Luteolin suppresses 5-hydroxytryptamine elevation in stimulated RBL-2H3 cells and experimental colitis mice.

Increased 5-hydroxytryptamine may be associated with the development and progression of inflammatory bowel disease. In this study, we examined the suppressive effect of flavonoids on the increased intra- and extracellular 5-hydroxytryptamine levels in rat mast RBL-2H3 cells, known to produce 5-hydroxytryptamine by the phorbol 12-myristate 13-acetate stimulation. Among the flavonoids examined, luteolin and quercetin significantly reduced the cellular 5-hydroxytryptamine concentration. Gene and protein expression analyses revealed that luteolin significantly suppressed cellular tryptophan hydroxylase 1 expression induced by phorbol 12-myristate 13-acetate stimulation. Mitogen-activated protein kinase/extracellular signal-regulated kinase signaling was also suppressed by luteolin, suggesting that this pathway is one of targets of 5-hydroxytryptamine modulation by luteolin. An in vivo experimental colitis model was prepared by administering 2.5% dextran sodium sulfate in drinking water to C57BL/6 mice for seven days. The ingestion of 0.1% dietary luteolin suppressed the increasing 5-hydroxytryptamine in the colorectal mucosa. In conclusion, luteolin possesses a suppressive effect on extensive 5-hydroxytryptamine formation in both experimental RBL-2H3 cells and colitis models.

Elevation of the serotonin-derived quinone, tryptamine-4,5-dione, in the intestine of ICR mice with dextran sulfate-induced colitis.

Inflammatory bowel diseases, including Crohn's disease and ulcerative colitis, are chronic inflammatory disorders associated with oxidative stress. The intestines produce 5-hydroxytryptamine that may negatively affect disease state under inflammatory conditions when overproduced. 5-Hydroxytryptamine is a substrate for myeloperoxidase and is converted into reactive tryptamine-4,5-dione. Here, an experimental colitis model was established through oral administration of 5% dextran sulfate sodium to ICR mice for 7 days. Furthermore, the formation of tryptamine-4,5-dione in the colorectal mucosa/submucosa and colorectal tissue was analyzed by chemical and immunochemical methodologies. First, free tryptamine-4,5-dione in the homogenate was chemically trapped by o-phenylenediamine and analyzed as the stable phenazine derivative. Tryptamine-4,5-dione localization as adducted proteins in the colorectal tissue was immunohistochemically confirmed, and as demonstrated by both methods, this resulted in the significant increase of tryptamine-4,5-dione in dextran sulfate sodium-challenged mice compared with control mice. Immunohistochemical staining confirmed tryptamine-4,5-dione-positive staining at the myeloperoxidase accumulation site in dextran sulfate sodium-challenged mice colorectal tissue. The tryptamine-4,5-dione locus in the mice was partly matched with that of a specific marker for myeloperoxidase, halogenated tyrosine. Overall, the results possibly indicate that tryptamine-4,5-dione is generated by neutrophil myeloperoxidase in inflammatory tissue and may contribute to the development of inflammatory bowel disease.

Development of a simple and rapid diagnosis method for swine edema disease to specifically detect Stx2e protein by immunochromatographic test.

Edema disease in piglets is caused by Shiga toxin 2e (Stx2e)-producing Escherichia coli. However, there is currently no available Stx2e-specific immunochromatographic test strip to differentiate Stx2e from other types of Shiga toxin 2. In the present study, to develop an Stx2e-specific immunochromatographic test strip, we isolated nine different monoclonal antibody-producing hybridoma clones from Stx2e toxoid-immunized mice and confirmed that six antibodies were A subunit-specific whereas three antibodies were B subunit-specific. Only one A subunit-specific monoclonal antibody (45B2) was cross-reactive with prototype Stx2 (Stx2a) at the same sensitivity, but the remaining eight monoclonal antibodies were not. In immunochromatographic tests using the highly sensitive antibodies, test strips using some combinations of gold colloid-conjugated monoclonal antibody with the B subunit-specific monoclonal antibody on the membrane detected Stx2e, but not other types of Shiga toxin 2. These test strips had the ability to detect Stx2e in the culture supernatant of clinically isolated Stx2e gene-positive strains, but not in those of Stx2e gene-negative strains. These results indicate that our test strip is practical for the specific detection of Stx2e to diagnose swine edema disease.

