Purification and characterization of a vulnificolysin-like cytolysin produced by Vibrio tubiashii

An extracellular cytolysin from Vibrio tubiashii was purified by sequential hydrophobic interaction chromatography with phenyl-Sepharose CL-4B and gel filtration with Sephacryl S-200. This protein is sensitive to heat and proteases, is inhibited by cholesterol, and has a molecular weight of 59,000 and an isoelectric point of 5.3. In addition to lysing various erythrocytes, it is cytolytic and/or cytotoxic to Chinese hamster ovary cells, Caco-2 cells, and Atlantic menhaden liver cells in tissue culture. Lysis of erythrocytes occurs by a multihit process that is dependent on temperature and pH. Twelve of the first 17 N-terminal amino acid residues (Asp-Asp-Tyr-Val-Pro-Val-Val-Glu-Lys-Val-Tyr-Tyr-Ile-Thr-Ser-Ser-Lys) are identical to those of the Vibrio vulnificus cytolysin.     

Isolation and Characterization of Vibrio tubiashii Outer Membrane Proteins and Determination of a toxR Homolog

Outer membrane proteins (OMPs) expressed by Vibrio tubiashii under different environmental growth conditions were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, N-terminal amino acid sequencing, and PCR analyses. Results showed the presence of a 38- to 40-kDa OmpU-like protein and ompU gene, a maltoporin-like protein, several novel OMPs, and a regulatory toxR homolog.     

Expression and characterization of a metalloprotease from a Vibrio parahaemolyticus isolate

The extracellular zinc metalloprotease from Vibrio parahaemolyticus (VPM) is a putative virulence factor for host infection. It is synthesized from the vpm gene of V. parahaemolyticus as a polypeptide of 814 amino acids with an estimated molecular mass of 89,833 Da, containing a zinc metalloprotease HEXXH consensus motif. To investigate the enzymatic properties of V. parahaemolyticus metalloprotease, the mature vpm gene was overexpressed in Escherichia coli, and the recombinant protein (rVPM) was purified by a His-binding metal affinity column (>95% purity). The activity of the recombinant protease produced in E. coli was examined by gelatin activity staining and proteolytic activity assays using gelatin and azocasein as substrates. rVPM showed maximum activity at about 37 degrees C and pH 8. The cytotoxicity against flounder gill cells and fish pathogenicity indicated a potential role in pathogenesis.     

The extracellular metalloprotease of Vibrio tubiashii is a major virulence factor for pacific oyster (Crassostrea gigas) larvae

Vibrio tubiashii is a recently reemerging pathogen of larval bivalve mollusks, causing both toxigenic and invasive disease. Marine Vibrio spp. produce an array of extracellular products as potential pathogenicity factors. Culture supernatants of V. tubiashii have been shown to be toxic to oyster larvae and were reported to contain a metalloprotease and a cytolysin/hemolysin. However, the structural genes responsible for these proteins have yet to be identified, and it is uncertain which extracellular products play a role in pathogenicity. We investigated the effects of the metalloprotease and hemolysin secreted by V. tubiashii on its ability to kill Pacific oyster (Crassostrea gigas) larvae. While V. tubiashii supernatants treated with metalloprotease inhibitors severely reduced the toxicity to oyster larvae, inhibition of the hemolytic activity did not affect larval toxicity. We identified structural genes of V. tubiashii encoding a metalloprotease (vtpA) and a hemolysin (vthA). Sequence analyses revealed that VtpA shared high homology with metalloproteases from a variety of Vibrio species, while VthA showed high homology only to the cytolysin/hemolysin of Vibrio vulnificus. Compared to the wild-type strain, a VtpA mutant of V. tubiashii not only produced reduced amounts of protease but also showed decreased toxicity to C. gigas larvae. Vibrio cholerae strains carrying the vtpA or vthA gene successfully secreted the heterologous protein. Culture supernatants of V. cholerae carrying vtpA but not vthA were highly toxic to Pacific oyster larvae. Together, these results suggest that the V. tubiashii extracellular metalloprotease is important in its pathogenicity to C. gigas larvae.     

Purification and Characterization of a Vulnificolysin-Like Cytolysin Produced by Vibrio tubiashii

An extracellular cytolysin from Vibrio tubiashii was purified by sequential hydrophobic interaction chromatography with phenyl-Sepharose CL-4B and gel filtration with Sephacryl S-200. This protein is sensitive to heat and proteases, is inhibited by cholesterol, and has a molecular weight of 59,000 and an isoelectric point of 5.3. In addition to lysing various erythrocytes, it is cytolytic and/or cytotoxic to Chinese hamster ovary cells, Caco-2 cells, and Atlantic menhaden liver cells in tissue culture. Lysis of erythrocytes occurs by a multihit process that is dependent on temperature and pH. Twelve of the first 17 N-terminal amino acid residues (Asp-Asp-Tyr-Val-Pro-Val-Val-Glu-Lys-Val-Tyr-Tyr-Ile-Thr-Ser-Ser-Lys) are identical to those of the Vibrio vulnificus cytolysin.     

