Expression of Cholera Toxin B Subunit in Transgenic Tomato Plants

Transgenic Research 11: 447–454, 2002. © 2002 Kluwer Academic Publishers. Printed in the Netherlands. 447 Expression of cholera toxin B subunit in transgenic tomato plants Dewal Jani1 , Laxman Singh Meena2 , Quazi Mohammad Rizwan-ul-Haq1 , Yogendra Singh2 , Arun K. Sharma1 & Akhilesh K. Tyagi1,∗ 1 Department of 2 Centre Plant Molecular Biology, University of Delhi South Campus, New Delhi -110021, India for Biochemical Technology, Mall Road, Delhi -110007, India Received and accepted: 10 January 2002 Key words: cholera toxin B subunit, edible vaccine, gene expression, GM1 -ELISA, transgenic plants Abstract Cholera toxin, secreted by Vibrio cholerae, consists of A and B subunits. The latter binds to GM1 -ganglioside receptors as a pentamer (∼55 kDa). Tomato plants were transformed with the gene encoding cholera toxin B subunit (ctxB) along with an endoplasmic reticulum retention signal (SEKDEL) under the control of the CaMV 35S promoter via Agrobacterium-mediated transformation. PCR and Southern analysis confirmed the presence of the ctxB gene in transformed tomato plants. Northern analysis showed the presence of the ctxB-specific transcript. Immunoblot assays of the plant-derived protein extract showed the presence of cholera toxin subunit B (CTB) with mobility similar to purified CTB from V. cholerae. Both tomato leaves and fruits expressed CTB at levels up to 0.02 and 0.04% of total soluble protein, respectively. The GM1 -ELISA showed that the plant-derived CTB bound specifically to GM1 -ganglioside receptor, suggesting that it retained its native pentameric form. This study forms a basis for exploring the utility of CTB to develop tomato-based edible vaccines against cholera. Introduction Cholera is a severe diarrheal disease caused by the bacterium Vibrio cholerae. The bacterium secretes cholera toxin that is responsible for the profuse watery diarrhea. The holotoxin comprises of one A and five B subunits (Zhang et al., 1995). The pentameric B moiety is a strong immunological adjuvant. The ideal vaccine for cholera would be one that provided antitoxin and anticolonizing immunity. Such vaccines are currently being tested. Since parenteral cholera vaccines are not considered to be very effective, both killed and live oral vaccines have been investigated. An oral vaccine composed of CTB mixed with inactivated V. cholerae cells gives protection against cholera (Sanchez et al., 1993). However, the cost of production of CTB is too high for developing countries to use it as a vaccine component. The development of genetic transformation technology has facilitated expression of foreign genes ∗ Author for correspondence: E-mail: aktpmb@hotmail.com in plants. Several institutions, including industries have begun experimenting to use transgenic plants as novel manufacturing systems for proteins of therapeutic value (Sharma et al., 1999; Fischer & Emans, 2000). Expression of antigens in edible tissues of plants offers an inexpensive source of vaccine, as this would avoid need for purification of the antigen. Various antigens like the rabies virus glycoprotein (McGarvey et al., 1995), heat labile enterotoxin B subunit of Escherichia coli (Haq et al., 1995; Lauterslager et al., 2001; Streatfield et al., 2001), Norwalk virus capsid protein (Mason et al., 1996), human insulincholera toxin B subunit fusion protein (Arakawa et al., 1998b) and hepatitis B surface antigen (Kapusta et al., 1999; Richter et al., 2000) have been produced in antigenically active form in edible tissues of plants. Cholera toxin B subunit has been expressed in transgenic tobacco (Hein et al., 1996; Wang et al., 2001) and potato (Arakawa et al., 1997) plants. The CTB protein, purified from transgenic tobacco plants was found to be antigenically similar to authentic protein (Wang et al., 2001). Efficacy of potato-based chol- 448 era toxin B subunit to elicit immune response in mice has also been established (Arakawa et al., 1998a). However, tobacco is not palatable because of high level of toxic compounds and potato needs cooking, which can denature antigens. Tomato plants, which bear palatable fruits can be grown throughout the year making them suitable for the continuous supply of the desired antigen. Therefore, the present investigation was undertaken to express CTB in transgenic tomato plants. To the best of our knowledge, the present report forms the first report of expression of cholera toxin B subunit in a major crop producing edible fruits. Materials and methods Plasmid construction The unmodified DNA sequence encoding for CTB of V. cholerae was