Identification and evaluation as a DNA vaccine candidate of a virulence-associated serine protease from a pathogenic Vibrio parahaemolyticus isolate

A putative serine protease gene was cloned from the genomic DNA of Vibrio parahaemolyticus FYZ8621.4. The gene consisted of 1779 base pairs and encoded a 592 amino acid protein. The gene was expressed in Escherichia coli. The expressed protease was purified by Ni-NTA His-Bind Resin column and showed a 63 kDa band on SDS-PAGE. The protease exhibited proteolytic activity on gelatin agar plate and showed maximal proteolytic activity at pH 8.0 and 37 °C. It hydrolyzed N-α-benzoyl-L-tyrosine p-nitroanilide (BAPNA), but did not N-benzoyl-L-arginine ethylester (BAEE), N-benzoyl-L-tyrosine ethylester (BTEE) and N-acetyl-L-tyrosine ethylester (ATEE). Mutants at conserved residues Asp(51) (Asp(51)-Asn), His(89) (His(89)-Asp) and Ser(318) (Ser(318)-Leu, Ser(318)-Pro) lost proteolytic activities completely. The protein was confirmed to belong to serine protease. The purified serine protease was toxic to zebrafish with a LD(50) of 15.4 μg/fish. A DNA vaccine was constructed by inserting the mutated serine protease (Ser(318)-Pro) gene into pEGFP-N1 plasmid. The pEGFP-N1/m-vps was transfected in HeLa cells. The serine protease was confirmed to be expressed by fluorescence microscopy observation and Western blotting analysis. The pEGFP-N1/m-vps was further observed to express in muscle of the injected turbot (Scophthalmus maximus) by Western blotting seven days after immunization. Efficient protection against lethal V. parahaemolyticus challenge was observed on the vaccinated turbot with pEGFP-N1/m-vps, with the highest relative percent survival (RPS) of 96.11%. Significant specific antibody responses were also observed in the turbot vaccinated with the DNA vaccine. The results indicated that the serine protease might be a potential virulence factor and could be used as an efficient vaccine candidate for the disease control caused by V. parahaemolyticus.     

Influence of salinity upon the phenotypic expression of antibiotic resistance in nonhalophilic and halophilic vibrios

The purpose of the present work is to demonstrate the influence of different NaCI concentrations included in the Mueller Hinton medium, upon the antibiotic susceptibility of 10 non-halophilic and 28 halophilic Vibrio strains. The highest number of resistance aspects were recorded at 1% NaCl concentration for V. cholerae O1/non O1 strains and at 3% for V. parahaemolyticus, V. algynolyticus, V. vulnificus, V. fisheri, V. anguillarum and V. metschnikovii.     

Virulence factors in Vibrios and Aeromonads isolated from seafood

Thirty-one isolates from seafood, identified as Aeromonas hydrophila (7), Aeromonas caviae (11), Vibrio parahaemolyticus (3), Vibrio fluvialis (5), Vibrio alginolytictus (3), Vibrio metschnikovii (1) and Vibrio damsela (1), were tested for possible virulence factors including extracellular hydrolytic enzymes, haemolysins, cytotoxins (VERO and HEp-2 cells) and adherence ability (HEp-2 cells). All the A. hydrophila strains were beta-haemolytic and produced cytotoxins as well as one strain of V. fluvialis. A. hydrophila and A. caviae strains, frequently adhesive, showed both aggregative and diffusive patterns, while five Vibrio strains only (three V. fluvialis, one V. parahaemolyticus and one V. alginolyticus) were adhesive with an aggregative pattern.     

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.     

Production of monoclonal antibodies against a hemagglutinin/protease of Vibrio cholerae non-01

Two hybridoma cell lines producing monoclonal antibodies (MAbs) against a hemagglutinin/protease (HA/P) from Vibrio cholerae non-01 were produced and characterized. The two MAbs contained the kappa light chain and were IgG1 type. They similarly neutralized HA/P protease activity derived from both V. cholerae non-01 and V. cholerae 01, whereas they were unable to neutralize the hemagglutinating activity of HA/P, suggesting that the epitopes for protease and hemagglutination activities are different. Western blotting analysis and the cross-neutralization test with the two MAbs confirmed the identity of HA/P produced by V. cholerae non-01 and 01. This study also suggests that HA/P of V. cholerae and a protease of V. parahaemolyticus are immunologically unrelated.     

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.     

