Structure and serological characterization of 5,7-diamino-3,5,7,9-tetradeoxy-non-2-ulosonic acid isolated from lipopolysaccharides of Vibrio parahaemolyticus O2 and O-untypable strain KX-V212

Lipopolysaccharides (LPS) of Vibrio parahaemolyticus O2 and O-untypable (OUT) strain (KX-V212) isolated from an individual patient were shown to contain 5,7-diamino-3,5,7,9-tetradeoxy-non-2-ulosonic acid (NonlA), which was readily released from LPS by mild acid hydrolysis. In the present study, we investigated the chemical and serological properties of NonlA isolated from LPS of V. parahaemolyticus O2 and OUT KX-V212. GC-MS and NMR analysis identified the NonlA from LPS of O2 to be 5,7-diacetamido-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic acid (5NAc7NAcNonlA) and that from LPS of KX-V212 to be 5-acetamido-7-(N-acetyl-D-alanyl)amido-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic acid (5NAc7NAlaNAcNonlA). In ELISA inhibition analysis, 5NAc7NAcNonlA inhibited the O2 LPS/anti-O2 antiserum system, whereas, 5NAc7NAlaNAcNonlA did not show any inhibitory activity. However, after N-deacylation of 5NAc7NAlaNAcNonlA followed by N-acetylation, the product (5NAc7NAcNonlA) inhibited the O2 LPS/anti-O2 antiserum system to the same extent as that of 5NAc7NAcNonlA obtained from O2 LPS. These results suggest that 5NAc7NAcNonlA might be related to the serological specificity of O2 LPS as one of main epitope(s) involved in O2 LPS.     

Structural analysis of the carbohydrate backbone of Vibrio parahaemolyticus O2 lipopolysaccharides

A structural investigation has been carried out on the carbohydrate backbone of Vibrio parahaemolyticus O2 lipopolysaccharides (LPS) isolated by dephosphorylation, O-deacylation and N-deacylation. The carbohydrate backbone is a short-chain saccharide consisting of nine monosaccharide units i.e., 1 mol each of D-galactose (Gal), D-glucose (Glc), D-glucuronic acid (GlcA), L-glycero-D-manno-heptose (L,D-Hep), D-glycero-D-manno-heptose (D,D-Hep), 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo), 5,7-diacetamido-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic acid (NonlA), and 2 mol of 2-amino-2-deoxy-D-glucose (D-glucosamine, GlcN). Based on the data obtained by NMR spectroscopy, fast-atom bombardment mass spectrometry (FABMS) and methylation analysis, a structure was elucidated for the carbohydrate backbone of O2 LPS. In the native O2 LPS, the 2-amino-2-deoxy-D-glucitol (GlcN-ol) at the reducing end of the nonasaccharide is present as GlcN. The lipid A backbone is a beta-D-GlcN-(1-->6)-D-GlcN disaccharide as is the case for many Gram-negative bacterial LPS. The lipid A proximal Kdo is substituted by the distal part of the carbohydrate chain at position-5. In the native O2 LPS, D-galacturonic acid, which is liberated from LPS by mild acid treatment or by dephosphorylation in hydrofluoric acid, is present although its binding position is unknown at present.     

Structural and genetic characterization of the O-antigen of Escherichia coli O161 containing a derivative of a higher acidic diamino sugar, legionaminic acid

The O-antigen is an essential component of lipopolysaccharide on the surface of Gram-negative bacteria and plays an important role in its pathogenicity. Composition and structure of the O-antigens of Escherichia coli are highly diverse mainly due to genetic variations in the O-antigen gene cluster. In this work, the chemical structure and the gene cluster of the O-antigen of E. coli O161 were studied. Chemical degradations, sugar analyses, and NMR spectroscopy showed that the O161 antigen possesses a trisaccharide O-repeating unit containing a 5-N-acetyl-7-N-(d-alanyl) derivative of 5,7-diamino-3,5,7,9-tetradeoxy-d-glycero-d-galacto-non-2-ulosonic (legionaminic) acid (Leg5Ac7Ala) and having the following structure:

