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.     

Flagellar basal body flg operon as a virulence determinant of Vibrio vulnificus

Vibrio vulnificus, a halophilic estuarine bacterium causing a rapidly progressing fatal septicemia, is highly cytotoxic to eukaryotic cells. To identify new virulence factors associated with cytotoxicity, we constructed a mariner-based transposon (Tn Himar1) library of the highly virulent clinical isolate MO6-24/O having a double mutation in the hemolysin and protease genes. The Himar1 mutant library was extensively screened for the mutants showing decreased cytotoxicity to HeLa cells. We selected a cytotoxicity defective mutant having a Himar1 insertion in an open reading frame showing 96% identity to Vibrio parahaemolyticus FlgC, a flagella basal body rod protein. The Tn Himar1 insertion mutation also resulted in a significant decrease in motility, adhesion, cytotoxicity, and lethality to mice. This is the first report showing that flg genes, which are components of the flagellum biogenesis gene cluster, might play an important role in the virulence of V. vulnificus.     

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.     

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.     

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.     

Suppression and inactivation of Vibrio vulnificus hemolysin in cirrhotic ascites, a human ex vivo experimental system

To elucidate the mechanisms underlying the in vivo suppression and inactivation of Vibrio vulnificus hemolysin (VvhA), we used cirrhotic ascites fluid as a human ex vivo experimental system. VvhA expression was suppressed in proportion to the amount of cirrhotic ascites. The expression of vvhA in undiluted cirrhotic ascites could be suppressed further by the addition of glucose, a constituent of cirrhotic ascites. VvhA was readily inactivated in the presence of cirrhotic ascites by a cholesterol-mediated oligomerization and interaction with an undefined constituent(s) of cirrhotic ascites. These results indicate that the expression of vvhA can be suppressed and that any VvhA produced is inactivated by the constituents of cirrhotic ascites. Our results suggest that only a very small portion of the VvhA that is produced in human body fluids may actually contribute to the pathogenesis of V. vulnificus septicemia. It is suggested that cirrhotic ascites could be used as a human ex vivo experimental system for the studies on the in vivo expression and the significance of V. vulnificus virulence factors.     

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.     

Outer membrane vesicles of Vibrio vulnificus deliver cytolysin-hemolysin VvhA into epithelial cells to induce cytotoxicity

The Gram-negative bacterium Vibrio vulnificus produces cytotoxins that induce the acute death of host cells. However, the secretory mechanisms of such cytotoxins have not been extensively studied. Previously, we reported that substantial amounts of V. vulnificus cytolysin-hemolysin (VvhA) are produced in vivo during the bacterial infection in mice and that this cytotoxin, in conjunction with RtxA1, mediates cytotoxicity. In this study, we investigated whether V. vulnificus cells release outer membrane vesicles (OMVs), which are used by some Gram-negative bacteria to deliver virulence factors into host cells. We found that V. vulnificus produce OMVs and that these vesicles can induce host cell death. This process appears to be mediated by VvhA, as evidenced by the finding that OMVs isolated from VvhA-null mutants do not induce cytotoxicity. In addition, cholesterol sequestration in the host cells prevents OMV-mediated VvhA delivery, indicating that VvhA-bearing OMVs interact with cholesterol on the host cell surface. Furthermore, intracellular expression experiments revealed that VvhA-mediated cytotoxicity is driven by its N-terminal leukocidin domain.     

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.     

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.     

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.     

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.     

Crystal structure of the Vibrio cholerae cytolysin (VCC) pro-toxin and its assembly into a heptameric transmembrane pore

Pathogenic Vibrio cholerae secrete V. cholerae cytolysin (VCC), an 80 kDa pro-toxin that assembles into an oligomeric pore on target cell membranes following proteolytic cleavage and interaction with cell surface receptors. To gain insight into the activation and targeting activities of VCC, we solved the crystal structure of the pro-toxin at 2.3A by X-ray diffraction. The core cytolytic domain of VCC shares a fold similar to the staphylococcal pore-forming toxins, but in VCC an amino-terminal pro-domain and two carboxy-terminal lectin domains decorate the cytolytic domain. The pro-domain masks a protomer surface that likely participates in inter-protomer interactions in the cytolytic oligomer, thereby explaining why proteolytic cleavage and movement of the pro-domain is necessary for toxin activation. A single beta-octyl glucoside molecule outlines a possible receptor binding site on one lectin domain, and removal of this domain leads to a tenfold decrease in lytic activity toward rabbit erythrocytes. VCC activated by proteolytic cleavage assembles into an oligomeric species upon addition of soybean asolectin/cholesterol liposomes and this oligomer was purified in detergent micelles. Analytical ultracentrifugation and crystallographic analysis indicate that the resulting VCC oligomer is a heptamer. Taken together, these studies define the architecture of a pore forming toxin and associated lectin domains, confirm the stoichiometry of the assembled oligomer as heptameric, and suggest a common mechanism of assembly for staphylococcal and Vibrio cytolytic toxins.     

The structure of a sulfated galactan from Porphyra haitanensis and its in vivo antioxidant activity

The sulfated galactan fraction F1 isolated from the red seaweed, Porphyra haitanensis, showed typical porphyran structure. It has a linear backbone of alternating 3-linked beta-D-galactosyl units and 4-linked alpha-L-galactosyl 6-sulfate and 3,6-anhydro-alpha-L-galactosyl units. The L-residues are mainly composed of alpha-L-galactosyl 6-sulfate units, and the 3,6-anhydrogalactosyl units are minor. Partial methylation occurred at the C-6 position of the D-galactosyl units and at the C-2 position of the 3,6-anhydro-alpha-L-galactosyl units. Intraperitoneal administration of F1 significantly decreased the lipid peroxidation in aging mice. F1 treatment increased the total antioxidant capacity and the activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in aging mice. The results indicated that F1 had significant in vivo antioxidant activity.     

Haemonchus contortus: in vitro and in vivo anthelmintic activity of aqueous and hydro-alcoholic extracts of Hedera helix

In vitro anthelmintic activity of crude extracts of the ripe fruits of Hedera helix was investigated on eggs and adult nematode parasites Haemonchus contortus. Aqueous extract of H. helix was also evaluated for in vivo anthelmintic activity at dose of 1.13 and 2.25 g/kg in sheep artificially infected with H. contortus. ED(50) for egg hatch inhibition was 0.12 and 0.17 mg/ml for aqueous and hydro-alcoholic extracts, respectively. There was no statistically significant difference in the activity of the two extract types (p>0.05). Hydro-alcoholic extract showed better in vitro activity against adult parasites compared to the aqueous extract. Significant faecal egg count reduction (FECR) was detected in groups treated with both doses of H. helix (p<0.05) on day 2 post-treatment. On day 7 post-treatment significant reduction was detected only for higher dose of H. helix (p<0.05) while on day 14 post-treatment there was no significant FECR in both groups treated with H. helix. The percentage of larvae recovered from culturing faeces obtained from groups of sheep treated with lower and higher doses of H. helix was 47.52% and 36.07%, respectively, which was significantly lower than (p<0.05) that recovered from the control group (60%). Significant (p<0.05), dose dependent total worm count reduction (WCR) was observed for groups of sheep treated with H. helix. Increasing the dose of H. helix improved the efficacy against the male than the female parasites. Treatment with both doses of H. helix helped the animals maintain their packed cell volume (PCV) unlike the untreated control group. The overall findings of the current study indicated that H. helix has a potential anthelmintic benefit and further in vitro and in vivo evaluation of the different parts and fractions is needed to make use of this plant for therapeutic purposes.