A potential bacterial biocontrol agent,strain S2V2 against pathogenic marine <Emphasis Type="Italic">Vibrio</Emphasis> in aquaculture

We isolated a marine bacterium strain S2V2 which inhibited the growth of pathogenic marine Vibrio spp. The aims of this research were to identify a new antibiotic-producing marine bacterium strain S2V2, and evaluate its spectrum activity and pathogenic property. Analysis of 16S rDNA sequence placed strain S2V2 in the genus Pseudoalteromonas, but the sequence similarity was low (95.46%) implying the strain might be a new species in this genus. Strain S2V2 inhibited the growth of 67.9% of 28 Vibrio strains tested. This strain inhibited V. alginolyticus, V. anguillarum, V. fluvialis, V. harveyi, V. metschnikovii, V. splendidus, V. ordalii, V. parahaemolyticus, and V. vulnificus, but inactive against V. campbellii, Aeromonas hydrophyla and Staphylococcus aureus. Strain S2V2 produced extracellular non proteinaceous antibacterial substances. The highest antibacterial activity was found when strain S2V2 was cultured for 96 h in ZoBell broth medium. An artificial infection to post larvae of Lithopenaeus vanname indicated that strain S2V2 was a non pathogenic bacterium. Non pathogenic property and specific antibacterial activity against a broad range of fish pathogenic marine Vibrio of strain S2V2 suggest that this strain is a prospective source of unique antibiotic and a potential biocontrol agent in marine aquaculture.     

Isolation and characterization of a serine protease-producing marine bacterium <Emphasis Type="Italic">Marinomonas arctica</Emphasis> PT-1

A serine protease-producing marine bacterial strain named as PT-1 was isolated and identified as a family of Marinomonas arctica, based on molecular characterization of 16S rRNA gene sequence, phylogenetic tree, and fatty acid composition analyses. Optimized culture conditions for growth of the bacterium PT-1 and production of protease (ProA) were determined to be pH 8.0 in the presence of 5 % NaCl, at 37 °C during 24 h of incubation in the presence of 1.0 % skim milk. The molecular weight of the purified ProA was estimated to be 63-kDa as a major band by SDS-PAGE. We were intrigued to find that the activity of ProA was not inhibited by pepstatin A, chymostatin, and leupeptin known as inhibitors for cysteine protease. However, phenylmethylsulfonyl fluoride (PMSF) completely inhibited protease activity, suggesting that the ProA is like a serine protease. To the best of our knowledge, this is the first report on serine protease of Marinomonas species.     

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.     

A novel alkaline protease from wild edible mushroom Termitomyces albuminosus

A protease with a molecular mass of 30 kDa and the N-terminal sequence of GLQTNAPWGLARSS, was isolated from fresh fruiting bodies of the wild edible mushroom Termitomyces albuminosus. The purification protocol included ion exchange chromatography on DEAE-cellulose, Q-Sepharose, SP-Sepharose and FPLC-gel filtration on Superdex 75. The protein was unadsorbed on DEAE-cellulose and Q-Sepharose, but adsorbed on SP-Sepharose. The optimal pH and temperature of the purified enzyme were 10.6 and 60 °C, respectively. The enzyme was stable in the presence of 2 % (v/v) Tween 80 and 4 M urea. More than 80 % of the enzyme activity was retained in 2 % (v/v) Triton X 100, 54 % in 10 mM EDTA and 31 % in 2 % (w/v) SDS. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride (PMSF), but not inhibited by dithiothreitol (DTT), pepstatin or lima bean trypsin inhibitor suggesting that it was a serine protease but not a trypsin-like one. The protease was inhibited by Hg(2+), Cu(2+), and Fe(3+) ions. The K(m) and V(max) values of the purified enzyme for casein were 8.26 mg ? ml(-1) and 0.668 mg ? ml(-1) ? min(-1), respectively.     

Effect and potential ecological significance of the interaction of humic acids with two aquatic extracellular proteases

1. The effects of humic acids and fulvic acids on an extracellular serine and metalloprotease purified from a strain of Aeromonas hydrophila isolated from a freshwater wetland were examined. The serine protease was inhibited by humic acids at pH and humic acid concentrations found naturally in the wetland where this strain of A. hydrophila was isolated. The metalloprotease was not inhibited by humic acids at any pH investigated. Fulvic acids had no effect upon either protease. 2. Inhibition of serine protease activity by humic acids was reversed by increasing the pH to 9, as well as increasing the ionic strength by addition of CaCl₂- It was concluded that the interaction between humic acids and the serine protease was primarily ionic. 3. The formation of a humic acid-serine protease complex increased the half-life of enzymatic activity in dilute solutions. Humic acids had no effect on the stability of the metalloprotease. 4. There was no clear effect of humic acids on the growth of A. hydrophila, indicating that either the serine protease is not involved in the rate-limiting step of growth or that sufficient activity exists even when the serine protease is inhibited by humic acids. 5. Increasing the enzymatic lifetime through association with humic acids may be an adaptive mechanism which could result in energy conservation due to a decreased requirement for protein synthesis.     

