Electrophoretic mobility and immune response of outer membrane proteins of Vibrio cholerae O139

Abstract The outer membrane (OM) protein components of a Vibrio cholerae O1 and four V. cholerae O139 strains, collected from cholera patients, were analysed by SDS-PAGE. A protein of 69 kDa molecular mass was observed only when the OMPs were prepared from strains grown in synthetic broth. As a result of passage in the rabbit ileal loop (RIL), virulence was enhanced, and a protein component around 18 kDa of the V. cholerae O139 OM became the major protein component. On immunoblot analysis with rabbit antiserum against V. cholerae O139 OM, it was shown that, apart from the major protein component of V. cholerae O1 OM of around 45 kDa and that of V. cholerae O139 OM of around 38 kDa, all other minor protein components were cross-reactive between the two serogroups. In immunoblot assays with convalescent sera obtained from V. cholerae O139-infected patients, it was observed that in addition to the lipopolysaccharide (LPS)-induced antibody, only the 38 kDa major protein component elicited considerable levels of antibody in the pateint. Minor OM components of 18 kDa were detected in the immunoblot analysis by LPS-directed antibody, however, as the OM proteins are known to be associated with LPS.     

副溶血性弧菌外膜蛋白K的B细胞线性表位预测

目的预测副溶血性弧菌外膜蛋白K(OmpK)的B细胞线性表位。方法 NCBI下载已登录的OmpK的基因序列,对其进行生物信息学分析,应用DNAStar protean软件综合分析OmpK蛋白的二级结构、柔性、表面可能性、亲水性和抗原指数等多种参数,预测其B细胞线性表位。结果 OmpK蛋白的优势B细胞线性表位位于肽链的第7-13、25-36、63-69、140-147、182-188、234-239区段。结论预测得到OmpK蛋白的6个优势B细胞线性表位,为进而克隆表达串联表位蛋白,研制副溶血性弧菌多表位疫苗奠定基础。     

Rapid and sensitive detection of Vibrio cholerae by loop-mediated isothermal amplification targeted to the gene of outer membrane protein ompW

Aims:  The present study was aimed to develop a loop-mediated isothermal amplification (LAMP) assay for rapid and specific detection of Vibrio cholerae .
Methods and Results:  A set of five designed primers that recognized specifically the V. cholerae ompW gene was used. The optimized time and temperature conditions for the LAMP assay were 75 min at 65°C, respectively. The LAMP method accurately identified 16 isolates of V. cholerae but did not detect 28 non- cholerae Vibrio isolates and 37 non- Vibrio bacterial isolates. The sensitivity of LAMP for V. cholerae detection in pure cultures was 2·2 × 10³ CFU ml⁻¹ or equivalent to 8 CFU per reaction. In the case of spiked shrimp samples without enrichment, the detection limit for V. cholerae was 2·2 × 10⁴ CFU g⁻¹ or equivalent to 20 CFU per reaction, while that of PCR was 100 CFU per reaction.
Conclusion:  The developed LAMP assay targeting ompW gene was rapid, specific and sensitive for V. cholerae detection.
Significant and Impact of the study:  The developed LAMP assay appears to be precise, accurate and a valuable tool for detection of V. cholerae . This assay can replace laborious biochemical tests for the identification of V. cholerae in contaminated food sample.     

TCP pilus biosynthesis in Vibrio cholera O1: gene sequence of tcpC encoding an outer membrane lipoprotein

The nucleotide sequence of the tcpC gene has been determined. It encodes a 53995-Da protein precursor with a signal sequence and cleavage site typical of a number of outer membrane lipoproteins, which are cleaved by the equivalent of signal peptidase II (Lsp) of Escherichia coli. The location of the tcpC gene is such that it is predicted to be translationally coupled to the 5' and 3' flanking genes, tcpY and tcpD, respectively, indicating that it forms part of an operon. Together with the lipoprotein signal sequence and the several hydrophobic domains it seems likely that TcpC is a surface-anchored trans-outer membrane lipoprotein.     

