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.     

Proteome analysis of sorbitol fermentation specific protein in Vibrio cholerae by 2-DE and MS

Vibrio cholerae can be differentiated into epidemic and non-epidemic strains by sorbitol fermentation speed, but little research has been done on its mechanisms. In this study, we investigated differential protein expression of the two strains in response to sorbitol metabolism. V. cholerae strains were cultured in media with and without sorbitol, respectively. Proteins were separated by 2-DE, and those that showed different expression in the two media were identified by MALDI-TOF MS. Fifteen proteins in epidemic strains and 11 proteins in non-epidemic strains showed a different expression in sorbitol medium. Among them, 4 proteins were common to epidemic and non-epidemic strains. Gene sequence analysis showed that some mutations occurred in these proteins between the two strains. Potential functions of these proteins included sugar uptake, amino acid uptake, electron transport, sulfate and thiosulfate transport.     

霍乱弧菌N16961超级整合子重复序列及基因盒分析

目的:确定O1群El Tor型霍乱弧菌N16961超级整合子(SI)中霍乱弧菌重复序列(VCR)的序列特点,以及VCR和基因盒的数量及位置。方法:用局部序列比对软件BLAST将VCR参考序列与霍乱弧菌N16961的Ⅱ号染色体进行比对,用Artemis Comparison Tool查看比对结果获得比对区域的位置信息,并采用perl语言脚本获得霍乱弧菌N16961的Ⅱ号染色体VCR相应区域的序列;用全局比对软件Clustal W将上一步获得的所有VCR序列进行多序列比对,采用perl语言脚本处理比对结果获得一致性序列;用MEGA4.0软件查看多序列比对结果,并采用perl语言脚本计算各位置变异频率,据此分析霍乱弧菌N16961的Ⅱ号染色体上VCR和基因盒的特点。结果:在N16961的超级整合子中有158个VCR,其核苷酸长度为117～124 bp;其一致性序列有126个核苷酸,其中37个为保守核苷酸位点,89个为可变核苷酸位点;139个VCR与相邻的VCR之间至少有1个基因,19个VCR相互之间没有任何基因;N16961的SI中共存在146个基因盒,基因盒大小为390～5924 bp不等,每个基因盒中整合的基因数目为1～9个不等。结论:建立了SI中VCR和基因盒的分析流程,分析了SI中VCR的保守及变异位点,明确了霍乱弧菌N16961的SI中VCR和基因盒的信息,为霍乱弧菌和其他细菌中SI的研究提供了分析基础。     

霍乱弧菌脂多糖O抗原基因在大肠杆菌中的克隆及表达

经典生物型及埃尔托生物型霍乱弧菌的染色体DNA片段分别与载体质粒pUC18,B.S(M13~+)进行克隆,从克隆株中筛选到能表达霍乱弧菌脂多糖O抗原基因的重组子。它们所表达的脂多糖O抗原具有很好的抗原性及免疫原性,其重组质粒pMG-301、pMG-302经酶切分析表明,外源片段大小分别为8.4kb,7.6kb,较文献报道的16kb要小,而且基因结构之间也存在很大差异。     

O1群和O139群霍乱弧菌主要抗原研究进展

霍乱弧菌是引起人和动物烈性肠道传染病霍乱的病原体。在霍乱弧菌的200多个血清群中,只有O1群和O139群霍乱弧菌能引起霍乱。快速准确检测O1群和O139群霍乱弧菌是霍乱防治的关键。表面抗原在O1群和O139群霍乱弧菌检测中发挥着重要作用。简要综述了O1群和O139群霍乱弧菌的脂多糖、霍乱肠毒素、外膜蛋白W、毒素共调菌毛和甘露糖敏感血凝素等5种主要抗原的研究进展。     

Effects of DNase production, plasmid size, and restriction barriers on transformation of Vibrio cholerae by electroporation and osmotic shock

Attempts to transform wild type strains of V. cholerae with plasmid DNA by traditional osmotic shock methods were not successful. A mutant of V. cholerae that was deficient in extracellular DNase was transformed with plasmid DNA by osmotic shock, demonstrating directly that extracellular DNase is a major barrier to transformation of V. cholerae. Transformation of wild type and DNase-negative strains of V. cholerae was accomplished by electroporation. Efficiency of transformation by electroporation increased with field strength, decreased with plasmid size, and was relatively insensitive to changes in the electrolyte composition of the buffer as long as isotonic sucrose was present. Host-controlled modification/restriction systems also affected transformation efficiency in V. cholerae.     