Isolation of B subunit-specific monoclonal antibody clones that strongly neutralize the toxicity of Shiga toxin 2.

Shiga toxin 2 (Stx2)-specific mAb-producing hybridoma clones were generated from mice. Because mice tend to produce small amounts of B subunit (Stx2B)-specific antibodies at the polyclonal antibody level after immunization via the parenteral route, mice were immunized intranasally with Stx2 toxoids with a mutant heat-labile enterotoxin as a mucosal adjuvant; 11 different hybridoma clones were obtained in two trials. Six of them were A subunit (Stx2A)-specific whereas five were Stx2B-specific antibody-producing clones. The in vitro neutralization activity of Stx2B-specific mAbs against Stx2 was greater than that of Stx2A-specific mAbs on HeLa229 cells. Furthermore, even at low concentrations two of the Stx2B-specific mAbs (45 and 75D9) completely inhibited receptor binding and showed in vivo neutralization activity against a fivefold median lethal dose of Stx2 in mice. In western blot analysis, these Stx2B-specific neutralization antibodies did not react to three different mutant forms of Stx2, each amino acid residue of which was associated with receptor binding. Additionally, the nucleotide sequences of the VH and VL regions of clones 45 and 75D9 were determined. Our Stx2B-specific mAbs may be new candidates for the development of mouse-human chimeric Stx2-neutralizing antibodies which have fewer adverse effects than animal antibodies for enterohemorrhagic Escherichia coli infection.

Evaluation of Shiga toxin 2e-specific chicken egg yolk immunoglobulin: production and neutralization activity.

Chicken egg yolk immunoglobulin (IgY) against Shiga toxin 2e (Stx2e), a major cause of swine edema disease, was prepared to evaluate its possible clinical applications. The titer of Stx2e-specific IgY in egg yolk derived from three chickens that had been immunized with an Stx2e toxoid increased 2 weeks after primary immunization and remained high until 90 days after this immunization. Anti-Stx2e IgY was found to neutralize the toxicity of Stx2e by reacting with its A and B subunits, indicating that IgY is a cost-effective agent to develop for prophylactic foods or diagnosis kits for edema disease.

Large-scale preparation of Shiga toxin 2 in Escherichia coli for toxoid vaccine antigen production.

Enterohemorrhagic Escherichia coli (EHEC) causes hemorrhagic colitis, and in more severe cases, a serious clinical complication called hemolytic uremic syndrome (HUS). Shiga toxin (Stx)is one of the factors that cause HUS. Serotypes of Stx produced by EHEC include Stx1 and Stx2. Although some genetically mutated toxoids of Stx have been developed, large-scale preparation of Stx that is practical for vaccine development has not been reported. Therefore, overexpression methods for Stx2 and mutant Stx2 (mStx2) in E. coli were developed. The expression plasmid pBSK-Stx2(His) was constructed by inserting the full-length Stx2 gene, in which a six-histidine tag gene was fused at the end of the B subunit into the lacZα fragment gene of the pBluescript II SK(+) vector. An E. coli MV1184 strain transformed with pBSK-Stx2(His) overexpressed histidine-tagged Stx2 (Stx2-His) in cells cultured in CAYE broth in the presence of lincomycin. Stx2-His was purified using TALON metal affinity resin followed by hydroxyapatite chromatography. From 1 L of culture, 68.8 mg of Stx2-His and 61.1 mg of mStx2-His, which was generated by site-directed mutagenesis, were obtained. Stx2-His had a cytotoxic effect on HeLa cells and was lethal to mice. However, the toxicity of mStx2-His was approximately 1000-fold lower than that of Stx2-His. Mice immunized with mStx2-His produced specific antibodies that neutralized the toxicity of Stx2 in HeLa cells. Moreover, these mice survived challenge with high doses of Stx2-His. Therefore, the lincomycin-inducible overexpression method is suitable for large-scale preparation of Stx2 vaccine antigens.