→8)-α-Legp5Ac7Ala-(2→4)-β-d-GlcpA-(1→3)-β-d-GlcpNAc-(1→     

5,7-Diamino-5,7,9-trideoxynon-2-ulosonic acid: a novel sugar from a phytopathogenic Pseudomonas lipopolysaccharide

Preliminary results on the structure of a novel sugar from a lipopolysaccharide from Pseudomonas corrugata, a plant pathogenic bacterium whose several aspects of phytopathogenic mechanism are under investigation, are described. This is a 5,7-diamino-5,7,9-trideoxynon-2-ulosonic acid, isolated as an O-glycoside from the Smith degradation of the O-chain. The structure was obtained both with NMR and MS methodologies. To the best of our knowledge, this is the first example of 3-hydroxylated non-2-ulosonic acid.     

Structural studies of the O-antigenic oligosaccharide from Vibrio salmonicida strain C2 isolated from Atlantic cod, Gadus morhua L

We report the chemical structure of the oligosaccharide part of Vibrio salmonicida lipopolysaccharide serotype C2, isolated from Atlantic cod (Gadus morhua L.). The structure was established by NMR spectroscopy and mass spectrometry. It is concluded that the oligosaccharide has the following structure in which L-alpha-D-Hepp is L-glycero-alpha-D-manno-heptopyranose, D-alpha-D-Hepp is D-glycero-alpha-D-manno-heptopyranose, alpha-NonA is 5-acetamidino-7-acetamido-3,5,7,9-tetradeoxy-L-glycero-alpha-D-galacto-nonulosonic acid, and PEA is phosphoethanolamine. [chemical structure: see text]     

Environmental Vibrio parahaemolyticus DNA signatures validation

Insignia is a novel DNA computational system which uses highly efficient algorithms to compare bacterial genomes and to identify specific DNA signatures to distinguish a target bacterium, or group of bacteria, from all other known bacterial species. It is currently being validated using different bacterial groups, including Vibrio spp. In this study, the genomic analysis by Insignia was conducted on Vibrio parahaemolyticus, a halophilic gram-negative bacteria which constitutes a leading cause of seafood-borne disease. Insignia was used to identify 37 V. parahaemolyticus-specific signatures and to design PCR assays to validate the representative signature sequences by TaqMan essays. The 37 assays targeted loci distributed around the genome and detected genes coding for hypothetical proteins and for proteins involved in adhesion, starvation and virulence. A panel of V. parahaemolyticus environmental strains isolated from the North Adriatic Sea (Italy) and from the Black Sea (Georgia) was used to validate the selected signatures. The signature assays revealed both sensitive and specific and the method allowed a more accurate identification of the tested bacterial strains at the species level when compared to biochemical and PCR standard methods. Using Insignia, it was possible to distinguish two different groups among the strains previously identified as V. parahaemolyticus: most of the strains were included in a "V. parahaemolyticus-like group" showing nearly all of the signatures assayed while a small group of 10 strains contained only a few of the signatures tested. By sequencing the 16S rDNA of this latter group, it was confirmed that they were not V. parahaemolyticus but in fact belonged to other Vibrio species. No significant genome-wide differences were detected between the strains isolated in Italy and in Georgia though the very different geographical origin.     

Isolation and characterization of a toxic intracellular protein from Vibrio parahaemolyticus

A toxic factor released from disrupted cells of Vibrio parahaemolyticus was partially purified by gel filtration after precipitation with (NH₄)₂SO₄ at 40% saturation. The factor, which was a thermostable protein of 63 kDa, lysed human erythrocytes at a concentration of 0.15 g ml^-1. Its LD₅₀ by intravenous injection into mice was 6.4 g. Fluid accumulated in suckling mice force-fed with the toxic material (1 to 25 g). Haemolytic activity, which occurred maximall at 37°C and pH 7.0 was enhanced by Ca²⁺, Cu²⁺ and Zn²⁺, each at 1 mm. Anti-toxic-factor serum agglutinated V. parahaemolyticus cells. The factor may play a role in the pathogenesis of V. parahaemolyticus infections and in the host's defence mechanisms against infection by the microorganism.     

