Fluorescently labeled liposomes for monitoring cholera toxin binding to epithelial cells

Vibrio cholerae, the causative agent for cholera, expresses a toxin required for virulence consisting of two subunits: the pentameric cholera toxin B (CTB) and cholera toxin A (CTA). CTB is frequently used as an indicator of the presence of pathogenic V. cholerae and binds to the G_M1 ganglioside on the surface of epithelial cells. To study V. cholerae virulence (CTB expression) in the presence of human epithelia, we devised an inexpensive, simple, and rapid method for quantifying CTB bound on epithelial surfaces in microtiter plates. G_M1 ganglioside was incorporated into the lipid bilayer of liposomes both encapsulating the fluorescent dye sulforhodamine B (SRB) and with SRB tagged to lipids in the bilayer (BEGs). In addition, G_M1-embedded liposomes encapsulating SRB only (EGs) and with SRB in their bilayers only (BGs) were synthesized. The three types of liposomes were compared with respect to their efficacy for both visualizing and quantifying CTB attached to the surface of Caco-2 cells. The BEGs were the most effective overall, providing both visualization under a fluorescence microscope and quantification after lysis in a microtiter plate reader. A limit of detection corresponding to 0.28 μg/ml applied CTB was attained for the on-cell assay using the microtiter plate reader approach, whereas as low as 2 μg/ml applied CTB could be observed under the fluorescence microscope.     

霍乱毒素B亚基基因具有自己的启动子

本研究发现并证实霍乱毒素B亚基基因上游Xba Ⅰ～Cla Ⅰ限制性片段内存在具有启动子活性的序列;在该启动子作用下,霍乱毒素B亚基表达水平可达200mg/L,氯霉素乙酰基转移酶基因表达水平随培养条件不同在0.3～10mg/L之间,大肠杆菌β-半乳糖苷酶基因的表达量达4100U/ml。在该启动子的控制下霍乱毒素B亚基基因可以高效表达,该启动子的存在可能是由于霍乱毒素操纵子中霍乱毒素B亚基表达量是A亚基的6倍。     

Very efficient extracellular production of cholera toxin B subunit using Bacillus brevis

Abstract We have constructed a very efficient synthesis and secretion system for cholera toxin B subunit (CTB) of Vibrio cholerae 569B using Bacillus brevis . The constructed expression-secretion vector has the multiple promoters and the signal peptide coding region of the mwp gene, a structural gene for one of the major cell wall proteins of B. brevis strain 47, directly followed by the gene encoding the mature CTB. A large amount of mature CTB (1.4 g per liter of culture) was secreted into the medium. It had the same amino terminal amino acid sequence as that of authentic CTB and was fully active in GM1 ganglioside binding assay.     

The capacity to produce cholera exotoxin in natural strains of Vibrio cholerae

The study of 27 V. cholerae strains, isolated from cholera patients and found to be hemolytically inactive, with a view to establish their capacity for the production of cholera toxin has revealed that 4 strains (V. cholerae cholerae Dacca 35, V. cholerae cholerae Dacca 3, V. cholerae cholerae B1307, V. cholerae cholerae J89) produce this protein. The quantitative determination of enterotoxin has been made with the use of GM1 ELISA technique. Strain Dacca 35 has been found to be highly toxigenic and, as regards the amount of exotoxin it produces, no different from V. cholerae cholerae strain 569B, a well-known producer of cholera toxin. In strain Dacca 35 correlation between the capacity of the cells for toxin production and the morphology of colonies has been established. The study has revealed that the chromosome of strain Dacca 35 contains two copies of gene vctAB responsible for the synthesis of cholera toxin.     

Reassessment of the prevalence of heat-stable enterotoxin (NAG-ST) among environmental Vibrio cholerae non-O1 strains isolated from Calcutta, India, by using a NAG-ST DNA probe.

A collection of 521 environmental isolates of Vibrio cholerae which were previously examined by the suckling mouse assay and found to be negative for the heat-stable enterotoxin NAG-ST were reassessed by a recently developed DNA probe for NAG-ST. A total of 12 (2.3%) of the isolates hybridized with the NAG-ST probe. By using a cholera toxin (CT) DNA probe, the CT gene was detected in six of the strains in the collection, although none of the isolates of V. cholerae non-O1 hybridized with both of the toxin probes. All of the NAG-ST and CT probe-positive strains were hemolysin positive. Thirty-fold-concentrated supernatants of the three representative NAG-ST DNA probe-positive V. cholerae non-O1 strains gave positive fluid accumulation ratios in the suckling mouse assay even after heating (100 degrees C for 5 min) and also inhibited the binding of a NAG-ST monoclonal antibody to the bound NAG-ST in a competitive enzyme-linked immunosorbent assay (ELISA). Likewise, all six CT probe-positive V. cholerae non-O1 strains produced in vitro CT when examined by the CT bead ELISA. HindIII digest patterns of chromosomal DNA from the representative NAG-ST gene-positive strains were visually indistinguishable. Between the groups of NAG-ST probe-positive strains examined, there was a variation in the hybridizable fragments, with one group of strains exhibiting a hybridizable fragment similar to that of the NRT 36 reference strain; a smaller HindIII fragment hybridized with the NAG-ST probe in the other group of strains.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reassessment of the prevalence of heat-stable enterotoxin (NAG-ST) among environmental Vibrio cholerae non-O1 strains isolated from Calcutta, India, by using a NAG-ST DNA probe.

