A human single-chain variable fragment targeting to Vibrio vulnificus RtxA toxin

Vibrio vulnificus secretes a multifunctional cytotoxin RtxA (VvRtxA), which plays a major role in the bacterial pathogenesis. The lack of an efficient VvRtxA detection tool has hampered the progress of V. vulnificus pathogenesis research. This study aims to isolate VvRtxA specific single-chain variable fragments (scFv) to serve as a detection agent. The VvRtxA C-terminal Gly-Asp (GD) repeat-containing region, which has been implicated for calcium binding and target cell recognition, was chosen as an antigen to screen a scFv phage display library. A scFv clone that reacted positively to VvRtxA was successfully obtained. Using the isolated scFv, a cell-based enzyme-linked immunosorbent assay was established for detecting cell-associated VvRtxA toxin in V. vulnificus infected HEp-2 cells. The result is consistent with previous observations that secretion of VvRtxA toxin is time-dependent on bacteria contacting with host cells. Utilization of scFv for VvRtxA toxin detection provides an applicable strategy devoid of conventional immunization.     

Crystal structure and functional studies reveal that PAS factor from Vibrio vulnificus is a novel member of the saposin-fold family

PAS factor is a novel putative bacterial secretion factor thought to induce secretion of periplasmic proteins. We solved the crystal structure of PAS factor from Vibrio vulnificus at 1.8A resolution and found it to be comprised of five alpha helices that form an antiparallel bundle with an up-and-down topology, and to adopt the saposin-fold characteristic of a family of proteins that bind to membranes and lipids. PAS factor lacks the disulfide bridge characteristic of mammalian saposin-fold proteins; in fact, it shows no sequence homology with mammalian proteins. Nevertheless, the molecular architectures are similar, and the shared propensity for membrane interaction suggests strongly that PAS factor is another member of the saposin-fold family. Analysis of the CD spectra showed that PAS factor binds to membranes directly, while measurement of calcein dye leakage showed that PAS factor interacts strongly with liposomes composed of anionic phospholipids, making them leaky, but binds very weakly with liposomes composed of zwitterionic phospholipids. Moreover, by analyzing tryptophan fluorescence emission from four single-tryptophan mutants (V10W, T22W, F35W, and L70W), we identified the putative phospholipid-binding site of PAS factor. The resultant membrane destabilization likely mediates secretion of periplasmic proteins required for the in vivo survival and pathogenesis of V.vulnificus.     

Sensitive and rapid identification of Vibrio vulnificus by loop-mediated isothermal amplification

Vibrio vulnificus is a serious bacterial pathogen for humans and aquatic animals. We developed a rapid, sensitive and specific identification method for V. vulnificus using loop-mediated isothermal amplification (LAMP) technique. A set of primers, composed of two outer primers and two inner primers, was designed based on the cytolysin gene sequence of V. vulnificus. The LAMP reaction was processed in a heat block at 65 °C for 60 min. The amplification products were detected by visual inspection using SYBR Green I, as well as by electrophoresis on agarose gels. Our results showed that the LAMP reaction was highly specific to V. vulnificus. This method was 10-fold more sensitive than conventional PCR. In conclusion, the LAMP assay was extremely rapid, simple, cost-effective, sensitive and specific for the rapid identification of V. vulnificus.     

Induction of nitric oxide synthase expression by Vibrio vulnificus cytolysin.

The pore-forming cytolysin of Vibrio vulnificus (VVC) causes severe hypotension and vasodilatation in vivo. Under the condition of bacterial sepsis, large amounts of nitric oxide (NO) produced by inducible NO synthase (iNOS) can contribute to host-induced tissue damage causing hypotension and septic shock. In this study, we investigated the effect of purified VVC on NO production in mouse peritoneal macrophages. VVC induced NO production in the presence of interferon-gamma. Increased NO production was not affected by polymyxin B, and heat inactivation of cytolysin abolished the NO-inducing capability. NO production was induced at the same concentration range of cytolysin for pore formation, as evidenced by the release of preloaded 2-deoxy-d-[(3)H]glucose. At the higher concentrations of cytolysin causing the depletion of cellular ATP, no NO production was observed. Increased expression of iNOS and activation of NFkappaB by VVC were confirmed by Western blotting and gel shift assay, respectively. These results suggest the role of cytolysin as an inducer of iNOS and NO production in macrophage and as a possible virulence determinant in V. vulnificus infection.     

