by MTT assay. Ninety microliters of a cell suspension (5xlOs cells/ml)and 10 fll of the crystal protein!; were placed in each well of a 96-well microplate. and incubated at 37°C for 3 hr.Then 0.5 flg/fll of MTT, 3-(4,5-dimethyl-?-thia70yl)-2j-diphenyl-2H-tetrazolium bromide (Sigma), was lddcdand incubated for 3 hr. After centrifugation, the suptrnatant was removed, and the precipitate of the converteddye were solubilized with acidic isopropanoL The absorbance of the converted dye was measured at 570 nm andthe survival rate of the cells was calculated. The average absorbance of mock-inoculated negative controls wastaken as a high value (1000/,: cell survival), and that of Triton-X I00 (Nacalai Tesque)-inoculated positive controlswas taken as a low value (0% cell survival).2.4 Evaluation of the cell membrane damageThe damage of cell membrane was cstimat(;d by measuring the activity of LDH, lactate dehydrogenase,leaked out of the damaged cells. One hundred and eighty microliters of a cell suspension containing Ix I05 cellsand 20 fll of the crystal proteins were placed in each well of a 96-well microplate, and incubated at 37°C for 3 hr.Then, the microplate was centrifuged at 250 x g for 10 Jilin and 100 fJI of the supernatant from each well weretransferred to a new microplate The activity of LDH in the supernatant was estimated by measuring theabsorbance at 490 nm using LDH cytotoxicity det ction kit (Takara). The average absorbance of mock-inoculated negative controls was taken as a low value (00/, of damage), and that of Triton-X 100 (Nacalai Tesqu )-inoculated positive controls was taken as a high value (I OO~ of damage).3. RESULTS3.1 Protein composition of the crystalThe purified crystal produced by A 1519 straincontained a variety of proteins ranging from 150 kDato 10 kDa in siLe (Fig. 1), The major c mponentsdetected in SDS-14% PAGE were polypeptides ofabout 150 kDa, 90 kDa, 70kDa, 20 kDa, and 10 kDa.The crystal produced by B. thuringiensis subsp.israelensis con~isted of Cry4A and Cry4B of 130 kDa,Cry II A of 70 kDa, and Cyt IA of 28 kDa, all of whichwere observed in Fig. I. Among them, Cyt 1A, themost abundant polypeptide, is known to have non-specific cytotoxicity.(kDa)220 -97-66 -45-30-'14.3-Fig. 1 Protein composition of the crystalsfrom A1519 strain and Bfi. Ten microgramsof the solubilized crystal proteins were analyzedby SDS-14'71 PAGE followed by CBB staining.M. molecular size markers.~larch 2002 Cytotoxicity of the Bacillus thlm:ngi~HsisCrystal Prot in against Mammalian ~ells 63(a) (b),0120 e.:... 100~ 90100 ro 80"0;-€ 80 E 70e.:... OJ 60<n E> 60 Q5 50"2: () 40:::J 40 '0en 30OJ20 g 20E 100 ro 000 5 10 15 20 25 1 5 10 20Crystal proteins [lJg/mlJ Crystal proteins [lJg/mlJ(C) (d)'0120 e.:... 100~ 90100 ro 8015'0 80 E 70e.:... OJ 60<n E> 60 Q5 50"2: () 40:::J 40 '0 30enOJ 2020 rnro 10E0 ro 00 5 10 15 20 25 0 1 5 10 20Crystal proteins [1Jg/ml] Crystal proteins [1Jg/mIJ(e)1009080<f!- 70<n 60> 50.~:J 40en 3020100MOlT-4IHeLa(f)100900' 80e.:. 70C 60"<3 50"§"0 40>. 30o 2010oMOLT-4 HelaFig. 2 The damage of cell membrane induced by the proteinase K-digested Bli crystal proteins. The Bricry tal proteins wcre solubilized and digested by proteinase K, and analyzed by MTT assay for the cytotoxicitytoward MOLT-4 (a) and HeLa (c). Cell survival in the presence of the solubilized (open box) and the proteinaseK-digested (solid box) crystal proteins were indicated. The damage of cell membrane of MOLT-4 (b) and HeLa(d) was also analyzed by LDH assay. The damage of cell membrane was indicated as the activities of LDHleaked out of the cell in the presence of the solubilized (open box) and the pwteinase K-digested (solid box)crystal proteins. MIT assay (e) and LDH assay (f) were done in the presence of ]00 flg/ml Bri crystal proteins.Results with the proteinase K-digested Bri crystal proteins (shaded box) and with proteinase K-digested BSA(open box) are shown.64 Masashi YAMAGIWA et al. MEM.FAC.ENG.OKA.UNI. VoI.36, No.23.2 The cytotoxic properties ofBti crystal proteinsTo investigate the cytotoxic effect of Bti crystal proteins on MOLT-4 cells, both MTT assay and LDH assaywere done. In Fig. 2a and