Hepatocyte growth factor activator inhibitor type 1 inhibits protease activity and proteolytic activation of human airway trypsin-like protease

Hepatocyte growth factor activator inhibitor type 1 (HAI-1) is a Kunitz-type transmembrane serine protease inhibitor initially identified as a potent inhibitor of hepatocyte growth factor activator (HGFA), a serine protease that converts pro-HGF to the active form. HAI-1 also has inhibitory activity against serine proteases such as matriptase, hepsin and prostasin. In this study, we examined effects of HAI-1 on the protease activity and proteolytic activation of human airway trypsin-like protease (HAT), a transmembrane serine protease that is expressed mainly in bronchial epithelial cells. A soluble form of HAI-1 inhibited the protease activity of HAT in vitro. HAT was proteolytically activated in cultured mammalian cells transfected with its expression vector, and a soluble form of active HAT was released into the conditioned medium. The proteolytic activation of HAT required its own serine protease activity. Co-expression of the transmembrane full-length HAI-1 inhibited the proteolytic activation of HAT. In addition, full-length HAI-1 associated with the transmembrane full-length HAT in co-expressing cells. Like other target proteases of HAI-1, HAT converted pro-HGF to the active form in vitro. These results suggest that HAI-1 functions as a physiological regulator of HAT by inhibiting its protease activity and proteolytic activation in airway epithelium.     

Purification and characterization of a secreted protease from the pathogenic marine bacterium Vibrio anguillarum

Vibrio anguillarum is a pathogenic marine bacterium which causes the disease vibriosis in salmonid fish, which is characterized by a fatal hemorrhagic septicemia accompanied by massive tissue destruction. In this paper, the purification of the major caseinolytic extracellular protease from V. anguillarum is presented. The purification steps include ammonium sulfate precipitation, DEAE-Sepharose chromatography, Sephacryl S-200 chromatography, and DEAE high-pressure liquid chromatography. The purified protease migrates with Mr = 38,000 upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A slightly larger protease of Mr 40,000 is also separated by this procedure, but accounts for only a minor fraction of the caseinolytic activity. The Mr 38,000 protease displays a broad pH activity profile in the neutral to basic range. It is not inhibited by serine, cysteine, or acid protease inhibitors, but is inhibited by EDTA and 1,10-phenanthroline, suggesting that it is a metalloprotease. The activity of the EDTA-inactivated protease could be partially restored by the addition of Ca2+ and Zn2+ together. The molecular weight and inhibition data show some similarities with proteases isolated from other Vibrio species such as Vibrio cholerae and Vibrio vulnificus.     

Optimal enrichment time for isolation of Vibrio parahaemolyticus from seafood.

The growth of Vibrio parahaemolyticus in a liquid medium was compared with that of human fecal flora and estuarine flora. No marked differences were noted between growth at 25 and 37 degrees C for V. parahaemolyticus. However, the marine organisms were strongly inhibited when incubated at 37 degrees C. Incubation for 8 h in an enrichment broth yielded V. parahaemolyticus growth, even with a small inoculum, whereas the marine and fecal floras were inhibited. Therefore, enrichment for 8 h at 37 degrees C appears to be optimal for isolation of V. parahaemolyticus, permitting more rapid results in seafood analysis.     

Molecular cloning and biochemical characterisation of proteases from Staphylococcus epidermidis.

We report the complete coding sequence and the partial amino acid sequence (determined by chemical sequencing) of Staphylococcus epidermidis extracellular cysteine (Ecp) and serine (Esp) proteases. The first enzyme shows an extended sequence similarity to Staphylococcus aureus cysteine protease (staphopain) and the second one resembles the serine protease produced by that species. The region directly upstream of the sequence coding for the mature protein in both enzymes displays significant homology to the profragments encoded by sspB and sspA, respectively, thus suggesting that the characterised enzymes may also be produced as proproteins. Furthermore, we report some biological properties of the cysteine protease, contributing to a better understanding of its role as a possible virulence factor. The proteolytic activity of this enzyme was rapidly and efficiently inhibited by human alpha-2-macroglobulin; however, human kininogen as well as cystatins (A, C and D) were not inhibitory. Moreover, the protease was capable of inactivating, by limited proteolysis, both alpha-1-antitrypsin and HMW-kininogen, but neither alpha-1-antichymotrypsin nor antithrombin III.     

