Tissue culture and expression of Escherichia coli heat-labile enterotoxin B subunit
in transgenic Peperomia pellucida
Nguyen Hoang Loc
a, Nguyen Hoang Bach
a, Tae-Geum Kim
b, Moon-Sik Yang
b,*a
Institute of Resources, Environment and Biotechnology, Hue University, Hue, Viet Nam
b
Division of Biological Sciences, Chonbuk National University and Jeonju Center, Korea Basic Science Institute, Jeonju, Chonbuk 561-756, Republic of Korea
a r t i c l e
i n f o
Article history:
Received 28 December 2009 and in revised form 16 February 2010 Available online 20 February 2010 Keywords:
Escherichia coli heat-labile enterotoxin B subunit (LTB) Edible vaccine Codon optimization Peperomia pellucida Synthetic gene
a b s t r a c t
The B subunit of Escherichia coli heat-labile enterotoxin (LTB), a non-toxic molecule with potent biological properties, is a powerful mucosal and parenteral adjuvant that induces a strong immune response against co-administered or coupled antigens. We synthesized a gene encoding the LTB adapted to the optimized coding sequences in plants and fused to the endoplasmic reticulum retention signal SEKDEL to enhance its expression level and protein assembly in plants. The synthetic LTB gene was located into a plant expression vector under the control of CaMV 35S promoter and was introduced into Peperomia pellucida by biolistic transformation method. The integration of synthetic LTB gene into genomic DNA of transgenic plants was confirmed by genomic DNA PCR amplification method. The assembly of plant-produced LTB was detected by western blot analysis. The amount of LTB protein produced in transgenic P. pellucida leaves was approximately 0.75% of the total soluble plant protein. Enzyme-linked immunosorbent assay indicated that plant-synthesized LTB protein bound specifically to GM1-ganglioside, which is receptor for LTB on the cell surface, suggesting that the LTB subunits formed biological active pentamers.
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Introduction
The enterotoxigenic Escherichia coli heat-labile enterotoxin B subunit (LTB) has been found to be of enterotoxigenic E. coli (ETEC) is a potent mucosal immunogen and immunoadjuvant for co-administered antigens. The results of numerous investigations have shown that LTB is a promising candidate to be a vaccine anti-gen against LT-producing ETEC[1–3]. It has already been expressed in several bacterial and plant systems [4–11]. ETEC is the most common cause of diarrhea, especially in many developing coun-tries, in which the sanitation and public hygiene systems are rudi-mentary, and where safe drinking water is unavailable. The major disease agent of ETEC is the LT. The LT is a plasmid-encoded, high molecular weight toxin, and immunologically and physicochemi-cally related to cholera toxin (CT)[12,13].
The crystal structure of LT revealed that it is composed of one A subunit (LTA) (27 kDa) and five non-covalently associated B sub-units (LTB) (11.6 kDa each) forming a ring-like pentamer. LTA has ADP-ribosylation activity that causes constitutive activation of adenylate cyclase, an increase in the intracellular cAMP and subse-quent severe diarrhea[10,14]. LTB is able to bind to GM1-ganglio-side, a glycosphingolipid found ubiquitously on the cell
membranes of mammals and to other related receptors, such as GD1b-ganglioside, asialo-GM1, lactosylceramide and certain galac-toproteins[15].
The LT and its related cholera toxin (CT) are extremely potent immunogens following mucosal or systemic delivery. It has been shown that LT acts as a strong mucosal adjuvant, which enhances serum and local immune responses to co-administered antigens, where most antigens are unable to induce immune responses [2,16,17]. Therefore, it is not surprising that LT has been incorpo-rated into putative mucosal vaccines to guard against a range of infectious agents. However, its inherent toxicity and allergenicity have hampered progress for human use [15]. One approach to overcome these problems is the use of a non-toxic derivative of LT, like LTB in isolation. Several studies with animal models and one human trial demonstrated that recombinant LTB (rLTB) can stimulate strong serum and mucosal immune responses against LT. Many studies have indicated that LTB could be used as a potent adjuvant[12,16,18]. LTB is highly resistant to proteolytic degrada-tion, and retains its pentameric quaternary structure in a pH as low as 2.0. This is additional evidence that supports LTB as a candidate antigen to be used in edible vaccines.