c are shown the results of MTT assay with MOLT-4 cells and HeLa cells, respectively.These results suggested that neither the solubilized nor the proteinase K-digested Bti crystal proteins induced theeffective cell death of MOLT-4 and HeLa, i.e. most cells survived from the treatment with the Bti crystal proteins.However, the results of LDH assay indicated that the proteinase K-digested Bti crystal proteins caused thesignificant damage of cell membrane of both MOLT-4 and HeLa and, again, the solely solubilized crystal wasnon-cytotoxic (Fig. 2b and 2d), suggesting that the major consequence by the Bti crystal proteins was the cellmembrane damage. Thus, it was strongly suggested that the cytotoxic effects shown above were caused by thenon-specific cytolysin CytlA. We also confirmed the cell death and membrane damage of both MOLT-4 andHeLa cells in the presence of a higher concentration (100 ).lglml) of Bti crystal proteins (Fig. 2e and t).3.3 The cytotoxic properties ofAISl9 crystal proteinsSimilar experiments using the A1519 crystal proteins were also done with MOLT-4 and HeLa cells. Theproteinase K-digested AI519 crystal proteins were toxic to MOLT-4 cells. Almost all the cells were killed at 5).lglml of the proteinase K-digested crystal proteins, although the non-processed crystal proteins exhibited nodetectable cytotoxicity against MOLT-4 cells even at a concentration of 20 ).lglml (data not shown).Interestingly, the results of LDH assay showed that the mortality of the MOLT-4 cells was only 40% even in thepresence of 20 ).lg/ml proteinase K-processed crystal proteins, and that the merely solubilized crystal proteinsexhibited, again, no detectable cytotoxicity (data not shown). This suggested that the cell death of MOLT-4occurred through some mechanisms other than the damage of cell membrane with pore formation caused by so farknown 8-endotoxins. We also investigated the cytotoxic effect of AI519 crystal proteins on HeLa cells. Theresult of MTT assay with HeLa cells suggested that both the solubilized and the proteinase K-digested crystalproteins exhibited very weak cytotoxicity against HeLa cells (data not shown). Moreover, neither the solubilizednor the proteinase K-digested crystal proteins caused detectable membrane damage of the HeLa cell as indicatedby LDH assay (data not shown). These results suggested that the A1519 crystal proteins were specifically activeagainst human leukemic T cell, MOLT-4. It was confirmed that proteinase K itself and the proteinase K-digested BSA exhibited no detectable cytotoxicity against MOLT-4 cells at the concentrations employed in thisstudy (data not shown). Furthermore, we investigated the cytotoxicity of the proteinase K-digested A1519crystal proteins against normal T cells, proving that it caused little cytotoxicity (data not shown). Therefore it issuggested, again, that the Al519 crystal proteins were specifically active against the human leukemic T cell.4. DISCUSSIONIn this study, we investigated the cytotoxicity of the crystal proteins produced by Bacillus thuringiensissubsp. israelensis and subsp. coreanensis A1519 strain. They are proved cytotoxic against the human leukemiccells. We found that the proteinase K-digested Bti crystal proteins caused the colloid-osmotic swelling and celllysis of MOLT-4 and HeLa, but the proteinase K-digested A1519 crystal proteins induced the specific cell deathof MOLT-4 through a mechanism other than that of Bti.Bti crystal proteins consist of Cry4A (130 kDa), Cry4B (130 kDa), CryllA (70 kDa), and CytlA (28 kDa)[1,7,8], which exhibited non-specific cytocidal activities against insect and mammalian cells in addition to thedipteran-specific insecticidal activity [15]. The cytotoxicity of Bti crystal proteins was attributed to Cytl A, abroad-spectrum cytolysin, active on a variety of vertebrate and invertebrate cells [4, 15]. Currently eleven Cyt l>-endotoxins are reported, and are believed to adopt a similar structure because they share a high degree of sequencesimilarity [5]. The structure of Cyt2Aa was revealed in aqueous solution [10]. Based on the structuralMarch 2002 Cytotoxicity of the Bacillus tlJuringiensis