Role of cell-associated N-acetyl-d-glucosamine specific haemagglutinin in the adhesion of Vibrio cholerae O1 to rabbit intestinal epithelial cells in vitro

Abstract Previously a N -acetyl- d -glucosamine specific cell-associated haemagglutinin (HA) had been purified from a Vibrio cholerae O1 strain. This study documents the role of this purified HA as an adhesin of V. cholerae O1. A significant inhibition in the adhesion of V. cholerae O1 bacterial cells to isolated rabbit intestinal brush borders (RIBB) was observed when the latter were pretreated with purified HA in ELISA. Antibody raised against purified HA and Fab (IgG) fragment of this serum inhibited adhesion of the bacteria to isolated rabbit intestinal epithelial cells (RIEC). V. cholerae O1 (both Ogawa and Inaba serovars) showed less adherence to isolated RIEC of animals immunised with the purified HA. Patients convalescing from V. cholerae O1 infection showed high ELISA titres against the purified HA indicating that it is expressed in the host during the disease process.     

Vibrio cholerae hemagglutinin/protease degrades chironomid egg masses

Cholera is a severe diarrheal disease caused by specific serogroups of Vibrio cholerae that are pathogenic to humans. The disease does not persist in a chronic state in humans or animals. The pathogen is naturally present as a free-living organism in the environment. Recently, it was suggested that egg masses of the nonbiting midge Chironomus sp. (Diptera) harbor and serve as a nutritive source for V. cholerae, thereby providing a natural reservoir for the organism. Here we report that V. cholerae O9, O1, and O139 supernatants lysed the gelatinous matrix of the chironomid egg mass and inhibited eggs from hatching. The extracellular factor responsible for the degradation of chironomid egg masses (egg mass degrading factor) was purified from V. cholerae O9 and O139 and was identified as the major secreted hemagglutinin/protease (HA/P) of V. cholerae. The substrate in the egg mass was characterized as a glycoprotein. These findings show that HA/P plays an important role in the interaction of V. cholerae and chironomid egg masses.     

Vibrio cholerae O139 produces a protease which is indistinguishable from the haemagglutinin/protease of Vibrio cholerae O1 and non-O1

Abstract Haemaglutinin/protease (HA/P) is one of the virulence factors of Vibrio cholerae O1 and pathogenic strains of V. cholerae non-O1. In this study, we examined protease activity of a new serogroup of Vibrio cholerae recently designated as O139 synonym Bengal. The protease activity was produced by all eight isolates of V. cholerae O139 from Bangladeshi patients. Purification and partial characterization of the protease from V. cholerae O139 demonstrated the purified protease (O139-P) was indistinguishable from that previously reported for HA/P of V. cholerae non-O1 (NAG-HA/P) and V. cholerae O1 (Vc-HA/P). These results prove that V. cholerae O139 produces a protease belonging to solHA/P, and suggest that the protease is another virulence factor found in newly emerged V. cholerae O139, as in V. cholerae O1.     

Vibrio cholerae Hemagglutinin/Protease Degrades Chironomid Egg Masses

Cholera is a severe diarrheal disease caused by specific serogroups of Vibrio cholerae that are pathogenic to humans. The disease does not persist in a chronic state in humans or animals. The pathogen is naturally present as a free-living organism in the environment. Recently, it was suggested that egg masses of the nonbiting midge Chironomus sp. (Diptera) harbor and serve as a nutritive source for V. cholerae, thereby providing a natural reservoir for the organism. Here we report that V. cholerae O9, O1, and O139 supernatants lysed the gelatinous matrix of the chironomid egg mass and inhibited eggs from hatching. The extracellular factor responsible for the degradation of chironomid egg masses (egg mass degrading factor) was purified from V. cholerae O9 and O139 and was identified as the major secreted hemagglutinin/protease (HA/P) of V. cholerae. The substrate in the egg mass was characterized as a glycoprotein. These findings show that HA/P plays an important role in the interaction of V. cholerae and chironomid egg masses.     

