Release of the Outer Membrane Vesicles from Vibrio cholerae and Vibrio parahaemolyticus

We found numerous small vesicles released from the cell by thin sectioning of the plate culture of Vibrio cholerae and V. parahaemolyticus fixed with the freeze-substitution technique. From the broth media of exponentially growing bacteria we could collect the vesicles by the centrifugation but not enough without fixation. The vesicles are encompassed with a membrane structure similar to the outer membrane of these bacteria. The anti-O (Inaba) serum reacted with the surface of the vesicles and the inside of the vesicle are generally filled with an electron-dense mass.     

Siderophore-Mediated Utilization of Iron Bound to Transferrin by Vibrio parahaemolyticus

Vibrio parahaemolyticus produces a structurally novel type of siderophore, termed vibrioferrin, in response to iron-limitation. This study was performed to examine whether vibrioferrin can assimilate iron from human iron-binding proteins for growth. Comparison of the growth rates between V. parahaemolyticus AQ 3354 and its spontaneously arising, vibrioferrin-deficient mutant revealed that vibrioferrin was able to sequester iron from 30% iron-saturated human transferrin for growth, but not from human lactoferrin even if fully saturated with iron. In both strains, iron limitation induced two high-molecular-weight outer membrane proteins with apparent molecular masses of approximately 78 and 83 kDa. Since only the outer membrane fraction including these proteins showed a binding capacity to ferric vibrioferrin complex, either of them may function as its cell surface receptor. These results suggested that the organism might utilize such a source of host iron through the action of vibrioferrin during in vivo survival and proliferation, although its importance in pathogenesis is unknown.     

Siderophore production of Vibrio parahaemolyticus strains from different sources.

Vibrio parahaemolyticus strains isolated from different sources were assayed for their ability to produce a siderophore, vibrioferrin, under iron-limited growth conditions. The mean value +/- standard error of mean (microM vibrioferrin in spent culture supernatant/optical density at 660 nm) was 832.3 +/- 66.9 for clinical isolates (n=44), which was significantly higher (P<0.01) than those for food isolates (461.0 +/- 66.5; n=37) and coastal isolates (378.8 +/- 37.2; n=26). This suggests that greater productivity of vibrioferrin by clinical isolates may be associated with a selective advantage for survival and proliferation under conditions of iron-limitation such as in the intestine [corrected].     

副溶血弧菌外膜铁蛋白受体pvuA基因的原核表达及产物的诱导条件优化

为构建弧菌铁蛋白受体pvuA重组质粒,提高其在大肠杆菌BL21中的表达产量,优化表达条件,并为其免疫原性研究奠定基础,从副溶血弧菌基因组DNA扩增了弧菌铁蛋白受体pvuA基因,构建了重组质粒pET-28a(+)-ferric vibrioferrin receptor,转入大肠杆菌BL21并经异丙基硫代半乳糖苷（isopropyl β-D-thiogalactoside,IPTG）诱导表达蛋白。在单因素试验的基础上,以菌体初始浓度、诱导时间、诱导温度、诱导剂浓度为自变量,菌体蛋白浓度为响应值,根据响应面法的Box-Benhnken中心设计原理,研究自变量及其交互作用对弧菌铁蛋白产量的影响,利用Design-Expert和响应面分析相结合的方法对诱导条件进行优化。IPTG诱导获得的重组蛋白以包涵体的形式存在,优化后最终确定重组弧菌铁蛋白受体pvuA最佳表达条件为菌体初始浓度OD600=0.6,诱导时间10 h,诱导温度37℃,IPTG浓度为1.0 mmol·L-1,此时包涵体沉淀中蛋白含量最高,为11.00 mg·mL-1。构建了弧菌铁蛋白受体pvuA的大肠杆菌重组表达质粒,通过优化表达...     

Cloning and Sequencing of the Vibrio parahaemolyticus fur Gene

A ferric uptake regulatory gene (fur) was cloned from Vibrio parahaemolyticus WP1 by a polymerase chain reaction-based technique followed by functional complementation of a fur mutation in Escherichia coli. A sequence analysis showed that, at the amino acid level, the V. parahaemolyticus Fur protein is 81% identical with the Fur protein from E. coli and over 90% identical with those of the Vibrio species.     