Unique biological activity of botulinum D/C mosaic neurotoxin in murine species.

Clostridium botulinum types C and D cause animal botulism by the production of serotype-specific or mosaic botulinum neurotoxin (BoNT). The D/C mosaic BoNT (BoNT/DC), which is produced by the isolate from bovine botulism in Japan, exhibits the highest toxicity to mice among all BoNTs. In contrast, rats appeared to be very resistant to BoNT/DC in type C and D BoNTs and their mosaic BoNTs. We attempted to characterize the enzymatic and receptor-binding activities of BoNT/DC by comparison with those of type C and D BoNTs (BoNT/C and BoNT/D). BoNT/DC and D showed similar toxic effects on cerebellar granule cells (CGCs) derived from the mouse, but the former showed less toxicity to rat CGCs. In recombinant murine-derived vesicle-associated membrane protein (VAMP), the enzymatic activities of both BoNTs to rat isoform 1 VAMP (VAMP1) were lower than those to the other VAMP homologues. We then examined the physiological significance of gangliosides as the binding components for types C and D, and mosaic BoNTs. BoNT/DC and C were found to cleave an intracellular substrate of PC12 cells upon the exogenous addition of GM1a and GT1b gangliosides, respectively, suggesting that each BoNT recognizes a different ganglioside moiety. The effect of BoNT/DC on glutamate release from CGCs was prevented by cholera toxin B-subunit (CTB) but not by a site-directed mutant of CTB that did not bind to GM1a. Bovine adrenal chromaffin cells appeared to be more sensitive to BoNT/DC than to BoNT/C and D. These results suggest that a unique mechanism of receptor binding of BoNT/DC may differentially regulate its biological activities in animals.

P19 embryonal carcinoma cells exhibit high sensitivity to botulinum type C and D/C mosaic neurotoxins.

Botulinum neurotoxins (BoNTs) inhibit neurotransmitter release at peripheral nerve terminals. They are serologically classified from A to G, C/D and D/C mosaic neurotoxins forming further subtypes of serotypes C and D. Cultured primary neurons, as well as neuronal cell lines such as PC12 and Neuro-2a, are often utilized in cell-based experiments on the toxic action of botulinum toxins. However, there are very few reports of the use of neural cell lines for studying BoNTs/C and D. In addition, the differentiated P19 neuronal cell line, which possesses cholinergic properties, has yet to be tested for its susceptibility to BoNTs. Here, the responsiveness of differentiated P19 cells to BoNT/C and BoNT/DC is reported. Both BoNT/C and BoNT/DC were shown to effectively bind to, and be internalized by, neurons derived from P19 cells. Subsequently, the intracellular substrates for BoNT/C and BoNT/DC were cleaved by treatment of the cells with the toxins in a ganglioside-dependent manner. Moreover, P19 neurons exhibited high sensitivity to BoNT/C and BoNT/DC, to the same extent as cultured primary neurons. These findings suggest that differentiated P19 cells possess full sensitivity to BoNT/C and BoNT/DC, thus making them a novel susceptible cell line for research into BoNTs.

Evaluation of immunochromatographic test of Shiga toxin 2e in enrichment cultures of swine edema disease clinical samples.

