The expression of biologically active cholera toxin in Escherichia coli.

Chromosomal DNA from Vibrio cholerae El Tor strain 1621 was digested with Hind III and the products fractionated by centrifugation through a sucrose gradient. A 15kb fragment containing the toxin gene of V. cholerae was identified by its homology with the heat labile toxin (LT) gene of toxigenic E. coli. This fragment was cloned in E. coli using pAT153 and subsequently characterised by digestion with different restriction endonucleases. Sequences homologous to the LT gene were identified by hybridisation and then sub-cloned using either pAT153 or pACYC184. Expression of the cloned CT gene in E. coli was detected using both cell culture and ELISA assays. One recombinant plasmid coded for the synthesis of an immunologically active but biologically inactive derivative of CT.     

Evolution and structure of two ADP-ribosylation enterotoxins, Escherichia coli heat-labile toxin and cholera toxin

Nucleotide sequence comparisons of the heat-labile enterotoxin (LTh) genes of E. coli pathogenic for humans with cholera toxin (CT) genes suggest that the two toxin genes have evolved from a common ancestry by a series of single base changes, while conserving the catalytic fragment A1 (ADP-ribose transferase). Based on the local hydrophilicity profiles of LTh and CT peptides, a transmembrane segment appears to be present in A1 in both toxins.     

Targeting of cholera toxin and Escherichia coli heat labile toxin in polarized epithelia: role of COOH-terminal KDEL

Vibrio cholerae and Escherichia coli heat labile toxins (CT and LT) elicit a secretory response from intestinal epithelia by binding apical receptors (ganglioside GM1) and subsequently activating basolateral effectors (adenylate cyclase). We have recently proposed that signal transduction in polarized cells may require transcytosis of toxin- containing membranes (Lencer, W. I., G. Strohmeier, S. Moe, S. L. Carlson, C. T. Constable, and J. L. Madara. 1995. Proc. Natl. Acad. Sci. USA. 92:10094-10098). Targeting of CT into this pathway depends initially on binding of toxin B subunits to GM1 at the cell surface. The anatomical compartments in which subsequent steps of CT processing occur are less clearly defined. However, the enzymatically active A subunit of CT contains the ER retention signal KDEL (RDEL in LT). Thus if the KDEL motif were required for normal CT trafficking, movement of CT from the Golgi to ER would be implied. To test this idea, recombinant wild-type (wt) and mutant CT and LT were prepared. The COOH- terminal KDEL sequence in CT was replaced by seven unrelated amino acids: LEDERAS. In LT, a single point mutation replacing leucine with valine in RDEL was made. Wt and mutant toxins displayed similar enzymatic activities and binding affinities to GM1 immobilized on plastic. Biologic activity of recombinant toxins was assessed as a Cl- secretory response elicited from the polarized human epithelial cell line T84 using standard electrophysiologic techniques. Mutations in K(R)DEL of both CT and LT delayed the time course of toxin-induced Cl- secretion. At T1/2, dose dependencies for K(R)DEL-mutant toxins were increased > or = 10-fold. KDEL-mutants displayed differentially greater temperature sensitivity. In direct concordance with a slower rate of signal transduction. KDEL-mutants were trafficked to the basolateral membrane more slowly than wt CT (assessed by selective cell surface biotinylation as transcytosis of B subunit). Mutation in K(R)DEL had no effect on the rate of toxin endocytosis. These data provide evidence that CT and LT interact directly with endogenous KDEL-receptors and imply that both toxins may require retrograde movement through Golgi cisternae and ER for efficient and maximal biologic activity.     

Characterization of the receptor for cholera toxin and Escherichia coli heat-labile toxin in rabbit intestinal brush borders.

