Identification and characterization of a virulence-associated protease from a pathogenic Pseudomonas fluorescens strain

Pseudomonas fluorescens is an aquaculture pathogen that can infect a number of fish species. The virulence mechanisms of aquatic P. fluorescens remain largely unknown. Many P. fluorescens strains are able to secrete an extracellular protease called AprX, yet no AprX-like proteins have been identified in pathogenic P. fluorescens associated with aquaculture. In this study, a gene encoding an AprX homologue was cloned from TSS, a pathogenic A fluorescens strain isolated from diseased fish. In TSS, AprX is secreted into the extracellular milieu, and the production of AprX is controlled by growth phase and calcium. Mutation of aprX has multiple effects, which include impaired abilities in interaction with cultured host cells, adherence to host mucus, modulation of host immune response, and dissemination and survival in host tissues and blood. Purified recombinant AprX exhibits apparent proteolytic activity, which is optimal at pH 8.0 and 50 degrees C. The protease activity of recombinant AprX is enhanced by Ca2+ and Zn2+ and reduced by Co2+. Cytotoxicity analyses showed that purified recombinant AprX has profound toxic effect on cultured fish cells. These results demonstrate that AprX is an extracellular metalloprotease that is involved in bacterial virulence. (C) 2009 Elsevier B.V. All rights reserved.

Diversity of Both the Cultivable Protease-Producing Bacteria and Their Extracellular Proteases in the Sediments of the South China Sea

Protease-producing bacteria are known to play an important role in degrading sedimentary particular organic nitrogen, and yet, their diversity and extracellular proteases remain largely unknown. In this paper, the diversity of the cultivable protease-producing bacteria and their extracellular proteases in the sediments of the South China Sea was investigated. The richness of the cultivable protease-producing bacteria reached 10(6) cells/g in all sediment samples. Analysis of the 16S rRNA gene sequences revealed that the predominant cultivated protease-producing bacteria are Gammaproteobacteria affiliated with the genera Pseudoalteromonas, Alteromonas, Marinobacter, Idiomarina, Halomonas, Vibrio, Shewanella, Pseudomonas, and Rheinheimera, with Alteromonas (34.6%) and Pseudoalteromonas (28.2%) as the predominant groups. Inhibitor analysis showed that nearly all the extracellular proteases from the bacteria are serine proteases or metalloproteases. Moreover, these proteases have different hydrolytic ability to different proteins, reflecting they may belong to different kinds of serine proteases or metalloproteases. To our knowledge, this study represents the first report of the diversity of bacterial proteases in deep-sea sediments.

Characterization of degQ(Vh), a serine protease and a protective immunogen from a pathogenic Vibrio harveyi strain

Vibrio harveyi is an important marine pathogen that can infect a number of aquaculture species. V. harveyi degQ (degQ(Vh)), the gene encoding a DegQ homologue, was cloned from T4, a pathogenic V. harveyi strain isolated from diseased fish. DegQ(Vh) was closely related to the HtrA family members identified in other Vibrio species and could complement the temperature-sensitive phenotype of an Escherichia coli strain defective in degP. Expression of degQVh in T4 was modulated by temperature, possibly through the sigma(E)-like factor. Enzymatic analyses demonstrated that the recombinant DegQVh protein expressed in and purified from E. coli was an active serine protease whose activity required the integrity of the catalytic site and the PDZ domains. The optimal temperature and pH of the recombinant DegQVh protein were 50 C and pH 8.0. A vaccination study indicated that the purified recombinant DegQVh was a protective immunogen that could confer protection upon fish against infection by V. harveyi. In order to improve the efficiency of DegQVh as a vaccine, a genetic construct in the form of the plasmid pAQ1 was built, in which the DNA encoding the processed DegQVh protein was fused with the DNA encoding the secretion region of AgaV, an extracellular beta-agarase. The E.coli strain harboring pAQ1 could express and secrete the chimeric DegQVh protein into the culture supernatant. Vaccination of fish with viable E. coli expressing chimeric degQ(Vh) significantly (P < 0.001) enhanced the survival of fish against V. harveyi challenge, which was possibly due to the relatively prolonged exposure of the immune system to the recombinant antigen produced constitutively, albeit at a gradually decreasing level, by the carrier strain.