衣原体主要外膜蛋白的研究进展

衣原体感染与多种慢性疾病密切相关,其主要外膜蛋白(MOMP)是一种多功能蛋白,分别与外膜结构的稳定性、生长代谢调节、抗原性和毒力密切相关。随着沙眼衣原体和肺炎衣原体基因组测序的完成,人们得以揭示其重要的生物合成、代谢途径,确定调控机制及其与致病的相关性。利用分子生物学技术在分子水平分析衣原体主要外膜蛋白的结构、抗原表位,对于免疫防御、免疫病理和免疫诊断均有重要意义。本文综述了衣原体主要外膜蛋白的分子结构、基因特性、抗原表位与应用前景。     

Utilization of Iron Sources and Its Possible Roles in the Pathogenesis of Vibrio parahaemolyticus

Vibrio parahaemolyticus is an important enteropathogen in Japan, Taiwan and other coastal regions. The influence of the regulation of iron on the pathogenesis of this pathogen has not been well characterized. The growth of pathogenic and non-pathogenic strains of V. parahaemolyticus on iron-limited agar plates was stimulated by ferritin, lactoferrin and transferrin at 30 μM , and also by hemin, hemoglobin and ferric ammonium citrate at 100 μM . Spontaneous iron-utilizing mutant strains (mutants) were derived from a clinical strain, ST550. Compared with the parent strain, lowered virulence was demonstrated for these mutants, as assayed by adult mouse and suckling mouse models. The in vivo growth and enterotoxigenicity of these mutants were also lower in the suckling mice. Adherence of the mutants to excised mouse intestine was lower as demonstrated by scanning electron microscopy. The iron-regulated outer membrane protein profile also changed in selected mutants. These results indicate that iron-regulated outer membrane proteins and other unknown factors associated with iron utilization may have profound influences, besides iron acquisition, on the pathogenesis of V. parahaemolyticus.     

Purification of a serine protease of Vibrio parahaemolyticus and its characterization

A 50 kDa protease designated as VPP1 was purified from the culture supernatant of a clinical strain of Vibrio parahaemolyticus by ammonium sulfate fractionation, Sephacryl S-200 HR gel filtration and Fractogel EMD TMAE 650 ion-exchange chromatography. VPP1 was inhibited by EDTA, EGTA and serine protease inhibitors, suggesting that it is a calcium-dependent serine protease. N-terminal amino acid sequence of VPP1 was quite similar to that of V. metschnikovii protease and antibody against VPP1 inhibited the activity of V. metschnikovii protease, suggesting the similarity of the two proteases. It was demonstrated that VPP1 or its related protease widely distribute in not only V. parahaemolyticus but also V. alginolyticus.     

PCR-based identification of pandemic group Vibrio parahaemolyticus with a novel group-specific primer pair

A PCR-based assay to identify pandemic group Vibrio parahaemolyticus has been developed. The assay employs an oligonucleotide primer pair derived from the group-specific sequence of an arbitrarily primed-PCR fragment, which is located in the genome encoding a "hypothetical protein," approximately 80% homologous to the Mn2+ and Fe2+ transporter of the NRAMP family of V. vulnificus. The assay distinguished the pandemic group from other V. parahaemolyticus strains by yielding a 235-bp specific amplicon, and can be a useful diagnostic tool for identification of pandemic group strains.     

A Filamentous Phage of Vibrio parahaemolyticus O3:K6 Isolated in Laos

A filamentous phage, ‘lvpf5’, of Vibrio parahaemolyticus O3:K6 strain LVP5 was isolated and characterized. The host range was not restricted to serotype O3:K6, but 7 of 99 V. parahaemolyticus strains with a variety of serotypes were susceptible to the phage. The phage was inactivated by heating at 80 C for 10 min and by treating with chloroform. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the phage exhibited a 3.8 kDa protein. The amino-terminal amino acid sequence of the coat protein was determined as AEGGAADPFEAIDLLGVATL. The phage genome consisted of a single-stranded DNA molecule. The activity of the phages was inhibited by anti-Na2 pili antibody.     

Ribotyping of Vibrio parahaemolyticus isolates obtained from food poisoning outbreaks in Taiwan.

Vibrio parahaemolyticus is a prevalent food-borne pathogen in Taiwan, Japan and other Asian countries. This work presents a novel ribotyping method for the molecular epidemiological examination of this pathogen. Genomic DNA was fragmented by HindIII digestion and hybridized with cDNA probe for Escherichia coli 16S and 23S RNA genes. A total of 121 isolates obtained from outbreaks during 1992 and 1994 in Taiwan were characterized by this ribotyping method. Four to seventeen restricted fragments were visualized in these isolates. After hierarchical cluster analysis, these isolates were grouped into thirty different ribotypes. In addition, A3, A7, E3 and F1 were the major ribotypes, consisting of 22.3, 13.2, 9.1, and 8.3% of the isolates, respectively. A, E, F, G and B were the major groups, consisting of 46.2, 14.0, 9.1, 6.7, and 6.7% of the isolates, respectively. The discriminatory ability of this ribotyping method, as determined by Simpson's index of diversity, was 0.93, which closely resembled that of a previously reported pulsed-field gel electrophoresis method.     

