Purification and partial characterization of a toxic serine protease produced by pathogenic Vibrio alginolyticus

An extracellular lethal toxin produced by Vibrio alginolyticus strain Swy originally isolated from diseased kuruma prawn (Penaeus japonicus) was purified using the AKTA purifier system with hydrophobic interaction chromatography, anion exchange and gel filtration columns. The toxin is an alkaline serine protease, inhibited by phenyl methylsulphonyl fluoride (PMSF), antipain and shows maximal activity at pH 8 to 11, having a pI of 4.3 and a molecular weight of approximately 33 kD. The toxin was completely inhibited by FeCl2 but partially inhibited by 3,4-dichloroisocoumarin (3,4-DCI), ethylenediamine tetraacetic acid (EDTA), ethylene glycol-bis(beta-amino-ethyl ether) N,N,N',N'-tetraacetic acid (EGTA), CuCl2 and ZnCl2. The purified protease was lethal for kuruma prawn at an LD50 of 0.29 microgram protein/g body weight. The haemolymph withdrawn from the moribund prawns injected with the toxic protease was unable to clot. The coagulogen in the kuruma prawn plasma showed an increased migration rate after incubation with this serine protease, and a plasma colour change from blue to pink was recorded. The addition of PMSF completely inhibited the lethal toxicity of the purified protease, indicating that this serine protease was a lethal toxin produced by the bacterium. The 33 kD protease was therefore a toxic protease produced by V. alginolyticus strain Swy.     

Lethal attribute of serine protease secreted by Vibrio alginolyticus strains in kuruma prawn Penaeus japonicus

Toxicity of the extracellular products (ECP) and the lethal attribute of serine protease secreted by five pathogenic Vibrio alginolyticus strains from various sources in kuruma prawn Penaeus japonicus were studied. The ECPs of organisms originally isolated from diseased kuruma prawn or small abalone Haliotis diversicolor supertexta were more lethal (LD50 value of 0.48 or 0.41 microg protein/g prawn) than those from diseased tiger prawn P. monodon, yellowfin porgy Acanthopagrus latus or horse mackerel (LD50 value of 0.98-1.17 microg protein/g prawn). All the ECPs manifested strong, weak and no activities against gelatin, sheep erythrocytes and chitin, respectively. In immunodiffusion tests using rabbit antiserum to a purified 33 kDa serine protease of strain Swy against ECP of each tested strain produced one single precipitation band in each treatment. Furthermore, the serine protease was suggested to be the dominant protease secreted by V. alginolyticus strains tested since the majority of enzymatic activity of the respective ECP was inhibited by phenylmethanesulfonyl fluoride (PMSF). A higher inhibition of serine protease activity by PMSF resulted in lower mortality rate of the ECPs injected into the prawns suggesting that the protease is one of the major lethal factor(s) secreted by V. alginolyticus.     

Cysteine protease is a major exotoxin of pathogenic luminous Vibrio harveyi in the tiger prawn, Penaeus monodon

The role of an extracellular cysteine protease, produced by pathogenic luminous Vibrio harveyi strain 820514 originally isolated from diseased tiger prawn (Penaeus monodon), in the disease process in the prawns was studied. The protease was lethal to P. monodon with an LD50 value of 0.3 microgram protein g-1 prawn. The lethal toxicity of the extracellular products (ECP) of the bacterium was neutralized by pre-incubation of the ECP with rabbit antiserum to the cysteine protease. Pre-incubation of ECP with CuCl2 (an inhibitor of cysteine protease) also inhibited toxicity. This suggests that cysteine protease is the major toxin produced by the bacterium. The present protease is the first toxic cysteine protease to be found in Vibrio species.     

Alkaline Serine Protease Is an Exotoxin of Vibrio alginolyticus in Kuruma Prawn,Penaeus japonicus

