Expression of Shigella sonnei lipopolysaccharide in Vibrio cholerae

Making use of a newly designed mobilizable suicide vector, the genetic determinants encoding Shigella sonnei lipopolysaccharide (LPS) were stably integrated into the chromosome of the live attenuated Vibrio cholerae vaccine strain CVD103-HgR. Expression studies showed that the production of complete S. sonnei O-polysaccharide (O-PS)-bearing LPS was limited in bivalent recombinant strains that were also proficient in the synthesis of the host-encoded Inaba O-PS. Conversely, high amounts of LPS carrying S. sonnei O-PS are produced in monovalent Inaba-deficient derivatives, even in those strains which do not co-express the compatible R1 LPS core. Thus, the non-enterobacterial V. cholerae LPS core efficiently acts as a receptor for covalent binding of S. sonnei O-PS provided that competition with the host O-PS is avoided. Expression of the R1 core interferes with cell division in recombinant V. cholerae without affecting other physiological properties of vaccine strain CVD103-HgR. Both monovalent and bivalent strains stimulated high serum-antibody titres specific for their respective O-serotype(s) when administered to rabbits. The potential of V. cholerae as an expression carrier for heterologous O-serotypes is discussed.     

Development and validation of a detection system for wild-type Vibrio cholerae in genetically modified cholera vaccine.

Orochol, a live oral cholera vaccine licensed in Switzerland and in other countries, is based on the genetically modified Vibrio cholerae strain CVD103-HgR. This strain is derived from the wild-type O1 strain Inaba 569B by deletion of a fragment internal to the ctxA gene encoding the A1 subunit of cholera toxin and by replacement of an internal fragment of the hlyA gene with a fragment carrying the mer operon mediating mercury resistance. In this study we describe a polymerase chain reaction (PCR) system for the detection of wild-type Vibrio cholerae and the identification of the vaccine strain for the quality control of production batches. A multiplex PCR system that targets the intact ctxA gene of the wild-type strain and simultaneously the integration site of the mer operon in the hlyA gene (hlyA::mer) of the vaccine strain CVD103-HgR was developed. To evaluate the detection limit of the system, vaccine suspensions were artificially contaminated with wild-type V. cholerae 569B cells and tested by PCR. The detection limit of the system was statistically evaluated and found to be at 11625 wild-type cells per vaccine sachet (95% confidence limit). This number is below the infective dose of wild-type Vibrio cholerae. In Switzerland this test is used in combination with other tests in the official batch-release procedure to assure the safety of each batch of the cholera vaccine Orochol.     

Use of stabilized luciferase-expressing plasmids to examine in vivo-induced promoters in the Vibrio cholerae vaccine strain CVD 103-HgR

Live, attenuated Vibrio cholerae vaccines can induce potent immune responses after only a single oral dose. The strategy of harnessing these strains to present antigens from heterologous pathogens to the mucosal immune system shows great promise. To fully realize this possibility, V. cholerae strains must be created that stably express antigens in vivo in sufficient quantity to generate an immune response. In vivo -induced promoters have been shown to increase the stability and immunogenicity of foreign antigens expressed from multicopy plasmids. We report the construction of a series of genetically stabilized plasmids expressing luciferase as a heterologous protein from the following in vivo -induced promoters: V. cholerae P _argC , P _fhuC and P _vca1008 , and Salmonella enterica serovar Typhi P _ompC . We demonstrate that several of these expression plasmids meet two critical criteria for V. cholerae live vector vaccine studies. First, the plasmids are highly stable in the V. cholerae vaccine strain CVD 103-HgR at low copy number, in the absence of selective pressure. Second, real-time bioluminescent imaging (BLI) demonstrates inducible in vivo expression of the promoters in the suckling mouse model of V. cholerae colonization. Moreover, the use of BLI allows for direct quantitative comparison of in vivo expression from four different promoters at various time points.     

Expression and morphology of enterotoxigenic Escherichia coli surface antigen CS31A in E. coli K12 and Vibrio cholerae

Enterotoxigenic Escherichia coli (ETEC) is known as a worldwide cause of diarrheal disease. The pathogenesis involves the attachment of the microorganisms to the mucosa and the production of enterotoxins. Surface expression of CS31A fimbriae was assessed by Western blots, dot blots, immunofluorescence, and electron microscopy using negative staining and immunogold labeling. These investigations revealed significant differences in both the morphology of the wild-type and recombinant strains and the antigen exposure of CS31A in the wild-type and recombinant strains. In the wild-type ETEC strain, expression of CS31A was subject to phase variation. The recombinant E. coli strain produced CS31A but was prone to epitope shedding. In Vibrio cholerae vaccine strain CVD 103-HgR, the recombinant CS31A antigen was expressed but was only found intracellularly. Thus, E. coli strains seem to lend themselves better to the development of recombinant vaccines expressing ETEC-specific antigens at the cell's surface than strains from other orders or genera such as V. cholerae.     