125I-labelled heat-labile toxin (from Escherichia coli) and 125I-labelled cholera toxin bound to immobilized ganglioside GM1 and Balb/c 3T3 cell membranes with identical specificities, i.e. each toxin inhibited binding of the other. Binding of both toxins to Balb/c 3T3 cell membranes was saturable, with 50% of maximal binding occurring at 0.3 nM for cholera toxin and 1.1 nM for heat-labile toxin, and the number of sites for each toxin was similar. The results suggest that both toxins recognize the same receptor, namely ganglioside GM1. In contrast, binding of 125I-heat-labile toxin to rabbit intestinal brush borders at 0 degree C was not inhibited by cholera toxin, although heat-labile toxin inhibited 125I-cholera toxin binding. In addition, there were 3-10-fold more binding sites for heat-labile toxin than for cholera toxin. At 37 degrees C cholera toxin, but more particularly its B-subunit, did significantly inhibit 125I-heat-labile toxin binding. Binding of 125I-cholera toxin was saturable, with 50% maximal of binding occurring at 1-2 nM, and was quantitatively inhibited by 10(-8) M unlabelled toxin or B-subunit. By contrast, binding of 125I-heat-labile toxin was non-saturable (up to 5 nM), and 2 X 10(-7) M unlabelled B-subunit was required to quantitatively inhibit binding. Neuraminidase treatment of brush borders increased 125I-cholera toxin but not heat-labile toxin binding. Extensive digestion of membranes with Streptomyces griseus proteinase or papain did not decrease the binding of either toxin. The additional binding sites for heat-labile toxin are not gangliosides. Thin-layer chromatograms of gangliosides which were overlayed with 125I-labelled toxins showed that binding of both toxins was largely restricted to ganglioside GM1. However, 125I-heat-labile toxin was able to bind to brush-border galactoproteins resolved by SDS/polyacrylamide-gel electrophoresis and transferred to nitrocellulose.     

Gene expression in the polycistronic operons of Escherichia coli heat-labile toxin and cholera toxin: a new model of translational control

A new model is proposed based on the suggestion that stable local secondary structures of mRNA interfere with ribosome movement on mRNA and consequently reduce the translation rate. This model accounts for a different level of translation for each cistron in the polycistronic mRNA of Escherichia coli heat-labile toxin (LT) and cholera toxin. We also conclude that the mRNA secondary structures have been conserved during the evolution of the toxin genes for its functional importance.     

Plasmid vectors encoding cholera toxin or the heat-labile enterotoxin from Escherichia coli are strong adjuvants for DNA vaccines

Two plasmid vectors encoding the A and B subunits of cholera toxin (CT) and two additional vectors encoding the A and B subunits of the Escherichia coli heat-labile enterotoxin (LT) were evaluated for their ability to serve as genetic adjuvants for particle-mediated DNA vaccines administered to the epidermis of laboratory animals. Both the CT and the LT vectors strongly augmented Th1 cytokine responses (gamma interferon [IFN-gamma]) to multiple viral antigens when codelivered with DNA vaccines. In addition, Th2 cytokine responses (interleukin 4 [IL-4]) were also augmented by both sets of vectors, with the effects of the LT vectors on IL-4 responses being more antigen dependent. The activities of both sets of vectors on antibody responses were antigen dependent and ranged from no effect to sharp reductions in the immunoglobulin G1 (IgG1)-to-IgG2a ratios. Overall, the LT vectors exhibited stronger adjuvant effects in terms of T-cell responses than did the CT vectors, and this was correlated with the induction of greater levels of cyclic AMP by the LT vectors following vector transfection into cultured cells. The adjuvant effects observed in vivo were due to the biological effects of the encoded proteins and not due to CpG motifs in the bacterial genes. Interestingly, the individual LT A and B subunit vectors exhibited partial adjuvant activity that was strongly influenced by the presence or absence of signal peptide coding sequences directing the encoded subunit to either intracellular or extracellular locations. Particle-mediated delivery of either the CT or LT adjuvant vectors in rodents and domestic pigs was well tolerated, suggesting that bacterial toxin-based genetic adjuvants may be a safe and effective strategy to enhance the potency of both prophylactic and therapeutic DNA vaccines for the induction of strong cellular immunity.     

Effects of cholera toxin, Escherichia coli heat stable toxin and sodium deoxycholate on neurotensin release from the ileum in vivo

Neurotensin (NT) is a biologically active peptide found in specialized epithelial cells (N-cells) in the distal small intestine. In this study we tested the hypothesis that NT may be released by luminal secretagogues, i.e., cholera toxin, Escherichia coli heat-stable toxin and sodium deoxycholate. Cholera toxin elicited net fluid secretion in anesthetized cats. This secretion was accompanied by an increased release of NT-like immunoreactivity (NTLI) into the mesenteric vein when NTLI was measured with either a C-terminally or a N-terminally directed antibody. An increasing plasma NTLI concentration (N-terminally directed antibody) was recorded in the mesenteric vein and femoral artery in cholera experiments. These results indicate that cholera toxin releases NT from the small intestine. Since neurotensin causes intestinal fluid secretion at least in part via an activation of enteric nerves we propose that the N-cell functions as a 'receptor cell' which activates an intramural secretory reflex upon luminal stimulation by cholera toxin. This study does not support a similar role for NT in the secretion elicited by the heat stable toxin of Escherichia coli or by sodium deoxycholate since we were unable to demonstrate any intestinal release of NTLI after exposing the intestine to these secretory agents.     