An extracellular lethal toxin produced by Vibrio alginolyticus strain Swy originally isolated from diseased kuruma prawn (Penaeus japonicus) was partially purified by Fast Protein Liquid Chromatography with hydrophobic interaction (Phenyl Sepharose High Performance) chromatography and gel filtration columns. The toxin is an alkaline serine protease, inhibited by phenyl-methylsulfonyl fluoride (PMSF), and showed maximal activity at pH 10, having a molecular weight of about 33 kDa estimated by SDS-PAGE and gel filtration chromatography. In addition, the toxin was also completely inhibited by FeCl₂ but partially inhibited by CaCl₂, CuCl₂, CoCl₂, MnCl₂, and ZnCl₂, and not inhibited by ethylenediamine tetraacetic acid (EDTA), ethylene glycol-bis(β-amino-ethyl ether) N,N,N′,N′-tetraacetic acid (EGTA), iodoacetamide, pepstatin A, sodium dodecyl sulfate (SDS), and N-tosyl-l-phenyl-alanine chloromethyl ketone (TPCK). Both the crude extracellular products (ECP) and the partially purified toxin are lethal for kuruma prawn at LD₅₀ values of 0.30 and 0.27 μg protein/g body weight, respectively. The addition of PMSF completely inhibited the lethal toxicity of both the ECP and the partially purified toxin, indicating that this serine protease is a lethal factor produced by the bacterium. The 33-kDa protease is, therefore, suggested to be a new toxic protease produced by V. alginolyticus strain Swy. Received: 12 April 1996 / Accepted: 31 July 1996     

Immunochemical study of the proteins of various tissues in Crustacea (Decapoda): nature, role, origin

The main proteins of the haemolymph of Crustacea Decapoda have been identified and analysed: haemocyanin, plasma coagulogen, heteroagglutinins, vitellogenins, and molt-related proteins. All these complex components exhibit a high molecular weight and as oligomeric fractions are able to aggregate or dissociate in subunits according to the composition of medium and experimental procedures. Besides their important r?le in the defense mechanism, some proteins are involved in the edification of diverse tissues. They are detected within different compartments: soft integument, calcified carapace and hepatopancreas. They are either in transit or sequestered or synthetized within these tissues. In the crayfish Astacus leptodactylus, some components have been identified in different compartments: --in aqueous extracts from soft integument: the haemocyanin, coagulogen and both fraction F1 (lipoprotein with an approximate molecular weight of 45 kdal) and fraction F2 related to the molt. Both coagulogen and fraction F2 appear sometimes as melanized. These two latter fractions exhibit some glucose-mannose residues and they occur with a higher relative amount than in the blood. --in soluble extracts from calcified cuticle: among the numerous fractions showing a high molecular weight, the haemocyanin and coagulogen are detected. --in aqueous extracts from hepatopancreas: both haemocyanin and coagulogen appear with a little relative amount. Components termed as Fa and Fb are found with a high concentration. One minor fraction is also detected. --in aqueous extracts from eggs: the haemocyanin and fraction Fb are present. Other proteins showing only some antigenic identities with those of the haemolymph are also detected in all these tissues. The haemolymph proteins are not present within these compartments following a passive diffusion. Indeed, their relative amount varies according to the tissue investigated and is different from that found in the blood. Except the haemocyanin detected in all tissues with different aggregation states, the haemolymph proteins identified vary in the organs studied. A qualitative and quantitative selection occurs when the blood proteins enter the other compartments. Perhaps some other proteins are not detected following alterations underwent either in the epithelial barriers or during the tannage process or the chitino-proteic complex formation or due to experimental procedures. On the other hand, each tissue has its own proteins. The integument contains crustacyanins alpha, beta, gamma; the eggs are mainly constituted of lipovitellins and the hepatopancreas is rich in small molecular weight proteins and digestive enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Pathogenesis of Gastroenteritis Caused by Vibrio carchariae in Cultured Marine Fish

Serious mortality among the cultured grouper Epinephelus coioides, characterized by a swollen intestine containing yellow fluid (gastroenteritis), occurred in 1993 in Taiwan. A bacterium isolated from the intestinal fluid and head kidney of moribund groupers was identified as Vibrio carchariae. Since then, the same Vibrio species has also been isolated from moribund black sea bream Acanthopagrus schlegeli, yellowfin sea bream A. latus, Japanese sea bass Lateolabrax japonicus, and red drum Sciaenops ocellatus suffering from the same syndrome. Each isolate was virulent to the respective fish. Recently, a similar syndrome, flounder infectious necrotizing enteritis, also caused by V. carchariae in summer flounder Paralichthys dentatus, was reported in Rhode Island. The extracellular products (ECPs) of V. carchariae strains EmI82KL (from grouper), Rd (from red drum), and SfUSA (from summer flounder, U.S.A.) were virulent to the grouper or red drum. A 33-kDa serine protease partially purified from the ECP of strain EmI82KL was lethal to the fish. All the moribund or killed fish exhibited gastroenteritis except those killed within 12 hours. This report is the first to show that intraperitoneal injection of the ECP or protease in the fish is virulent and can reproduce gastroenteritis. The serine protease was suggested as a major toxin in the grouper or red drum secreted by V. carchariae.     

An evaluation of chromogenic substrates for characterization of serine protease produced by pathogenic Vibrio alginolyticus.