Strains of Vibrio cholerae serogroups O1 and O139 that produce the basic protective antigens

To find out stable and effective producers of major protective antigens intended for use as components of cholera chemical vaccine against V. cholerae strains of serogroups O and O139, the comparative analysis of the production of cholera toxin, toxin-coregulated pili (TCP), antigens O1 and O139, polysaccharide capsule and outer membrane protein OmpU in different V. cholerae strains groups O1 and O139 has been made. V. cholerae strain KM68, serogroup O1, has been found capable of the production of antigen O1, serovar Ogawa, protein OmpU at a sufficiently high level and the hyperproduction of cholera toxin and TCP, and thus suitable for use in the manufacture of cholera bivalent vaccine as the source of these antigens. Specially selected alysogenic noncapsular strain KM137 of serogroup O139, characterized by a high and stable level of the biosynthesis of this somatic antigen when grown in both laboratory and production conditions, may serve as the produces of antigen O139.     

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.     

01群与非01群0139型霍乱弧菌毒力的对比研究

非０１群０１３９型霍乱弧菌是近年引起南亚次大陆霍乱流行的新型病原体,将其与０１群霍乱弧菌的毒力特性进行对比研究对于了解其特性及研制相关的菌苗具有重要意义。本文报告了４株０１３９型霍乱弧菌与０１群霍乱弧菌菌株的对比测定结果。发现０１３９型霍乱弧菌与０１群霍乱弧菌有所不同,呈不透明的菌落形态,光学显微镜及电子显微镜检显示有荚膜的表型,在体内具有较高的繁殖能力并产生肠毒素,体内侵袭试验结果表明所有４株０     

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.     

Structure and arrangement of the cholera toxin genes in Vibrio cholerae O139

Abstract The sequence of the ctxB gene encoding the B subunit of cholera toxin has been determined for a strain of Vibrio cholerae of the novel O139 serotype associated with recent outbreaks of severe cholera throughout South-East Asia and found to be identical to the ctxB gene in V. cholerae O1 of the E1 Tor biotype. Analyses by Southern hybridization and PCR showed that all strains of the O139 serotype V. cholerae tested carried cholera toxin genes and other gene associated with a virulence cassette DNA region at two loci identical or homologous to those identified in the Classical rather than the E1 Tor biotype of V. cholerae serotype O1 although these loci in O139 could reside on restriction fragments of variable size.     

Construction of cholera toxin B subunit-producing Vibrio cholerae strains using the Mariner-FRT transposon delivery system

The most widely used oral whole-cell-recombinant B subunit cholera vaccine contains the nontoxic cholera toxin B subunit (CTXB) and either heat- or formalin-killed Vibrio cholerae O1 strains. Vibrio cholerae O1 strains in the vaccine provide antibacterial immunity, and CTXB contributes to the vaccine's efficacy by stimulating production of anti-CTXB antibody. Various attempts have been made to increase CTXB production. In this study, the mariner-FRT transposon delivery system developed by Chiang and Mekalanos was used to place the ctxB gene under the control of a strong chromosomal promoter in a nontoxigenic V. cholerae El Tor strain, M7922. The expression level of CTXB in transposon insertion mutant clones was screened by ganglioside-dependent enzyme-linked immunosorbent assay. Among CTXB-producing V. cholerae clones that were isolated, M7922-C1 produced the highest amount of CTXB (3.17+/-1.69 microg mL(-1)). M7922-C1 harbors a single insertion of ctxB into VC0972, which encodes a putative porin protein. Although the level of CTXB expression in this strain was not exceptionally high, this study indicates the possibility of using this delivery system to construct vaccine strains that overexpress specific antigens.     

O1或O139霍乱疫苗对异群霍乱缺乏交叉保护

O1和O139霍乱弧菌是引起急性腹泻的病原微生物,用这两群菌的灭活全菌体与重组霍乱毒素B亚单位构建的亚单位／菌体型疫苗免疫队CA小鼠,对本群菌的攻击可提供良好免疫保护,而对异群霍乱菌则缺乏交叉保护作用。     

宋内Ⅰ相O抗原及霍乱毒素B亚基在福氏2a痢疾菌中的表达及其免疫保护效果的观察

本文利用基因重组的方法,将宋内Ｉ相Ｏ抗原基因以及霍乱毒素Ｂ亚单位基因（ｃｔｘ－Ｂ）克隆至带链球菌的ａｓｄ基因的表达载体,然后转化至ａｓｄ－痢疾菌苗株福氏２ａＴ３２。脂多糖银染以及Ｗｅｓｔｅｒｎｂｌｏｔｔｉｎｇ实验证实以上两基因都能在宿主菌中稳定表达。动物（小白鼠）保护实验表明,该重组菌对福氏２ａ、宋内氏痢疾菌的保护效率达１００％,对霍乱弧菌的保护效率也达７０％。该菌具有稳定、无抗生素标记、多价的特点。     

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.     