Peperomia pellucida, a small herbaceous species of the Pipera-ceae family, distributes mainly in Central and South America, Africa, South-East Asian countries, and Australia. It grows in clumps, thriving in loose, humid soils and a tropical to subtropical climate with bright green heart-shaped shiny succulent leaves
1046-5928/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2010.02.010
*Corresponding author. Fax: +82 63 270 4334.
E-mail addresses:[email protected](T.-G. Kim), [email protected](M.-S. Yang).
Contents lists available atScienceDirect
Protein Expression and Purification
used in salad. P. pellucida has been used as a food item as well as a medicinal herb. Anti-inflammatory, chemotherapeutic, and analge-sic properties have been found in crude extracts of P. pellucida. It may have potential as a broad spectrum antibiotic, as demon-strated in tests against Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, and Escherichia coli[19]. The aim of this study was to efficiently produce recombinant LTB in P. pellucide plants. The gene encoding LTB was introduced and expressed in P. pellucida by biolistic transformation method, and the product was found to have a pentameric form with the ability to bind the cell receptor, GM1-ganglioside (monosialotetrahexosylganglio-side). According to Kang et al.[1]and Lesieur et al.[20], pentamer-ization of LTB is essential for binding to its natural receptor GM1. Materials and methods
Plant materials and culture conditions
Seeds of P. pellucida were surface-sterilized with 0.1% mercury chloride for 1 min and rinsed three times with sterile distilled water. Sterilized seeds were then germinated on the MS medium [21]consisting 2% sucrose and 0.8% agar in Erlenmayer flask. The stem segments (1 cm in length) were cut from 4-week-old plant-lets and cultured on the MS medium supplemented with 15% CW (coconut water), and different concentrations of BAP (benzylamino purine) and NAA (naphthaleneacetic acid) for shoot regeneration. Regenerating shoots (3 cm in length) were then excised from mul-tiple-shoot and rooted on the MS medium without plant growth regulators.
The pH of medium was adjusted to 5.8, and then it was auto-claved at 121 °C for 15 min. The cultures were incubated at 25 ± 2 °C under an intensity of 2000–3000 lux with a photoperiod of 10-h day light.
Construction of plant expression vector
The synthetic LTB (sLTB) gene was modified based on optimized codon usage of a plant using the overlap extension PCR method[2]. The plant expression vector used in our study, pMYO51, consists of a sLTB, a signal peptide, and the ER retention signal (SEKDEL), un-der the control of CaMV 35S (Cauliflower Mosaic Virus 35S RNA) promoter (Fig. 1).
Plant transformation
The stems of P. pellucida grown aseptically on the MS medium described in section Plant materials and culture conditions were cut and placed on filter paper on top of the MS agar medium before bombardment. The stems were then bombarded with gold (1
l
m) particles using a Biolistic PDS 1000/He Particle Delivery System (Bio-rad, Hercules, CA) following the manufacturer’s instructions and placed in a culture room at 25 °C. Two to 3 days after bom-bardment, the stems were placed on the selection mediumcon-taining 50 mg/L kanamycin. The shoots generated from the selection medium after 5–6 weeks were isolated and transferred to the MS medium without antibiotic and plant growth regulator to stimulate root formation. The putative transgenic P. pellucida plantlets formed roots in 2–3 weeks.
Genomic DNA isolation and PCR analysis
Total genomic DNA was extracted from leaves of putative trans-genic and wild-type P. pellucida by the method of Kang and Fawley [22]. PCR analysis was accomplished using the forward primer (5’-GGATCCGCCACCATGGTGAAGGTGAAG-3’) and the reverse primer (5’-GGTACCTCATAGCTCATCTTTC-3’), which are specific for sLTB gene. PCR amplification was performed as follows: a genomic denaturation at 95 °C for 10 min, followed by 30 cycles of 95 °C for 30 s, 55 °C for 30 s, 72 °C for 30 s; then a final extension at 72 °C for 10 min. PCR products were separated by electrophoresis on 1% agarose gel and stained with ethidium bromide.