To simplify the diagnosis of swine edema disease, overnight culture supernatants of swine clinical samples were assayed using immunochromatographic test strips we developed previously. Small-intestinal contents, mesenteric lymph nodes, and fecal samples were cultured in casamino acid-yeast extract broth overnight, after which supernatants were loaded onto immunochromatographic test strips to determine whether they could detect Shiga toxin 2e (Stx2e). Among 23 clinical samples in which PCR-identified stx2e-positive E. coli were isolated, samples from seven of ten small-intestinal contents, one of three mesenteric lymph nodes and six of ten fecal samples showed Stx2e-positive reactions in the protein-based immunochromatographic test. Additionally, one small-intestinal content sample, in which stx2e-positive E. coli were not isolated, showed an Stx2e-positive reaction. Furthermore, the immunochromatographic test results of the samples were associated with the toxin concentration determined by sandwich ELISA and cytotoxicity assay results on Vero cells. The toxin concentration range of the samples with positive and negative reactions were 2.1-196.2 ng/ml and 0-12.8 ng/ml, respectively. The sensitivity and specificity of this immunochromatographic test strip calculated from all clinical samples analyzed in this study were 60.9% and 94.4%, respectively. Our immunochromatographic test strip has strong potential for simple and accurate diagnosis for edema disease by detecting toxin expression, complementing the PCR method.

Development of a homogeneous time-resolved FRET (HTRF) assay for the quantification of Shiga toxin 2 produced by E. coli.

Shiga toxin-producing Escherichia coli (STEC) is a major intestinal pathogen and causes serious gastrointestinal illness, which includes diarrhea, hemorrhagic colitis, and life-threatening hemolytic uremic syndrome. The major virulence factors of STEC are Shiga toxins (Stx1 and Stx2), which belong to the AB-type toxin family. Among several subtypes of Stx1 and Stx2, the production of Stx2a is thought to be a risk factor for severe STEC infections, but Stx2a production levels vary markedly between STEC strains, even strains with the same serotype. Therefore, quantitative analyses of Stx2 production by STEC strains are important to understand the virulence potential of specific lineages or sublineages. In this study, we developed a novel Stx2 quantification method by utilizing homogeneous time-resolved fluorescence resonance energy transfer (HTRF) technology. To determine suitable "sandwich" assay conditions, we tested 6 combinations of fluorescence-labeled monoclonal antibodies (mAbs) specific to Stx2 and compared the HTRF signal intensities obtained at various incubation times. Through this analysis, we selected the most suitable mAb pair, one recognizing the A subunit and the other recognizing the B subunit, thus together detecting Stx holotoxins. The optimal incubation time was also determined (18 h). Then, we optimized the concentrations of the two mAbs based on the range for linearity. The established HTRF assay detected 0.5 ng/ml of the highly purified recombinant Stx2a and Stx2e proteins and the working range was 1-64 ng/ml for both Stx2a and Stx2e. Through the quantification analysis of Stx proteins in STEC cell lysates, we confirmed that other Stx2 subtypes (Stx2b, Stx2c, Stx2d and Stx2g) can also be quantified at a certain level of accuracy, while this assay system does not detect Stx2f, which is highly divergent in sequence from other Stx2 subtypes, and Stx1. As the HTRF protocol we established is simple, this assay system should prove useful for the quantitative analysis of Stx2 production levels of a large number of STEC strains.

Passive oral immunization by egg yolk immunoglobulin (IgY) to Vibrio cholerae effectively prevents cholera.

In an attempt to prepare egg yolk immunoglobulin (IgY) to treat and prevent cholera, hens were immunized by a mixture of heat- or formalin-killed Vibrio cholerae O1 and O139 organisms, or by the recombinant cholera toxin B subunit (CTB). The IgYs were partially purified from egg yolk and orally administered to suckling mice before or after challenge with live O1 or O139 cells. The anti-O1 and O139 IgYs and the mixture of either IgY with anti-CTB IgY significantly protected the occurrence of cholera caused by both O1 and O139 infection. Since large amounts of IgY can be prepared very easily and at low cost, this seems to be a useful procedure for preventing and treating cholera.

Molecular analysis of an extrachromosomal element containing the C2 toxin gene discovered in Clostridium botulinum type C.