Four chromogenic substrates for characterizing serine protease of Vibrio alginolyticus were evaluated. The protease activity of bacterial extracellular products, or the fractions of 33 kD protease purified by the AKTA purifier system with various columns, was completely inhibited by ethylenediamine tetra-acetic acid, ethylene glycol-bis(beta-amino-ethyl ether) N,N,N',N'-tetraacetic acid (EGTA), antipain and phenylmethylsulphonyl fluoride (PMSF) using water-soluble substrates (azoalbumin and azocasein). It was only completely inhibited by antipain and PMSF using water-insoluble substrates (azocoll and hide powder azure). The protease activity was not, or only partially, inhibited by 1,10-phenanthroline and sodium dodecyl sulphate (SDS) using all four substrates. Since chelating agents and 1,10-phenanthroline are commonly employed as inhibitors to identify metalloprotease, the two water-soluble substrates may not be appropriate for this purpose, except for using 1,10-phenanthroline as an inhibitor. Chelating agents may be still applicable as inhibitors using water-insoluble substrates and 1,10-phenanthroline is highly recommended in the characterization for metalloprotease to avoid confusion. In the present study, the 33 kD protease was further confirmed as an SDS-resistant serine protease and not a metalloprotease.     

White shrimp (Litopenaeus vannamei) recombinant lysozyme has antibacterial activity against Gram negative bacteria: Vibrio alginolyticus, Vibrio parahemolyticus and Vibrio cholerae

C-type lysozyme has been described as an antibacterial component of the shrimp innate defence system. We determined quantitatively the antibacterial activity of white shrimp (Litopenaeus vannamei) recombinant lysozyme against three Gram negative bacteria: Vibrio alginolyticus, Vibrio parahemolyticus and Vibrio cholerae, using a turbidimetric assay with live bacteria and differential bacterial viable count after interaction with the protein. In conclusion, the antibacterial activity of recombinant shrimp lysozyme against Vibrio sp. is at least equal to the values against the Gram positive M. luteus and more active against the shrimp pathogens V. alginolyticus and V. parahemolyticus.     

Characterization of extracellular alkaline proteases and collagenase induction in Vibrio alginolyticus

The number and approximate molecular weights of extracellular alkaline proteases produced by Vibrio alginolyticus were determined by gelatin-PAGE. Three major bands of protease activity with apparent molecular weights of approximately 28 000, 22 500 and 19 500 (proteases 1, 2 and 3, respectively) and two minor bands of protease activity with apparent molecular weights of approximately 15 500 and 14 500 (proteases 4 and 5, respectively) were obtained after gelatin-PAGE. The activities of the five proteases were inhibited by serine protease inhibitors but their activities were not affected by inhibitors of trypsin-like enzymes. Histidine, which inhibited V. alginolyticus collagenase, did not inhibit the activities of the alkaline serine proteases. The production of protease 1, however, was enhanced by histidine. Protease 1 production was also affected by temperature and production was depressed at 37 degrees C. Gelatin-PAGE of a commercial V. alginolyticus collagenase preparation revealed four bands of activity which were identified as collagenases with apparent molecular weights of approximately 45 000, 38 500, 33 500 and 31 000. The collagenase preparation was contaminated with two serine proteases. The release of [3H]proline from collagen matrices produced by smooth muscle cells was shown to be a sensitive assay for bacterial collagenases and was used to show that V. alginolyticus produced a basal constitutive level of extracellular collagenase. The constitutive levels of collagenase were affected by aeration.     

Sequencing and preliminary characterization of the Na+-translocating NADH:ubiquinone oxidoreductase from Vibrio harveyi

The Na(+)-translocating NADH: ubiquinone oxidoreductase (Na(+)-NQR) generates an electrochemical Na(+) potential driven by aerobic respiration. Previous studies on the enzyme from Vibrio alginolyticus have shown that the Na(+)-NQR has six subunits, and it is known to contain FAD and an FeS center as redox cofactors. In the current work, the enzyme from the marine bacterium Vibrio harveyi has been purified and characterized. In addition to FAD, a second flavin, tentatively identified as FMN, was discovered to be covalently attached to the NqrC subunit. The purified V. harveyi Na(+)-NQR was reconstituted into proteoliposomes. The generation of a transmembrane electric potential by the enzyme upon NADH:Q(1) oxidoreduction was strictly dependent on Na(+), resistant to the protonophore CCCP, and sensitive to the sodium ionophore ETH-157, showing that the enzyme operates as a primary electrogenic sodium pump. Interior alkalinization of the inside-out proteoliposomes due to the operation of the Na(+)-NQR was accelerated by CCCP, inhibited by valinomycin, and completely arrested by ETH-157. Hence, the protons required for ubiquinol formation must be taken up from the outside of the liposomes, which corresponds to the bacterial cytoplasm. The Na(+)-NQR operon from this bacterium was sequenced, and the sequence shows strong homology to the previously reported Na(+)-NQR operons from V. alginolyticus and Haemophilus influenzae. Homology studies show that a number of other bacteria, including a number of pathogenic species, also have an Na(+)-NQR operon.     

