Cell-Free Systems Enable the Production of AB5 Toxins for Diagnostic Applications.

Cell-free protein synthesis (CFPS) represents a versatile key technology for the production of toxic proteins. As a cell lysate, rather than viable cells, is used, the toxic effects on the host organism can be circumvented. The open nature of cell-free systems allows for the addition of supplements affecting protein concentration and folding. Here, we present the cell-free synthesis and functional characterization of two AB5 toxins, namely the cholera toxin (Ctx) and the heat-labile enterotoxin (LT), using two eukaryotic cell-free systems based on Chinese hamster ovary (CHO) and Spodoptera frugiperda (Sf21) cells. Through an iterative optimization procedure, the synthesis of the individual AB5 toxins was established, and the formation of multimeric structures could be shown by autoradiography. A functional analysis was performed using cell-based assays, thereby demonstrating that the LT complex induced the characteristic cell elongation of target cells after 24 h. The LT complex induced cell death at higher concentrations, starting at an initial concentration of 5 nM. The initial toxic effects of the Ctx multimer could already be detected at 4 nM. The detection and characterization of such AB5 toxins is of utmost importance, and the monitoring of intracellular trafficking facilitates the further identification of the mechanism of action of these toxins. We showed that the B-subunit of LT (LTB) could be fluorescently labeled using an LTB-Strep fusion protein, which is a proof-of-concept for future Trojan horse applications. Further, we performed a mutational analysis of the CtxA subunit as its template was modified, and an amber stop codon was inserted into CtxA's active site. Subsequently, a non-canonical amino acid was site-specifically incorporated using bio-orthogonal systems. Finally, a fluorescently labeled CtxA protein was produced using copper-catalyzed click reactions as well as a Staudinger ligation. As expected, the modified Ctx multimer no longer induced toxic effects. In our study, we showed that CFPS could be used to study the active centers of toxins by inserting mutations. Additionally, this methodology can be applied for the design of Trojan horses and targeted toxins, as well as enabling the intracellular trafficking of toxins as a prerequisite for the analysis of the toxin's mechanism of action.

Preparation and evaluation of chitosan nanoparticles containing CtxB antigen against Vibrio cholera.

Vibrio cholera is a Gram-negative pathogen that causes diarrheal disease. The B subunit of Chlora toxin (CtxB) is one of the most important antigens of Vibrio cholera in which mediates the attachment of the bacteria to target cells. The aim of this study was to prepare chitosan nanoparticles containing CtxB and evaluate the effect of the antigen entrapment on the immunogenicity of this antigen. For this, the pET28a vector was induced using IPTG. Recombinant CtxB protein was expressed and purified using Ni-NTA column and finally was confirmed by western blotting. Following the confirmation of the protein entrapment onto the chitosan nanoparticles, the formulation was prescribed to BALB/c mice in three groups, including oral, oral-injection and injection groups. Serum and fecal IgA and IgG were evaluated by ELISA test. Finally, challenge of immunized mice was performed using Ctx toxin and rabbit ileal loop test. Using SDS-PAGE and western blotting, the 17.5 kDa recombinant CtxB was confirmed. Size electrical charge and of nanoparticles were determined and approved by Zetasizer. Nanoparticles prescription showed 1/102400 IgG endpoint titers for injection groups and 1/1600, 1/6400 for oral, oral-injection groups respectively and Serum and fecal IgA endpoint titers showed above 1/160 in all groups. Furthermore, immunized mice were able to neutralize Ctx toxin by ileal loop test. The CtxB is a suitable immunogen of V. cholera to be incorporated in both protective and preventive vaccines. Chitosan nanoparticles improve the immune responses and it may be used as a carrier for vaccine delivery.

Uptake and intracellular fate of cholera toxin subunit b-modified mesoporous silica nanoparticle-supported lipid bilayers (aka protocells) in motoneurons.