霍乱疫苗中残余霍乱毒素检测方法的建立及验证

采用间接酶联免疫法,即用神经节苷脂包被,加入待检样品,再加入兔抗霍乱毒素B亚单位抗体,用标准样品的吸光值(A值)对标准样品的浓度绘制4-参数拟合曲线,根据标准曲线计算出待测样品中的CT浓度。结果显示,在浓度范围(0.6～16)ng/ml之间,CT标准浓度和检测浓度成线性关系,r2=0.9986。精确度在浓度范围(0.6～16)ng/ml,CT的平均回收率在96.24%～114.44%之间。精密度:批内变异CV%≤12.98%,批间变异CV%≤18.48%。特异性CT浓度在10ng/ml时,平均回收率为102.6%;CT浓度在5ng/ml时,平均回收率为111.17%;CT浓度在2.5ng/ml时,平均回收率为123.83%。实验表明该方法可检测霍乱疫苗原液中CT的含量。     

Priming immunization against cholera toxin and E. coli heat-labile toxin by a cholera toxin short peptide-beta-galactosidase hybrid synthesized in E. coli.

A synthetic oligodeoxynucleotide encoding for a small peptide was employed for the expression of this peptide in a form suitable for immunization. The encoded peptide, namely, the region 50-64 of the B subunit of cholera toxin (CTP3), had previously been identified as a relevant epitope of cholera toxin. Thus, multiple immunizations with its conjugate to a protein carrier led to an efficient neutralizing response against native cholera toxin. Immunization with the resulting fusion protein of CTP3 and beta-galactosidase, followed by a booster injection of a sub-immunizing amount (1 microgram) of cholera toxin, led to a substantial level of neutralizing antibodies against both cholera toxin and the heat-labile toxin of Escherichia coli.     

Expression in Escherichia coli of two mutated genes encoding the cholera toxin B subunit

To allow subsequent genetically mediated fusion of foreign antigens to cholera toxin B subunit (CTB), two mutated CTB encoding genes (ctxB) were constructed and overexpressed in Escherichia coli. The signal peptide coding sequence was deleted and restriction sites were created at both ends of the modified sequence. Both synthesized CTBs contain additional amino acid(s) at the N terminus (one and three). They were purified as insoluble products and refolded into the natural pentameric CTB structure by a denaturation-renaturation cycle. After renaturation, both recombinant proteins recovered CTB antigenicity and the ability to bind to GM1 gangliosides, as shown by in vitro analysis. Preliminary data indicated that both properties were unaltered by fusion of a foreign peptide to the mutated CTBs.     

Structure and function of cholera toxin and the related Escherichia coli heat-labile enterotoxin.

Cholera and the related Escherichia coli-associated diarrheal disease are important problems confronting Third World nations and any area where water supplies can become contaminated. The disease is extremely debilitating and may be fatal in the absence of treatment. Symptoms are caused by the action of cholera toxin, secreted by the bacterium Vibrio cholerae, or by a closely related heat-labile enterotoxin, produced by Escherichia coli, that causes a milder, more common traveler's diarrhea. Both toxins bind receptors in intestinal epithelial cells and insert an enzymatic subunit that modifies a G protein associated with the adenylate cyclase complex. The consequent stimulated production of cyclic AMP, or other factors such as increased synthesis of prostaglandins by intoxicated cells, initiates a metabolic cascade that results in the excessive secretion of fluid and electrolytes characteristic of the disease. The toxins have a very high degree of structural and functional homology and may be evolutionarily related. Several effective new vaccine formulations have been developed and tested, and a growing family of endogenous cofactors is being discovered in eukaryotic cells. The recent elucidation of the three-dimensional structure of the heat-labile enterotoxin has provided an opportunity to examine and compare the correlations between structure and function of the two toxins. This information may improve our understanding of the disease process itself, as well as illuminate the role of the toxin in studies of signal transduction and G-protein function.     

