Synthesis and assembly of Escherichia coli heat-labile enterotoxin B subunit in transgenic lettuce (Lactuca sativa)

Escherichia coli heat-labile enterotoxin B subunit (LTB) strongly induces immune responses and can be used as an adjuvant for co-administered antigens. Synthetic LTB (sLTB) based on optimal codon usage by plants was introduced into lettuce cells (Lactuca sativa) by Agrobacterium tumefaciens-mediated transformation methods. The sLTB gene was detected in the genomic DNA of transgenic lettuce leaf cells by PCR DNA amplification. Synthesis and assembly of the sLTB protein into oligomeric structures of pentameric size was observed in transgenic plant extracts using Western blot analysis. The binding of sLTB pentamers to intestinal epithelial cell membrane glycolipid receptors was confirmed by G(M1)-ganglioside enzyme-linked immunosorbent assay (G(M1)-ELISA). Based on the results of ELISA, sLTB protein comprised approximately 1.0-2.0% of total soluble protein in transgenic lettuce leaf tissues. The synthesis and assembly of sLTB monomers into biologically active oligomers in transgenic lettuce leaf tissues demonstrates the feasibility of the use of edible plant-based vaccines consumed in the form of raw plant materials to induce mucosal immunity.     

Alterations at the carboxyl terminus change assembly and secretion properties of the B subunit of Escherichia coli heat-labile enterotoxin

The gene encoding the B subunit of heat-labile enterotoxin (etxB) was mutated at its 3' end by targeted addition of random nucleotide sequences. Gene products from five mutated etxB genes, all of which were shown to encode B subunits with short carboxy-terminal amino acid extensions, were analyzed with respect to a range of functional and structural properties. One class of altered B subunits, exemplified by EtxB124 and EtxB138, which both have seven extra amino acid residues, were found to be specifically defective in their ability to stably associate with A subunits and form holotoxin. Other altered B subunits were less subtlely affected by extensions at their C termini and were, in addition to their failure to associate with A subunits, unable to translocate into the periplasm of Escherichia coli, to pentamerize, or to bind to GM1 ganglioside. This suggests that the carboxy-terminal domain of EtxB mediates A subunit-B subunit interaction.     

Synthesis of a precursor to the B subunit of heat-labile enterotoxin in Escherichia coli.

Escherichia coli K-12 minicells were employed to investigate the biosynthesis of plasmid-encoded, heat-labile enterotoxin of E. coli. Two polypeptide species related to the B subunit of the toxin were expressed in the minicells. One of these polypeptides (molecular weight, 11,500) was immunoprecipitated by antiserum to cholera toxin. Because the B subunits of heat-labile enterotoxin and cholera toxin have common antigenic sites, we concluded that this species was the mature B subunit. The larger polypeptide (molecular weight, 13,000) is likely to be a precursor of the B subunit because it could be chased into the mature form. This conversion was inhibited by compounds which dissipate proton motive force, suggesting that processing requires energy.     

Mass production of somatic embryos expressing Escherichia coli heat-labile enterotoxin B subunit in Siberian ginseng

The B subunit of Escherichia coli heat-labile toxin (LTB) is a potent mucosal immunogen and immunoadjuvant for co-administered antigens. In order to produce large scale of LTB for the development of edible vaccine, we used transgenic somatic embryos of Siberian ginseng, which is known as medicinal plant. When transgenic somatic embryos were cultured in 130L air-lift type bioreactor, they were developed to mature somatic embryos through somatic embryogenesis and contained approximately 0.36% LTB of the total soluble protein. Enzyme-linked immunosorbent assay indicated that the somatic embryo-synthesized LTB protein bound specifically to GM1-ganglioside, suggesting the LTB subunits formed active pentamers. Therefore, the use of the bioreactor system for expression of LTB proteins in somatic embryos allows for continuous mass production in a short-term period.     