Characterization of degQ(Vh), a serine protease and a protective immunogen from a pathogenic Vibrio harveyi strain

Vibrio harveyi is an important marine pathogen that can infect a number of aquaculture species. V. harveyi degQ (degQ(Vh)), the gene encoding a DegQ homologue, was cloned from T4, a pathogenic V. harveyi strain isolated from diseased fish. DegQ(Vh) was closely related to the HtrA family members identified in other Vibrio species and could complement the temperature-sensitive phenotype of an Escherichia coli strain defective in degP. Expression of degQVh in T4 was modulated by temperature, possibly through the sigma(E)-like factor. Enzymatic analyses demonstrated that the recombinant DegQVh protein expressed in and purified from E. coli was an active serine protease whose activity required the integrity of the catalytic site and the PDZ domains. The optimal temperature and pH of the recombinant DegQVh protein were 50 C and pH 8.0. A vaccination study indicated that the purified recombinant DegQVh was a protective immunogen that could confer protection upon fish against infection by V. harveyi. In order to improve the efficiency of DegQVh as a vaccine, a genetic construct in the form of the plasmid pAQ1 was built, in which the DNA encoding the processed DegQVh protein was fused with the DNA encoding the secretion region of AgaV, an extracellular beta-agarase. The E.coli strain harboring pAQ1 could express and secrete the chimeric DegQVh protein into the culture supernatant. Vaccination of fish with viable E. coli expressing chimeric degQ(Vh) significantly (P < 0.001) enhanced the survival of fish against V. harveyi challenge, which was possibly due to the relatively prolonged exposure of the immune system to the recombinant antigen produced constitutively, albeit at a gradually decreasing level, by the carrier strain.

Rapid detection of virulent protease secreted by Vibrio anguillarum by dot enzyme-linked immunosorbent assay

Dot enzyme-linked immunosorbent assay (dot-ELISA), indirect ELISA and Western blot were performed to detect the virulent protease secreted by Vibrio anguillarum which was isolated from the diseased left-eyed flounder, Paralichthys olivaceous. Sensitivity results showed that dot-ELISA is a more sensitive, rapid and simple technique for the protease detection. The minimal detectable amount of protease is about 7 pg in the dot-ELISA test, while 7.8 ng in the indirect ELISA and 6.25 ng in the Western blot respectively. Protease could be detected 2 h after incubation of V. anguillarum in the 2216E liquid medium but enzyme activity was very low at that period. From 6 to 12 h, the amount and enzyme activity of protease increased markedly and reached maximum at stationary phase. Analysis of serum samples periodically collected from the infected flounders showed that after 2 h of infection by V. anguillarum, the pathogenic bacteria could be detected in the blood of the infected flounders but no protease was found. It was 5 similar to 6 h after infection that the protease was detected in blood and then the amount increased as infection advanced. Quantitative detection of protease either incubation in the medium or from the blood of infected flounders could be accomplished in virtue of positive controls of quantificational protease standards ("marker") so that the alterations of protease secretion both in vitro and in vivo could be understood generally. In addition, the indirect ELISA and dot-ELISA were also performed to detect V. anguillarum cells. Results indicated that the sensitivity of indirect ELISA to bacteria cells is higher than that of the dot-ELISA, and that the minimal detectable amount is approximately 10(4) cell/mL in the indirect ELISA, while 10(5) cell/mL in the dot-ELISA.

Trypsin inhibitory loop is an excellent lead structure to design serine protease inhibitors and antimicrobial peptides

The disulfide-bridged hendecapeptide ( CWTKSIPPKPC) loop, derived from an amphibian skin peptide, is found to have strong trypsin inhibitory capability. This loop, called the trypsin inhibitory loop ( TIL), appears to be the smallest serine protease inhib

Purification and characterization of an irreversible serine protease inhibitor from skin secretions of Bufo andrewsi

Amphibian skin secretions contain many bioactive compounds. In the present work, an irreversible serine protease inhibitor, termed baserpin, was purified for the first time from the skin secretions of toad Bufo andrewsi by Successive ion-exchange and gelfiltration chromatography. Baserpin is a single chain glycoprotein, with an apparent molecular weight of about 60 kDa in SDS-PAGE. Baserpin is an irreversible inhibitor and effectively inhibits the catalytic activity of trypsin, chymotrypsin and elastase. SDS-stable baserpin-trypsin complex could be seen in SDS-PAGE indicates that it possibly belongs to the serpin superfamily. According to the association rates determined, baserpin is a potent inhibitor of bovine trypsin (4.6 X 10(6) M-1 S-1), bovine chymotrypsin (8.9 X 10(6) M-1 s(-1)) and porcine elastase (6.8 X 10(6) M-1 s(-1)), whereas it shows no inhibitory effect on thrombin. The N-terminal sequence of baserpin is HTQYPDILIAKPXDK, which shows no similarity with other known serine protease inhibitors. (c) 2005 Elsevier Ltd. All rights reserved.