Cholera toxin B (CTB) modified mesoporous silica nanoparticle supported lipid bilayers (CTB-protocells) are a promising, customizable approach for targeting therapeutic cargo to motoneurons. In the present study, the endocytic mechanism and intracellular fate of CTB-protocells in motoneurons were examined to provide information for the development of therapeutic application and cargo delivery. Pharmacological inhibitors elucidated CTB-protocells endocytosis to be dependent on the integrity of lipid rafts and macropinocytosis. Using immunofluorescence techniques, live confocal and transmission electron microscopy, CTB-protocells were primarily found in the cytosol, membrane lipid domains and Golgi. There was no difference in the amount of motoneuron activity dependent uptake of CTB-protocells in neuromuscular junctions, consistent with clathrin activation at the axon terminals during low frequency activity. In conclusion, CTB-protocells uptake is mediated principally by lipid rafts and macropinocytosis. Once internalized, CTB-protocells escape lysosomal degradation, and engage biological pathways that are not readily accessible by untargeted delivery methods.

Self-encapsulating Poly(lactic-co-glycolic acid) (PLGA) Microspheres for Intranasal Vaccine Delivery.

Herein we describe a formulation of self-encapsulating poly(lactic-co-glycolic acid) (PLGA) microspheres for vaccine delivery. Self-healing encapsulation is a novel encapsulation method developed by our group that enables the aqueous loading of large molecules into premade PLGA microspheres. Calcium phosphate (CaHPO4) adjuvant gel was incorporated into the microspheres as a protein-trapping agent for improved encapsulation of antigen. Microspheres were found to have a median size of 7.05 ± 0.31 µm, with a w/w loading of 0.60 ± 0.05% of ovalbumin (OVA) model antigen. The formulation demonstrated continuous release of OVA over a 49-day period. Released OVA maintained its antigenicity over the measured period of >21 days of release. C57BL/6 mice were immunized via the intranasal route with prime and booster doses of OVA (10 µg) loaded into microspheres or coadministered with cholera toxin B (CTB), the gold standard of mucosal adjuvants. Microspheres generated a Th2-type response in both serum and local mucosa, with IgG antibody responses approaching those generated by CTB. The results suggest that this formulation of self-encapsulating microspheres shows promise for further study as a vaccine delivery system.

[Effect of plant extracts on cytotoxic activity of Vibrio cholerae hemolysin].

AIM: Study of plant extracts that have the ability to neutralize cytotoxic activity of hemolysin. MATERIALS AND METHODS: Preparations of purified and recombinant V. cholerae eltor hemolysin as well as supernatants of V. cholerae strains were used. Determination ofcytotoxic activity of hemolysin and neutralizing activity of plant extracts were carried out by using cell cultures CHO-K1 and CaCo2. RESULTS: Out of 9 water extracts only 3 - extracts of Rhei rhizome, Limonium gmelinii and Quercus robur neutralized hemolysin in cell culture CHO-K1 and CaCo2, whereas the other extracts--Humulus lupulus, Ocimum basilicum, Chelidonium majus, Juglans regia, Achillea milefolium and Hypericum perforatum did not have anti-cytotoxic effect. Neutralizing properties of extracts are exhibited during their co-incubation with hemolysin preparations and supernatants of V. cholerae strains already within 10 minutes. CONCLUSION: Plant extracts that have anti-cytotoxic activity against hemolysin are perspective for development oftherapeutic-prophylaxis preparations.

Grape extracts inhibit multiple events in the cell biology of cholera intoxication.

Vibrio cholerae produces cholera toxin (CT), an AB5 protein toxin that is primarily responsible for the profuse watery diarrhea of cholera. CT is secreted into the extracellular milieu, but the toxin attacks its Gsα target within the cytosol of a host cell. Thus, CT must cross a cellular membrane barrier in order to function. This event only occurs after the toxin travels by retrograde vesicular transport from the cell surface to the endoplasmic reticulum (ER). The catalytic A1 polypeptide then dissociates from the rest of the toxin and assumes an unfolded conformation that facilitates its transfer to the cytosol by a process involving the quality control system of ER-associated degradation. Productive intoxication is blocked by alterations to the vesicular transport of CT and/or the ER-to-cytosol translocation of CTA1. Various plant compounds have been reported to inhibit the cytopathic activity of CT, so in this work we evaluated the potential anti-CT properties of grape extract. Two grape extracts currently sold as nutritional supplements inhibited CT and Escherichia coli heat-labile toxin activity against cultured cells and intestinal loops. CT intoxication was blocked even when the extracts were added an hour after the initial toxin exposure. A specific subset of host-toxin interactions involving both the catalytic CTA1 subunit and the cell-binding CTB pentamer were affected. The extracts blocked toxin binding to the cell surface, prevented unfolding of the isolated CTA1 subunit, inhibited CTA1 translocation to the cytosol, and disrupted the catalytic activity of CTA1. Grape extract could thus potentially serve as a novel therapeutic to prevent or possibly treat cholera.