Immunoblot detection of LTB protein in transgenic plants
Leaf samples (approximately 0.5 g) from transgenic and wild-type P. pellucida were ground adding liquid nitrogen, and resus-pended in 1 mL of extraction buffer (50 mM HEPES, pH 7.5, 10 mM potassium acetate, 5 mM magnesium acetate, 1 mM EDTA, 1 mM dithiothreitol, and 2 mM phenylmethanesulfonyl fluoride). An aliquot (20
l
g) of total soluble protein, as determined by Brad-ford protein assay (Bio-rad), from transgenic and wild-type plants were separated on 12% SDS-polyacrylamide gel electrophoresis. Purified bacterial LTB was also loaded in the range of 0.5l
g. The separated protein bands were transferred from the gel to Hybond C membrane (Bio-rad) using a Trans-blotÒSD semi-dry transfercell (Bio-rad) at 15 V for 30 min. Nonspecific antibody reactions were blocked by incubating the membranes in 25 mL of 3% bovine serum albumin (BSA) in TBST buffer (TBS plus 0.05% Tween-20) with gentle agitation overnight. The membrane was incubated for 2 h with gentle agitation in 10 mL of 1:5000 dilution of rabbit anti-LTB antiserum (Immunology Consultants Lab. Inc., OR) in TBST antibody dilution buffer containing 1.5% BSA and then washed three times with TBST buffer. The membrane was incubated for 2 h in 1:5000 dilution of anti-rabbit IgG conjugated with alkaline phosphatase (Promega S3731, Madison, WI) in TBST buffer and washed three times with TBST buffer, and once with TMN buffer. After washing, the color was developed with BCIP/NBT in TMN buffer.
Quantification of LTB protein level by ELISA
LTB protein levels expressed in transgenic P. pellucida plants were determined using a quantitative ELISA assay. Total soluble protein from transgenic and wild-type plants were loaded into a 96-well microtiter plate (Nunc, Roskilde, Denmark) with 100
l
L/ well of selected concentration and incubated overnight at 4 °C.The plate was washed three times with washing buffer PBST. The background was blocked by incubation in 1% BSA in PBS (300
l
L/ well) at 37 °C for 2 h, and then the wells were washed three times with PBST. The plate was incubated with a 1:5000 dilution of rab-bit anti-LTB antibody (Immunology Consultants Lab. Inc., OR) (100l
L/well) in 0.01 M PBS containing 0.5% BSA for 2 h at 37 °C, and washed four times with PBST. The wells were incubated with 1:10,000 dilution of goat anti-rabbit IgG conjugated with horserad-ish peroxidase (Sigma G-7641, St. Louis, MO) (100l
L/well) in 0.01 M PBS containing 0.5% BSA for 2 h at 37 °C, and washed four times with PBST. The plate was finally incubated with 100l
L/well TMB substrates (Pharmingen 2606KC and 2607KC, San Diego, CA) for 30 min in the dark to maximize the reaction rate. After incuba-tion, the reaction was measured at 405 nm in an automated ELISA system (Bio-rad).GM1-ganglioside binding assay
To determine the ability of plant-produced LTB to bind to gan-gliosides, microtiter plate was coated with monosialoganglioside GM1 (Sigma) by incubating the plate with 100
l
L/well GM1 (3.0l
g/mL) in bicarbonate buffer, pH 9.6 at 4 °C overnight. Afterthree washes with PBST, the plate was blocked with 1% BSA in 0.01 M PBS. The plate was then washed three times with PBST, and was incubated with various concentrations of total soluble protein from transgenic and wild-type P. pellucida plants in PBS (100
l
L/well) for 2 h at 37 °C. The primary and secondary antibody treatments were as described above. As a control, plate was coated with 100l
L/well BSA (3.0l
g/mL).Results and discussion P. pellucida tissue culture
Stems of P. pellucida plantlets were cultured on the MS medium supplemented with various concentrations of BAP and NAA. Our results showed that regeneration of shoots occurred from nearly 100% of stem explants after 2–3 weeks of culture on both media containing 1.0 mg/L BAP and 0.1 mg/L NAA (approximately 12 shoots/explant), and 1.0 mg/L BAP and 15% CW (approximately 24 shoots/explant). However, the former stimulated stronger growth of the shoots. The explants did not regenerate shoots on the other media (data not shown). In vitro shoots were then iso-lated and transferred onto the MS medium without plant growth
regulators. The rooting response was observed around the two weeks of culture. Preliminary work showed that 90% shoots rooted with 2–3 cm in length (Fig. 2). Generally, we have cultured suc-cessfully P. pellucida plant in in vitro condition, and to our knowl-edge there is no information available concerning tissue culture of this plant species.