A collection of 521 environmental isolates of Vibrio cholerae which were previously examined by the suckling mouse assay and found to be negative for the heat-stable enterotoxin NAG-ST were reassessed by a recently developed DNA probe for NAG-ST. A total of 12 (2.3%) of the isolates hybridized with the NAG-ST probe. By using a cholera toxin (CT) DNA probe, the CT gene was detected in six of the strains in the collection, although none of the isolates of V. cholerae non-O1 hybridized with both of the toxin probes. All of the NAG-ST and CT probe-positive strains were hemolysin positive. Thirty-fold-concentrated supernatants of the three representative NAG-ST DNA probe-positive V. cholerae non-O1 strains gave positive fluid accumulation ratios in the suckling mouse assay even after heating (100 degrees C for 5 min) and also inhibited the binding of a NAG-ST monoclonal antibody to the bound NAG-ST in a competitive enzyme-linked immunosorbent assay (ELISA). Likewise, all six CT probe-positive V. cholerae non-O1 strains produced in vitro CT when examined by the CT bead ELISA. HindIII digest patterns of chromosomal DNA from the representative NAG-ST gene-positive strains were visually indistinguishable. Between the groups of NAG-ST probe-positive strains examined, there was a variation in the hybridizable fragments, with one group of strains exhibiting a hybridizable fragment similar to that of the NRT 36 reference strain; a smaller HindIII fragment hybridized with the NAG-ST probe in the other group of strains.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunizing Adult Female Mice with a TcpA-A2-CTB Chimera Provides a High Level of Protection for Their Pups in the Infant Mouse Model of Cholera

Vibrio cholerae expresses two primary virulence factors, cholera toxin (CT) and the toxin-coregulated pilus (TCP). CT causes profuse watery diarrhea, and TCP (composed of repeating copies of the major pilin TcpA) is required for intestinal colonization by V. cholerae. Antibodies to CT or TcpA can protect against cholera in animal models. We developed a TcpA holotoxin-like chimera (TcpA-A2-CTB) to elicit both anti-TcpA and anti-CTB antibodies and evaluated its immunogenicity and protective efficacy in the infant mouse model of cholera. Adult female CD-1 mice were immunized intraperitoneally three times with the TcpA-A2-CTB chimera and compared with similar groups immunized with a TcpA+CTB mixture, TcpA alone, TcpA with Salmonella typhimurium flagellin subunit FliC as adjuvant, or CTB alone. Blood and fecal samples were analyzed for antigen-specific IgG or IgA, respectively, using quantitative ELISA. Immunized females were mated; their reared offspring were challenged orogastrically with 10 or 20 LD₅₀ of V. cholerae El Tor N16961; and vaccine efficacy was assessed by survival of the challenged pups at 48 hrs. All pups from dams immunized with the TcpA-A2-CTB chimera or the TcpA+CTB mixture survived at both challenge doses. In contrast, no pups from dams immunized with TcpA+FliC or CTB alone survived at the 20 LD₅₀ challenge dose, although the anti-TcpA or anti-CTB antibody level elicited by these immunizations was comparable to the corresponding antibody level achieved by immunization with TcpA-A2-CTB or TcpA+CTB. Taken together, these findings comprise strong preliminary evidence for synergistic action between anti-TcpA and anti-CTB antibodies in protecting mice against cholera. Weight loss analysis showed that only immunization of dams with TcpA-A2-CTB chimera or TcpA+CTB mixture protected their pups against excess weight loss from severe diarrhea. These data support the concept of including both TcpA and CTB as immunogens in development of an effective multivalent subunit vaccine against V. cholerae.     