Cloning and partial characterization of a novel hemolysin gene of Vibrio tubiashii and the development of a PCR-based detection assay

Vibrio tubiashii expresses virulence factors, such as a vulnificolysin-like hemolysin or cytolysin and a zinc metalloprotease, similar to those of other pathogenic vibrios. In this study, we report the cloning of a novel hemolysin gene of V.?tubiashii in Escherichia coli . A V.?tubiashii gene library was screened for hemolytic activity on sheep blood agar. Three hemolytic clones pGem:hly1, pGem:hly2, and pGem:hly3 were sequenced, and the sequences showed a strong homology to the ribA gene coding for guanosine triphosphate cyclohydrolase II (GCH II), required for riboflavin biosynthesis and reported to be responsible for hemolytic activity in Helicobacter pylori . The plasmids pGem:hly1 and pGem:hly3 when introduced into E. coli BSV18 (ribA18::Tn5) were able to restore growth of strain BSV18 in a medium without riboflavin and also produced hemolytic activity on blood agar. PCR primers based on the cloned hly-ribA sequence were tested using 23 different Vibrio strains representing 10 different species. Amplification of ribA gene locus only occurred with V.?tubiashii strains. In summary, our results indicate that we have cloned a ribA homolog of V.?tubiashii that imparts hemolytic activity to E.?coli clones, and primers based on this gene locus might be useful as a species-specific identification tool for V.?tubiashii.     

Mortalities of Eastern and Pacific Oyster Larvae Caused by the Pathogens Vibrio coralliilyticus and Vibrio tubiashii

Vibrio tubiashii is reported to be a bacterial pathogen of larval Eastern oysters (Crassostrea virginica) and Pacific oysters (Crassostrea gigas) and has been associated with major hatchery crashes, causing shortages in seed oysters for commercial shellfish producers. Another bacterium, Vibrio coralliilyticus, a well-known coral pathogen, has recently been shown to elicit mortality in fish and shellfish. Several strains of V. coralliilyticus, such as ATCC 19105 and Pacific isolates RE22 and RE98, were misidentified as V. tubiashii until recently. We compared the mortalities caused by two V. tubiashii and four V. coralliilyticus strains in Eastern and Pacific oyster larvae. The 50% lethal dose (LD₅₀) of V. coralliilyticus in Eastern oysters (defined here as the dose required to kill 50% of the population in 6 days) ranged from 1.1 × 10⁴ to 3.0 × 10⁴ CFU/ml seawater; strains RE98 and RE22 were the most virulent. This study shows that V. coralliilyticus causes mortality in Eastern oyster larvae. Results for Pacific oysters were similar, with LD₅₀s between 1.2 × 10⁴ and 4.0 × 10⁴ CFU/ml. Vibrio tubiashii ATCC 19106 and ATCC 19109 were highly infectious toward Eastern oyster larvae but were essentially nonpathogenic toward healthy Pacific oyster larvae at dosages of ≥1.1 × 10⁴ CFU/ml. These data, coupled with the fact that several isolates originally thought to be V. tubiashii are actually V. coralliilyticus, suggest that V. coralliilyticus has been a more significant pathogen for larval bivalve shellfish than V. tubiashii, particularly on the U.S. West Coast, contributing to substantial hatchery-associated morbidity and mortality in recent years.     

Isolation and characterization of a novel extracellular metalloprotease from Bacillus subtilis.

We have isolated and characterized two minor extracellular proteases from culture supernatants of a strain of Bacillus subtilis containing deletion mutations of the genes for the extracellular proteases subtilisin (apr) and neutral protease (npr) and a minor extracellular protease (epr) as well as intracellular serine protease-I (isp-1). Characterization studies have revealed that one of these enzymes is the previously described protease bacillopeptidase F. The second enzyme, the subject of this report, is a novel metalloprotease, which we designate Mpr. Mpr is a unique metalloprotease that has been purified to apparent homogeneity by using both conventional and high-performance liquid chromatography procedures. Mpr has a molecular mass of approximately 28 kilodaltons on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a basic isoelectric point of 8.7. The enzyme showed maximal activity against azocoll at pH 7.5 and 50 degrees C. Mpr was inhibited by dithiothreitol and a combination of beta-mercaptoethanol and EDTA. Activity was moderately inhibited by beta-mercaptoethanol and EDTA alone as well as by cysteine and citrate and only marginally by phosphoramidon 1,10-phenanthroline and N-[N-(L-3-trans-carboxyoxiran-2-carbonyl)-L-leucyl]-agmatine. Mpr was not inhibited by phenylmethylsulfonyl fluoride. In addition, Mpr showed esterolytic but not collagenolytic activities. Our studies suggest that Mpr is a secreted metalloprotease containing cysteine residues that are required for maximal activity.     