Pathogenic and vaccine significance of toxin-coregulated pili of Vibrio cholerae E1 Tor.

Vibrio cholerae O1 strains are classified into one of two biotypes, classical and E1 Tor, the latter being primarily responsible for cholera cases worldwide since 1961. Recent studies in our laboratory have focused upon the pathogenic and vaccine significance of the toxin-coregulated pili (TCP) produced by strains of E1 Tor biotype. Mutants in which the tcpA gene (encoding the pilin subunit protein) has been inactivated are dramatically attenuated in the infant mouse cholera model, showing markedly reduced colonisation potential in mixed-infection competition experiments. Significantly, in the vaccine context, antibodies to TCP are sufficient to prevent experimental infection, although our data suggest that this protective effect might be limited to strains of homologous biotype. Since we have shown that tcpA sequences are conserved within a biotype but differ between biotypes, this latter observation suggests that the biotype-restricted pilin epitopes might have greater vaccine significance. Similar studies indicate that TCP also play a critical role in colonisation by strains of the recently-recognised O139 serogroup, which is thought to have evolved from an O1 E1 Tor strain. In contrast to the effect of introducing mutations in the tcpA gene, strains carrying inactivated mshA genes (encoding the subunit of the mannose-sensitive haemagglutinin pilus) show unaltered in vivo behaviour. Consistent with this finding is our inability to demonstrate any protective effect associated with antibodies to MSHA. Ongoing approaches to vaccine development are variously aimed at improving the immunogenicity of the current inactivated whole-cell vaccine, or assessing the field efficacy of a promising live attenuated strain. The possible implications of our findings are discussed in relation to both of these options.     

霍乱O139型菌苗的试制

对来自孟加拉、泰国、印度、中国四地区Ｏ１３９型霍乱菌株进行了毒力、免疫原性、免疫力与相互交叉保护力试验,结果显示不同地区分离的Ｏ１３９型霍乱弧菌其所试特性相互间无差异。用中国（９３－３）株试制的菌体菌苗,其抗原性、毒性、免疫力安全性等经检定符合霍乱菌苗规程要求。鉴于Ｏ１３９型霍乱弧菌存在荚膜的特性,而现有的几种荚膜多糖菌苗都显示有明显的保护作用,因而,使用Ｏ１３９型菌苗有可能在一定程度上达到控制Ｏ１３９型霍乱流行的目的。     

多重PCR方法检测霍乱弧菌的研究

霍乱弧菌是霍乱的病原体,可以分为O1群、O139群和非O1/非O139群。O1群和O139群霍乱弧菌产生的霍乱肠毒素(也称霍乱毒素)是产生霍乱的主要原因,也只有O1群和O139群霍乱弧菌可引起霍乱。其他群的霍乱弧菌毒性不高,但在食品中也不允许被检出。实验以霍乱胶原酶基因和霍乱毒素基因为目的基因,试图建立一种PCR方法对霍乱弧菌进行检测研究,结果表明此方法可以用于食品中的霍乱弧菌检测。     

Activity of 22 antibacterials against O1 and O139 serogroup Vibrio cholerae strains isolated from humans within 1927-2005 in various regions of the world]

Analysis of antibioticograms of 390 O1 and O139 serogroup Vibrio cholerae strains isolated from humans within 1927-2005 in various regions of the world showed that the strains of V. cholerae isolated within 1927-1966 were susceptible to 22 antibacterials, the strains isolated within 1938-1993 possessed 1-3 resistance markers and the strains isolated within 1994-2005 had 3-8 resistance markers including resistance to fluoroquinolones. All the strains of O139 serogroup V. cholerae isolated in 1993 and 1994 possessed 3 resistance markers. Studies on albino mice with generalized experimental cholera due to the V. cholerae eltor 1 strain (P-18826, 2005) isolated from a cholera patient, which was highly resistant to nalidixic acid, streptomycin, ampicillin and trimethoprim/sulfamethoxazole and showed cross resistance to fluoroquinolones (ciprofloxacin, ofloxacin, pefloxacin and norfloxacin) and moderate resistance to ceftriaxone and cefotaxime, revealed that the only efficient antibiotics were tetracyclines and aminoglycosides (except streptomycin). The investigation demonstrated an extension of the antibiotic resistance spectra of the epidemically significant strains of the cholera pathogen and the necessity of using antibacterial drugs in strict accordance with the antibioticograms in emergent prophylaxis and therapy of cholera and immediate replacement of the drug by a more active one.     