Sequence analysis of the heat-labile enterotoxin subunit B gene originating in human enterotoxigenic Escherichia coli

In this study, we determined the amino-terminal coding sequence, covering the signal peptide and the amino-terminus of the mature peptide, of the heat-labile enterotoxin subunit B (LT-B) gene originating in human enterotoxigenic Escherichia coli. Neither the signal sequence nor the amino-terminal sequence of the mature LT-B was identical to those sequences from porcine enterotoxigenic E. coli, but there was an extensive homology.     

Hemagglutinating activity of the B subunit(s) of the heat-labile enterotoxin isolated from chicken enterotoxigenic Escherichia coli

The hemagglutinating activity of the B subunit(s) of the heat-labile enterotoxin (LTc-B) produced by chicken enterotoxigenic Escherichia coli was studied by hemagglutination and hemagglutination inhibition. No or weak hemagglutination of intact human erythrocytes was found by the LTc-B at the highest concentration used, whereas strong hemagglutination of both neuraminidase- and pronase-treated human erythrocytes was found. Enhancement in hemagglutination of treated human erythrocytes induced by the LTc-B was over 2 to 120-fold for type A and B erythrocytes and over 8-fold for type O erythrocytes, respectively. With intact and treated sheep erythrocytes, on the other hand, no hemagglutination was found by the LTc-B at the highest concentration used. Hemagglutination of pronase-treated human type B erythrocytes by the LTc-B was inhibited by methyl-alpha-D-galactopyranoside, galactose, melibiose, hog A + H, asialo-bovine salivary mucin and asialo-thyroglobulin among mono-, di- and polysaccharides and glycoproteins used as inhibitors. These results suggest that the LTc-B is a galactose-specific bacterial lectin.     

Binding and hemagglutinating properties of the B subunit(s) of heat-labile enterotoxin isolated from human enteroxigenic Escherichia coli

The binding and hemagglutinating activities of the B subunit(s) of the heat-labile enterotoxin (LTh-B) isolated from human enterotoxigenic Escherichia coli were investigated. The binding of 125I-labeled LTh-B to neuraminidase-treated human type B erythrocytes was most effectively inhibited by ganglioside GM1. A number of mono-, di- and polysaccharides, as well as several glycoproteins were at least 500 times less potent inhibitors. However, hemagglutination was effectively inhibited by galactose, melibiose and hog A + H but not by ganglioside GM1. Preincubation of the LTh-B with ganglioside GM1 gave much stronger hemagglutination than LTh-B alone. These results suggest that the predominant binding substance for LTh-B on neuraminidase-treated human type B erythrocytes is ganglioside GM1, but indicate that the interaction of LTh-B with ganglioside GM1 is different in hemagglutination.     

Expression of Escherichia coli heat-labile enterotoxin b subunit (LTB) in carrot (Daucus carota L.)

We expressed the B subunit of enterotoxigenic Escherichia coli heat-labile enterotoxin (LTB) encoded by a synthetic codon-optimized gene in carrot. An Agrobacterium-mediated transformation method was used. Thirty independent transgenic lines were regenerated via somatic embryogenesis after 6 months in culture and were transferred to a greenhouse. GM1-ELISA assay was used to assess LTB protein content in mature taproots. Some transgenic lines expressed LTB up to 0.3% of the total soluble protein, which is tenfold higher than the expression levels reported earlier using the native bacterial gene in plants. Immunological assay confirmed proper assembly of the pentameric complex and in vitro activity of the recombinant LTB protein, suggesting that it can be functional in prevention of diarrhea.     