Isolation and characterization of a cold-induced nonculturable suppression mutant of Vibrio vulnificus

The viable but nonculturable (VBNC) suppression mutant formed platable cells at low temperature stress after inoculation in artificial seawater (ASW). Suppression subtractive hybridization was used to identify differentially expressed genes among cDNAs of the VBNC suppression mutant and the wild-type Vibrio vulnificus strain. Glutathione S-transferase was identified as a responsive gene of the VBNC suppression mutant in our assay, and was highly expressed from the VBNC suppression mutant at low temperature stress. Culturability tests revealed that the wild-type cells were sensitive to oxidative stress in the hydrogen peroxide (H(2)O(2)) and to 1-chloro-2,4-dinitrobenzene (CDNB) compared with the VBNC suppression mutant cells. Adding glutathione showed that many wild-type V. vulnificus cells maintained culturability in cold ASW. These results suggest that non-nutritional growth inhibitors, such as peroxide that accumulates at low temperatures, influence VBNC in V. vulnificus cells.     

Structure of vulnibactin,a new polyamine-containing siderophore from Vibrio vulnificus

A new siderophore named vulnibactin has been isolated from low iron cultures of Vibrio vulnificus, a human pathogen. The structure was established as N-[3-(2,3-dihydroxybenzamido)propyl]-1,3-bis[2-(2-hydroxyphenyl)-trans-5-methyl-2-oxazoline-4-carboxamido]propane by a combination of acid hydrolysis, nuclear magnetic resonance spectroscopy and positive fast atom bombardment mass spectrometry. Vulnibactin is characterized as containing one residue of 2,3-dihydroxybenzoic acid as well as two residues of salicylic acid, both of which are involved in the formation of oxazoline rings with l-threonine bound to a norspermidine backbone. In addition, two other compounds with siderophore activity were purified and their structures were also determined. These two compounds provided further support for the structure of vulnibactin.     

Cytotoxic mechanism of Vibrio vulnificus cytolysin in CPAE cells

Vibrio vulnificus is an estuarian bacterium that causes septicemia and serious wound infection. The cytolysin, one of the important virulence determinants in V. vulnificus infection, has been reported to have lethal activity primarily by increasing pulmonary vascular permeability. In the present study, we investigated the cytotoxic mechanism of V. vulnificus cytolysin in cultured pulmonary artery endothelial (CPAE) cells, which are possible target cells of cytolysin in vivo. V. vulnificus cytolysin caused the CPAE cell damages with elevation of the cytosolic free Ca2+, DNA fragmentation, and decrease of the cellular NAD+ and ATP level. These cytotoxic effects of V. vulnificus cytolysin were prevented by EGTA and aminobenzamide, but were not affected by verapamil or catalase. These results indicate that the elevation of cytosolic free Ca2+ induced by V. vulnificus cytolysin causes the increase of DNA fragmentation and the damaged DNA activates nuclear poly(ADP-ribose) synthetase, which depletes the cellular NAD+ and ATP, resulting in cell death.     

Fluorescent intensity of a novel NADPH-binding protein of Vibrio vulnificus can be improved by directed evolution

Blue fluorescent protein, BfgV, found from Vibrio vulnificus CKM-1, fluoresces through augmenting the intrinsic fluorescence of bound NADPH. Random mutagenesis and DNA shuffling were applied to increase the fluorescent intensity of BfgV. The wild type bfgV gene was subjected to four cycles of mutagenesis processes. A prominent D7 mutant protein had fluorescent intensity four times larger than wild type BfgV. The emission wavelength of this mutant protein appeared at 440 nm, which was 16 nm shorter than that of BfgV. There were eight amino acid substitutions in D7. As these substitutions were assigned to the modeled 3D structure of BfgV, three of them, V83M, G176S, and E179K, were shown to be located around NADPH-binding site. Time course analysis indicated the synthesis of D7 protein and fluorescent expression in Escherichia coli transformants were synchronic. This property was different from that of wild type GFP.     