amplified with the help of CTBF (forward) primer (5′ -GAA TTA AGG ATC CAC CAT GAT TAA ATT AAA ATT TGG TG-3′ ) which added BamH I site and Kozak sequence and CTBR (reverse) primer (5′ -C TGG AGC TCA TAG CTC ATC TTT CTC AGA ATT TGC CAT ACT AAT TGC GG-3′ ), which added a sequence coding for SEKDEL and a Sac I site. The amplified gene was cloned into pUC18. The ctxB gene was analyzed by DNA sequencing. The recombinant plasmid was designated pUCCTB. The ctxB gene from pUCCTB was excised as a BamH I/Sac I fragment and was used to replace a BamH I/Sac I fragment encoding the gusA gene in the binary vector pBI121 to generate vector pBICTB. The cassette for ctxB gene along with the CaMV 35S promoter and nos terminator was excised from pBICTB using Hind III/EcoR I and cloned into another binary vector pCAMBIA2301, digested with the same enzymes. The resulting plasmid was designated as pCAMBIACTB. were then cocultivated with a 48 h culture of Agrobacterium tumefaciens strain LBA4404 harboring pCAMBIACTB at a density of 1 × 108 cells/ml. The explants were incubated in the bacterial suspension for 30 min, blotted gently on a sterile filter paper, transferred to the preculture medium and incubated at 28◦C for 48 h. The explants were washed thoroughly using MS-B5 medium and transferred to MS-B5 solid medium containing kanamycin (100 mg/l), cefotaxime (500 mg/l) and trans-zeatin (1 mg/l). The explants formed visible calli on selection medium after 15–20 days. After 30– 40 days incubation, regenerated shoots were excised at the base from the calli and transferred to MSB5 basal medium without kanamycin or hormones to stimulate root formation. Plants were transferred to soil and maintained at 28◦C under a 12 h photoperiod. Fluorescent lights (Philips India Ltd) at an intensity of 100–125 µmol/m2 /s were used. Detection of CTB gene in transformed plants Genomic DNA was isolated from leaves as described by Dellaporta et al. (1983). The presence of ctxB was determined by PCR analysis using the primers CTBF and CTBR. 100 ng genomic DNA was used as the template and the PCR cycling conditions were as follows: 94◦ C for 30 s, 54◦ C for 40 s, and 72◦ C for 45 s for a total of 35 cycles. The PCR products were analyzed on a 1% agarose gel. For Southern analysis, 10 µg of genomic DNA from the untransformed control plants and each of the putative transformed plants was digested with Hind III. This enzyme cuts just once outside the promoter region controlling ctxB expression. The digested DNA samples were analyzed on a 1.2% agarose gel, blotted on nylon membrane and hybridized using 32 P-labeled ctxB gene as a probe, following standard procedure (Sambrook et al., 1989). Plant transformation Analysis of RNA from transformed tomato plants Tomato (Lycopersicon esculentum Mill var. Pusa Ruby) seeds were surface-sterilized with 4% sodium hypochlorite solution for 12 min and germinated on a Murashige and Skoog medium, supplemented with the organic components of B5 medium (MS-B5). Cotyledons from 11-day-old seedlings were cut at the tip and near the petiolar end and precultured for 48 h at 22◦C on MS-B5 solid medium supplemented with 2 mg/l BAP on a 12 h day photoperiod regime. These explants Total RNA from the leaves of transformed tomato plants was isolated according to the method of Logemann et al. (1987) or Westoff et al. (1991). Twenty five microgram of total RNA was run on 1.2% formaldehyde agarose gel and transferred to a nylon membrane by capillary blotting. The blot was hybridized with a 32 P-labeled probe encompassing the ctxB coding region following standard procedures (Sambrook et al., 1989). 449 Immunoblot detection of CTB protein in leaves and fruits of transformed tomato plants Transgenic tomato leaves were evaluated for the presence of CTB protein by immunoblot analysis using ECLPLUS chemiluminiscence kit (Amersham Pharmacia, RPN 2132). Fresh or frozen tomato leaves (∼1 g) were ground to a fine powder in liquid nitrogen and added to 1 ml extraction buffer (200 mM Tris–HCl pH 8.0, 100 mM NaCl, 400 mM sucrose, 10 mM EDTA, 5 mM DTT, 1 mM phenylmethyl sulfonyl fluoride, 0.05% Tween-20). For extraction of protein from fruit, the fruits were peeled off and seeds were removed. Fruit pulp (∼1 g), was homogenized with 500 µl of 2 × extraction buffer. The tissue homogenate was centrifuged twice at 12000 × g for 15 min at 4◦ C to remove insoluble debris. An aliquot of supernatant containing 50–100 µg total soluble protein, as determined by Bradford protein assay (Bradford, 1976), from transformed and untransformed plants was resolved on a 15% sodium dodecyl sulfate polyacrylamide gel. Purified CTB protein (Sigma C-9903) in