Overexpression of a Mutant B Subunit in Toxigenic Vibrio cholerae Diminishes Production of Active Cholera Toxin In Vivo

A mutant cholera toxin B subunit containing a G33E substitution was constructed and expressed in V. cholerae. The G33E amino acid substitution did not affect the amount of recombinant CTB secreted to the culture medium. The overexpression of the mutant B subunits in wild-type toxigenic cholera vibrios led to an 80% decrease in production of active cholera toxin in vitro and in vivo. Overexpression of B^G33E subunits could be instrumental in the increase of the biosafety of live attenuated cholera candidate vaccine strains. Received: 29 January 1998 / Accepted: 30 April 1998     

Detection of biological toxins on an active electronic microchip

An electric-field-driven assay for fluorescein-labeled staphylococcal enterotoxin B and cholera toxin B was developed on an active electronic microchip. An array of microlocations was transformed into an immunoassay array by electronically biasing electrodes at each microlocation to attract biotinylated capture antibodies. The electric field generated on the array directed the transport, concentration, and binding of biotinylated capture antibodies to streptavidin-coated microlocations. Subsequently, solutions of fluorescein-labeled staphylococcal enterotoxin B and fluorescein-labeled cholera toxin B were electronically addressed to the assay sites by an applied electric field. Each toxin was specifically bound to microlocations containing the appropriate capture antibody with little nonspecific binding to assay sites lacking the appropriate capture antibody. It was possible to detect both toxins from a mixture in a single electronic addressing step; detection was accomplished after a 1-min application of the electric field followed by washing. The ability to perform a rapid, electric field-mediated immunoassay for multiple analytes may provide an advantage over existing approaches.     

Cholera toxin modulates the systemic immune responses against Vibrio cholerae surface antigens after repeated inoculations

The immunomodulating properties of a low cholera toxin (CT) dose over the systemic antibody response against Vibrio cholerae antigens after a comparatively extensive period of time were evaluated. Groups of 10 mice were injected intraperitoneally three times at 0, 30 and 86 days with 500 microl of buffer or 10(8) viable recombinant V. cholerae bacteria (lacking cholera toxin A subunit) with or without 100 ng of CT. Sera were obtained from inoculated mice at 0, 14, 28, 37, 58, 80, 93, 114, 236 and 356 days after the first injection. Vibriocidal activity and IgM and IgG anti-lipopolysaccharide (LPS) or outer membrane protein (OMP) antibodies levels were estimated by ELISA in sera of inoculated mice. Anti-LPS IgG subclasses were measured 2 weeks after each immunization by ELISA. Treatment of mice with CT markedly influenced the immune response to LPS but not against OMP of V. cholerae. Simultaneous intraperitoneal administration of CT with V. cholerae resulted in marked enhancement of both IgM anti-LPS and vibriocidal titers which subsisted for a relatively extensive period of time after repeated antigen administration. No differences were observed in IgM and IgG anti-OMP titers after extended periods of time between CT and control treatments. A similar pattern of IgG anti-LPS subclasses was observed in the serum samples analyzed. These results suggest that long term CT administration modulates the IgM anti-V. cholerae LPS response and the serum vibriocidal activity.     

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.     

Development and validation of a mismatch amplification mutation PCR assay to monitor the dissemination of an emerging variant of Vibrio cholerae O1 biotype El Tor

A mismatch amplification mutation PCR assay was developed and validated for rapid detection of the biotype specific cholera toxin B subunit of V. cholerae O1. This assay will enable easy monitoring of the spread of a new emerging variant of the El Tor biotype of V. cholerae O1.     

Single multiplex polymerase chain reaction for environmental surveillance of toxigenic—Pathogenic O1 and Non-O1vibrio cholerae

A multiplex PCR assay was developed for the detection of toxigenic and pathogenic V. cholerae from direct water sources using specific primers targeting diverse genes, viz. outer membrane protein (ompW), cholera toxin (ctxB), ORF specific for O1 (rfbG), zonula occludens (zot) and toxin co-regulated pilus (tcpB); among these genes, ompW acts as internal control for V. cholerae, the ctx gene as a marker for toxigenicity and tcp for pathogenicity. The sensitivity of multiplex PCR was 5 x 10(4) V. cholerae cells per reaction. The procedure was simplified as direct bacterial cells were used as template and there was no need for DNA extraction. The assay was specific as no amplification occurred with the other bacteria used. Toxigenic V. cholerae were artificially spiked in different water samples, filtered through a 0.45 microm membrane, and the filters containing bacteria were enriched in APW for 6 h. PCR following filtration and enrichment could detect as little as 8 V. cholerae cells per mL in different spiked water samples. Various environmental potable water samples were screened for the presence of V. cholerae using this assay procedure. The proposed method is rapid, sensitive and specific for environmental surveillance for the presence of toxigenic-pathogenic and nonpathogenic V. cholerae.     