N-Acetyl-d-glucosamine-specific lectin purified from Vibrio cholerae 01

An N-acetyl-D-glucosamine-specific cell associated hemagglutinin (HA) was isolated and purified from a strain of Vibrio cholerae 01 by chitin affinity chromatography followed by separation on Bio Gel P-150. A single stained protein band of 47 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was observed with the purified HA. HA-antisera produced a single precipitin band against the purified HA in an immunodiffusion test without exhibiting any reactivity towards purified lipopolysaccharide (LPS). Purified HA, used as solid-phase antigen in an enzyme-linked immunosorbent assay (ELISA), reacted strongly with HA-antisera but cross-reacted negligibly with antisera raised against purified LPS. Hemagglutinating activity of the purified HA was highly sensitive to N-acetyl-D-glucosamine. The immunogold-labelling method using HA-antisera confirmed the location of the HA on the surface of the bacterial cells. The HA-antisera reacted with a protein component of the homologous outer membrane preparation. A significant inhibition was observed in the adhesive capability of the V. cholerae 01 strain to isolated rabbit intestinal epithelial cells (RIEC) in vitro when the later were pre-treated with the purified HA.     

Characterization of a clinical Vibrio cholerae O139 isolate from Mexico

Pathogenic strains of Vibrio cholerae O139 possess the cholera toxin A subunit (ctxA) gene as well as the gene for toxin co-regulated pili (tcpA). We report the isolation of a ctxA-negative, tcpA-negative V. cholerae O139 strain (INDREI) from a patient in Mexico diagnosed with gastrointestinal illness. Certain phenotypic characteristics of this strain were identical to those of V. cholerae O1 biotype El Tor. Unlike ctxA-positive V. cholerae O139 strains, this strain was sensitive to a wide panel of antibiotics, including ampicillin, chloramphenicol, ciprofloxacin, gentamicin, furazolidone, nalidixic acid, nitrofurantoin, tetracycline, trimethoprim-sulfamethoxazole, and streptomycin, but was resistant to polymyxin B. Ribotype and pulsed-field gel electrophoresis profiles of INDRE1 differed from those of ctxA-positive V. cholerae O139 and other V. cholerae strains. Phenotypic characteristics of the Mexico strain were similar to those reported for V. cholerae O139 isolates from Argentina and Sri Lanka.     

Purification and characterization of Vibrio cholerae O139 fimbriae

Abstract A Vibrio cholerae O139 (strain Al-1841) isolated from a patient with a cholera-like disease in Bangladesh predominantly produced new curved, wavy fimbriae (Al-1841 fimbriae) and small numbers of previously reported V. cholerae non-O1 S7-like pili. The former was purified and characterized. The molecular mass of the Al-1841 fimbrial subunit was less than 2.5 kDa, and it was immunologically different from that of V. cholerae non-O1 S7 pili. This novel fimbrial antigen was detected in all 182 Gram-negative strains from five genera tested but was absent from the Gram-positive bacteria tested. The purified Al-1841 fimbriae did not agglutinate human or rabbit erythrocytes.     

Studies on adhesion, haemagglutination and other biological properties of Vibrio cholerae O139

Abstract The adhesive capabilities of eight Vibrio cholerae O139 epidemic strains to isolated rabbit intestinal epithelial cells (RIEC) were observed to be high similar to those observed with a Vibrio cholerae O1 strain isolated from patients. Toxin production by the strains, measured by accumulation of fluid in rabbit ileal loop model, was high and the toxin was lethal as the animal expired within 6 h. Culture filtrates of the strains exhibited the presence of vascular permeability factor which produce induration and necrosis in the adult rabbit and guinea pig skin. All the strains showed high to moderate haemagglutinin titres against chicken erythrocytes and produced El Tor-like haemolysin. SDS-PAGE of the outer membrane preparation of the strains showed the presence of major protein component at 38 kDa region. The lethality of the toxin, high adhesive activity, shifting of the major outer membrane protein band and production of thermolabile haemolysin on Wagatsuma agar were the major variations of these epidemic strains from V. cholerae O1 and V. cholerae non-O1 strains isolated previously.     

Depolymerization of the capsular polysaccharide from Vibrio cholerae O139 by a lyase associated with the bacteriophage JA1.