Escherichia coli heat-labile enterotoxin. Nucleotide sequence of the A subunit gene

We report the complete DNA sequence of the Escherichia coli elt A gene, which codes for the A subunit of the heat-labile enterotoxin, LT. The amino acid sequence of the LT A subunit has been deduced from the DNA sequence of elt A. The LT A subunit starts with methionine, ends with leucine, and comprises 254 amino acids. The computed molecular weight of LT A is 29,673. The A subunit of cholera toxin (CT A) has been shown to be structurally and functionally related to the LT A subunit. Comparison of the primary structure of LT A with the known partial amino acid sequence of CT A indicates that the 2 polypeptides share considerable homology throughout their sequences. The NH2-terminal regions exhibit the highest degree of homology (91%), while the COOH-terminal region, containing the sole cystine residue in each toxin is less conserved (approximately 52%). Alignment of homologous residues in the COOH-terminal regions of LT A and CT A indicates that a likely site for proteolytic cleavage of LT A is after Arg residue 188. The resulting A2 polypeptide would be 46 amino acids long, would contain a single cysteine residue, and have Mr = 5261. The elt A nucleotide sequence further predicts that the LT A protein is synthesized in a precursor form, possessing an 18-amino acid signal sequence at its NH2 terminus.     

Recombinant Escherichia coli heat-labile enterotoxin B subunit humoral adjuvant effect depends on dose and administration route

Information on the use of Escherichia coli heat-labile enterotoxin B subunit (LTB) as a parenteral adjuvant is scarce. We evaluate the adjuvant effect of different concentrations of recombinant LTB (rLTB), as well as the influence of administration route (intramuscular and subcutaneous) on mice immune response. The use of 10 μg/dose of rLTB as adjuvant of an inactivated vaccine composed by Suid herpesvirus type 1 (SuHV-1), used to immunize mice intramuscularly, induced the highest average titers of anti-SuHV-1 antibodies (P < 0.05). The same vaccines used subcutaneously induced lower titers of antibodies. The lower the anti-rLTB humoral response determined by ELISA, the higher was its adjuvant activity. In the challenge experiment with SuHV-1, 56% (14/25) (P < 0.05) of the animals inoculated intramuscularly and 32% (8/25) inoculated subcutaneously survived, highlighting the influence of the concentration and the route of administration of rLTB on its performance as an adjuvant. Therefore, rLTB can significantly help in the induction of immunity against SuHV-1 in mice, especially if used intramuscularly in the concentration of 10 μg/dose, representing the best cost-benefit ratio.     

B subunit of Escherichia coli heat-labile enterotoxin as adjuvant of humoral immune response in recombinant BCG vaccination

The B subunit of Escherichia coli heat-labile enterotoxin (LTB), a nontoxic molecule with potent biological properties, is a powerful mucosal and parenteral adjuvant that induces a strong immune response against co-administered or coupled antigens. In this paper, the effect of LTB on the humoral immune response to recombinant BCG (rBCG) vaccination was evaluated. Isogenic mice were immunized with rBCG expressing the R1 repeat region of the P97 adhesin of Mycoplasma hyopneumoniae alone (rBCG/R1) or fused to LTB (rBCG/LTBR1). Anti-R1 systemic antibody levels (IgG1, IgG2a, IgG2b, IgG3, IgM, and IgA) were measured by ELISA using recombinant R1 as antigen. With the exception of IgM, LTB doubled the anti-R1 antibody levels in rBCG vaccination. The IgG1/IgG2a mean ratio showed that both rBCG/LTBR1 and rBCG/R1 induced a mixed Th1/Th2 immune response. Interestingly, anti-R1 serum IgA was induced only by rBCG/LTBR1. These results demonstrate that LTB has an adjuvant effect on the humoral immune response to recombinant antigens expressed in BCG.     

Crystallization and preliminary X-ray diffraction study of the heat-labile enterotoxin B subunit produced by enterotoxigenic Escherichia coli

Heat-labile enterotoxin LT produced by enterotoxigenic Escherichia coli is composed of A and B subunits. The A subunit is enzymatically active; whereas, through the action of the B subunit, the toxin binds to the receptor, a GM1 ganglioside present on the cell surface. Crystals of the LT-B subunit were formed at room temperature by vapor diffusion with polyethylene glycol in the presence of the non-ionic detergent beta-octylglucoside. The crystals were characterized by X-radiation as orthorhombic, space group P2(1)2(1)2(1), with unit cell dimensions of a = 224.1 A, b = 65.3 A, c = 118.4 A. They diffract X-rays to a resolution of at least 2.5 A and are stable to X-rays.     