Lipopolysaccharide Isolated from a New O-Antigenic Form (O13) of Vibrio parahaemolyticus

The chemical properties of a lipopolysaccharide (LPS) isolated from a new O-antigenic form (O13) of Vibrio parahaemolyticus were investigated. The LPS contained glucose, galactose, L -glycero-D -manno-heptose and glucosamine. 2-Keto-3-deoxy-octonate (KDO) was not detected in the LPS by the periodate-thiobarbituric acid test (Weissbach's reaction) under conventional hydrolysis conditions. Instead, phosphorylated KDO (X1 and X2) was found in its strong-acid hydrolysate. This sugar composition was identical to that of V. parahaemolyticus O3, O5 and O11 LPS, indicating that, based on the sugar composition, O13 LPS belongs to Chemotype III to which O3, O5 and O11 belong. In addition, structural study demonstrated the presence of KDO 4-phosphate in its inner-core region.     

Characterization of Vibrio parahaemolyticus manganese-resistant mutants in reference to the function of the ferric uptake regulatory protein

In many bacteria, the ferric uptake regulatory protein (Fur) has a central role in the negative regulation of genes affected by iron limitation. In this study, Vibrio parahaemolyticus strains carrying mutations in the fur gene encoding Fur were isolated by the manganese selection method to assess the function of Fur in connection with alternations in the coordinate expression of the siderophore vibrioferrin (VF) and iron-repressible outer membrane proteins (IROMPs). Ten out of 25 manganese-resistant mutants constitutively produced VF and expressed at least two IROMPs irrespective of the iron concentration in the medium. PCR-direct DNA sequencing of the fur genes in these mutants identified four different point mutations causing amino acid changes. Moreover, a fur overexpressing plasmid was constructed to prepare antiserum against V. parahaemolyticus Fur. Western blotting with this antiserum revealed that the intracellular abundance of the wild-type Fur was not significantly affected by the iron concentrations in the growth medium, and that the Fur proteins of the mutant strains occurred at substantially smaller amounts and/or migrated more rapidly in sodium dodecyl sulfate-polyacrylamide gel electrophoresis than the wild-type Fur. These data afford an additional insight into the structure-function relationship of Fur and imply its involvement in the iron acquisition systems of V. parahaemolyticus, although it is yet unknown whether its action on the target genes is direct or indirect.     

Emergence and serovar transition of Vibrio parahaemolyticus pandemic strains isolated during a diarrhea outbreak in Vietnam between 1997 and 1999

We characterized 523 Vibrio parahaemolyticus strains isolated during a survey of diarrhea patients in Khanh Hoa province, Vietnam between 1997 and 1999. Forty-nine percent of the strains were judged to belong to the pandemic strains that emerged around 1996 and spread to many countries. These strains were positive in the GS-PCR assay and carried the tdh gene. The ORF8 of the f237 phage genome, a possible marker of the pandemic clone, was absent in 10% of these strains. Eleven O: K serovars were detected among the pandemic strains and the strains representing all 11 serovars of pandemic strains were shown to be closely related regardless of the ORF8 genotype using arbitrarily primed PCR and pulsed field gel electrophoresis analyses. It was clear that a transition of major serovars occurred among the pandemic strains represented by the emergence of O3: K6 in 1997, O4: K68 in 1998, and O1: K25 in 1998 and 1999.     

Chemical and Immunological Characterization of a Low Molecular Weight Outer Membrane Protein of Salmonella typhi

A new immunogenic outer membrane protein, Omp-28 (MW 28,000 and pI 4.6), was isolated from smooth Salmonella typhi cells by the use of an extracting medium containing 6 m urea, 1% deoxycholate and 5 mM EDTA. The purification of Omp-28 was performed by gel filtration and fast ion exchange chromatography. This protein showed to be the prevalent component isolated by the latter methodology. Omp-28 is formed by three identical subunits (MW 9,000), not linked by disulfide bonds. The partial N-terminal amino acid sequence of Omp-28 presented great homology with part of the sequence of an Escherichia coli protein found in a precursor whose sequence was predicted by c-DNA. ELISA and Western blotting identified Omp-28 as the major antigenic protein present in the outer membrane protein fraction, isolated by gel filtration. Antibodies against Omp-28 were detected by ELISA in 43% of 28 sera from typhoid fever convalescent patients. The antisera from mice immunized with Omp-28 and the highest positive typhoid fever convalescent serum gave a positive bactericidal test, killing 50% of Salmonella typhi cells in serum dilutions of 1/80 and 1/320, respectively. These results indicate the immunogenic importance of Omp-28 isolated from Salmonella typhi outer membrane and strongly suggest it should be used in further studies of animal protection against the disease caused by this pathogenic bacteria.     