Isolation and characterization ofVibrio vulnificus inhabiting the marine environment of the southwestern area of Taiwan

Vibrio vulnificus, a marine bacterium, is of concern in Taiwan because it causes wound infections and sepsis with a high mortality rate every year. To examine forV. vulnificus, 13 samples of seawater or oysters were collected from nine sites in Yunlin, Chiayi, and Tainan. Seventy-seven strains ofV. vulnificus were isolated from 11 samples. Among these environmental isolates, 72 (91%) were indole-positive, a characteristic of biotype 1. The remaining five strains although indole-negative, a characteristic previously found exclusively in biotype 2 strains, were all ornithine decarboxylase- and mannitol-positive, which has never been reported for biotype 2 strains. Based on the overall biochemical reactions obtained using a commercial identification system, these indole-negative strains appeared to be more like biotype 1. Fifty-seven ribotypes were identified among these isolates, indicating the great genetic divergence in this species. Of the 30 environmental isolates tested, 17 (56.7%) exhibited virulence comparable to the clinical isolates in the mouse, implying that a high proportion of theV. vulnificus strains in the marine environments might be pathogenic to humans.     

Complete Sequence of Plasmid pMP1 from the Marine Environmental Vibrio vulnificus and Location of Its Replication Origin

A novel cryptic plasmid, pMP1, from an environmental Vibrio vulnificus MP-4 isolated from Mai Po Nature Reserve in Hong Kong, has been characterized. The 7.6-kb plasmid had guanine–cytosine content of 40.03% and encoded four open reading frames (ORFs) with >100 amino acids. The predicted protein of ORF1 contained 478 amino acids showing 29% identity and 50% similarity over 309 amino acids to the integrase of Vibrio cholerae phage VP2. ORF2 encoded a putative protein of 596 amino acids, which were 23% identity and 42% similarity over 455 amino acids to the tail tape measure protein TP901 of Chromohalobacter salexigens phage. ORF3 and ORF4 encoded putative proteins of 103 and 287 amino acids, respectively, but showed no homologies to any known proteins. Further experiments indicated that a 3.2-kb fragment from EcoRI digestion could self-replicate. Analysis indicated that a sequence upstream of ORF4 had the features characteristic of theta-type replicons: AT-rich region, six potential direct repeats (iterons) spaced approximately two DNA helical turn apart (about 23 bp), two copies of 9 bp dnaA boxes, three Dam methylation sites, and five inverted repeats. Complementation experiments confirmed that the protein encoded by ORF4 was required for plasmid replication. We propose that ORF4 encode a new type of Rep protein and pMP1 is a new type of theta plasmid.     

Identification of Vibrio spp. in vibriosis Penaeus vannamei using developed monoclonal antibodies

Immunohistochemical study using monoclonal antibodies specific to various shrimp viruses and Vibrio spp. was performed in shrimp samples died from unknown cause with symptoms of black stripes on lateral sides of cephalothorax or smoky body coloration. The positive results in muscular tissue were obtained with MAb VAL57 (specific to Vibrio spp.) and in hepatopancreas tissues with MAbs VVB158 (specific to V. vulnificus) and VPC701 (specific to V. parahaemolyticus). Twelve isolates of Vibrio spp. isolated from shrimp tissues were identified with various MAbs by dot blotting, biochemical tests and 16S rRNA gene. The results revealed three groups of V. vulnificus and one group of V. shilonii. All four groups of isolated Vibrio spp. were immunologically and biochemically different. None of the V. parahaemolyticus-like bacterium was isolated. The results demonstrated that the mortality in shrimp is accompanied by the presence of Vibrio spp.     

Characterization of Vibrio mimicus phospholipase A (PhlA) and cytotoxicity on fish cell

Vibrio mimicus is a typical strain of Vibrio cholerae and produces a phospholipase (PhlA) which shares a highly conserved amino acid sequence with the lecithinase (Lec) of V. cholerae. The recombinant protein (rPhlA) produced from the phlA gene of V. mimicus was expressed in Escherichia coli as His-tag fused protein. The rPhlA was purified by gel filtration and Ni-metal affinity chromatographies. When the action mode was investigated by TLC and GC-MS, the purified rPhlA protein showed a phospholipase A activity, which cleaved the fatty acids at the sn-1 and sn-2 positions of phosphatidylcholine. However, it did not show lysophospholipase, sphingomyelinase, and phospholipase C activities. The rPhlA showed maximum activity at temperature of about 40 degrees C and pH around 8-9. Some divalent cations could affect the activity of PhlA. The addition of Co(2+) increased the activity, whereas Mg(2+) and Zn(2+) did not enhance the enzyme activity. The rPhlA could lyse the erythrocytes obtained from the fish such as rainbow trout and tilapia. A significant cytotoxic activity on a fish cell line, CHSE-214, was observed after 24h exposure to 40 microg rPhlA protein.     