Colonization of professional divers by toxigenic Vibrio cholerae O1 and V. cholerae non-O1 at dive sites in the United States, Ukraine and Russia

Abstract Vibrio cholerae , recognized as the causative agent of epidemic cholera, was isolated from healthy professional divers and from water samples collected at dive sites in the United States, Ukraine and Russia. Swabs of nose, ear and throat of divers and their tank regulators, i.e. the divers and their diving gear, were taken before and after routine dives. Blood samples were collected before and 30–60 days after each dive to measure IgG and IgA titers against the whole cell antigen of V. cholerae O1. Nine strains of V. cholerae O1 and nine strains of V. cholerae non-O1 were isolated during this study. These isolates were identified by conventional biochemical tests and indirect fluorescent antibody staining methods, using fluorescein isothiocyanate-labeled monoclonal antibody, COLTA, prepared against the 'A' antigenic factor of the lipopolysaccharide of V. cholerae O1, and serotyped by slide agglutination. Seven of the nine strains of V. cholerae O1 isolated and successfully cultured during the studies, were toxigenic by enzyme-linked immunosorbent assay and polymerase chain reaction. Analyses of IgG and IgA antibodies of the divers showed that most of the divers had prior exposure to V. cholerae O1. V. cholerae serotype non-O1 strains isolated during the study were found to be non-toxigenic.     

Antibodies (IgG) to lipopolysaccharide of Vibrio cholerae O1 mediate protection through inhibition of intestinal adherence and colonisation in a mouse model

An antiserum raised against purified lipopolysaccharide (LPS) of a Vibrio cholerae O1 strain (Co366) induced passive protection against challenge with the parent as well as other O1 organisms but not against O139 or non-O1/non-O139 organisms. A considerable level of protection against O1 strains was also observed with the IgG fraction of the antiserum which inhibited intestinal adherence and colonisation. The monovalent Fab(IgG) fragment, on the other hand, showed only a low level of protection. Interestingly, purified LPS failed to inhibit intestinal colonisation by the parent strain (Co366), thereby suggesting that the cell surface LPS moieties of vibrios may not be directly involved in the colonisation process. It may be concluded that the anti-LPS antibodies induce passive protection through microagglutination and/or immobilisation of vibrios which do not allow the organisms to adhere to and colonise the intestine.     

Characterization of the Melanogenic System in Vibrio cholerae,ATCC 14035

The nature of the pigment formed by Vibrio cholerae and the characterization of its biosynthetic pathway is shown. This microorganism is able to synthesize melanin-like pigment when cultured in the presence of L-tyrosine. Other phenolic chemicals related to L-tyrosine do not lead to pigment production. The microorganism has no tyrosine hydroxylase activity, and the levels of dopa oxidase activity are very low, making the existence of a tyrosinase very unlikely. However, Vibrio cholerae contained transami-nases that transforms L-tyrosine into p-hydroxyphenylpyruvate. Moreover, Vibrio cholerae is able to go further in the catabolic pathway, releasing a great amount of homogentisic acid. This acid can spontaneously be oxidized to its p-quinone form, which subsequently polymerizes leading to pigment formation. It is concluded that the pigment formed by Vibrio cholerae is not synthesized by the Raper-Mason pathway, but by a L-tyrosine catabolism pathway leading to homogentisic acid. Some simple properties of that melanin are compared to model eu- and pheomelanin, but no clear distinction could be stated, indicating the similarity between all these pigments.     

Effect of Vibrio cholerae non-O1 protease on lysozyme, lactoferrin and secretory immunoglobulin A

Abstract The effect of Vibrio cholerae non-O1 protease on host defense proteins (lysozyme, secretory immunoglobullin A and lactoferrin) was studied in relation to its virulence mechanism. The proteins treated with the protease were analysed by SDS-PAGE. There was no influence of the protease on lysozyme. The protease cleaved lactoferrin into two fragments of 50 kDa and 34 kDa. N-terminal amino acid sequencing of these fragments revealed that the cleavage site was near the hinge region, between serine 420 and serine 421. This cleavage could affect the transition from open to closed configuration which is involved in iron binding and release. The anti-bacterial activity of lactoferrin was not affected by protease treatment. Secretory immunoglobulin A yielded a 42-kDa protein as the cleavage product. The susceptibility of secretory immunoglobulin A to V. cholerae non-O1 protease suggests a mechanism by which bacteria might evade the effect of this immunoglobulin.     

