Purification and Characterization of Vibrio vulnificus Protease

Abstract

A protease was purified from a strain of Vibrio vulnificus isolated from the blood of a septicemic human. The vibrio was cultured in bacto peptone-yeast extract medium, and the protease was purified by a purification procedure including ultrafiltration of the culture supernatant with an Amicon YM 5 membrane, diethylaminoethyl-Sephacel column chromatography, Sephacryl S-200 column chromatography and fast protein liquid chromatography on Mono Q column. The protease preparation revealed homogeneity on polyacrylamide gel electrophoresis and about 30,000-fold purification was achieved, with a yield of about 30% . The isoelectric point of the purified V. vulnificus protease was about 5.80 and its molecular weight was ca. 45,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The optimum pH of the protease activity was 8.0. The V. vulnificus protease was inhibited by a metalloprotease inhibitor and zinc ion and/or ferrous ion were essential for its enzyme activity. No cysteine residue was detected in the V. vulnificus protease. The protease had caseinolytic, elastolytic and collagenolytic activities.The illnesses caused by Vibrio vulnificus, a marine bacterium, are divided into two groups, wound-infection group and primary septicemia group (2, 3). In the former, edema and erythema are formed around a new wound in contact with sea water. In the latter group, cutaneous lesions, such as bullae, erythema or necrotic ulcer, often appear on one or more extremities.V. vulnificus is known to produce many toxic or pathogenic factors, such as cytolysin (8, 13, 23), protease (11, 13, 25), phospholipase A2 (27), and siderophores (24). However, in the pathogenesis of V. vulnificus infection, the actual role of any product has not been clarified yet. Kreger and Lockwood (13) reported that V. vulnificus cytolysin activity in the culture filtrate decreased in the stationary phase of the growth and the decrease correlated with the appearance of protease in the culture filtrate. We also observed significant excretion of protease from many strains of V. vulnificus isolated from patients. The pathogenic significance of proteases by some bacteria, such as pseudomonal protease (7,10,12) and serratial protease (9,16,19), has been reported. These proteases have vascular permeability-enhancing and hemorrhagic activities which are closely related to the above mentioned cutaneous lesions. We also found that the crude V. vulnificus protease preparation

Production of Antigenically Related Exocellular Elastolytic Proteases Mediating Hemagglutination by Vibrios

“…Similar patterns of inactivations were observed with the proteases from V mimicus and V vulnificus, as shown previously (3,10). As shown in Table 1, the caseinolytic activities of the proteases from V fluvialis, V furnissii and V campbellii, but not that of V metschnikovii, were inhibited by the heavy metal ions tested.…”

“…Later, it was demonstrated to be a metalloprotease containing zinc as an essential ligand (1). Similarly, proteases from a number of pathogenic vibrios were also shown to be metalloproteases (3,(8)(9)(10)14). Conversely, the protease from a hyperproducing mutant of the pathogen V parahaemolyticus was shown to be a serine protease (Hata et al, personal communication).…”

Structural basis for the autoprocessing of zinc metalloproteases in the thermolysin family

“…For example, λ-toxin from Clostridium perfringens can degrade various human immune defense proteins (8). Vibriolysin from Vibrio vulnificus (9) and pseudolysin from Pseudomonas aeruginosa (10) can be lethally blood-poisonous to humans. Therefore, understanding the catalytic and maturation mechanisms of TLPs is very important for developing therapeutics to treat such infectious diseases.…”

Purification of a Serine Protease of Vibrio parahaemolyticus and Its Characterization

“…The protease fraction eluted was concentrated by Ultrafiltration Membrane YM10 and applied on a HiLoad 16/60 Sephacryl S-200 HR gel filtration column (Amersham Pharmacia Biotech) equilibrated with TBS. Protease activity of each fraction was determined as described previously using azocasein (Sigma Aldrich, St. Louis, Mo., U.S.A.) as the substrate (13). One protease unit (PU) was defined as the amount of protease which digests 1 µg of azocasein in 1 min.…”