Relationship between structure and antigenicity of O1 Vibrio cholerae lipopolysaccharides

The relationship between the release of fructose from O1 Vibrio cholerae lipopolysaccharides (LPS) by dilute acetic acid hydrolysis and the decrease in their antigenicity was examined. Decrease in the antigenicity of LPS was not parallel with the release of fructose, and occurred very much later than the latter. Periodate oxidation of LPS resulted in the total elimination of the fructose and glucose, and two-thirds of the heptose constituents, but no difference in the antigenicity of LPS was observed before and after oxidation. These findings indicate that the fructose present in O1 V. cholerae LPS is not substantially involved in their specific antigenicity. In the O1 V. cholerae LPS, the fructose is in the branch structure, most probably in the core region.     

霍乱弧菌O139多糖抗原的提取方法及优化

目的:研究霍乱弧菌O139多糖抗原的提取方法,以获得高纯度的多糖抗原。方法:采用热酚水法提取霍乱弧菌O139的脂多糖(LPS),并增加了DNaseⅠ、RNase消化步骤,以去除DNA及RNA的污染;进一步采用酸水解法获得脱毒的O特异性多糖(O-SP)。结果:经生化方法检测,证实提纯的LPS和O-SP纯度较高,能满足进行霍乱免疫研究的要求。结论:该方法简便可行,值得推广。     

Phase Variable O Antigen Biosynthetic Genes Control Expression of the Major Protective Antigen and Bacteriophage Receptor in Vibrio cholerae O1

The Vibrio cholerae lipopolysaccharide O1 antigen is a major target of bacteriophages and the human immune system and is of critical importance for vaccine design. We used an O1-specific lytic bacteriophage as a tool to probe the capacity of V. cholerae to alter its O1 antigen and identified a novel mechanism by which this organism can modulate O antigen expression and exhibit intra-strain heterogeneity. We identified two phase variable genes required for O1 antigen biosynthesis, manA and wbeL. manA resides outside of the previously recognized O1 antigen biosynthetic locus, and encodes for a phosphomannose isomerase critical for the initial step in O1 antigen biosynthesis. We determined that manA and wbeL phase variants are attenuated for virulence, providing functional evidence to further support the critical role of the O1 antigen for infectivity. We provide the first report of phase variation modulating O1 antigen expression in V. cholerae, and show that the maintenance of these phase variable loci is an important means by which this facultative pathogen can generate the diverse subpopulations of cells needed for infecting the host intestinal tract and for escaping predation by an O1-specific phage.     

Vibrio cholerae O139 bacteriophages

Cholera bacteriophages have been isolated from 27 lysogenic cultures of V. cholerae O139. As shown the pages under study belong to two morphological groups A1 and F1 and serological types II and XII. The use of prophage typing and the sensitivity test to specific phage made it possible to differentiate V. cholerae strains, serogroup O139.     

宋内Ⅰ相抗原和霍乱CT-B共表达的免疫保护效果观察

将编码宋内氏痢疾菌(Shigella sonnei)I相O抗原的基因和霍乱弧菌(Vibrio choler-ae)的CT-B基因克隆至带asd基因的质粒PYA248,得重组质粒PMGL105。将该重组质粒转入asd基因缺失的减毒伤寒沙门氏菌X4072,构成了一个不带抗药性基因的载体-宿主平衡致死系统。一系列实验表明,该重组菌X4072(PMGL105)能稳定地表达宋内I相O抗原和霍乱弧菌的CT-B抗原。小鼠免疫保护实验表明,该重组菌对有毒的宋内氏I相痢疾杆菌及霍乱弧菌的攻击均具有良好保护作用。     

Pili of a Vibrio cholerae O139

The pili of a strain of Vibrio cholerae O139 were purified and characterized. They were morphologically, electrophoretically and immunologically indistinguishable from the pili with 16 kDa subunit protein of V. cholerae O1. All 22 strains of V. cholerae O139 examined possessed the pili. The pili were different in hemagglutination inhibition pattern from V. cholerae O1 16K pili.     

Serological Reactivity of Humans to a Vibrio cholerae Common Antigen

Over the course of seven pandemics, Vibrio cholerae serotypes have varied. In 1992 the appearance of a new serotype, O139 Bengal, began the eighth cholera pandemic. Several new O139 antigens have been identified, yet a common V. cholerae antigen has not been described. In this study, a monoclonal antibody specific against an 18.7-kDa outer membrane antigen reacted in dotblot analysis with 292 epidemiologically diverse V. cholerae isolates including O1, non-O1, and O139 serotypes. Serum collected from volunteers experimentally challenged with V. cholerae O139, and rabbit antisera to V. cholerae O1, were reactive with the 18.7-kDa antigen by Western immunoblot. This is the first report that the 18.7-kDa antigen is present in V. cholerae O139. Received: 11 August 1997 / Accepted: 22 September 1997     

Multiplex polymerase chain reaction to detect toxigenic Vibrio cholerae and to biotype Vibrio cholerae O1

A multiplex polymerase chain reaction (PCR) was developed to identify cholera toxin-producing Vibrio cholerae and to biotype V. cholerae O1. Enterotoxin-producing V. cholerae strains were identified with a primer pair that amplified a fragment of the ctxA2-B gene. Vibrio cholerae O1 strains were simultaneously differentiated into biotypes with three primers specified for the hlyA gene in the same reaction. The hlyA amplicon in the multiplex PCR serves as an internal control when testing toxin-producing strains, as hlyA gene sequences exist in all tested V. cholerae strains. Enrichment of V. cholerae present on oysters for 6 h in alkaline peptone water before detection by a nested PCR with internal primers for ctxA2-B gene yielded a detection limit lower than 3 colony-forming units (cfu) per gram of food.     