Enhancement of recombinant cholera toxin B subunit production in Escherichia coli by applying a fed-batch control strategy

Summary High production levels of recombinant cholera toxin B subunit in Escherichia coli were obtained with the design of an efficient fed-batch process and control strategy. The fed-batch results demonstrated a biomass production of 58 g/L (Cell Dry Weight) attaining production levels of heterologous protein of 4.7g/L in the intracellular fraction, 0.96 g/L exported into the periplasm and 0.27 g/L secreted into the culture supernatant.     

Targeted vaccine adjuvants based on modified cholera toxin

The present review describes immunomodulation with targeted adjuvants that will allow for the development of efficacious mucosal vaccines. We have studied cholera toxin (CT) and derivatives thereof, to rationally design vaccine adjuvant vectors that are both highly efficacious as well as safe and non-toxic. Two strategies were exploited; the first using CT or the enzymatically inactive receptor-binding B-subunit of CT (CTB) and the second, using CTA1 or an enzymatically inactive mutant CTA1R7K., that was linked, in a fusion protein, to the B-cell targeting moiety, DD, from Staphylococcus areus proteinA. Our studies provide compelling evidence that delivery of Ag in the absence of ADP-ribosylation can promote tolerance, whereas, ADP-ribosyltransferase-active conjugates, prevent tolerance but induce IgA immunity. Our analysis revealed unique subsets of mucosal and systemic DC that appeared to be responsible for the ADP-ribosyltransferase sensitive dichotomy between tolerance and IgA immunity. Whether targeting of B cells suffice for tolerance-induction or requires participation of DCs, is at present an unresolved issue. Nevertheless, enzymatic modulation differentiates and matures the DC to promote CD4 T cell help for IgA B cell development. Ag-presentation in the absence of enzyme, as seen with CTA1R7K-DD, expands specific T cells to a similar extent as enzymatically active CTA1-DD, but fails to recruit help for germinal center expansion of activated B cells. We have given special attention to the genes that adjuvants turn on using Affymetrix technology. In particular, modulation of the expression of co-stimulatory molecules on the targeted APC; CD80, CD86, CD83 and B7RP-1, play important roles for the effect of the ADP-ribosylating CTA1-based adjuvants for the development of tolerance or active IgA immunity.     

Synthesis of cholera toxin B subunit gene: cloning and expression of a functional 6XHis-tagged protein in Escherichia coli

Cholera toxin B subunit (CTB) has been extensively studied as immunogen, adjuvant, and oral tolerance inductor depending on the antigen conjugated or coadministered. It has been already expressed in several bacterial and yeast systems. In this study, we synthesized a versatile gene coding a 6XHis-tagged CTB (359bp). The sequence was designed according to codon usage of Escherichia coli, Lactobacillus casei, and Salmonella typhimurium. The gene assembly was based on a polymerase chain reaction, in which the polymerase extends DNA fragments from a pool of overlapping oligonucleotides. The synthetic gene was amplified, cloned, and expressed in E. coli in an insoluble form, reaching levels about 13 mg of purified active pentameric rCTB per liter of induced culture. Western blot and ELISA analyses showed that recombinant CTB is strongly and specifically recognized by polyclonal antibodies against the cholera toxin. The ability to form the functional pentamers was observed in cell culture by the inhibition of cholera toxin activity on Y1 adrenal cells in the presence of recombinant CTB. The 6XHis-tagged CTB provides a simple way to obtain functional CTB through Ni(2+)-charged resin after refolding and also free of possible CTA contaminants as in the case of CTB obtained from Vibrio cholerae cultures.     

Immunogenicity and efficacy of oral vaccines in developing countries: lessons from a live cholera vaccine

Oral vaccines, whether living or non-living, viral or bacterial, elicit diminished immune responses or have lower efficacy in developing countries than in developed countries. Here I describe studies with a live oral cholera vaccine that include older children no longer deriving immune support from breast milk or maternal antibodies and that identify some of the factors accounting for the lower immunogenicity, as well as suggesting counter-measures that may enhance the effectiveness of oral immunization in developing countries. The fundamental breakthrough is likely to require reversing effects of the 'environmental enteropathy' that is often present in children living in fecally contaminated, impoverished environments.