Clostridium botulinum cultures are classified into seven types, types A to G, based on the antigenicity of the neurotoxins produced. Of these seven types, only types C and D produce C2 toxin in addition to the neurotoxin. The C2 toxin consists of two components designated C2I and C2II. The genes encoding the C2 toxin components have been cloned, and it has been stated that they might be on the cell chromosome. The present study confirmed by using pulsed-field gel electrophoresis and subsequent Southern hybridization that these genes are on a large plasmid. The complete nucleotide sequence of this plasmid was determined by using a combination of inverse PCR and primer walking. The sequence was 106,981 bp long and contained 123 potential open reading frames, including the c2I and c2II genes. The 57 products of these open reading frames had sequences similar to those of well-known proteins. It was speculated that 9 these 57 gene products were related to DNA replication, 2 were responsible for the two-component regulatory system, and 3 were sigma factors. In addition, a total of 20 genes encoding proteins related to diverse processes in purine catabolism were found in two regions. In these regions, there were 9 and 11 genes rarely found in plasmids, indicating that this plasmid plays an important role in purine catabolism, as well as in C2 toxin production.

Lincomycin-induced over-expression of mature recombinant cholera toxin B subunit and the holotoxin in Escherichia coli.

Cholera toxin (CT) B subunit (CTB) was overproduced using a novel expression system in Escherichia coli. An expression plasmid was constructed by inserting the gene encoding the full-length CTB and the Shine-Dalgarno (SD) sequence derived from CTB or from the heat-labile enterotoxin B subunit (LTB) of enterotoxigenic E. coli into the lacZalpha gene fragment in the pBluescript SK(+) vector. The E. coli strain MV1184 was transformed with each plasmid and then cultured in CAYE broth containing lincomycin. Recombinant CTB (rCTB) was purified from each cell extract. rCTB was overproduced in both transformants without obvious toxicity and was structurally and biologically identical to that of CT purified from Vibrio cholerae, indicating that the original SD and CTB signal sequences were also sufficient to express rCTB in E. coli. Lincomycin-induced rCTB expression was inhibited by mutating the lac promoter, suggesting that lincomycin affects the lactose operon. Based on these findings, we constructed a plasmid that contained the wild-type CT operon and successfully overproduced CT (rCT) using the same procedure for rCTB. Although rCT had an intact A subunit, the amino-terminal modifications and biological properties of the A and B subunits of rCT were identical to those of CT. These results suggest that this novel rCTB over-expression system would also be useful to generate both wild-type and mutant CT proteins that will facilitate further studies on the characteristics of CT, such as mucosal adjuvant activity.

Clinical application of Clostridium botulinum type A neurotoxin purified by a simple procedure for patients with urinary incontinence caused by refractory destrusor overactivity.

Type A neurotoxin of Clostridium botulinum was purified by a simple procedure using a lactose gel column. This procedure was previously reported for type B neurotoxin. Hemagglutinin-positive toxins (19S and 16S) were bound to the column under acid conditions, and the neurotoxin alone was dissociated from these hemagglutinin-positive toxins by changing the pH of the column to an alkaline condition. The toxicity of this purified toxin preparation was retained for at least 1 year at -30 degrees C by supplementing it with either 0.1% albumin or 0.05% albumin plus 1% trehalose. This preparation was used to treat 18 patients with urinary incontinence caused by refractory idiopathic and neurogenic detrusor overactivity; 16 of the patients showed excellent improvement. Improvements started within 1 week after injection in most cases and lasted 3-12 months [corrected]

Immunochromatographic detection of the heat-labile enterotoxin of enterotoxigenic Escherichia coli with cross-detection of cholera toxin.