Neuroanatomical basis for acupuncture point PC8 in the rat: neural tracing study with cholera toxin subunit B.

OBJECTIVES: This study was performed to investigate the innervations related to acupuncture point PC8 in rats using a neural tracing technique. METHODS: After 6 µL of 1% cholera toxin subunit B (CTB) was injected into the site between the second and third metacarpal bone in rats, a corresponding site to acupuncture point PC8 in the human body, CTB labelling was examined with immunofluorescence and immunohistochemistry in the dorsal root ganglia (DRG), spinal cord and brainstem. RESULTS: All CTB labelling appeared on the ipsilateral side of the injection. The labelled sensory neurons distributed from cervical (C)6 to thoracic (T)1 DRG, while the labelled motor neurons were located on the dorsolateral part of the spinal ventral horn ranging from the C6 to T1 segments. In addition, the transganglionically-labelled axonal terminals were found to be dense in the medial part of laminae 3-4 from C6 to the T1 spinal dorsal horn, as far as in the cuneate nucleus. CONCLUSIONS: These results indicate that sensory and motor neurons associated with PC8 distribute in a distinct segmental pattern. The sensory information from PC8 could be transganglionically transported to the spinal dorsal horn and cuneate nucleus.

Cholera toxin: a paradigm of a multifunctional protein.

Cholera toxin (CT) was discovered exactly half a century ago by S.N. De. We have come a long way since this epoch-making discovery. Retrospectively, science had to wait a long time since Koch's prediction of the existence of a toxin, and its actual discovery by De. CT is not just another enterotoxin that causes the signs and symptoms of the dreaded disease, cholera. It is unique in many respects, starting from its structure to its functions. CT is a multifunctional protein that is capable of influencing the immune system in many ways. It not only has remarkable adjuvant properties, but also acts as an anti-inflammatory agent, by modulating specific signal transduction pathways. Its immunomodulatory properties can be harnessed for treatment of various autoimmune disorders, and have shown great promise in the area of immunotherapeutics. CT can truly be considered as a paradigm of a multifunctional protein.

Cholera toxin B subunit-domain III of dengue virus envelope glycoprotein E fusion protein production in transgenic plants.

Envelope glycoprotein E of the dengue virus, which plays a crucial role in its entry into host cells, has an immunogenic domain III (EIII, amino acids 297-394), which is capable of inducing neutralizing antibodies. However, mice immunized with EIII protein without adjuvant elicited low immune responses. To improve low immune responses, a DNA fragment, consisting of cholera toxin B subunit and EIII gene (CTB-EIII), was constructed and introduced into tobacco plant cells (Nicotiana tabacum L. cv. MD609) by Agrobacterium tumefaciens-mediated transformation methods. The integration and transcription of CTB-EIII fusion gene were confirmed in transgenic plants by genomic DNA PCR amplification and Northern blot analysis, respectively. The results of immunoblot analysis with anti-CTB and anti-dengue virus antibodies showed the expression of the CTB-EIII fusion protein in transgenic plant extracts. Based on the G(M1)-ELISA results, the CTB-EIII protein expressed in plants showed the biological activity for intestinal epithelial cell membrane glycolipid receptor, G(M1)-ganglioside, and its expression level was up to about 0.019% of total soluble protein in transgenic plant leaf tissues. The feasibility of using a plant-produced CTB-EIII fusion protein to generate immunogenicity against domain III will be tested in future animal experiments.

[Neuroanatomical characteristics of acupoint "Chengshan" (BL 57) in the rat: a cholera toxin subunit B conjugated with Alexa Fluor 488 method study].