the range of 10–30 ng was also loaded on the gel. The resolved proteins were transferred from the gel to a PVDF membrane (Amersham Pharmacia, RPNF L/98/03) by electroblotting on Hoefer TransphorTM apparatus with active cooling. Non-specific antibody binding sites were blocked by incubation in 5% non-fat milk containing Tris buffer saline (TBS; 10 mM Tris–HCl, pH 7.5, 500 mM NaCl) for 1 h at room temperature with shaking at 40 r.p.m. Subsequently, washing was done in TBS buffer for 5 min with gentle agitation. The membrane was incubated for 4 h in 25 ml of 1:5000 dilution of rabbit anti-cholera toxin antiserum (Sigma C-3062) in TBS-T (TBS with 0.05% Tween-20) containing 1% fat-free milk followed by three washes for 5 min each with TBS-T. The membrane was further incubated with 1:10,000 dilution of goat anti-rabbit IgG, conjugated to peroxidase (Sigma A-9169) in TBS-T with 1% fat-free milk for 1 h at room temperature with gentle agitation. The membrane was washed three times in TBST and once in TBS followed by incubation with ECLPLUS substrate buffer for 5 min at room temperature. The excess solution was drained and the membrane was covered in saran wrap and placed in an autoradiography cassette on a Kodak X-OMAT film. The film was exposed for 15 s–30 min at room temperature and processed in the dark for image development. CTB-GM1 -ganglioside receptor binding assay To determine the affinity of plant-derived CTB protein for the GM1 -ganglioside receptor, GM1 -ELISA was performed. A 96 well microtiter plate was coated with GM1 -ganglioside receptor (Sigma G-7641) by incubating 100 µl GM1 (10 µg/ml) in bicarbonate buffer, (15 mM Na2 CO3 , 35 mM NaHCO3 , pH 9.6) at 4◦ C overnight. The wells were blocked with 300 µl of 3% fat-free milk in PBST for 2 h followed by three washes with PBST. The plate was incubated with various concentrations of total soluble protein from leaves of transformed and untransformed plants in bicarbonate buffer (100 µl per well) overnight at 4◦ C. The plate was washed three times with PBST (phosphate buffer saline with 0.05% Tween-20), the wells were blocked with 300 µl per well of 3% fat-free milk in PBST at 37◦ C for 2 h followed by three washes with PBST. The plate was incubated with 1:2000 dilution of rabbit anti-cholera toxin antibody for 3 h followed by three washes with PBST, followed by incubation with 1:5000 dilution of anti-rabbit IgG, conjugated to horseradish peroxidase. Color was developed using orthophenylenediamine as substrate. Absorbance at 490 nm was recorded. Results and discussion The most common route of vaccination remains that of parenteral administration. However, for the prevention of diseases that are confined to the mucosal system such as cholera, oral delivery of the antigen would be the best means to prime the immune system. Oral delivery has often been resisted because of the likelihood of protein degradation in the gut. Previous reports have shown that plants can express antigens at high levels in their native form (Fischer & Emans, 2000). The antigen remains encapsulated in the plant tissue and therefore is subjected to a less harsh environment in the digestive system. However, expression of antigenic proteins in tissues of transgenic plants that cannot be fed raw is undesirable since for example, boiling potato tubers expressing CTB has been shown to destroy CTB pentamers by 50% (Arakawa et al., 1998a). Therefore, the ideal transgenic plant tissue expressing antigenic proteins would be the one that can be consumed raw. These studies prompted us to create transgenic tomato plants expressing CTB. 450 Figure 1. Structure of the plasmid pCAMBIACTB. The construct carries the left and right borders of the T DNA that demarcates the sequences normally incorporated into the plant genomic DNA via Agrobacterium-mediated transformation. The ctxB coding sequence lies downstream of the CaMV 35S promoter and is followed by the nopaline synthase gene (nos) terminator. The nptII gene provides kanamycin resistance in the transformed plants. The gusA gene is a screenable marker for the transformed plants. KZ and ER are the Kozak sequence and endoplasmic retention signal, respectively. Transformation of tomato plants with ctxB The plasmid pCAMBIACTB (Figure 1), which incorporated ctxB, gusA, nptII genes driven by 35S promoter was constructed as described in Materials and methods and used for transformation of tomato plants. Tomato cotyledons (138) were transformed by co-cultivation with A. tumefaciens strain LBA4404 containing vector pCAMBIACTB. Out of several shoots regenerated on selection medium, four plants (A, B, C, and D) were established