Vibrio cholerae O22 might be a putative source of exogenous DNA resulting in the emergence of the new strain of Vibrio cholerae O139

The new epidemic strain O139 of Vibrio cholerae, the etiologic agent of cholera, has probably emerged from the pandemic strain O1 El Tor through a genetic rearrangement involving the horizontal transfer of exogenous O-antigen- and capsule-encoding genes of unknown origin. In V. cholerae O139, these genes are associated with an insertion sequence designated IS1358_O139. In this work, we studied the distribution of seven genes flanking the IS1358_O139 element in 13 serovars of V. cholerae strains. All these O139 genes and an IS1358 element designated IS1358_O22-1 were only found in V. cholerae O22 with a similar genetic organization. Sequence analysis of a 4.5-kb fragment containing IS1358_022-1 and the adjacent genes revealed that these genes are highly homologous to those of V. cholerae O139. These results suggest that strains of V. cholerae O22 from the environment might have been the source of the exogenous DNA resulting in the emergence of the new epidemic strain O139.     

Geographical variation in the distribution of phage types of Vibrio cholerae O1 and non-O1

A Vibrio cholerae O1 phage-typing scheme, developed in the USSR, has been used to type 120 strains of V. cholerae O1 isolated in Africa and Asia, and 56 non-O1 V. cholerae isolated in England and Wales from infections contracted abroad. 90.9% of V. cholerae O1 were typable. Phage type 13 predominated among African strains whereas types 11 and 15 were more common among strains from Asia. Only 14.3% of non-O1 V. cholerae reacted with the phages.     

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.     

不同来源RNA聚合酶对霍乱弧菌分型噬菌体VP3启动子的作用

目的:研究不同来源的RNA聚合酶对预测的霍乱弧菌分型噬菌体VP3启动子的作用。方法:以含有预测的VP3启动子区的片段取代质粒pRL-null的T7启动子区,以海肾萤光素酶基因Rluc为报告基因,在霍乱弧菌N16961内检测霍乱弧菌RNA聚合酶对克隆的启动子区的作用;将上述重组质粒和表达VP3 RNA聚合酶的质粒共转化大肠杆菌JM109,检测大肠杆菌和VP3的RNA聚合酶对克隆的启动子区的作用。结果:N16961的RNA聚合酶不能识别并作用于启动子P1、P2、P5、P6、P10和P12,JM109的RNA聚合酶可能识别并作用于启动子P7和P11;只有P2、P7、P8、P9、P13、P16和P17在JM109内可以被克隆表达的VP3 RNA聚合酶识别转录。结论:宿主菌N16961与非宿主菌JM109的RNA聚合酶识别转录VP3启动子的能力不同,可能与噬菌体的宿主特异性有关;VP3的RNA聚合酶对大部分有活性的VP3启动子具有直接启动转录作用,但部分启动子可能需要VP3或宿主蛋白的辅助作用才能表现出更强的活性;VP3启动子对VP3 RNA聚合酶的特异性也不同,P1、P2和P12对VP3的RNA聚合酶具有高度特异性,P7和P11的特异性较弱。     

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.     

Diversity in the arrangement of the CTX prophages in classical strains of Vibrio cholerae O1

This study reports the results of a molecular analysis of the CTX prophages in classical biotype strains of Vibrio cholerae O1 of clinical origin isolated between 1970 and 1979 in India. All strains were sensitive to group IV classical phage and polymyxin B but resistant to group 5 El Tor phage. These phenotypic traits are consistent to that exhibited by the classical biotype. PCR studies reconfirmed their biotype assignment and showed the presence of intact CTX prophages and the presence of the recently described toxin linked cryptic plasmid. Restriction fragment length polymorphism of rRNA genes and pulsed-field gel electrophoresis showed clonal diversity among the strains. The most notable observation was the finding that one strain (GP13) has three CTX prophages while another (GP147) has four CTX prophages. This is the first time heterogeneity is reported in the arrangement of the CTX prophages among classical strains of V. cholerae O1.     