Cytotoxicity and enterotoxicity of the thermostable direct hemolysin-deletion mutants of Vibrio parahaemolyticus

The thermostable direct hemolysin (TDH) has been proposed to be a major virulence factor of Vibrio parahaemolyticus. We have recently completed the genome sequence of a TDH-producing V. parahaemolyticus strain, RIMD2210633. In this study, we constructed tdh-deletion mutants from the sequenced strain by homologous recombination and analyzed their phenotypes. Although the deletion of both copies of tdh completely abolished the hemolytic activity of the wild-type strain, the deletion did not affect the cytotoxicity to HeLa cells. Enterotoxicity, assayed by the rabbit ileal loop test, was lowered by tdh deletion, but the mutant still showed partial fluid accumulation in rabbit intestine. These results indicate that the cytotoxicity and enterotoxicity of TDH-producing V. parahaemolyticus are not explained by TDH alone, and suggest that an unknown virulence factor(s) could be involved in these pathogenic activities.     

A Peptidomimetic Antibiotic Targets Outer Membrane Proteins and Disrupts Selectively the Outer Membrane in Escherichia coli

Increasing antibacterial resistance presents a major challenge in antibiotic discovery. One attractive target in Gram-negative bacteria is the unique asymmetric outer membrane (OM), which acts as a permeability barrier that protects the cell from external stresses, such as the presence of antibiotics. We describe a novel β-hairpin macrocyclic peptide JB-95 with potent antimicrobial activity against Escherichia coli. This peptide exhibits no cellular lytic activity, but electron microscopy and fluorescence studies reveal an ability to selectively disrupt the OM but not the inner membrane of E. coli. The selective targeting of the OM probably occurs through interactions of JB-95 with selected β-barrel OM proteins, including BamA and LptD as shown by photolabeling experiments. Membrane proteomic studies reveal rapid depletion of many β-barrel OM proteins from JB-95-treated E. coli, consistent with induction of a membrane stress response and/or direct inhibition of the Bam folding machine. The results suggest that lethal disruption of the OM by JB-95 occurs through a novel mechanism of action at key interaction sites within clusters of β-barrel proteins in the OM. These findings open new avenues for developing antibiotics that specifically target β-barrel proteins and the integrity of the Gram-negative OM.     

重组土拉弗朗西斯菌外膜蛋白FopA的融合表达及抗原性分析

目的:探索一种大量表达功能性土拉弗朗西斯菌外膜蛋白FopA的方法。方法:采用SignalP 3.0 Server进行信号肽预测,将土拉弗朗西斯菌外膜蛋白FopA信号肽的基因序列(75bp)去除,将1107bp的核心序列克隆至原核表达载体pET32a,并在大肠杆菌BL21(DE3)中诱导表达。结果:构建了pET32a-fopA载体,重组蛋白FopA表达量约占菌体总蛋白量60%,Western blot分析显示重组FopA蛋白有较好的抗原性。结论:获得了高效表达FopA的pET32a-fopA表达载体,为下一步土拉弗朗西斯菌外膜蛋白FopA应用研究奠定了基础。     

Evaluation of a Recombinant 27-kDa Outer Membrane Protein of Coxiella burnetii as an Immunodiagnostic Reagent

The 27-kDa outer membrane protein from eight strains of Coxiella burnetii was expressed in the pET-21c protein expression system. Two fusion proteins with molecular masses of 30 and 32 kDa were evident in all eight of the recombinants by SDS-PAGE and immunoblotting. A protein having an approximate size of 30 kDa was purified from the Escherichia coli lysates by one-step affinity purification. The utility of the purified recombinant protein in ELISA was also evaluated by testing its reactivity with human sera and comparing this reactivity with that of Nine Mile phase II antigen. All of the 40 IF-positive serum samples were ELISA-positive for both the Nine Mile phase II and recombinant antigens, and negative serum controls were negative for both antigens. These results suggest that ELISA with the 27-kDa recombinant antigen is a sensitive and specific method for detecting anti-C. burnetii antibodies in human sera.