and the presence of ctxB was confirmed by PCR amplification of the genomic DNA. All four plants showed the presence of a 413 base pair band corresponding to the ctxB gene amplified from the plasmid pCAMBIACTB (Figure 2). Integration of CTB gene into the tomato genome was confirmed by Southern hybridization. The results show that the gene was integrated into the Figure 2. PCR amplification of ctxB from transformed tomato plants. Genomic DNA was isolated from transformed and wild type (WT) leaf tissue and PCR amplification was performed using CTBF and CTBR primers. Ten nanograms of pCAMBIACTB (ctxB containing plasmid) was used as a positive control. genome of the host plant (Figure 3), thereby indicating that the ctxB gene-specific band obtained by PCR (Figure 2) was not due to persisting cells of Agrobacterium containing the plasmid. DNA from wild type plants did not hybridize with the probe to any significant level. In addition, results also show that plants A and B had at least two copies of the gene integrated, whereas a single copy was integrated into the genome of plants C and D (Figure 3). The progeny of plant C also showed the presence of a single band for the transgene (data not shown). Figure 3. Integration of the ctxB gene in transformed tomato plants. Genomic DNA (10 µg) from a wild type (WT) plant and four transformed plants (A, B, C, D) was restricted with Hind III and hybridized with [32 P]-labeled-ctxB fragment. The minus (−) sign indicates undigested samples while the plus sign (+) indicates digested DNA samples. 451 Figure 4. Detection of ctxB mRNA in transgenic tomato plants. (A) Total RNA (25 µg) from wild type (WT) and transformed (A, B, C, D) tomato plants was separated on 1.2% formaldehyde agarose gel followed by northern transfer and hybridization with [32 P]-labeled ctxB fragment. (B) rRNA quality in ethidium bromide stained gel. Detection of ctxB-specific mRNA in transgenic plants Total RNA from wild type and transgenic plants was assayed for the presence of ctxB-specific mRNA by northern blotting. All transgenic plants analyzed showed the presence of the ctxB-specific transcript (Figure 4). The signal was variable among different transformants indicating possible position effect due to random insertions of the foreign gene into the host plant genome. Interestingly, transcript level was higher in plants containing a single copy of the transgene. This indicates that the transgenic plants expressed mRNA of ctxB gene and the mRNA was stable. The wild type control leaves showed no detectable signal. Immunoblot analysis of CTB protein in transgenic plants In order to confirm the presence of CTB protein in transgenic plants, western blot analysis of total protein from leaves and fruits of transgenic plants along with known amounts of purified CTB was performed. The samples were analyzed by 15% SDS-PAGE followed by transfer to PVDF membrane and incubation with anti-cholera toxin antibody raised in rabbit. As shown in Figures 5 and 6, total soluble protein from leaves and fruits of transgenic plants C and D showed the presence of oligomeric CTB, which was slightly retarded in mobility compared to purified CTB protein. This difference in migration may be due to the presence of the hexapeptide SEKDEL, or possibly due to failure of plant cells to remove the leader peptide, as reported earlier (Arakawa et al., 1997). Transgenic plants A and B did not show expression of CTB proteins although Southern analysis indicated the presence of two integrated copies of ctxB gene and accumulation of significant amounts of mRNA (Figures 3 and 4). Abrogated expression of CTB protein could be due to gene silencing as neither mRNA nor protein were found to accumulate in mature plants A and B and the presence of multiple copies of a gene is known to lead to gene silencing (Matzke & Matzke, 1995). Homogenates from wild type plants did not cross-react significantly with anti-cholera toxin antibody. CTB protein was quantitatively estimated in leaves and fruits of the transgenic plants by densitometric analysis. The maximum expression level in leaves of these plants was 0.02% of total soluble protein as inferred from comparison with known amounts of purified CTB (Figure 5). Tomato fruits from plants C 452 Figure 5. Immunoblot detection of CTB protein expressed in leaf tissue. One hundred micrograms total soluble protein extract from leaves of wild type (WT) and two transformed plants (C, D) along with 20, 30, and 40 ng of purified CTB protein were separated on 15% SDS-PAGE. Rabbit anti-cholera toxin antiserum was used for immunodetection. Figure 6. Immunoblot detection of CTB protein expressed