Isolation and characterization of a cytolysin produced by Vibrio cholerae serogroup non-O1

A thermolabile toxin (molecular weight, 52 711; isoelectric point, 8.65) produced by a clinical isolate of Vibrio cholerae serogroup non-O1 was cytotoxic for Y-1 mouse adrenal cells and Chinese hamster ovary cells. The toxin lysed rabbit red blood cells and produced a hemorrhagic zone in rabbit skin. When injected intravenously into adult mice, the cytolysin was rapidly lethal and caused fluid accumulation in both 5- and 18-h rabbit ileal loops. Strains of V. cholerae that produced cytolysin but no cholerae enterotoxin were able to cause fluid accumulation in rabbit intestinal loops.     

Isolation and preliminary characterization of bacteriocins produced by Vibrio vulnificus

AIMS: The aim of this work was to isolate bacteriocins from the environment that would be effective in neutralizing Vibrio vulnificus in seafood. METHODS AND RESULTS: Water samples from Wilmington (NC, USA) were plated to determine total viable counts and to isolate presumptive Vibrio spp. Isolates containing plasmids were checked for antimicrobial activity which was not due to lytic bacteriophage or small, non-specific molecules. Three bacteriocin producers were detected and their inhibitory spectra determined: IW1 inhibited few strains of V. vulnificus; BC1 inhibited several strains of V. vulnificus, V. cholerae and V. parahaemolyticus and BC2 inhibited all tested Vibrio spp., Plesiomonas shigelloides and Escherichia coli. Loss of inhibitory activity coincided with loss of the bacteriocinogenic plasmid. The bacteriocins were found to be between 1.3 and 9.0 kDa. IW1 was heat labile, while BC1 was moderately stable except at extreme temperatures. BC2 was very stable and maintained its activity when frozen, autoclaved or exposed to extreme pH values. CONCLUSIONS: Bacteriocins have been isolated from environmental isolates of V. vulnificus and V. cholerae. BC2, with its broad spectrum and stability, may be useful in neutralizing V. vulnificus. SIGNIFICANCE AND IMPACT OF THE STUDY: The results have significance in relation to reducing the occurrence of food poisoning caused by V. vulnificus.     

Characterization of the zinc-containing metalloprotease encoded by zpx and development of a species-specific detection method for Enterobacter sakazakii

Enterobacter sakazakii causes a severe form of neonatal meningitis that occurs as sporadic cases as well as outbreaks. The disease has been epidemiologically associated with consumption of reconstituted, dried infant formulas. Very little information is available regarding pathogenicity of the organism and production of virulence factors. Clinical and environmental strains were screened for production of factors which have activity against Chinese hamster ovary (CHO) cells in tissue culture. Polymyxin B lysate and sonicate preparations but not culture supernatants from the strains caused "rounding" of CHO cells. Subsequent studies showed that the CHO cell-rounding factor is a proteolytic enzyme that has activity against azocasein. The cell-bound protease was isolated by using a combination of polymyxin B lysis, followed by sonication of cells harvested from tryptone broth. The protease was purified to homogeneity by sequential ammonium sulfate precipitation, gel filtration chromatography with Sephadex G-100, hydrophobic interaction chromatography with phenyl-Sepharose CL-4B, and a second gel filtration with Sephadex G-100. In addition to activity against azocasein, the purified protease also exhibits activity against azocoll and insoluble casein but not elastin. The protease has a molecular weight of 38,000 and an isoelectric point of 4.4. It is heat labile and for maximal activity against azocasein has an optimum temperature of 37 degrees C and a pH range of 5 to 7. Proteolytic activity is inhibited by ortho-phenanthroline and Zincov but is not affected by phenylmethylsulfonyl fluoride, N-ethylmaleimide, and trypsin inhibitors, which demonstrates that the protease is a zinc-containing metalloprotease. The metalloprotease does not hemagglutinate chicken or sheep erythrocytes. Twenty-three to 27 of the first 42 N-terminal amino acid residues of the metalloprotease are identical to proteases produced by Serratia proteamaculans, Pectobacterium carotovorum, and Anabaena sp. PCR analysis using primers designed from a consensus nucleotide sequence showed that 135 E. sakazakii strains possessed the metalloprotease gene, zpx, and 25 non-E. sakazakii strains did not. The cloned zpx gene of strain 29544 consists of 1,026 nucleotides, and the deduced amino acid sequence of the metalloprotease has 341 amino acid residues, which corresponds to a theoretical protein size of 37,782 with a theoretical pI of 5.23. The sequence possesses three well-characterized zinc-binding and active-site motifs present in other bacterial zinc metalloproteases.     