The in vitro stem segments have been used as explants to trans-fer LTB gene by biolistic transformation. MS medium supple-mented with 1.0 mg/L BAP and 0.1 mg/L NAA have been used to regenerate shoot from transgenic stem segments.
Transformation of LTB gene and PCR analysis
The regenerated transgenic plants showed no morphological changes in comparison to wild-type plants (data not shown). Six plants resulting from independent transformation events were se-lected and maintained in in vitro condition. The presence of the sLTB gene in transgenic plants was confirmed by genomic DNA PCR amplification method followed by gel electrophoresis of the amplified fragments (Fig. 3). The expected size (414 bp) of PCR products was amplified (lanes 1–6) in all transformed plants. The PCR products with same size was obtained with pMYO51 vector as a positive control (PC) template. The PCR product was not de-tected in non-transgenic plants (NC).
Immunoblot analysis of plant-synthesized LTB protein
Six P. pellucida transgenic plants were used for LTB protein char-acterization. The total soluble proteins (TSPs) were extracted from leaves of the individual transgenic P. pellucida plants. Purified bac-terial LTB was used as a positive control to detect antibody-specific protein in the transgenic plants. Immunoblot analysis of these plants revealed an oligomeric LTB protein with a molecular weight of approximately 45 kDa (five non-covalently associated B sub-units (LTB) of 11.6 kDa each forming a ring-like pentamer) (Fig. 4). This result is similar with the results obtained from differ-ent plant expression systems, such as that of tobacco[1]and let-tuce [3]. However, there were only two transgenic plants expressing LTB protein (# 3 and 4). The others and non-transgenic plant did not cross-react with LTB antibody, therefore the specific signal band corresponding to LTB protein did not occur on the membrane. According to Dekeyser et al.[23], expression of a gene integrated at different genomic locations was often influenced by position effects.
ELISA quantification of LTB protein
An ELISA analysis was used to quantify the expression level of LTB protein in the leaf tissues of transgenic plants (# 3 and 4). The percentage of LTB protein in each plant was calculated from the TSP used in three replicates of the assay. According to this method, the concentrations of TSP loaded in the microtiter plate
wells yielded LTB protein levels are approximately 0.75 and 0.55% of TSPs in the respective transgenic plants (Fig. 5).
Binding assay of LTB protein to GM1-ganglioside
To study the oligomerization of LTB protein produced in trans-genic plant, its binding ability to GM1-ganglioside receptor was tested using 96-well plates coated with ganglioside. GM1-ganglioside has been shown to be the receptor for biological active LTB protein in vivo, and pentameric structure is required for appre-ciable receptor binding. In the GM1-ELISA binding assays, LTB pro-tein produced in transgenic plants demonstrated a strong affinity for GM1-ganglioside, but not for BSA (Fig. 6). Based on the
absor-Fig. 3. Genomic DNA PCR amplification analysis to detect the sLTB gene in the genomes of transgenic plants. The PCR products were separated on 1.0% agarose gel. Lane SM is DNA size marker (lambda/Hind III); lane PC is pMYO51 vector harboring LTB gene was used as positive control; lane NC is genomic DNA of non-transgenic plant was used as template; lanes 1–6 are genomic DNA of transgenic plants was used as template.