Expression, characterization and immunogenicity of a major outer membrane protein from Vibrio alginolyticus

Vibrio alginolyticus is one of the Vibrio pathogens common to humans and marine animals.During infection and induction of the host immune response,outer membrane proteins of bacteria play animportant role.In this study,an outer membrane protein gene(ompW)was cloned from V.alginolyticus andexpressed in Escherichia coli.The 645 bp open reading frame(ORF)encodes a protein of 214 amino acidresidues with a predicted molecular weight of 23.3 kDa.The amino acid sequence showed a high identitywith that of Photobacterium damselae(96.2%)and Vibrio parahaemolyticus(94.4%).The alignment analy-sis indicated that OmpW was highly conserved.Sodium dodecyl sulfate-polyacrylamide gel electrophoresisshowed that the gene was over-expressed in E.coli BL21(DE3).Western blot analysis revealed that theexpressed protein had immunoreactivity.The recombinant protein was purified by affinity chromatographyon Ni-NTA Superflow resin.Large yellow croaker vaccinated with the purified OmpW showed significantlyincreased antibody to OmpW,which could resist the infection by V.alginolyticus.A specific antibody wasdetected by enzyme-linked immunosorbent assay.This study suggested that the conserved OmpW could bean effective vaccine candidate against infection by V.alginolyticus.     

The properties and structure of the membrane ATPase from Vibrio alginolyticus

Abstract Membrane ATPase of marine alkali-tolerant bacterium Vibrio alginolyticus has been studied. The ATPase was inhibited by diethylstilbestrol, DCCD, NBD-Cl and sodium azide, but it was insensitive to vanadate. The ATPase was solubilized by EDTA-treatment and partially purified by ion exchange chromatography. Two major polypeptides with molecular weights of 55 kDa and 58 kDa were found in this preparation. The molecular weight of the soluble ATPase was estimated to be 360 kDa. These data indicate, that the ATPase of Vibrio alginolyticus is likely to belong to F₀–F₁ type.     

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.     

Heat shock protein DnaK — Substrate of actin-specific bacterial protease ECP32

It has been found that actin-specific bacterial protease ECP32 cleaves prokaryotic heat shock protein DnaK, which belongs to the family of heat shock proteins with molecular weight 70 kDa. We propose a new one-step method for DnaK purification using heat treatment. The technique yields ∼1 mg of partially purified DnaK from 25 g of wet bacterial biomass. Polyclonal antibodies against DnaK were obtained. The degree of ECP32 catalyzed proteolysis of partially purified DnaK and that of DnaK in initial cell extracts was compared.     

Characteristics of adhesion of epiphytic bacteria on leaves of the seagrass Zostera marina and on abiotic surfaces

A comparative study of the adhesion of epiphytic bacteria and marine free-living, saprophytic, and pathogenic bacteria on seagrass leaves and abiotic surfaces was performed to prove the occurrence of true epiphytes of Zostera marina and to elucidate the bacterium-plant symbiotrophic relationships. It was shown that in the course of adhesion to the seagrass leaves of two taxonomically different bacteria, Cytophaga sp. KMM 3552 and Pseudoalteromonas citrea KMM 461, isolated from the seagrass surface, the number of viable cells increased 3-7-fold after 60 h of incubation, reaching 1.0-2.0 x 10(5) cells/cm2; however, in the case of adhesion of these bacteria to abiotic surfaces, such as glass or metal, virtually no viable cells were observed after 60 h of incubation. Such selectivity of cell adhesion was not observed in the case of three other bacterial species studied, viz., Vibrio alginolyticus KMM 3551, Bacillus subtilis KMM 430, and Pseudomonas aeruginosa KMM 433. The amount of viable cells of V. alginolyticus KMM 3551 adsorbed on glass and metal surfaces increased twofold after 40 h of incubation. The cells of saprophytic B. subtilis KMM 430 and pathogenic P. aeruginosa KMM 433 adsorbed on three studied substrata remained viable for 36 h and died by the 60th hour of incubation.