A serogroup of non-O1 Vibrio cholerae possessing the Inaba antigen of Vibrio cholerae O1

A serogroup of non-O1 Vibrio cholerae, tentatively named Hakata, possessing the C (Inaba) factor but not the B (Ogawa) and A factors of V. cholerae O1 is described. Strains of this serogroup were isolated from river and estuarine waters and from frozen shrimps.     

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.     

A serogroup of non-O1 Vibrio cholerae possessing the Inaba antigen of Vibrio cholerae O1

A serogroup of non-O1 Vibrio cholerae , tentatively named Hakata, possessing the C (Inaba) factor but not the B (Ogawa) and A factors of V. cholerae O1 is described. Strains of this serogroup were isolated from river and estuarine waters and from frozen shrimps.     

A simple and convenient microtiter plate assay for the detection of bactericidal antibodies to Vibrio cholerae O1 and Vibrio cholerae O139

It is believed that the correlate of protection for cholera can be determined by the serum vibriocidal assay. The currently available vibriocidal assays, based on the conventional agar plating technique, are labor intensive. We developed a simple and convenient microtiter plate assay for the detection of vibriocidal antibodies that is equally as efficient for Vibrio cholerae O1 and for V. cholerae O139. The addition of succinate and neotetrazolium made it possible to measure the growth of surviving bacterial target cells by monitoring a color change. We evaluated assay parameters (target strains, growth of target cells, complement source and concentration) that may affect the reproducibility of the method for V. cholerae O139. The results obtained with the microtiter plate assay were uniformly similar to those obtained with the conventional agar plating assay, when testing both the Inaba and Ogawa serotypes of V. cholerae O1. The microtiter plate assay was also convenient for measuring the activity of animal sera and mouse monoclonal antibodies.     

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.     

Antigenic specificity of Vibrio cholerae O139 nitrosoguanidine mutants

The action of nitrosoguanidine (NG) on the culture of V. cholerae O139 P-16064 resulted in the appearance of mutant 16064 NG6, not agglutinating with commercial diagnostic serum O139. Its incapacity of agglutination was due to the sorption of the specific serum with strains V. cholerae O22 and R-variant RCA-385, which caused the loss of antibodies to common determinants. Experiments with the sorption of immune sera made it possible to suggest that one of the determinants of LPS O139, phosphate-galactose, was absent in NG mutant.     

Comparative characteristics of the preparations of hemolysin of ctx+ and ctx- Vibrio cholerae eltor and Vibrio cholerae O139 strains

Hemolysin of ctx+ Vibrio cholerae strains was obtained and studied. Ctx+ Vibrio cholerae O1 and O139 strains produced the hemolysin during cultivation in triptone medium without FeCl3. Mol.wt. and the spectrum of lytic activities of hemolysins of ctx+ Vibrio cholerae did not differ from hemolysins of ctx- strains.     

Quantitative evaluation of colonizing ability of Vibrio cholerae O1

A new method to evaluate the adhesive ability of Vibrio cholerae O1 was proposed. Broth cultured V. cholerae O1 and a piece of formalin-fixed rabbit intestinal wall were incubated together in KRT buffer and the number of adhered organisms was counted under a scanning electron microscope. This method was much less laborious than other methods that have been used so far, and most significantly, constant results were obtained in repeated experiments. The adhesive properties of toxigenic V. cholerae O1 evaluated by this method correlated well with its observed experimental pathogenicity.     

Adhesive Property of Toxin-Coregulated Pilus of Vibrio cholerae O1

The adhesive property of toxin-coregulated pilus (TCP) to the human intestine (jejunum), and whether or not TCP mediates the adhesion of Vibrio cholerae 395 organisms to the intestinal epithelium were investigated using visually proving methods. The purified TCP did not agglutinate human erythrocytes nor adhere to the surface of human intestinal epithelium. V. cholerae 395 adhered to the epithelium, but the adhesion was not inhibited by blocking the pili with the Fab fraction of anti-TCP IgG. The organisms adhered to the intestine treated with purified TCP in advance, as well as to the intact intestine. These findings suggest that TCP is not involved in the adhesion of these organisms to the intestinal epithelium.     

Hemolytic activity of Vibrio cholerae eltor and V. cholerae O139 toxigenic and nontoxigenic strains

A total of 20 ctx- and 16 ctx+ V. cholerae eltor strains, 20 ctx- and 22 ctx+ V. cholerae O139 strains were under study. Hemolytic activity was tested in modified Greig test with sheep, guinea pig and rabbit red blood cells. The comparative study of the hemolytic properties of V. cholerae O1 and O139 under different conditions of cultivation demonstrated their capacity of lysing sheep red blood cells (SRBC) irrespective of the presence of toxigenic properties. A wider spectrum of lytic activity of ctx- strains in Greig test with respect to red blood cells of different animals and the capacity of lysing SRBC, most resistant to the action of toxin, may be due to a considerably greater content of Hly+ clones in their population.