We have studied the interaction between the Vibrio cholerae O139 specific phage JA1, belonging to the Podoviridae family, and the capsular polysaccharide (CPS) of the parent strain from which the phage was isolated. Upon incubation of the JA1 phage with the CPS, oligosaccharides were isolated and purified. The oligosaccharides derived from one (shown below) and two repeating units of the CPS were characterized using NMR spectroscopy, mass spectrometry and sugar analysis (structure: see text). The cleavage was found to occur by beta-elimination at the 4-substituted alpha-linked galacturonic acid, which results in a 4-deoxy-beta-L-threo-hex-4-enopyranosyl uronic acid group (Sug). The enzyme associated with the JA1 phage responsible for the depolymerization of the V. cholerae O139 CPS is thus a lyase.     

Involvement of the hap gene (mucinase) in the survival of Vibrio cholerae O1 in association with the blue-green alga,Anabaena sp

Mucinase is a soluble haemagglutinin protease, which may be important for the survival of Vibrio cholerae in association with mucilaginous blue-green algae (cyanobacteria). A comparative survival study was carried out with an Anabaena sp. and a wild-type V. cholerae O1 strain hap+ gene (haemagglutinin-protease), together with its isogenic mutant hap (hap-deleted gene). A simple spread plate technique was followed to count culturable V. cholerae O1 on taurocholate tellurite gelatin agar plate. The fluorescent antibody technique of Kogure et al. (1979) was used for the microscopical viable count of V. cholerae O1. Polymerase chain reaction (PCR) and Southern blot hybridization were carried out to detect a lower number of viable but nonculturable (VBNC) V. cholerae O1 from the laboratory-based experiments. The wild and mutant V. cholerae O1 strains survived in culturable form for 22 and 10 days. respectively, in association with the Anabaena sp., with the difference being statistically significant (P < 0.01). The fluorescent antibody technique, PCR, and hybridization results also showed that the wild strain survived better in the VBNC state than did the mutant VBNC strain in association with an Anabaena sp. These results indicate that the enzyme mucinase may play an important role in the association and long-term survival of V. cholerae O1 with a mucilaginous blue-green alga, Anabaena sp.     

Detection of heat-stable enterotoxin in a cholera toxin gene-positive strain of Vibrio cholerae O1.

DNA colony hybridization with a polynucleotide clonal DNA probe for heat-stable enterotoxin of Vibrio cholerae non-O1 (NAG-ST) was used to screen 197 isolates of V. cholerae O1. Under stringent hybridizing and washing conditions, one strain (GP156) reacted with the probe. The concentrated supernatant from V. cholerae O1 GP156, heated at 100 degrees C for 5 min, elicited fluid accumulation in the suckling mice and that could be completely neutralized by an anti-NAG-ST monoclonal antibody (mAb2F). The preparation from V. cholerae O1 GP156 also inhibited the binding of mAb2F to NAG-ST in a competitive ELISA. V. cholerae O1 GP156 was confirmed to possess a gene encoding cholera toxin (CT). These results indicate that a heat-stable enterotoxin is produced by certain strains of CT-producing V. cholerae O1.     

A new type of conjugative transposon encodes resistance to sulfamethoxazole, trimethoprim, and streptomycin in Vibrio cholerae O139.

Vibrio cholerae O139 is the first non-O1 serogroup of V. cholerae to give rise to epidemic cholera. Apparently, this new serogroup arose from an El Tor O1 strain of V cholerae, but V. cholerae O139 is distinguishable from V. cholerae El Tor O1 by virtue of its novel antigenic structure and also its characteristic pattern of resistances to the antibiotics sulfamethoxazole, trimethoprim, streptomycin, and furazolidone. We found that the first three of these antibiotic resistances are carried on an approximately 62-kb self-transmissible, chromosomally integrating genetic element which we have termed the SXT element. This novel conjugative transposon-like element could be conjugally transferred from V. cholerae O139 to V cholerae O1 and Escherichia coli strains, where it integrated into the recipient chromosomes in a site-specific manner independent of recA. To study the potential virulence properties of the SXT element as well as to improve upon the live attenuated O139 vaccine strain Bengal-2, a large internal deletion in the SXT element was crossed on to the Bengal-2 chromosome. The resulting strain, Bengal-2.SXT(s), is sensitive to sulfamethoxazole and trimethoprim and colonizes the intestines of suckling mice as well as wild-type strains do, suggesting that the SXT element does not encode a colonization factor. Derivatives of Bengal-2.SXT(s) are predicted to be safe, antibiotic-sensitive, live attenuated vaccines for cholera due to the O139 serogroup.     