Cellular location of heat-labile enterotoxin in Escherichia coli.

We demonstrated that both the A and B subunits of heat-labile enterotoxin from Escherichia coli are located in the periplasm. The toxin was shown to form aggregates in Tris-EDTA buffers which are routinely used for isolating membranes. The aggregates pellet upon centrifugation, and this may explain why several previous investigators have concluded that enterotoxin is associated with membranes.     

Inhibition of microtubule assembly in vitro by anti-tubulin monoclonal antibodies

Five monoclonal antibodies against N-terminal domains of alpha- or beta-tubulin were tested for their ability to interfere with the in vitro formation of microtubules. Although all the antibodies exhibited similar association constants for immobilized tubulin, they differed in their inhibitory effect on microtubule assembly. For the most potent antibody, TU-13, the antibody/tubulin molar ratio of about 1:320 was sufficient for a 50% inhibition. The data indicate that the surface regions of N-terminal domains of tubulin are involved in the formation of microtubules.     

Export and processing analysis of a fusion between the extracellular heat-stable enterotoxin and the periplasmic B subunit of the heat-labile enterotoxin in Escherichia coli

As an initial approach in the study of the mechanism of secretion of the extracellular heat-stable enterotoxin of Escherichia coli (STA), and in order to use this polypeptide as an extracellular carrier we previously constructed a fusion between the complete STA toxin (pre-pro-STA) and the mature B subunit of the periplasmic heat-labile enterotoxin (LTB); the resulting STA-LTB hybrid was not secreted to the extracellular environment, and cells expressing the hybrid lysed at temperatures above 35 degrees C. In this work we have established that the hybrid is initially detected as pre-pro-STA-LTB and converted to pro-STA-LTB, which lacks the 19 amino acids that share the properties of a signal peptide; the sequenced 17 amino-terminal residues of pro-STA-LTB defined the processing site of pre-pro-STA-LTB at pro-3phe-2ala-1 decreases gln+1. This process was sensitive to an energy uncoupler (CCCP) and was correlated with translocation of pro-STA-LTB across the inner membrane. Additionally, we are able to show that although pre-pro-STA-LTB is processed at 37 degrees C and 29 degrees C, it is more efficiently processed at the latter temperature. At 37 degrees C, pro-STA-LTB was poorly released into the periplasm, resulting in accumulation of this protein, pre-pro-STA-LTB, and pre-beta-lactamase in the inner membrane, and in cell lysis. In contrast, at 29 degrees C pro-STA-LTB was localized in the periplasm and in the inner membrane, and pre-pro-STA-LTB and pre-beta-lactamase did not accumulate; however, translocation of periplasmic pro-STA-LTB across the outer membrane still did not occur, and a second processing step that would eliminate the pro segment from pro-STA-LTB was never observed. Thus, the fusion of pre-pro-STA and LTB resulted in a polypeptide that, while incompatible with secretion to the extracellular medium, is exported to the periplasm in a temperature-conditional fashion. This latter observation is consistent with an STA secretion pathway whereby pre-pro-STA is first processed to periplasmic pro-STA by the removal of a 19-amino-acid signal peptide.     

Release of heat-labile enterotoxin subunits by Escherichia coli.

Most of the heat-labile enterotoxin (LT) synthesized by Escherichia coli is cell associated; however, a small portion of LT (approximately 10%) is released by bacterial cells into the culture supernatant. The LT subunit B (LT-B) produced by a cloned LT-B gene (tox B) was released in amounts equal to the parent LT release. In contrast, no release of LT subunit A (LT-A) or its smaller derivatives was observed in strains containing cloned toxA genes. The data suggest that LT-B is necessary for the release of LT-A across the bacterial membrane.