Here, we report the development of an immunochromatographic test strip that can detect heat-labile enterotoxin (LT) produced by enterotoxigenic Escherichia coli. Five types of monoclonal antibody (mAb)-producing hybridomas were isolated: three mAbs were A subunit specific and two were B subunit specific. Four mAbs also cross-reacted with both LT proteins derived from swine and human E. coli strains, but only one mAb 57B9 additionally cross-reacted with cholera toxin. Thus, mAb 57B9 was used to form a gold colloid-conjugated antibody for the immunochromatographic test by combination with polyclonal anti-LT rabbit IgG. This test strip detected not only LT in the culture supernatant of LT gene-positive strains, but also cholera toxin in the culture supernatant of Vibrio cholerae. These results indicate that this test strip is suitable for the diagnosis of both enterotoxigenic E. coli and V. cholerae infection.

Characterisation of monoclonal antibodies against haemagglutinin associated with Clostridium botulinum type C neurotoxin.

Of 11 monoclonal antibodies (MAbs) prepared against the non-toxic component of type C Clostridium botulinum 16S toxin to clarify the function of the non-toxic component, seven recognised HA1, three recognised HA3b and one recognised HA2. Results of epitope mapping indicated that three of the seven anti-HA1 MAbs recognised the region between amino acid residues 121 and 140 and four recognised the three-dimensional structure of HA1. Three anti-HA3b MAbs recognised different regions between (approximately) amino acids 405-430, 180-270 and 275-297. The ability of these MAbs to interfere with binding of 16S toxin or non-toxic component, HA1 or HA3b to erythrocytes and to intestine tissue sections of guinea-pig was observed. MAbs against HA3b and HA2 did not inhibit 16S toxin binding to either erythrocytes or epithelial cells, whereas some MAbs against HA1 did inhibit binding. The seven anti-HA1 MAbs can be classified into four groups based on their binding inhibition activities. The anti-HA1 MAbs that inhibited the binding of 16S toxin to the epithelial cells also neutralised or reduced the oral toxicity in mice, indicating that HA may play an important role in the absorption of the 16S toxin from the small intestine.

Mucosal immunisation with Clostridium botulinum type C 16 S toxoid and its non-toxic component.

Clostridium botulinum types C and D produce a 16 S (500 kDa) toxin that is formed by conjugation of neurotoxin with a non-toxic component (nonTox). The amino acid sequences of type C and D nonTox components are almost identical. In a previous report it was proposed that nonTox is necessary for the effective absorption of the toxin from the small intestine. This suggested the hypothesis that mucosal immunity against nonTox in the small intestine might prevent the absorption of both C- and D-16 S toxins. The nonTox was purified from a mutant strain, (C)-N71, that does not produce neurotoxin. This nonTox or detoxified C-16 S toxin were mixed with adjuvant (a mutant form of heat-labile toxin of Escherichia coli), and inoculated into mice via the nasal or oral route, or both. The mice inoculated nasally four times with nonTox or toxoid produced high levels of antibodies (including IgA) against the immunogens, both in intestinal fluids and sera. When these nonTox-immunised mice were challenged orally with 2 and 20 oral minimum lethal doses (MLD) of C- or D-16 S toxins, the same results were obtained with both C and D; the mice survived after challenge with 2 MLD of either C or D but were killed by 20 MLD of either toxin although the time to death was significantly longer than in the control non-immunised mice. These results indicate that the local anti-nonTox antibodies reduce absorption of both C- and D-16 S toxins from the small intestine. The C-16 S toxoid-immunised mice showed similar behaviour with type D toxin challenge, probably due to the same mechanism, but were protected against 20 MLD of C-16 S toxin.

Simple method for Shiga toxin 2e purification by affinity chromatography via binding to the divinyl sulfone group.