Structure of the O-polysaccharide from the lipopolysaccharide from Vibrio cholerae O6

The O-polysaccharide from Vibrio cholerae O6 was isolated from the LPS by mild-acid hydrolysis and has been investigated by sugar and methylation analysis and NMR spectroscopy. The polysaccharide was also depolymerized with aqueous hydrofluoric acid to give the repeating unit and multiples thereof. The O-polysaccharide had the following tetrasaccharide repeating unit. Two O-acetyl groups are present, one of them making the GlcNAc residue fully substituted and the steric crowding considerable at the branching residue.     

Identification and characterization of the pathogenic effect of a Vibrio parahaemolyticus-related bacterium isolated from clam Meretrix meretrix with mass mortality

A mass mortality of clam, Meretrix meretrix, occurred in Jiangsu Province of China in the late September of 2007. Of the isolates obtained from the diseased clams, MM21 had the strongest virulence to the clam in the virulence test, with a LD50 value of ∼6 × 10⁶ CFU ml⁻¹. MM21 was identified as Vibrio parahaemolyticus by the VITEK 2 Compact system and 16S rDNA sequencing. Detection of virulence-associated genes by PCR indicated that MM21 was positive for toxR and tlh, and negative for tdh. Compared with control group, histiocytes from MM21-infected clams displayed a variety of cytopathological changes by transmission electron microscopy examination, which included increased lipid droplets in hepatocytes, deposition of granules in the mantle, excessive secretion in the gill. The results of our study suggested that MM21 may have been an etiological element in the mass mortalities of hard clam (M. meretrix) in Jiangsu Province of China in 2007.     

基于颜色判定的环介导恒温扩增法快速检测副溶血性弧菌

利用DNA环介导恒温核酸扩增法（LAMP）针对副溶血性弧菌特异基因tlh基因设计4条引物,通过引物特异性识别tlh基因上的6个独立区域来快速检测副溶血性弧菌.LAMP反应的过程中会产生白色沉淀焦磷酸镁,故可以通过监测浊度来判定反应结果.实时浊度仪监测反应结果表明,LAMP反应在60～65℃恒温条件下50min内完成;如果在反应前添加羟基萘酚兰（HNB）,蓝色的阳性结果很明显区别于紫色阴性结果;LAMP方法的最低检出限为9.74pg/μL,PCR方法最低检出限为97.4pg/μL,LAMP方法检测灵敏度是PCR方法检测灵敏度的10倍,且具有良好的特异性.LAMP方法用于快速检测副溶血性弧菌具有检测过程简单、实验装置简便、反应结果肉眼可辨别、灵敏度高和特异性强的特点,所以LAMP方法检测副溶血性弧菌特别适合用于现场和基层检疫及医疗单位的快速诊断.     

Isolation and characterization of Vibrio parahaemolyticus from seafoods along the southwest coast of India

The work was aimed to study the microbial quality of the seafood sold in the domestic markets and incidence of Vibrio parahaemolyticus. Samples comprising of shellfish, finfish, and cephalopods were collected from various fish markets in and around Cochin. Presumed V. parahaemolyticus were identified by standard biochemical tests, and further confirmed by polymerase chain reaction targeting species-specific tl gene (450 bp). About 81% of the samples were found to exceed the limits specified for total plate count while total presumptive V. parahaemolyticus count was above the limit in 71% of the samples ranging from 5.5 × 10⁵ to 9.7 × 10⁷ and 0.31 × 10² to 7.8 × 10⁶ cfu/g, respectively. Pathogenicity of the identified isolates was confirmed by Kanagawa phenomenon and urease activity. A total of 10% of the isolates exhibited weak haemolysis on Wagatsuma agar, and 1% of the isolates showed urease activity using Christensen’s urea agar. Random amplified polymorphic DNA analysis revealed two major clusters based on the species rather than seasonality. The gel pattern revealed 8–10 bands ranging from 0.45 to 3.0 kb. Antibiogram results revealed 85% of the strains sensitive to chloramphenicol and nitrofurantoin. Multiple antibiotic resistance index was found to be 0.4 thus suggesting the risk potential involved in consuming seafoods. The present study has clearly demonstrated the need to adopt seafood safety measures for the products meant for human consumption.     

