Investigation of the Expression of Serine Protease in Vibrio vulnificus.

Vibrio vulnificus is a Gram-negative estuarine bacterium that causes infection in immuno-compromised patients, eels, and shrimp. V. vulnificus NCIMB2137, a metalloprotease-negative strain isolated from a diseased eel, produces a 45-kDa chymotrypsin-like alkaline serine protease known as VvsA. The gene encoding vvsA also includes another gene, vvsB with an unknown function; however, it is assumed to be an essential molecular chaperone for the maturation of VvsA. In the present study, we used an in vitro cell-free translation system to examine the maturation pathway of VvsA. We individually expressed the vvsA and vvsB genes and detected their mRNAs. However, the sample produced from vvsA did not exhibit protease activity. A sodium dodecyl sulfate (SDS) analysis detected the VvsB protein, but not the VvsA protein. A Western blotting analysis using a histidine (His)-tag at the amino terminus of proteins also showed no protein production by vvsA. These results suggested the translation, but not the transcription of vvsA. Factors derived from Escherichia coli were used in the in vitro cell-free translation system employed in the present study. The operon of the serine protease gene containing vvsA and vvsB was expressed in E. coli. Although serine proteases were produced, they were cleaved at different sites and no active mature forms were detected. These results indicate that the operon encoding vvsA and vvsB is a gene constructed to be specifically expressed in V. vulnificus.

Functional analysis of N-terminal propeptide in the precursor of Vibrio vulnificus metalloprotease by using cell-free translational system.

Vibrio vulnificus is a human pathogen causing fatal septicemia with edematous and hemorrhagic skin damage. Among multiple virulence factors, an extracellular metalloprotease termed as V. vulnificus protease (VVP) is known to play a crucial role in eliciting the skin damage. The mature VVP (413 aa) is composed of two domains, the N-terminal core domain with proteolytic activity and the C-terminal domain mediates efficient attachment to protein substrates. However, VVP is produced as an inactive precursor (609 aa) with a signal peptide (24 aa) and propeptide (172 aa). In order to clarify the function of propeptide, a series of DNA fragments encoding the VVP precursor and its various domains were designed and the proteins were expressed in vitro by using cell-free translational system. The results indicated that the propeptide might function as an intramolecular chaperon to promote the proper folding of both N-terminal and C-terminal domains. The obtained results also suggest that the propeptide, itself was unstable and thus digested easily by the enzymes present in cell lysate used for cell-free system. Additionally, the C-terminal domain in VVP found to inhibit the folding of the N-terminal domain in absence of propeptide.

Defensive effects of human intestinal antimicrobial peptides against infectious diseases caused by Vibrio mimicus and V. vulnificus.

Of human pathogenic Vibrio species, V. mimicus causes gastroenteritis whereas V. vulnificus causes fatal septicemia after consumption of contaminated seafood. These two pathogens produce hemolytic toxins termed V. mimicus hemolysin (VMH) and V. vulnificus hemolysin (VVH), respectively. These toxins elicit the cytolysis of various eukaryotic cells, as well as erythrocytes. The human intestine secretes cationic antimicrobial peptides (AMPs) to prevent infectious diseases. Paneth cells in the small intestine secrete α-defensin 5 (HD-5) and epithelial cells in the large intestine produce LL-37. In the present study, we examined the bactericidal activities of AMPs against V. mimicus and V. vulnificus. Although HD-5 showed no bactericidal activity, LL-37 revealed significant activity against both Vibrio species, suggesting that neither V. mimicus nor V. vulnificus can multiply in the large intestine. We also tested whether AMPs had the ability to inactivate the hemolytic toxins. Only HD-5 was found to inactivate VMH, but not VVH, in a dose-dependent manner through the direct binding to VMH. Therefore, it is considered that V. mimicus cannot penetrate the small intestinal epithelium because the cytolytic action of VMH is inactivated by HD-5.

Uniform culture in solid-state fermentation with fungi and its efficient enzyme production.