Bactericidal properties of hemo-cytolysin from Vibrio cholerae non O1 P-11702 strain in a panel of indicator cultures for detection of vibriocins

The influence of the preparation of hemo-cytolysin, obtained from V. cholerae non O1 strain P-11702 and inducing lysis of both red blood cells and V. cholerae cultures using a panel of indicator cultures for the detection of vibriocins, was studied. The set of indicator cultures contained 2 Shigella flexneri strains, 1 S. dysenteriae strain, 3 S. sonnei strains, 3 Escherichia coli strains and 2 V. cholerae strains, one of them being atypical. Hemo-cytolysin exhibited lytic activity with respect to S. dysenteriae, S. sonnei strains and 1 V. cholerae strain. i.e. to 4 out of 11 indicator strains. V. cholerae atypical strain proved to be resistant to the preparation in contrast to 33 V. cholerae typical strains, studied previously.     

Serogroup conversion of Vibrio cholerae in aquatic reservoirs

The environmental reservoirs for Vibrio cholerae are natural aquatic habitats, where it colonizes the chitinous exoskeletons of copepod molts. Growth of V. cholerae on a chitin surface induces competence for natural transformation, a mechanism for intra-species gene exchange. The antigenically diverse O-serogroup determinants of V. cholerae are encoded by a genetically variable biosynthetic cluster of genes that is flanked on either side by chromosomal regions that are conserved between different serogroups. To determine whether this genomic motif and chitin-induced natural transformation might enable the exchange of serogroup-specific gene clusters between different O serogroups of V. cholerae, a strain of V. cholerae O1 El Tor was co-cultured with a strain of V. cholerae O139 Bengal within a biofilm on the same chitin surface immersed in seawater, and O1-to-O139 transformants were obtained. Serogroup conversion of the O1 recipient by the O139 donor was demonstrated by comparative genomic hybridization, biochemical and serological characterization of the O-antigenic determinant, and resistance of O1-to-O139 transformants to bacteriolysis by a virulent O1-specific phage. Serogroup conversion was shown to have occurred as a single-step exchange of large fragments of DNA. Crossovers were localized to regions of homology common to other V. cholerae serogroups that flank serogroup-specific encoding sequences. This result and the successful serogroup conversion of an O1 strain by O37 genomic DNA indicate that chitin-induced natural transformation might be a common mechanism for serogroup conversion in aquatic habitats and for the emergence of V. cholerae variants that are better adapted for survival in environmental niches or more pathogenic for humans.     

A method for studies of an El Tor-associated antigen of Vibrio cholerae O1

A method for studying the biotype El Tor associated mannose-sensitive haemagglutinin (MSHA) of V. cholerae O1 has been developed. By using crude MSHA adsorbed to chicken erythrocytes as solid phase antigen in an enzyme-linked immunosorbent assay (ELISA), antisera against V. cholerae of the El Tor biotype reacted in high titre with the MSHA-coated cells, whereas antisera against vibrios of the classical biotype did not bind significantly, i.e. in higher titre than pre-immune sera. The binding of anti-MSHA serum, or a monoclonal antibody against MSHA, to the MSHA-coated erythrocytes could be efficiently inhibited by crude MSHA as well as by El Tor vibrios whereas neither V. cholerae lipopolysaccharide nor different strains of classical vibrios had any inhibitory effect. These results support the existence of an El Tor-associated immunogen. They also suggest a possibility of determining antibodies against different haemagglutinins in ELISA without having access to purified antigens.     

O1群霍乱弧菌实时荧光定量PCR快速检测方法的建立

目的:建立针对O1群霍乱弧菌的实时荧光定量TaqMan PCR快速检测方法,并进行模拟粪便标本的检测评价。方法:根据O1群霍乱弧菌O抗原编码基因rfb的特异性序列设计引物和TaqMan探针,建立检测O1群霍乱弧菌的实时荧光定量TaqMan PCR快速检测方法,对所建立的方法分别进行实验室内的灵敏度及特异性评价;将O1群霍乱弧菌灭活菌株悬液倍比稀释后与健康成人新鲜粪便混匀,制备成模拟带菌者粪便标本,提取DNA,进行Taq-Man PCR检测,用以评价该方法。结果:建立了快速检测O1群霍乱弧菌的实时荧光定量TaqMan PCR方法,灵敏度为每反应体系104拷贝;该方法对其他14种肠道菌DNA没有扩增;该方法对模拟粪便标本的检测灵敏度为每反应体系102 CFU。结论:建立了一种快速、高效检测O1群霍乱弧菌的荧光定量PCR检测方法,该方法可用于O1群霍乱弧菌临床粪便标本的检测。     