Here we describe a simple affinity purification method for Shiga toxin 2e (Stx2e), a major causative factor of edema disease in swine. Escherichia coli strain MV1184 transformed with the expression plasmid pBSK-Stx2e produced Stx2e when cultivated in CAYE broth containing lincomycin. Stx2e bound to commercial D-galactose gel, containing α-D-galactose immobilized on agarose resin via a divinyl sulfone linker, and was eluted with phosphate-buffered saline containing 4.5 M MgCl2. A small amount of Stx2e bound to another commercial α-galactose-immobilized agarose resin, but not to ß-galactose-immobilized resin. In addition, Stx2e bound to thiophilic adsorbent resin containing ß-mercaptoethanol immobilized on agarose resin via a divinyl sulfone, and was purified in the same manner as from D-galactose gel, but the Stx2e sample contained some contamination. These results indicate that Stx2e bound to D-galactose gel mainly through the divinyl sulfone group on the resin and to a lesser extent through α-D-galactose. With these methods, the yields of Stx2e and attenuated mutant Stx2e (mStx2e) from 1 L of culture were approximately 36 mg and 27.7 mg, respectively, and the binding capacity of the D-galactose gel and thiophilic adsorbent resin for Stx2e was at least 20 mg per 1 ml of resin. In addition, using chimeric toxins with prototype Stx2 which did not bind to thiophilic adsorbent resin and some types of mutant Stx2e and Stx2 which contained inserted mutations in the B subunits, we found that, at the least, asparagine (amino acid 17 of the B subunits) was associated with Stx2e binding to the divinyl sulfone group. The mStx2e that was isolated exhibited vaccine effects in ICR mice, indicating that these methods are beneficial for large-scale preparation of Stx2e toxoid, which protects swine from edema disease.

Single chain variable fragment antibodies against Shiga toxins isolated from a human antibody phage display library.

Shiga toxins (Stxs) are involved in the pathogenesis of hemolytic-uremic syndrome and other severe systemic complications following enterohemorrhagic Escherichia coli infection in humans. Passive immunotherapies using monoclonal antibodies have been shown to be effective for neutralizing the toxic effects of Stxs. However, animal-derived monoclonal antibodies are sometimes immunogenic and their production is both laborious and expensive. We here report the isolation of single-chain variable fragment antibodies against Stxs by screening a phage display library constructed from a naïve human repertoire. An antibody among the selected clones designated B22 bound to the binding subunits of both Stx-1 and Stx-2, and strongly neutralized the cytotoxicity of Stx-1. This is the first example of a monovalent antibody showing Stx-neutralizing activity. The B22 antibody is also completely naturally occurring in human, which reduces the possibility of adverse immunological effects, and can be easily produced using bacterial protein synthesis systems.

Nucleotide sequence analysis of the enterotoxigenic Escherichia coli Ent plasmid.

We report here the complete nucleotide sequence of pEntH10407 (65 147 bp), an enterotoxigenic Escherichia coli enterotoxin plasmid (Ent plasmid), which is self-transmissible at low frequency. Within the plasmid, we identified 100 open reading frames (ORFs) which could encode polypeptides. These ORFs included regions encoding heat-labile (LT) and heat-stable (STIa) enterotoxins, regions encoding tools for plasmid replication and an incomplete tra (conjugation) region. The LT and STIa region was located 13.5 kb apart and was surrounded by three IS1s and an IS600 in opposite reading orientations, indicating that the enterotoxin genes may have been horizontally transferred into the plasmid. We identified a single RepFIIA replication region (2.0 kb) including RepA proteins similar to RepA1, RepA2, RepA3 and RepA4. The incomplete tra region was made up of 17 tra genes, which were nearly identical to the corresponding genes of R100, and showed evidence of multiple insertions of ISEc8 and ISEc8-like elements. These data suggest that pEntH10407 has the mosaic nature characteristic of bacterial virulence plasmids, which contains information about its evolution. Although the tra genes might originally have rendered pEntH10407 self-transferable to the same degree as R100, multiple insertion events have occurred in the tra region of pEntH10407 to make it less mobile. Another self-transmissible plasmid might help pEntH10407 to transfer efficiently into H10407 strain. In this paper, we suggest another possibility: that the enterotoxigenic H10407 strain might be formed by auto-transfer of pEntH10407 at a low rate using the incomplete tra region.