Solid-state fermentation (SSF) has attracted a lot of interest for carrying out high-level protein production in filamentous fungi. However, it has problems such as the fermentation heat generated during the culture in addition to the reduced mobility of substances. These conditions lead to a nonuniform state in the culture substrate and result in low reproducibility. We constructed a non-airflow box (NAB) with a moisture permeable fluoropolymer membrane, thereby making it possible to control and maintain uniform and optimal conditions in the substrate. For the NAB culture in Aspergillus oryzae, temperature and water content on/in the whole substrate were more consistent than for a traditional tray box (TB) culture. Total weight after the culture remained constant and dry conditions could be achieved during the culture. These data demonstrate the possibility of growing a uniform culture of the whole substrate for SSF. The NAB is advantageous because it allows for the control of exact temperature and water content in the substrate during the culture by allowing vapor with latent heat to dissipate out of the box. In addition, several enzymes in the NAB culture exhibited higher production levels than in the TB culture. We believe that culturing in the constructed NAB could become a standard technique for commercial SSF.

Generation of active fragments from human zymogens in the bradykinin-generating cascade by extracellular proteases from Vibrio vulnificus and V. parahaemolyticus.

Vibrio vulnificus is an opportunistic human pathogen causing septicemia, and the infection is characterized by formation of the edematous skin lesions on limbs. This pathogenic species secretes a thermolysin-like metalloprotease as a virulence determinant. The metalloprotease was confirmed to activate human factor XII-plasma kallikrein-kinin cascade that results in liberation of bradykinin, a chemical mediator enhancing the vascular permeability, from high-molecular weight kininogen. Namely, the metalloprotease showed to generate active fragments by cleavage of Arg-Ile, Arg-Val or Gly-Leu peptide bond in human zymogens (plasma prekallikrein and factor XII). In spite of induction of the sufficient vascular permeability-enhancing and edema-forming reaction in the guinea pig model, a serine protease from V. parahaemolyticus, a human pathogen causing primarily watery diarrhea, showed far less ability to activate and to cleave the human zymogens. These results in part may explain why only V. vulnificus often causes serious edematous skin damages in humans.

Regulation system for protease production in Vibrio vulnificus.

Vibrio vulnificus is a causative agent of serious food-borne diseases in humans related to consumption of raw seafoods. This human pathogen secretes a metalloprotease (VVP) that evokes enhancement of the vascular permeability and disruption of the capillaries. Production of microbial proteases is generally induced at early stationary phase of its growth. This cell density dependent regulation of VVP production in V. vulnificus known to be the quorum-sensing. When V. vulnificus was cultivated in Luria-Bertani (LB) medium, accumulation of the autoinducer, the signal molecule operating the quorum-sensing system, was detected. Moreover, expression of the vvp gene encoding VVP was found to be closely related with expression of the luxS gene that encode the synthase of the autoinducer precursor (luxS). These findings may indicate VVP production is controlled by the quorum-sensing system in LB medium. Furthermore, this system functioned more effectively at 26 degrees C than at 37 degrees C. When incubated at 37 degrees C in human serum supplemented with ferric chloride, production of VVP and expression of vvp increased in proportion to the concentration of ferric ion; whereas, expression of luxS was not increased. This suggests that VVP production in human serum containing ferric ion may be regulated mainly by the system other than the quorum-sensing system.

High growing ability of Vibrio vulnificus biotype 1 is essential for production of a toxic metalloprotease causing systemic diseases in humans.

Vibrio vulnificus biotype 1, a causative agent of fatal septicemia or wound infection in humans, is known to produce a toxic metalloprotease as an important virulence determinant. V. vulnificus biotype 2 (serovar E), a primary eel pathogen, was found to elaborate an extracellular metalloprotease that was indistinguishable from that of biotype 1. The potential of V. vulnificus biotype 1 for production of the metalloprotease was compared with biotype 2 and other human non-pathogenic Vibrio species (Vibrio anguillarum and Vibrio proteolyticus). When cultivated at 25 degrees C in tryptone-yeast extract broth supplemented with 0.9% NaCl, all bacteria multiplied sufficiently and secreted significant amounts of the metalloprotease. However, at 37 degrees C with 0.9% NaCl, V. anguillarum neither grew nor produced the metalloprotease. In human serum, only V. vulnificus biotype 1 revealed a steady multiplication accompanied with production of the extracellular metalloprotease. This prominent ability of biotype 1 in growth and protease production may contribute to cause serious systemic diseases in humans.