Crystal Protein against Mammalian Cells 65information of Cyt2Aa together with the characterization of CytlAa mutants [16] and the proteolysis ofmembrane-bound CytlAa and Cyt2Aa [6], it is suggested that Cyt toxin form a ~-barrel pore via insertion of ~5,~6, and ~7 into the membrane. On the other hand, Butko et at. reported that the effect of CytlA is a general anddetergent-like perturbation of the membrane surface rather than creation of a proteinaceous channels or pores [2,3). No matter which incident, formation of the pore or detergent-like perturbation effect on the membrane, theCyt toxin causes, the consequence of the Cyt toxin action is the cell membrane damage leading to the colloid-osmotic cell lysis [9]. The data obtained in this study also strongly suggested that the proteinase K-digested Bticrystal proteins, major active component of which is CytlA, caused membrane damage of mammalian cells (Fig.2). However, the proteinase K-digested A1519 crystal proteins exhibited the strong cell-killing activity notaccompanied with the cell membrane damage. It is, therefore, suggested that the major effect of A1519 crystalproteins is not the cell membrane damage, but some other incident such as apoptosis. But we cannot rule out thepossibility that the A1519 crystal proteins cause the pore formation or a detergent-like perturbation effect on themembrane. One of the most plausible interpretation for the mode of action of A1519 crystal proteins is asfollows; a receptor on the membrane of susceptible cells for the A 1519 crystal proteins was by chance a cell-deathinducing membrane protein, so the binding of the A1519 crystal proteins to such a membrane protein set on thecell-death inducing (apoptotic) signa!. The cytotoxic effect through membrane damage by the A1519 crystalproteins is, if any, negligible at the concentration employed in this study.In this study we have reported the distinctive biological activity of A1519 crystal proteins. Furtherinvestigation is in progress to reveal what the active component of A1519 crystal proteins is, what the receptormolecule of mammalian cells for the A1519 crystal proteins is, and how A1519 crystal proteins induce the deathof the mammalian cells.ACKNOWLEDGEMENTThis study was performed through Special Coordination Funds for Promoting Science and Technology(Leading Research Utilizing Potential of Regional Science and Technology) of the Ministry of Education, Culture,Sports, Science and Technology of the Japanese Government.REFERENCES[I] Aronson, A. I., W. Beckman, and P. Dunn, Microbio!. Rev., 50(1986), 1-24.[2] Butko, P., F. Huang, M. Pusztai-Carey, and W. K. Surewicz, Biochemistry, 36(1997), 12862-12868.[3] Butka, P., F. Huang, M. Pusztai-Carey, and W. K. Surewicz, Biochemistry, 35(1996), 11355-11360.[4] Chilcott, C. N., and D. J. Ellar, J.. Gen. Microbiol., 134(1988), 2551-2558.[5J Crickmore, N., D. R. Zeigler, 1. Feitelson, E. Schnepf, J. Van Rie, D. Lereclus, J. Baum, and D. H. Dean,Microbio!. Mol. Bio!. Rev., 62(1998), 807-813.[6J Du, J., B. H. Knowles, J. Li, and D. J. Ellar, Biochem. J., 338(1999), 185-193.[71 HOfte, H., and H. R. Whiteley, Microbio!. Rev., 53(1989),242-255.[8J Insell, 1. P., and P. C. Fitz-James, App!. Environ. Microbiol., 50(1985), 56-62.[9J Knowles, B. H., and D. 1. Ellar, Biochim. Biophys. Acta, 924(1987), 509-518.[10] Li, J., P. A. Koni, and D. J. Ellar, 1. Mol. BioI., 257(1996), 129-152.[II] Meadows, M. P., D. J. Ellis, J. Butt, P. Jarrett, and H. D. Burges, App!. Environ. Microbiol., 58(1992), 1344-1350.[12] Mizuki, E., M. Ohba, T. Akao, S. Yamashita, H. Saitoh, and Y. S. Park, J. App!. Microbial., 86(1999),477-486.66 Masashi YAMAGIWA et ai. MEM.FAC.ENG.OKA.UNI. Vo1.36. No.2[13] Nishimoto, T., H. Yoshisue, K. Ihara, H. Sakai, and T. Komano, FEBS Lett., 348(1994), 249-254.[14] Ohba, M., and K. Aizawa, J. Invertebr. Pathol., 47(1986),12-20.[15] Thomas, W. E., and D. J. Ellar, J. Cell Sci., 60(1983),181-197.[16] Ward, E. S., D. J. 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Cytotoxicity of the Bacillus thuringiensis Crystal Protein against Mammalian Cells

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