Comparison of outer membrane protein profiles of Vibrio vulnificus biotypes 1 and 2

Abstract The outer membrane proteins of 17 Vibrio vulnificus biotype 2 strains from Japanese and European cels, and 12 biotype 1 strains from clinical and environmental sources have been compared. The overall profile in both biotypes was similar, and a major protein band of molecular mass 36 kDa was detected in the majority of the strain. Differences in the minor bands allowed differentiation of strains from different origins, suggesting that outer membrane protein profiles could be useful as epidemiological markers in the species V. vulnificus . Immunoblotting with antisera to whole cells of selected strains of biotypes 1 and 2 showed a strong antigenic response to outer membrane proteins 66, 60, 48, 46 and 44 kDa; these were common to all strains examined, independent of their biotypes and origins. These results demonstrate the presence of antigenically related outer membrane proteins in both biotypes of V. vulnificus .     

A novel multiplex PCR for the identification of Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus

AIM: To establish a simple multiplex polymerase chain reaction (PCR) that will identify Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus. METHODS AND RESULTS: A total of 429 Vibrio spp. from various origins were tested with the novel primers targeting toxR. The reverse primers were all designed to be species specific, while the forward primer was universal. The primers correctly identified all the V. parahaemolyticus, V. cholerae and V. vulnificus isolates tested. CONCLUSIONS: The toxR multiplex PCR works well when the initial colony morphology is known. If not, Vibrio alginolyticus might represent a diagnostic obstacle. SIGNIFICANCE AND IMPACT OF THE STUDY: The method provides a fast and reliable way of identifying the main Vibrio spp. involved in food-borne disease. The method could prove very useful for laboratories working with identification of these Vibrio spp.     

Comparative immunogenicity of outer membrane protein K and whole-cell antigens of Vibrio parahaemolyticus for diagnosis

The immunogenicity of soluble outer membrane protein K (OmpK)- small ubiquitin-like modifier, OmpK inclusion bodies, formalin, and heat-killed Vibrio parahaemolyticus cells were prepared and studied in a mouse model. The results of whole-cell ELISA and Western blot (WB) revealed that the serum against soluble OmpK and OmpK inclusion bodies reacted only with homologous V. parahaemolyticus. Furthermore, recombinant OmpK proteins were not recognized by the serum against whole-cell V. parahaemolyticus antigens. Unexpectedly, the serum against formalin and heat-killed V. parahaemolyticus reacted broadly with homologous (an immunization strain) and heterologous (non-immunization strains) V. parahaemolyticus and Vibrio species. The WB results revealed that the serum against the two V. parahaemolyticus whole-cell antigens primarily reacted with proteins that were approximately 100, 70, 36, 28, and 22 kDa in the cell lysates from different Vibrio strains, rather than the recombinant OmpK. The 70 and 28 kDa proteins exhibited specificity to Vibrio species, while the 22 kDa protein was more specific to V. parahaemolyticus. This study showed the limitation of recombinant OmpK to prepare diagnostic antibodies and revealed several specific Omps of Vibrio sp. and V. parahaemolyticus that were promising in diagnosis and vaccine development.     

Analysis of some factors involved in the virulence mechanism of Vibrio cholerae non-01

Abstract A study was carried out to evaluate the potential intestinal toxicity of 188 samples of Vibrio cholerae non-01 isolated from seawater found along the beaches of Rio de Janeiro city. Three different assays were carried out involving: (a) detection of vascular permeability factor (PF) in guinea pigs (together with assessment of two culture media for production of the toxin); (b) intestinal fluid accumulation (FA) in suckling mice; and (c) detection of haemolysin. The results demonstrated that both culture media gave a similar level of performance. In the animal assays, 43% of the samples induced PF in guinea pigs, 28.7% caused intestinal fluid accumulation in suckling mice, and 63.28% contained haemolysin. Only 4.25% of the samples gave positive results in all three tests.     