Fig. 4. Western blot analysis of LTB protein in transgenic P. pellucida plants. (A) Unboiled TSPs of transgenic plants with LTB (lines 1–6); (B) boiled TSPs of transgenic plants with LTB (lines 3 and 4). Lane WM is protein weight marker (97– 14.4 kDa); lane PC is purified bacterial LTB protein as positive control; line NC is protein extract of non-transgenic plant; P is pentamer; M is monomer.
bance measurement used to determine GM1 binding, the LTB pro-tein expressed in the two transgenic plants (#3 and 4) was very similar. The strong relative binding efficacy of plant-produced LTB for GM1 indicates that plant-derived LTB subunit interacting with GM1.
In conclusion, we have successfully transformed sLTB gene into P. pellucida plant. This edible transgenic P. pellucida is a useful sys-tem for expressing other antigen proteins for mucosal immuniza-tion by oral consumpimmuniza-tion of the raw plant material. The ability of the LTB protein produced in P. pellucida to generate both immuno-genicity and adjuvanticity will be the subject of analysis in future mucosal immunization experiments in animals.
Acknowledgment
This research was supported by a grant of Higher Education Project from Vietnam Ministry of Education and Training (2008– 2011).
References
[1] T.J. Kang, N.H. Loc, M.O. Jang, Y.S. Jang, Y.S. Kim, J.E. Seo, M.S. Yang, Expression of the B subunit of E. coli heat-labile enterotoxin in the chloroplasts of plants and its characterization, Transgenic Res. 12 (2003) 683–691.
[2] T.J. Kang, S.C. Han, M.O. Jang, K.H. Kang, Y.S. Jang, M.S. Yang, Enhanced expression of B subunit of Escherichia coli heat-labile enterotoxin in tobacco by optimization of coding sequence, Appl. Biochem. Biotechnol. 117 (2004) 175– 187.
[3] T.G. Kim, M.Y. Kim, B.G. Kim, T.J. Kang, Y.S. Kim, Y.S. Jang, C.J. Arntzen, M.S. Yang, Synthesis and assembly of Escherichia coli heat-labile enterotoxin B
subunit in transgenic lettuce (Lactuca sativa), Protein Expr. Purif. 51 (2007) 22– 27.
[4] H.S. Mason, T.A. Haq, J.D. Clements, C.J. Arntzen, Edible vaccine protects mice against Escherichia coli heat-labile enterotoxin (LT): potatoes expressing a synthetic LT-B gene, Vaccine 16 (1998) 1336–1343.
[5] M.A. Rezaee, A. Rezaee, S.M. Moazzeni, A.H. Salmanian, Y. Yasuda, K. Tochikubo, S.N. Pirayeh, M. Arzanlou, Expression of Escherichia coli heat-labile enterotoxin B subunit (LTB) in Saccharomyces cerevisiae, J. Microbiol. 43 (2005) 354–360.
[6] T.J. Kang, W.S. Lee, E.G. Choi, J.W. Kim, B.G. Kim, M.S. Yang, Mass production of somatic embryos expressing Escherichia coli heat-labile enterotoxin B subunit in Siberian ginseng, J. Biotechnol. 121 (2006) 124–133.
[7] N.V. Ravin, V.V. Kuprianov, L.A. Zamchuk, A.V. Kochetov, Y.L. Dorokhov, J.G. Atabekov, K.G. Skryabin, Highly efficient expression of Escherichia coli heat-labile enterotoxin B subunit in plants using potato virus X-based vector, Biochem. (Mosc.) 73 (2008) 1108–1113.
[8] S. Rosales-Mendoza, R.E. Soria-Guerra, M.T. de Jesús Olivers-Flores, R. López-Revilla, G.R. Argüello-Astorga, J.F. Jiménez-Bremont, R.F. García-de la Cruz, J.P. Loyola-Rodríguez, A.G. Alpuche-Solís, Expression of Escherichia coli heat-labile enterotoxin b subunit (LTB) in carrot (Daucus carota L.), Plant Cell Rep. 26 (2007) 969–976.