Rapid detection and identification of odontoglossum ringspot virus by polymerase chain reaction amplification

Abstract A hemolysis gene ( hlx ) which lyses sheep erythrocytes on blood agar plates when expressed in Escherichia coli was cloned from Vibrio cholerae . The cloned gene is predicted to encode a polypeptide of 92 amino acid residues with a deduced molecular mass of 10451. E. coli transformed with this gene lysed sheep, goose, horse and chicken erythrocytes but not those of guinea pig and human. The hlx gene was observed in classical- and El Tor-biotype V. cholerae O1, V. cholerae non-O1, and V. mimicus , but not in V. parahaemolyticus .     

Production and cross-reactivity patterns of a panel of high affinity monoclonal antibodies to Vibrio cholerae O139 Bengal

Abstract A series of monoclonal antibodies of different isotypes specific for Vibrio cholerae O139, the new pandemic strain of cholera, was produced. These mAbs reacted only with the reference strain (MO45) representing serovar O139 but did not react with any of the other reference strains representing serovars O1 to O140. Significantly, the mAbs did not agglutinate the R-cultures of V. cholerae (CA385, 20–93) which demonstrated the exceptional specificity of these mAbs and indicated that the mAbs recognized antigenic determinants unique for the O139 serovar. There was heterogeneity in the intensity of reactivity of the mAbs with strains of V. cholerae O139 isolated from diverse sources. Apart from 4H6, the other mAbs agglutinated all the O139 strains examined. 2D12 and 2F8 were the best mAbs based on the intensity of agglutination with all the O139 strains. Evaluation of 3A10 in comparison with a polyclonal anti-O139 antibody raised in rabbit using the slide agglutination format revealed that 3A10 fared as well as the polyclonal antibody for the laboratory identification of the O139 serovar. The acquisition of these mAbs provide reagents which would be very useful in the development of simple immunodiagnostic assays for the diagnosis of V. cholerae O139 infections.     

Production of monoclonal antibodies against a hemagglutinin/protease of Vibrio cholerae non-01

Two hybridoma cell lines producing monoclonal antibodies (MAbs) against a hemagglutinin/protease (HA/P) from Vibrio cholerae non-01 were produced and characterized. The two MAbs contained the kappa light chain and were IgG1 type. They similarly neutralized HA/P protease activity derived from both V. cholerae non-01 and V. cholerae 01, whereas they were unable to neutralize the hemagglutinating activity of HA/P, suggesting that the epitopes for protease and hemagglutination activities are different. Western blotting analysis and the cross-neutralization test with the two MAbs confirmed the identity of HA/P produced by V. cholerae non-01 and 01. This study also suggests that HA/P of V. cholerae and a protease of V. parahaemolyticus are immunologically unrelated.     

The absence of a flagellum leads to altered colony morphology, biofilm development and virulence in Vibrio cholerae O139

Throughout most of history, epidemic and pandemic cholera was caused by Vibrio cholerae of the serogroup O1. In 1992, however, a V. cholerae strain of the serogroup O139 emerged as a new agent of epidemic cholera. Interestingly, V. cholerae O139 forms biofilms on abiotic surfaces more rapidly than V. cholerae O1 biotype El Tor, perhaps because regulation of exopolysaccharide synthesis in V. cholerae O139 differs from that in O1 El Tor. Here, we show that all flagellar mutants of V. cholerae O139 have a rugose colony morphology that is dependent on the vps genes. This suggests that the absence of the flagellar structure constitutes a signal to increase exopolysaccharide synthesis. Furthermore, although exopolysaccharide production is required for the development of a three-dimensional biofilm, inappropriate exopolysaccharide production leads to inefficient colonization of the infant mouse intestinal epithelium by flagellar mutants. Thus, precise regulation of exopolysaccharide synthesis is an important factor in the survival of V. cholerae O139 in both aquatic environments and the mammalian intestine.