A variant type of Vibrio cholerae SXT element in a multidrug-resistant strain of Vibrio fluvialis

Vibrio fluvialis strain H-08942 was isolated from an infant aged 6 months who was suffering from cholera-like diarrhea in India. This strain showed the typical multidrug-resistance phenotype of an SXT element. It was resistant to sulfamethoxazole (Su), trimethoprim (Tm), chloramphenicol (Cm) and streptomycin (Sm), in addition to other antibiotics such as ampicillin (Am), furazolidone (Fz), nalidixic acid (Na), and gentamicin (Gm). The SXT element is a Vibrio cholerae-derived integrative and conjugative element (ICE) that has also been referred to as a conjugative transposon. Our goal was to find a relationship between these resistant phenotypes and the presence of the SXT element in this unique strain. By using PCR, we detected the antibiotic resistance genes, the integrase gene and the attP attachment site of SXT element. Cloning and DNA sequencing results showed that both the SXT integrase gene and its attP site of V. fluvialis were similar but not identical to those of V. cholerae. The SXT integrase gene of V. fluvialis has a 99% identity to that of V. cholerae, and the attP site of SXT of V. fluvialis is variant and shorter (641 bp) than that of V. cholerae (785 bp). It was possible for the SXT of V. fluvialis to be transferred by conjugation to a laboratory strain of Escherichia coli. Here, we report the detection of a variant SXT element in species other than V. cholerae, with molecular characterization and analysis of its integrase gene and its attP site.     

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.     

Major outer membrane proteins in the phytopathogenic bacteria Erwinia carotovora subsp. carotovora and subsp. atroseptica

Abstract Immunological similarities of heat-labile Escherichia coli enterotoxins pathogenic for man (LTh) and piglets (LTp) and cholera enterotoxin (CT) were examined quantitatively by the reversed Mancini test. The following results were obtained by analysis of rabbit antisera against these toxins. (1) 86% and 61% of the immunoglobulins in anti-CT antisera were antibodies cross-reacting with LTh and LTp, respectively; (2) 77% and 66% of the immunoglobulins in anti-LTh antisera were antibodies cross-reacting with LTp and CT, respectively; (3) 75% and 59% of the immunoglobulins in anti-LTp antisera were antibodies cross-reacting with LTh and CT, respectively.     

非O1群、非O139群霍乱弧菌致败血症1例报道

对襄阳市中心医院分离的2株疑似霍乱弧菌进行鉴定及药物敏感性试验。利用MicroScan WalkAway 40鉴定仪进行生化鉴定及药物敏感性试验,玻片凝集法确定血清型别,PCR扩增16SrRNA保守区基因并将产物进行测序分析。此2株疑似霍乱菌株O1群及O139群霍乱弧菌诊断血清均不凝集,16SrRNA扩增产物测序Blast比对分析与数据库中霍乱弧菌相似性达100％,药敏结果显示对氨苄西林、庆大霉素、环丙沙星、阿米卡星、氯霉素、复方新诺明（SXT）、四环素均敏感。该病例为非O1、非O139群霍乱弧菌导致的败血症,可能经胃肠道途径传播。     

Expression, characterization and immunogenicity of a major outer membrane protein from Vibrio alginolyticus

Vibrio alginolyticus is one of the Vibrio pathogens common to humans and marine animals.During infection and induction of the host immune response,outer membrane proteins of bacteria play animportant role.In this study,an outer membrane protein gene(ompW)was cloned from V.alginolyticus andexpressed in Escherichia coli.The 645 bp open reading frame(ORF)encodes a protein of 214 amino acidresidues with a predicted molecular weight of 23.3 kDa.The amino acid sequence showed a high identitywith that of Photobacterium damselae(96.2%)and Vibrio parahaemolyticus(94.4%).The alignment analy-sis indicated that OmpW was highly conserved.Sodium dodecyl sulfate-polyacrylamide gel electrophoresisshowed that the gene was over-expressed in E.coli BL21(DE3).Western blot analysis revealed that theexpressed protein had immunoreactivity.The recombinant protein was purified by affinity chromatographyon Ni-NTA Superflow resin.Large yellow croaker vaccinated with the purified OmpW showed significantlyincreased antibody to OmpW,which could resist the infection by V.alginolyticus.A specific antibody wasdetected by enzyme-linked immunosorbent assay.This study suggested that the conserved OmpW could bean effective vaccine candidate against infection by V.alginolyticus.     