[9] J.G. Lim, J.A. Kim, H.J. Chung, T.G. Kim, J.M. Kim, K.R. Lee, S.M. Park, M.S. Yang, D.H. Kim, Expression of functional pentameric heat-labile enterotoxin B subunit of Escherichia coli in Saccharomyces cerevisiae, J. Microbiol. Biotechnol. 19 (2009) 502–510.
[10] S. Kozuka, Y. Yasuda, M. Isaka, Efficient extracellular production of recombinant Escherichia coli heat-labile enterotoxin B subunit by using the expression/secretion system of Bacillus brevis and its mucosal immunoadjuvanticity, Vaccine 18 (2000) 1730–1737.
[11] R. Chikwamba, J. McMurray, H. Shou, B. Frame, S.E. Pegg, P. Scott, H. Mason, K. Wang, Expression of a synthetic E. coli heat-labile enterotoxin B subunit (LTB) in maize, Mol. Breed. 10 (2002) 253–265.
[12] J.J. Guidry, L. Cardenas, E. Cheng, J.D. Clements, Role of receptor binding in toxicity, immunogenicity and adjuvanticity of E. coli heat-labile enterotoxin, Infect. Immun. 65 (1997) 4943–4950.
[13] J.M. Fleckenstein, L.E. Lindler, E.A. Elsinghorst, J.B. Dale, Identification of a gene within a pathogenicity island of enterotoxigenic Escherichia coli H10407 required for maximal secretion of the heat-labile enterotoxin, Infect. Immun. 68 (2000) 2766–2774.
[14] J.P. Nataro, J.B. Kaper, Diarrheagenic Escherichia coli, Clin. Microbiol. Rev. 11 (1998) 142–201.
[15] N.A. Williams, T.R. Hirst, T.O. Nashar, Immune modulation by the cholera-like enterotoxins: from adjuvant to therapeutic, Immunol. Today 20 (1999) 95– 101.
[16] B.L. Dickinson, Use of Escherichia coli heat-labile enterotoxin as an oral adjuvant, in: H. Kiyono, P.L. Ogra, J.R. McGhee (Eds.), Mucosal Vaccines, Academic Press, San Diego, CA, 1996, pp. 73–87.
[17] T. Arakawa, D.K. Chong, J.L. Merritt, W.H. Langride, Expression of cholera toxin B subunit oligomers in transgenic potato plants, Transgenic Res. 6 (1997) 403– 413.
[18] K. Tochikubo, Y. Yasuda, Principles of mucosal immunity and development of mucosal vaccines using cholera toxin B subunit and its related adjuvants, Rec. Res. Devel. Microbiol. 4 (2000) 387–405.
[19] L.D. Moi, T.M. Hoi, D.D. Huyen, T.H. Thai, N.K. Ban, Plant resources of Vietnam-Bioactive plants, vol. 1, Agriculture Publishing House, Ha Noi, Vietnam, 2005. [20] C. Lesieur, M.J. Cliff, R. Carter, R.F.L. James, A.R. Clarke, T.R. Hirst, A kinetic model of intermediate formation during assembly of cholera toxin B-subunit pentamers, J. Biol. Chem. 277 (2002) 16697–16704.
[21] T. Murashige, F. Skoog, A revised medium for rapid growth and bioassays with tobacco tissue culture, Physiol. Plant 15 (1962) 473–497.
[22] T.J. Kang, M.W. Fawley, Variable (CA/GT)n simple sequence repeat DNA in the alga Chlamydomonas, Plant Mol. Biol. 35 (1997) 943–948.
[23] R.A. Dekeyser, B. Claes, R.M.U. De Rycke, M.E. Habets, M.C. Van Montagu, A.B. Caplan, Transient gene expression in intact and organized rice tissues, Plant Cell 2 (1990) 591–602.