Isolation and characterization of a novel glycosyl-phosphatidylinositol-anchored glycoconjugate expressed by developing neurons.

In search of new markers for studying thymic and nervous system ontogeny, we raised rat monoclonal antibodies against glycosyl-phosphatidylinositol-anchored molecules among which larger groupings have been shown to be ectoenzymes and adhesion molecules. Two of these monoclonal antibodies (H193-4 and H194-563, IgG) were found to recognize glycosyl-phosphatidylinositol-anchored glycoconjugates of 28-33 kDa (P31) and 50-70 kDa in developing mouse brain and thymus respectively, when these tissues were analysed by immunoblot experiments. P31 antigen was found to be transiently expressed by neurons in neural primary cultures [Rougon, G., Alterman, L., Dennis, K., Guo, X. J. & Kinnon, K. (1991) Eur. J. Immunol. 21, 1397-1402]. We show in this report that, in developing mouse brain, a maximal expression occurred between embryonic day 17 and post-natal day 5, a period that corresponds to the formation of neuronal networks. P31 antigen was immunopurified and found to possess the following properties: (a) it was soluble in alkaline solvents; (b) it bound to DEAE-cellulose and was eluted by a salt gradient of 0-1 M NaCl; (c) it was sensitive to endoglycosidase F digestion; (d) it was insensitive to heparinase, hyaluronidase, chondroitinase ABC, endo-beta-galactosidase and sialidase treatment; (e) it was labile to mild acid hydrolysis without loss of immunoreactivity; (f) it contained phosphate; (g) it lost its immunoreactivity after treatment with phosphatidylinositol phospholipase C and treatment. These characteristics combine to suggest that P31 is an anionic glycoconjugate sharing similarities with Leishmania donovani lipophosphoglycan and with the heat-stable antigen recognized by J11d antibody on murine hematopo?etic cells. This last hypothesis was further confirmed by the observation that oligonucleotide probes derived from the heat-stable antigen-encoding cDNA detect, in developing brain, a 1.8-kb mRNA species similar in size to that reported for the heat-stable antigen mRNA and following the same developmental expression as P31 antigen.     

Isolation and characterization of tributyltin chloride-resistant marine Vibrio

Tributyltin chloride (TBTCl)-resistant marine bacteria were isolated from coastal sea water. One of these bacteria (Vibrio M-1) was highly resistant when grown in medium containing 125 microM of TBTCl. This strain was sensitive to other metals. Two polypeptides, 30 kDa and 12 kDa, increased when the strain was cultured in the medium supplemented with TBTCl. Initially TBTCl was taken up by the cell; however, the amount of TBTCl determined in the cellular fraction was low after the exponential growth phase of Vibrio M-1, suggesting the existence of a TBTCl-efflux system.     

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.     

Isolation and characterization of exopolysaccharide produced by Vibrio harveyi strain VB23

AIMS: The aim of the study was to isolate and characterize exopolysaccharide (EPS) produced by Vibrio harveyi strain VB23. METHODS AND RESULTS: Growth and EPS production by V. harveyi strain VB23, was studied in mineral salts medium supplemented with NaCl (1.5%) and glucose (0.2%). The rate of EPS production in batch cultures was highest during the late log phase of growth when compared with stationary growth phase. The exopolymer was recovered from the culture supernatant by using a cold ethanol precipitation-dialysis procedure. Chemical analyses of EPS revealed that it is primarily composed of neutral sugars, uronic acids, proteins and sulfates. The purified EPS revealed prominent functional reactive groups, such as hydroxyl, carboxylic and amides, which correspond to a typical heteropolymeric polysaccharide and the EPS, also possessed good emulsification activity. The gas chromatographic analysis of an alditol acetate-derivatized sample of EPS revealed that it is composed primarily of galactose and glucose. Minor components found were rhamnose, fucose, ribose, arabinose, xylose and mannose. CONCLUSIONS: The EPS produced by V. harveyi strain VB23 is a heteropolysaccharide possessing good emulsification activity. EPS was readily isolated from culture supernatants, which suggests that the EPS was a slime-like EPS. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of EPS characterization in luminous V. harveyi bacteria, which describes the isolation and characterization of an EPS expressed by V. harveyi. The results of the study contributes significantly towards an understanding of the chemical composition and applications of the EPS in environmental biotechnology and bioremediation.     

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.