用粘粒载体克隆霍乱弧菌染色体基因方法的建立

本文采用我国 El Tor 型霍乱弧菌805菌株(稻叶血清型,噬菌体-生物型 ld)为实验菌株,对粘粒载体(Cosmid)基因克隆的方法,进行了探索.首先从感染噬菌体λ突变体的溶原菌(HB 2688,HB2690)制备包装抽提物。制备方案有二:一是直接制备 HB2688,HB2690混合包装抽提物;另一是用超声波处理制备 HB2690抽提物;用冻融法制备 HB2688抽提物.包装时再将两者混合.本文认为以 pHC79为载体进行包装时,两方案效果一样,第一方案操作较简便些.在核酸操作中,从805菌株提取染色体 DNA,要求 DNA 的长度约为100kb;进行染色体 DNA部分消解时,应使大部分 DNA 片段在30～40kb 之间;连接时,碱性磷酸酶处理过的载体量应为30～40kb DNA 的10倍.本文以 pHC79为载体,建立805菌株染色体 DNA 无性繁殖系,得到500多个克隆子。     

The ability of two different Vibrio spp. bacteriophages to infect Vibrio harveyi, Vibrio cholerae and Vibrio mimicus

AIMS: To determine the host range of the Vibrio harveyi myovirus-like bacteriophage (VHML) and the cholera toxin conversion bacteriophage (CTX Phi) within a range of Vibrio cholerae and V. mimicus and V. harveyi, V. cholerae and V. mimicus isolates respectively. METHODS AND RESULTS: Three V. harveyi, eight V. cholerae and five V. mimicus isolates were incubated with VHML and CTX Phi. Polymerase chain reaction (PCR) was used to determine the presence of VHML and CTX Phi in infected isolates. We demonstrated that it was possible to infect one isolate of V. cholerae (isolate ACM #2773/ATCC #14035) with VHML. This isolate successfully incorporated VHML into its genome as evident by positive PCR amplification of the sequence coding part of the tail sheath of VHML. Attempts to infect all other V. cholerae and V. mimicus isolates with VHML were unsuccessful. Attempts to infect V. cholerae non-01, V. harveyi and V. mimicus isolates with CTX Phi were unsuccessful. CONCLUSIONS: Bacteriophage infection is limited by bacteriophage-exclusion systems operating within bacterial strains and these systems appear to be highly selective. One system may allow the co-existence of one bacteriophage while excluding another. VHML appears to have a narrow host range which may be related to a common receptor protein in such strains. The lack of the vibrio pathogenicity island bacteriophage (VPI Phi) in the isolates used in this study may explain why infections with CTX Phi were unsuccessful. SIGNIFICANCE AND IMPACT OF THE STUDY: The current study has demonstrated that Vibrio spp. bacteriophages may infect other Vibrio spp.     

A gene for the enterotoxin zonula occludens toxin is present in Vibrio mimicus and Vibrio cholerae O139

Abstract The presence of the zonula occludens toxin (ZOT) gene, which encodes an enterotoxin produced by serotype O1 strains of the pathogenic bacterium, Vibrio cholerae , in addition to cholera toxin, was investigated in selected strains of V. mimicus and the new pandemic V. cholerae non-O1 serotype O139. The zot gene was detected by polymerase chain reaction (PCR) amplification, using sets of primers based on the sequence of the V. cholerae O1 zot sequence. PCR amplification of genomic DNAs of both cholera toxin gene ( ctx ) positive and ctx⁻ strains of V. mimicus detected the presence of zot gene. An Acc -I- Eco RV V. cholerae zot gene fragment designed to overlap PCR products was used as a probe. Southern hybridization studies confirmed that the PCR fragments from V. mimicus and V. cholerae O139 were strongly homologous to the V. cholerae O1 zot gene. The zot gene was found with 3 to 5 strains of V. mimicus of which only one strain harbored the ctx gene. The presence of a zot gene in ctx⁻ toxigenic V. mimicus indicates a possible role of ZOT in the toxigenicity of this species. We conclude that, in addition to ctx, V. mimicus and V. cholerae O139 have the potential to produce ZOT.     

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.