Bactericidal effect of lactoferrin and lactoferrin chimera against halophilic Vibrio parahaemolyticus

Infections caused by Vibrio parahaemolyticus, an halophilic member of the genus Vibrio, have increased globally in the last 5 years. Diarrhea caused by V. parahaemolyticus results from eating raw or undercooked seafood. The aim of this work was to investigate whether lactoferrin and some lactoferrin-peptides have bactericidal activity against Vibrio parahaemolyticus ATCC 17802, the pandemic strain O3:K6, and the multidrug resistant isolate 727, as well as against Vibrio cholerae strains O1 and non-O1. Whereas both peptides lactoferricin (17-30) and lactoferrampin (265-284) did not have bactericidal activity, 40 microM of lactoferrin chimera (a fusion of the two peptides) inhibited the growth of all Vibrio tested to the same extent as the antibiotic gentamicin. The cidal effect of LFchimera showed a clear concentration response in contrast to bovine lactoferrin which showed higher inhibition at 10 microM than at 40 microM. FITC-labeled LFchimera bound to the bacterial membranes. Moreover LFchimera permeabilized bacterial cells and membranes were seriously damaged. Finally, in experiments with the multidrug resistant isolate 727, sub-lethal doses of LFchimera strongly reduced the concentrations of ampicillin, gentamicin or kanamicin needed to reach more than 95% growth inhibition, suggesting synergistic effects. These data indicate that LFchimera is a potential candidate to combat the multidrug resistant pathogenic Vibrio species.     

Effect of the addition of four potential probiotic strains on the survival of pacific white shrimp (Litopenaeus vannamei) following immersion challenge with Vibrio parahaemolyticus

Four bacterial strains isolated from the gastrointestinal tract of adult shrimp Litopenaeus vannamei, Vibrio alginolyticus UTM 102, Bacillus subtilis UTM 126, Roseobacter gallaeciensis SLV03, and Pseudomonas aestumarina SLV22, were evaluated for potential use as probiotics for shrimp. In vitro studies demonstrated antagonism against the shrimp-pathogenic bacterium, Vibrio parahaemolyticus PS-017. Feeding shrimp with diets containing the potential probiotics showed the best feed conversion ratio in comparison with the control groups. After feeding with the potential probiotics for 28 days, challenge by immersion indicated effectiveness at reducing disease caused by V. parahaemolyticus in shrimp.     

Structural characterization of the O-specific polysaccharide from the lipopolysaccharide of the fish pathogen Aeromonas bestiarum strain P1S

The O-specific polysaccharide obtained by mild-acid degradation of lipopolysaccharide of Aeromonas bestiarum P1S was studied by sugar and methylation analyses along with ¹H and ¹³C NMR spectroscopy. The sequence of the sugar residues was determined using ¹H,¹H NOESY and ¹H,¹³C HMBC experiments. The O-specific polysaccharide was found to be a high-molecular-mass polysaccharide composed of tetrasaccharide repeating units of the structureSince small amounts of a terminal Quip3N residue were identified in methylation analysis, it was assumed that the elucidated structure also represented the biological repeating unit of the O-specific polysaccharide.     

Structural and functional peculiarities of the lipopolysaccharide of Azospirillum brasilense SR55, isolated from the roots of Triticum durum

Azospirillum brasilense SR55, isolated from the rhizosphere of Triticum durum, was classified as serogroup II on the basis of serological tests. Such serogroup affiliation is uncharacteristic of wheat-associated Azospirillum species. The lipid A of A. brasilense SR55 lipopolysaccharide contained 3-hydroxytetradecanoic, 3-hydroxyhexadecanoic, hexadecanoic and octadecenoic fatty acids. The structure of the lipopolysaccharide's O polysaccharide was established, with the branched octasaccharide repeating unit being represented by l-rhamnose, l-3-O-Me-rhamnose, d-galactose and d-glucuronic acid. The SR55 lipopolysaccharide induced deformations of wheat root hairs. The lipopolysaccharide was not involved in bacterial cell aggregation, but its use to pretreat wheat roots was conducive to cell adsorption. This study shows that Azospirillum bacteria can utilise their own lipopolysaccharide as a carbon source, which may give them an advantage in competitive natural environments.     