Molecular analysis of Vibrio cholerae O1, O139, non-O1, and non-O139 strains: clonal relationships between clinical and environmental isolates

A total of 26 strains of Vibrio cholerae, including members of the O1, O139, and non-O1, non-O139 serogroups from both clinical and environmental sources, were examined for the presence of genes encoding cholera toxin (ctxA), zonula occludens toxin (zot), accessory cholera enterotoxin (ace), hemolysin (hlyA), NAG-specific heat-stable toxin (st), toxin-coregulated pilus (tcpA), and outer membrane protein (ompU), for genomic organization, and for the presence of the regulatory protein genes tcpI and toxR in order to determine relationships between epidemic serotypes and sources of isolation. While 22 of the 26 strains were hemolytic on 5% sheep blood nutrient agar, all strains were PCR positive for hlyA, the hemolysin gene. When multiplex PCR was used, all serogroup O1 and O139 strains were positive for tcpA, ompU, and tcpI. All O1 and O139 strains except one O1 strain and one O139 strain were positive for the ctxA, zot, and ace genes. Also, O1 strain VO3 was negative for the zot gene. All of the non-O1, non-O139 strains were negative for the ctxA, zot, ace, tcpA, and tcpI genes, and all of the non-O1, non-O139 strains except strain VO26 were negative for ompU. All of the strains except non-O1, non-O139 strain VO22 were PCR positive for the gene encoding the central regulatory protein, toxR. All V. cholerae strains were negative for the NAG-specific st gene. Of the nine non-ctx-producing strains of V. cholerae, only one, non-O1, non-O139 strain VO24, caused fluid accumulation in the rabbit ileal loop assay. The other eight strains, including an O1 strain, an O139 strain, and six non-O1, non-O139 strains, regardless of the source of isolation, caused fluid accumulation after two to five serial passages through the rabbit gut. Culture filtrates of all non-cholera-toxigenic strains grown in AKI media also caused fluid accumulation, suggesting that a new toxin was produced in AKI medium by these strains. Studies of clonality performed by using enterobacterial repetitive intergenic consensus sequence PCR, Box element PCR, amplified fragment length polymorphism (AFLP), and pulsed-field gel electrophoresis (PFGE) collectively indicated that the V. cholerae O1 and O139 strains had a clonal origin, whereas the non-O1, non-O139 strains belonged to different clones. The clinical isolates closely resembled environmental isolates in their genomic patterns. Overall, there was an excellent correlation among the results of the PCR, AFLP, and PFGE analyses, and individual strains derived from clinical and environmental sources produced similar fingerprint patterns. From the results of this study, we concluded that the non-cholera-toxin-producing strains of V. cholerae, whether of clinical or environmental origin, possess the ability to produce a new secretogenic toxin that is entirely different from the toxin produced by toxigenic V. cholerae O1 and O139 strains. We also concluded that the aquatic environment is a reservoir for V. cholerae O1, O139, non-O1, and non-O139 serogroup strains.     

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.     

Characterization and immunogenicity of Vibrio cholerae ghosts expressing toxin-coregulated pili

Bacterial ghosts are attractive for use as non-living vaccines and as carriers of heterologous antigens of vaccine relevance. Ghosts were prepared from Vibrio cholerae strains of O1 or O139 serogroup after growth under culture conditions, which favor or repress the production of toxin-coregulated pili (TCP). Immunoblotting confirmed the TCP status of these V. cholerae ghosts (VCG), which retained the cellular morphology and envelope sub-component profile of viable bacteria. Rabbits were immunized with VCGs prepared from O139 bacteria with TCP-positive or TCP-negative phenotypes and the resulting sera assayed for antibodies to lipopolysaccharide (LPS) and to TCP. Regardless of the TCP status of the VCG preparations used for immunization, all animals produced antibodies to LPS as demonstrated in bactericidal assays. These antibodies were probably responsible for the capacity of the antisera to confer passive immunity to challenge with the homologous O139 strain in the infant mouse cholera model (IMCM). Only following immunization with TCP-positive VCG, however, were antibodies to TCP generated, as judged by the potential of antisera to mediate protection against a challenge strain of heterologous serogroup.