Detection of Vibrio cholerae by real-time nucleic acid sequence-based amplification

A multitarget molecular beacon-based real-time nucleic acid sequence-based amplification (NASBA) assay for the specific detection of Vibrio cholerae has been developed. The genes encoding the cholera toxin (ctxA), the toxin-coregulated pilus (tcpA; colonization factor), the ctxA toxin regulator (toxR), hemolysin (hlyA), and the 60-kDa chaperonin product (groEL) were selected as target sequences for detection. The beacons for the five different genetic targets were evaluated by serial dilution of RNA from V. cholerae cells. RNase treatment of the nucleic acids eliminated all NASBA, whereas DNase treatment had no effect, showing that RNA and not DNA was amplified. The specificity of the assay was investigated by testing several isolates of V. cholerae, other Vibrio species, and Bacillus cereus, Salmonella enterica, and Escherichia coli strains. The toxR, groEL, and hlyA beacons identified all V. cholerae isolates, whereas the ctxA and tcpA beacons identified the O1 toxigenic clinical isolates. The NASBA assay detected V. cholerae at 50 CFU/ml by using the general marker groEL and tcpA that specifically indicates toxigenic strains. A correlation between cell viability and NASBA was demonstrated for the ctxA, toxR, and hlyA targets. RNA isolated from different environmental water samples spiked with V. cholerae was specifically detected by NASBA. These results indicate that NASBA can be used in the rapid detection of V. cholerae from various environmental water samples. This method has a strong potential for detecting toxigenic strains by using the tcpA and ctxA markers. The entire assay including RNA extraction and NASBA was completed within 3 h.     

Detection of toxigenic Vibrio cholerae from environmental water samples by an enrichment broth cultivation-pit-stop semi-nested PCR procedure

A pit-stop semi-nested PCR assay for the detection of toxigenic Vibrio cholerae in environmental water samples was developed and its performance evaluated. The PCR technique amplifies sequences within the cholera toxin operon specific for toxigenic V. cholerae. The PCR procedure coupled with an enrichment culture detected as few as four V. cholerae organisms in pure culture. Treated sewage, surface, ground and drinking water samples were seeded with V. cholerae and following enrichment, a detection limit of as few as 1 V. cholerae cfu ml(-1) was obtained with amplification reactions from crude bacterial lysates. The proposed method, which includes a combination of enrichment, rapid sample preparation and a pit-stop semi-nested PCR, could be applicable in the rapid detection of toxigenic V. cholerae in environmental water samples.     

Alteration of cholera toxin biosynthesis in Vibrio cholerae 01 as a result of temperate phage 139 integration into bacterial chromosome

Infection of V. cholerae 01 (classical and eltor biovars) cells with the temperate cholera phage 139 derived from V. cholerae serogroup 0139 followed by integration of the phage genome into the bacterial chromosome significantly increased the production of cholera toxin, the main virulence factor. The level of toxin biosynthesis in the lysogenic V. cholerae classical strain increased 3-fold and that in V. eltor thirty times in comparison with the parental strains. Increased production of cholera toxin was not associated with an increase in the number of copies of genes involved in its biosynthesis but seemed to be due to changes in toxinogenesis regulation.     

Increased sensitivity of cholera toxin detection by enzyme-linked immunosorbent assay with chemical immobilization of antibody onto nylon surface providing hydrophobicity

We developed an improved method to chemically immobilize antibodies on a nylon surface using a styrene maleic anhydride copolymer having an aryl group, which provides hydrophobicity to the nylon surface. We applied it to a modified enzyme-linked immunosorbent assay (slip-ELISA) for the detection of cholera toxin (CT). The sensitivity of slip-ELISA for CT detection was 1,000 times higher than that of conventional methods of physical adsorption using polystyrene plates, and 10 times higher than that of the method of chemical immobilization using maleic anhydride methylvinyl ether copolymer.     

Generation of antibodies reactive with fumonisins B1, B2, and B3 by using cholera toxin as the carrier-adjuvant.

Murine polyclonal antibodies reactive with fumonisins B1, B2, and B3 were produced after a novel immunization procedure with cholera toxin as both a hapten carrier protein and adjuvant. Immunization of mice with two 7.5-micrograms doses of fumonisin B1-cholera toxin conjugate without adjuvant resulted in the production of fumonisin B1-specific antibodies in all mice within 15 days when intraperitoneal, subcutaneous, and intravenous routes were used. In contrast, conventional immunization procedures with fumonisin B1-bovine serum albumin conjugates with and without Freund's adjuvant were largely ineffective. Fumonisin antibodies could be readily mass-produced in ascites fluid by using cholera toxin as a carrier-adjuvant. A competitive indirect enzyme-linked immunosorbent assay (ELISA) was devised whereby immobilized fumonisin B1-ovalbumin and free fumonisin B1 competed for antibody binding. The detection limit for fumonisin B1 in the ELISA was 100 ng/ml. The antiserum cross-reacted with fumonisins B2 and B3 but not with the hydrolyzed backbone of fumonisin B1 and tricarballylic acid. Concentrations of fumonisins B1, B2, and B3 required for 50% binding inhibition were 260, 300, and 650 ng/ml, respectively. These polyclonal antibodies should find wide usage in the ELISA for fumonisins in foods, feeds, and tissues.