Structural characterization of the O-chain polysaccharide of Aeromonas caviae ATCC 15468 lipopolysaccharide

The O-chain polysaccharide produced by a mild acid degradation of Aeromonas caviae ATCC 15468 lipopolysaccharide was found to be composed of L-rhamnose, 2-acetamido-2-deoxy-D-glucose, 2-acetamido-2-deoxy-D-galactose and phosphoglycerol. Subsequent methylation and CE-ESIMS analyses and 1D/2D NMR ((1)H, (13)C and (31)P) spectroscopy showed that the O-chain polysaccharide is a high-molecular-mass acidic branched polymer of tetrasaccharide repeating units with a phosphoglycerol substituent having the following structure: [structure: see text] where Gro represents glycerol and P represents a phosphate group.     

Structural elucidation of the core-lipid A backbone from the lipopolysaccharide of Acinetobacter radioresistens S13, an organic solvent tolerant Gram-negative bacterium

The structure of the core oligosaccharide of the lipopolysaccharide from an organic solvent tolerant Gram-negative bacterium, Acinetobacter radioresistens S13, was investigated by chemical analysis, NMR spectroscopy and MALDI-TOF mass spectrometry. All the experiments were performed on the oligosaccharides obtained either by alkaline degradation or mild acid hydrolysis. The data showed the presence of two novel oligosaccharide molecules containing a trisaccharide of 3-deoxy-D-manno-octulopyranosonic acid in the inner core region and a glucose rich outer core whose structure is the following: [structure: see text] R=H in the main oligosaccharide and beta-Glc in the minor product. The bacterium was grown on aromatic (phenol and benzoic acid) and nonaromatic carbon sources and the core oligosaccharide resulted to occur always with this novel structure.     

Generation and Characterization of a scFv Antibody Against T3SS Needle of Vibrio parahaemolyticus

Vibrio parahaemolyticus, a halophilic gram-negative bacterium, is a food-borne pathogen that largely inhabits marine and estuarine environments, and poses a serious threat to human and animal health all over the world. The hollow “needle” channel, a specific assemble of T3SS which exists in most of gram-negative bacteria, plays a key role in the transition of virulence effectors to host cells. In this study, needle protein VP1694 was successfully expressed and purified, and the fusion protein Trx-VP1694 was used to immunize Balb/c mice. Subsequently, a phage single-chain fragment variable antibody (scFv) library was constructed, and a specific scFv against VP1694 named scFv-FA7 was screened by phage display panning. To further identify the characters of scFv, the soluble expression vector pACYC-scFv-skp was constructed and the soluble scFv was purified by Ni²⁺ affinity chromatography. ELISA analysis showed that the scFv-FA7 was specific to VP1694 antigen, and its affinity constant was 1.07 × 10⁸ L/mol. These results offer a molecular basis to prevent and cure diseases by scFv, and also provide a new strategy for further research on virulence mechanism of T3SS in V. parahaemolyticus by scFv.     

Comparative genomic analysis of clinical and environmental strains provides insight into the pathogenicity and evolution of Vibrio parahaemolyticus

Background

Vibrio parahaemolyticus is a Gram-negative halophilic bacterium. Infections with the bacterium could become systemic and can be life-threatening to immunocompromised individuals. Genome sequences of a few clinical isolates of V. parahaemolyticus are currently available, but the genome dynamics across the species and virulence potential of environmental strains on a genome-scale have not been described before.

Results

Here we present genome sequences of four V. parahaemolyticus clinical strains from stool samples of patients and five environmental strains in Hong Kong. Phylogenomics analysis based on single nucleotide polymorphisms revealed a clear distinction between the clinical and environmental isolates. A new gene cluster belonging to the biofilm associated proteins of V. parahaemolyticus was found in clincial strains. In addition, a novel small genomic island frequently found among clinical isolates was reported. A few environmental strains were found harboring virulence genes and prophage elements, indicating their virulence potential. A unique biphenyl degradation pathway was also reported. A database for V. parahaemolyticus (http://kwanlab.bio.cuhk.edu.hk/vp) was constructed here as a platform to access and analyze genome sequences and annotations of the bacterium.

Conclusions

We have performed a comparative genomics analysis of clinical and environmental strains of V. parahaemolyticus. Our analyses could facilitate understanding of the phylogenetic diversity and niche adaptation of this bacterium.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1135) contains supplementary material, which is available to authorized users.