Occurrence and distribution of the human pathogen Vibrio vulnificus in a subtropical Gulf of Mexico estuary

Water and sediment samples from Charlotte Harbor, Florida were examined for the autochthonous human pathogen, Vibrio vulnificus, for 1 year (March 1997–February 1998). Within the estuary, mean water column levels of V. vulnificus ranged between 58 CFU/100 ml and 1.21×10³ CFU/100 ml while sediment levels were up to 2 orders of magnitude greater.Vibrio vulnificus was detected throughout the year in Charlotte Harbor. The highest concentrations (5.14×10³ CFU/100 ml) of the year were found at warm temperatures and moderate salinities in September. The lowest mean concentration occurred in March at 26 CFU/100 ml. Although concentrations of Vibrio vulnificus were positively correlated with temperature, salinity was a more important factor influencing variability of this organism. In Charlotte Harbor, an optimal salinity of 15 psu (practical salinity units) was found for recovery of high concentrations of the pathogen. There were significant positive and negative correlations above and below 15 psu, respectively. Results from this study suggest that unlike temperate estuaries, in regions of moderate year round temperatures, such as the tropics or subtropics, salinity strongly controls the geographical and seasonal distribution of V. vulnificus between sediment and water column.     

Computational modeling of differences in the quorum sensing induced luminescence phenotypes of Vibrio harveyi and Vibrio cholerae

Vibrio harveyi and Vibrio cholerae have quorum sensing pathways with similar design and highly homologous components including multiple small RNAs (sRNAs). However, the associated luminescence phenotypes of strains with sRNA deletions differ dramatically: in V. harveyi, the sRNAs act additively; however, in V. cholerae, the sRNAs act redundantly. Furthermore, there are striking differences in the luminescence phenotypes for different pathway mutants in V. harveyi and V. cholerae. However, these differences have not been connected with the observed differences for the sRNA deletion strains in these bacteria. In this work, we present a model for quorum sensing induced luminescence phenotypes focusing on the interactions of multiple sRNAs with target mRNA. Within our model, we find that one key parameter - the fold-change in protein concentration necessary for luminescence activation - can control whether the sRNAs appear to act additively or redundantly. For specific parameter choices, we find that differences in this key parameter can also explain hitherto unconnected luminescence phenotypes differences for various pathway mutants in V. harveyi and V. cholerae. The model can thus provide a unifying explanation for observed differences in luminescence phenotypes and can also be used to make testable predictions for future experiments.     

Virulence and molecular typing of Vibrio harveyi strains isolated from cultured dentex,gilthead sea bream and European sea bass

Vibrio harveyi was isolated from internal organs or ulcers of diseased and apparently healthy gilthead sea bream (Sparus aurata) and European sea bass (Dicentrarchus labrax) cultured in several fish farms located on the Spanish Mediterranean coast. The prevalence of the bacterium was significantly higher in European sea bass than in gilthead sea bream, and was closely related to the season in both fish species, occurring almost exclusively on warm months (June to November). After phenotypic characterization, a selection of forty five isolates from gilthead sea bream, sea bass, and several isolates previously obtained from common dentex (Dentex dentex) of the same area, were molecularly typed by automated ribotyping and random amplified polymorphic DNA (RAPD) analysis. Cluster analysis of data established 8 RAPD types and 13 ribotypes among wild isolates, and the combination of both techniques allowed to define fourteen different groups and a clear discrimination of all outbreaks and samplings. Several strains isolated from diseased gilthead sea bream and sea bass and also from asymptomatic sea bream, were tested for virulence in both fish species by intracoelomic injection. All the isolates (11) were pathogenic for sea bass, with nine out of the eleven LD50 values ranging from 1.5 x 10(5) to 1.6 x 10(6) cfu/fish. Gilthead sea bream was unaffected by the seven tested strains, even by those more virulent for sea bass, and only one strain caused a 10% mortality at 4.2 x 10(7) cfu/fish. This is the first report on virulence of V. harveyi for sea bass.     

Vibrio navarrensis biotype pommerensis: a new biotype of V. navarrensis isolated in the German Baltic Sea

The determination of phenotypic and genotypic traits of a group of closely related Vibrio strains from the Baltic Sea did not allow species designation. DNA-DNA hybridization and fatty acid analysis revealed them as Vibrio navarrensis. Therefore we suggest these Vibrios to represent a new biotype, named V. navarrensis biotype pommerensis.