溶藻弧菌中类霍乱弧菌毒力岛转座酶基因及侧翼序列分子生物学特征分析

[目的]调查类似霍乱弧菌毒力岛(VPI)转座酶(vpiT)的基因是否在溶藻弧菌中分布,并了解其全序列及侧翼序列的分子生物学特征.[方法]对94株溶藻弧菌是否携带类似VPI的vpiT基因进行PCR检测,对阳性株进行了PCR产物直接测序,根据获得的部分已知序列,设计引物,通过反向PCR扩增出全长类似vpiT的基因valT及部分侧翼片段,对反向PCR产物进行克隆测序,然后对获得的valT及侧翼序列进行生物信息学分析.[结果]发现94株溶藻弧菌中只有从粤东对虾池水分离的2个株E06011、E0612在PCR检测中产生了预期扩增片段.测序表明两者序列(valT-S1)完全一致.根据反向PCR及克隆最终获得的溶藻弧菌E0601全长valT基因及部分侧翼序列valT-S3.对valT-S3生物信息学分析表明:valT是一个高度类似于霍乱弧菌毒力岛vpiT的转座酶基因.[结论]根据上述结果及相关文献,有理由相信valT基因及其侧翼片段是异源获得,霍乱弧菌VPI元件或整体很可能在包括溶藻弧菌在内的弧菌种间转移.     

Development of an improved synthetic medium for a better production of the new cholera toxin and its immunological relationship with the toxin produced by Vibrio cholerae O139 strains

Abstract An improved synthetic medium (M4) comprising syncase medium supplemented with sodium chloride (1%) and sucrose (0.5%) pH adjusted to 7.4 was developed for a better production of the new cholera toxin (NCT). The culture filtrates prepared in the M4 medium caused significantly ( P < 0.05) more fluid accumulation than that in syncase medium. Crude toxin, prepared in the M4 medium with V. cholerae O1 strains (X-392 and 2740-80) caused a reaction similar to that of the same amount of NCT (32 μg) prepared in the syncase medium. The neutralization of the optimal loop reacting dose of the NCT prepared in the M4 medium by anti-NCT raised against syncase prepared toxin indicates the release of the same kind of toxin in both media. These observations indicate that the modified M4 medium may be used for NCT preparation and further characterization. All the strains of Vibro cholerae O139 used in this study produced a toxin antigenically similar to NCT.     

4株霍乱弧菌非产毒株溶源性噬菌体pre-CTXΦ 的基因组结构分析

霍乱弧菌溶源性噬菌体CTXΦ携带霍乱毒素基因ctxAB,通过其结构基因gⅢ编码产生的PⅢ蛋白识别霍乱弧菌毒素共调菌毛(toxin co-regulated pilus, TCP)的主要结构亚单位TcpA,从而感染具有TCP的霍乱弧菌,使之成为产毒菌株。CTXΦ还有不携带ctxAB的前体pre-CTXΦ,根据CTXΦ基因组中调控基因rstR序列型不同,可分成不同的型别。在不同霍乱弧菌菌株的基因组中,已发现CTXΦ/pre-CTXΦ基因组及其亚型的多种组合排列方式。研究该噬菌体家族的基因组多样性,能够分析其进化及在霍乱弧菌产毒株形成中的作用。本研究发现了4株O1和O139群霍乱弧菌非产毒株具有pre-CTXΦ基因组及多样的rstR序列型,进一步对pre-CTXΦ在4株菌株中的基因组特征进行了分析。利用第3代基因测序法(短读长测序技术和单分子长读长测序技术),获得了4株菌株的基因组序列。利用长读长测序和拼接分析,精确地获得了具有长片段重复序列结构的pre-CTXΦ基因组排列,明确了4株测序菌株中多样的pre-CTXΦ基因组排列。在非产毒株基因组菌株VC3193中发现了携带古典型pre-CTXΦ;还在菌株VC702的pre-CTXΦ基因组中首次发现了肺炎克雷白菌的转座子结构(Gen Bank序列号：SRIL00000000)。在这4株测序菌株中,受体TcpA以及pre-CTXΦ的PⅢ蛋白也具有明显差异的序列,有 TcpA和PⅢ新序列型,这提示了CTXΦ家族感染宿主菌的受体-配体相互识别的复杂对应关系。本研究丰富了对CTXΦ/pre-CTXΦ家族基因组及其整合排列的多样化认识,也为分析该溶源性噬菌体在不同遗传特征霍乱弧菌菌株间的水平转移和促使新产毒克隆形成方面提供了更多的证据。