OBJECTIVE: To investigate neuroanatomical characteristics of the primary sensory afferent and the motor neurons coming from and innervating acupoint "Chengshan" (BL 57) area in the rat by using cholera toxin subunit B conjugated with Alexa Fluor 488 (CTB-Alexa 488), a new generation of fluorescent neural tracing reagent. METHODS: Four male SD rats were used in the present study. Under anesthesia, 0.05% CTB-Alexa 488 (5 ML) was injected into the central part of the rear of the hind leg, a corresponding site of "Chengshan" (BL 57) in the human body. After 40-48 surviving hours, the rat's brain, spinal cord and dorsal root ganglia (DRGs) of the lumbar segments (L1-L6) were dissected following perfusion with 4% paraformaldehyde, cut into sections and observed under fluorescent microscope equipped with a digital camera. The neurons labeled by CTB-Alexa 488 were counted. RESULTS: All CTB-Alexa 488 labeled neurons appeared in green under fluorescent filters of 450-490 and were located ipsilaterally on the injection side. The labeled primary sensory neurons were found in the DRGs at L4 (11 neurons) and L5 (35 neurons). Among them, 29 neurons (63.04%) were bigger, with their cell body diameters being 35-50 microm and 17 (36.96%) smaller, with their body diameters being lower than 35 microm. The labeled motor neurons were found to distribute in the mediolateral portion of lamina IX, forming a longitudinal column from L4 to L5. Of the observed 316 motor neurons, 259 (81.96%) belong to alpha type with their body diameters being 25-40 microm and 57 (18.04%) to gamma type with their body diameters being lower than 25 microm. CONCLUSION: The CTB-Alexa 488-labeled primary sensory and motor neurons innervating acupoint "Cheng-shan" (BL 57) distribute in the DRGs of L4-L5. The present fluorescent tracing technique may be quite useful for investigating the neural characteristics of acupoints.

Expression and assembly of cholera toxin B subunit (CTB) in transgenic carrot (Daucus carota L.).

We expressed the cholera toxin B subunit (CTB) fused to an endoplasmic reticulum retention signal (SEKDEL) in carrot roots using an Agrobacterium-mediated transformation method. Fourteen independent transgenic lines were regenerated via somatic embryogenesis after 6 months of culture. The sCTB gene was detected in the genomic DNA of transgenic carrot by PCR amplification. Expressions and assembly of sCTB protein into oligomeric structures of pentameric size were observed in transgenic plant extracts by Western blot analysis. The sCTB produced by transgenic carrot roots demonstrated strong affinity for GM1-ganglioside, suggesting that the sCTB conserved the antigenic sites for binding and proper folding of the pentameric sCTB structure. The expression level of sCTB comprised approximately 0.48% of total soluble protein (TSP) in root of transgenic carrot.

Efficient induction of oral tolerance by fusing cholera toxin B subunit with allergen-specific T-cell epitopes accumulated in rice seed.

Cholera toxin B (CTB) subunit is an efficient mucosal carrier molecule for induction of oral tolerance to antigens and allergens. Here, T-cell epitopes of Cry j 1 and Cry j 2, major allergens in Japanese cedar pollen, were expressed in rice seed as a fusion protein with either CTB or rice glutelin as a control. Feeding mice with rice seed containing CTB-fused T-cell epitopes suppressed allergen-specific IgE responses and pollen-induced clinical symptoms at 50-fold lower doses of T-cell epitopes than required when using control seed. Our findings present a novel potential strategy for immunotherapy of type-I allergy.

Rapid detection of the Vibrio cholerae ctx gene in food enrichments using real-time polymerase chain reaction.