in fruit tissue. Fifty micrograms total soluble protein samples from fruits of wild type (WT) and four transgenic plants, A, B, C and D along with purified protein standards were separated on 15% SDS-PAGE, transferred to PVDF membrane and developed with rabbit anti-cholera toxin antiserum. and D expressed significant amounts of CTB (0.03 and 0.04% of the total soluble protein, respectively (Figure 6)). A tomato weighing 100 g contains approximately 1.1 g of total soluble protein (Jones, 1999) and, therefore, would express approximately 440 µg CTB. In an earlier report CTB:SEKDEL fusion protein expression in potato was found to be 0.3% of total soluble protein, which is higher than the levels reported here (Arakawa et al., 1997). Higher yield of CTB protein achieved by Arakawa et al. (1997) could be due to the use of the stronger mas P2 promoter. However, use of the same promoter to drive an insulin gene resulted in expression levels of 0.05% of total soluble protein (Arakawa et al., 1998b), indicating that sta- bility of RNA or protein could contribute significantly towards achieving high expression levels. Binding of plant-derived CTB to GM1-ganglioside receptor The oligomerization of transgenic plant-expressed CTB protein was also tested by its ability to bind to GM1 -ganglioside receptor. GM1 -ganglioside has been shown to be the receptor for CTB protein in vivo (Haan et al., 1998) and for appreciable receptor binding a pentameric structure is required (Tsuji et al., 1995). In addition, GM1 -ganglioside receptor binding in vitro has been used to assess the native pentameric 453 be designed that employ stronger promoters. Viral leader sequences that act as translational enhancers can also be utilized to increase the amount of protein made. One of the methods, which can lead to expression of antigens in plants at high level, and also helps in containment of the transgenes is chloroplast transformation. Daniell et al. (2001) have achieved accumulation of CTB at the level of 4.1% of total soluble protein in tobacco leaves. Recent development in transformation of tomato chloroplasts (Ruf et al., 2001) can be of great significance for development of edible vaccines. It should be investigated whether the CTB expressed in plants is immunogenic in the mouse model system to validate the suitability of the edible vaccine against cholera. Acknowledgements Figure 7. GM1 binding assay of plant-derived CTB. The ELISA was performed by coating the microtitre plates with GM1 -ganglioside. Ten micrograms total soluble protein from transformed and wild type (WT) plants was added to selected wells. C is the parent plant and C1–C9 are the offsprings of C. Standard deviation is marked at the top of each bar. form of recombinant CTB (Arakawa et al., 1997). The total soluble protein from plant C expressing CTB, its progeny (C1-C9) and wild type plant were analyzed for their ability to bind GM1 -ganglioside receptor using ELISA. The protein samples were added to wells precoated with GM1 and assayed using CTB-specific antibody. Protein samples isolated from plant C and its progeny bound GM1 ; whereas the total protein from wild type plants did not bind (Figure 7). The experiment was repeated four times and results of a representative experiment have been given. The results suggest that the CTB protein expressed in tomato plants retained its native pentameric form to interact with GM1 -ganglioside. Thus, there is no inherent limitation in the production of this recombinant protein in plants and this expressed protein will have antigenic properties similar to the protein expressed in bacteria. Since pentamerization is essential for binding of GM1 -ganglioside, this assay also suggests that the recombinant protein is able to fold in its native conformation. To test the utility of these plants as edible vaccine by feeding the tomato fruits to animal models, plants producing CTB at higher levels may be required. To achieve this goal, expression vectors could The research work has been supported by financial assistance from the Department of Biotechnology, Government of India, New Delhi. Senior research fellowship by CSIR, New Delhi, to DJ is also acknowledged. Thanks are also due to Dr RA Jefferson, CAMBIA, Australia, for providing pCAMBIA 2301 vector. References Arakawa T, Chong DKX, Merrit JL and Langridge WHR (1997) Expression of cholera toxin B subunit oligomers in transgenic potato plants. Transgenic Res 6: 403–413. Arakawa T, Chong DKX and Langridge WHR (1998a) Efficacy of a food plant-based cholera toxin B subunit vaccine. Nat Biotech 16: 292–297. 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