An anticoagulant serine protease from the wasp venom of Vespa magnifica

Wasp is an important venomous animal that can induce human fatalities. Coagulopathy is a clinical symptom after massive wasp stings, but the reason leading to the envenomation manifestation is still not known. In this paper, a toxin protein is purified and characterized by Sephadex G-75 gel filtration, CM-Sephadex C-25 cationic exchange and fast protein liquid chromatography (FPLC) from the venom of the wasp, Vespa magnifica (Smith). This protein, named magnvesin. contains serine protease-like activity and inhibits blood coagulation. The cDNA encoding magnvesin is cloned from the venom sac cDNA library of the wasp. The deduced protein from the cDNA is composed of 305 amino acid residues. Magnvesin shares 52% identity with allergen serine protease from the wasp Polistes dominulus. Magnvesin exerted its anti-coagulant function by hydrolyzing coagulant factors TF, VII, VIII, IX and X. (c) 2008 Elsevier Ltd. All rights reserved.

Two serine protease inhibitors from the skin secretions of the toad, Bombina microdeladigitora

Two serine protease inhibitors (named BMSI 1 and BMSI 2, respectively) were identified from the skin secretions of the toad, Bombina microdeladigitora. The cDNAs encoding BMSIs were cloned from a cDNA library prepared from the toad skin. The deduced complete amino acid sequences of BMSIs indicate that mature BMSI1 and BMSI2 are composed of 60 amino acids including 10 half-cystines to form 5 disulfide bridges. A FASTA search in the databanks revealed that BMSIs exhibit sequence similarity with other serine protease inhibitors from amphibians of the genus Bombina. BMSI1 potently inhibited trypsin and thrombin with a K(i) value of 0.02 mu M and 0.15 mu M, respectively. Sequence analysis revealed that all serine protease inhibitors from five amphibians of the genus Bombina share highly conserved primary structures. (c) 2007 Elsevier Inc. All rights reserved.

Isolation, expression and characterization of a novel dual serine protease inhibitor, OH-TCI, from king cobra venom

Snake venom Kunitz/BPTI members are good tools for understanding of structure-functional relationship between serine proteases and their inhibitors. A novel dual Kunitz/BPTI serine proteinase inhibitor named OH-TCI (trypsin- and chymotrypsin-dual inhibito

A unique group of inactive serine protease homologues from snake venom

A number of inactive serine protease homologues (SPHs), which have poorly understood functions, have been identified in invertebrates and vertebrates. Recently, several SPH transcripts have been reported from snake venom glands, which provide potential ne

Purification and characterization of jerdofibrase, a serine protease from the venom of Trimeresurus jerdonii snake

A fibrin(ogen)olytic serine protease from Trimeresurus jerdonii venom was identified and purified to SDS-polyacrylamide gel electrophoresis homogeneity. It is a single chain polypeptide with a molecular weight of 32 kDa under reduced condition and 28 kDa

Jerdonase, a novel serine protease with kinin-releasing and fibrinogenolytic activity from Trimeresurus jerdonii venom

A novel kinin-releasing and fibrin (ogen)olytic enzyme termed jerdonase was purified to homogeneity from the venom of Trimeresurus jerdonii by DEAE Sephadex A-50 anion exchange, Sephadex G-100 (superfine) gel filtration and reverse-phase high performance liquid chromatography (RP-HPLC). Jerdonase migrated as a single band with an approximate molecular weight of 55 kD under the reduced conditions and 53 kD under the non-reduced conditions. The enzyme was a glycoprotein containing 35.8% neutral carbohydrate. The N-terminal amino acid sequence of jerdonase was determined to be IIGGDECNINEHPFLVALYDA, which showed high sequence identity to other snake venom serine proteases. Jerdonase catalyzed the hydrolysis of BAEE, S-2238 and S-2302, which was inhibited by phenymethylsulfonyl fluoride (PMSF), but not affected by ethylenediaminetetraacetic acid (EDTA). Jerdonase preferentially cleaved the Aalpha-chain of human fibrinogen with lower activity towards Bbeta-chain. Moreover, the enzyme hydrolyzed bovine low-molecular-mass kininogen and releasing bradykinin. In conclusion, all results indicated that jerdonase was a multifunctional venom serine protease.