A real-time polymerase chain reaction (qPCR) assay for the detection of the ctxA gene of toxigenic Vibrio cholerae (Vc) was validated against standard culture techniques. The first experimental phase determined optimal enrichment conditions for detection by culture and qPCR of Vc in shrimp, bottled water, milk, and potato salad. The conditions tested included temperature (35 and 42 degrees C), time (6 and 18 h), and effect of shaking (0 and 100 rpm). No definitive trends were found with enrichment temperature or shaking on Vc isolation frequency or detection by qPCR. Generally, Vc was detected by qPCR more frequently than Vc was isolated, but this difference was significant only in the 35 degrees C 6 h enrichment without shaking. In the second phase of experiments, shrimp, bottled water, milk, potato salad, and oysters were inoculated with each of 3 toxigenic Vc strains (Latin American O1 strain, an O139 strain, and an O1 strain from the U.S. Gulf Coast) and enriched under static conditions at 42OC for 6 and 18 h. Overall, detection frequency of ctx by qPCR was 98% (88/90) and 100% (90/90) after 6 and 18 h enrichments, respectively, while Vc isolation frequency was 87% (78/90) and 83% (75/90) after 6 and 18 h, respectively. Toxigenic Vc can be detected by qPCR within an 8 h work day using the 6 h enrichment procedure, assuming an initial level of at least 1-2 colony-forming units/g; however, overnight enrichment may be necessary to detect lower levels. These data indicate that the qPCR assay for ctx is a more reliable, sensitive, and rapid alternative to standard Vc culture methods and is applicable to diverse food products.

Synthesis and assembly of a cholera toxin B subunit-rotavirus VP7 fusion protein in transgenic potato.

A gene encoding VP7, the outer capsid protein of simian rotavirus SA11, was fused to the carboxyl terminus of the cholera toxin B subunit gene. A plant expression vector containing the fusion gene under control of the mannopine synthase P2 promoter was introduced into Solanum tuberosum cells by Agrobacterium tumefaciens-mediated transformation. The CTB::VP7 fusion gene was detected in the genomic DNA of transformed potato leaf cells by polymerase chain reaction (PCR) amplification methods. Immunoblot analysis of transformed potato tuber tissue extracts showed that synthesis and assembly of the CTB::VP7 fusion protein into oligomers of pentameric size occurred in the transformed plant cells. The binding of CTB::VP7 fusion protein pentamers to sialo-sugar containing GM1 ganglioside receptors on the intestinal epithelial cell membrane was quantified by enzyme-linked immunosorbent assay (ELISA). The ELISA results showed that the CTB::VP7 fusion protein made up approx 0.01% of the total soluble tuber protein. Synthesis and assembly of CTB::VP7 monomers into biologically active pentamers in transformed potato tubers demonstrates the feasibility of using edible plants as a mucosal vaccine for the production and delivery system for rotavirus capsid protein antigens.

A plant-based cholera toxin B subunit-insulin fusion protein protects against the development of autoimmune diabetes.

Oral administration of disease-specific autoantigens can prevent or delay the onset of autoimmune disease symptoms. We have generated transgenic potato plants that synthesize human insulin, a major insulin-dependent diabetes mellitus autoantigen, at levels up to 0.05% of total soluble protein. To direct delivery of plant-synthesized insulin to the gut-associated lymphoid tissues, insulin was linked to the C-terminus of the cholera toxin B subunit (CTB). Transgenic potato tubers produced 0.1% of total soluble protein as the pentameric CTB-insulin fusion, which retained GM1-ganglioside binding affinity and native antigenicity of both CTB and insulin. Nonobese diabetic mice fed transformed potato tuber tissues containing microgram amounts of the CTB-insulin fusion protein showed a substantial reduction in pancreatic islet inflammation (insulitis), and a delay in the progression of clinical diabetes. Feeding transgenic potato tissues producing insulin or CTB protein alone did not provide a significant reduction in insulitis or diabetic symptoms. The experimental results indicate that food plants are feasible production and delivery systems for immunotolerization against this T cell-mediated autoimmune disease.

Study of lectin-ganglioside interactions by high-performance liquid affinity chromatography.

A high-performance affinity column containing immobilized modified GM1 (lyso-GM1) was used to study the binding of an endogenous human brain lectin (HBL) in comparison with other carbohydrate-binding proteins. The proteins are previously converted into biotinylated derivatives. Detection of biotinylated proteins in the eluates by a microtitre plate assay ensures good sensitivity. The maximum binding capacity of the adsorbent for HBL is obtained in Tris buffer supplemented with beta-mercaptoethanol. The binding is inhibitable by specific sugar. It is concluded that the use of immobilized glycolipids in analytical high-performance liquid affinity chromatographic methods may serve as models in the study of interactions between gangliosides and carbohydrate-binding proteins.