The epidermolytic toxins are serine proteases

Certain strains of Staphylococcus aureus usually belonging to phage group II produce epidermolytic toxins (ETA and ETB) which cause intraepidermal splitting in mice, neonates and occasionally adults. Amino acid sequences of ETA and ETB have been reported but the mechanism of epidermolysis remains unknown. A search of the NBRF-PIR computer database showed the toxins to have significant sequence similarity with staphylococcal V8 protease and that the catalytic triad of V8 protease is present in ETA and ETB. Comparison of ETA, ETB and V8 protease with other members of the trypsin-like serine protease family revealed little homology save for the immediate vicinity of the residues constituting the catalytic triad. The toxins, therefore, exhibit a distant relationship to mammalian serine proteases. A potential Ca2(+)-binding loop was identified in ETA (but not ETB) on the basis of sequence similarity with the second calcium-binding loop of rat intestinal calcium-binding protein. Epidermolysis produced by ETA in the mouse bioassay was shown to be inhibited by the presence of EDTA consistent with a Ca2(+)-dependent mechanism.     

Kinetic studies on the inhibitions of mast cell chymase by natural serine protease inhibitors: indications for potential biological functions of these inhibitors

We have found that degranulation from mast cells is specifically inhibited by the inhibitors of chymase (10). Among the natural serine protease inhibitors tested, Bowman-Birk soybean protease inhibitor, Eglin C, and human alpha 1-antichymotrypsin inhibited chymase more strongly than did chymostatin, Kunitz soybean protease inhibitor, and phosphatidylserine. Of the inhibitors tested, Bowman-Birk soybean protease inhibitor was the strongest inhibitor of chymase, its Ki value being 13.2 X 10(-9) M. Kinetic studies showed that these inhibitors were all noncompetitive inhibitors of chymase. Bowman-Birk and Kunitz soybean protease inhibitors inhibited both chymotrypsin-type and trypsin-type serine proteases but Eglin C specifically inhibited chymotrypsin-type proteases.     

极端嗜盐古生菌(Natrinema sp.)R6-5胞外嗜盐蛋白酶的纯化和性质研究

采用bacitracin-Sepharose 4B亲和层析的方法得到凝胶电泳均一的来自极端嗜盐古生菌(Natrinema sp.)R6-5的胞外嗜盐蛋白酶。经SDS-PAGE分析该酶亚基分子量为62kDa。PMSF对它的活性完全抑制,表明它是一种丝氨酸蛋白酶,该酶反应的最适NaCl浓度为3mol/L,最适温度为45℃,最适pH值为8.0。在高盐条件下能维持高活性并十分稳定,具有重要的潜在应用价值。     

Activation of the silkworm cytokine by bacterial and fungal cell wall components via a reactive oxygen species-triggered mechanism

The insect cytokine paralytic peptide (PP) induces muscle contraction in silkworm larvae. Here we demonstrate that bacterial and fungal cell wall components peptidoglycan and glucan stimulate muscle contraction via activation of PP in the hemolymph. Anti-PP antibody suppressed the muscle contraction induced by PP, peptidoglycan, or glucan. The contraction was also inhibited by free radical scavengers and serine protease inhibitors. Moreover, injecting live silkworms with peptidoglycan or glucan generated the active form of PP. The active form of PP was also produced in vitro when peptidoglycan or glucan was incubated with hemolymph containing the PP precursor. Generation of the active form of PP was suppressed by free radical scavengers and serine protease inhibitors. Furthermore, PP activation in isolated hemolymph was inhibited by potassium cyanide, suggesting that cellular activity is involved. Stimulation by peptidoglycan promoted the generation of reactive oxygen species by silkworm hemocytes. The addition of either the active form of PP or anti-PP antibody to Staphylococcus aureus injected into silkworm larvae delayed or enhanced, respectively, the killing effect of S. aureus, suggesting that activated PP contributes to host resistance to infectious pathogens. These findings suggest that immunologic stimulants such as peptidoglycan or glucan induce reactive oxygen species production from larval hemocytes, followed by the activation of serine protease, which mediates the PP processing reaction and leads to defensive responses.