Purification and characterization of a putative virulence factor, serine protease, from Vibrio parahaemolyticus

Abstract

A protease (protease A) was successfully purified from the extracellular proteins of Vibrio parahaemolyticus no. 93, a clinical strain carrying neither tdh nor trh genes, using phenyl‐Sepharose CL‐4B hydrophobic interaction chromatography. The molecular mass of protease A was 43 kDa using gel filtration, which was in agreement with the results obtained from SDS–PAGE, suggesting that protease A was a monomeric protein. Additionally, the isoelectric point of this protein was 5.0. The optimum temperature and pH of protease A ranged from 40°C to 50°C and pH 8, respectively. Protease A activity was inhibited by serine protease inhibitors, such as phenylmethylsulfonyl fluoride and soybean trypsin inhibitor; moreover, the activity could be blocked by treatment with 20 mM of 1,10‐phenanthroline, but could not be restored by adding metal ions. These results indicated that protease A is a serine protease that requires metal. The 12 N‐terminal residues of protease A showed a high degree of identity (81%) to the sequence of Vibrio metschnikovii VapT serine protease. The purified protease had significant effects on the growth of Chinese hamster ovary, HeLa, Vero and Caco‐2 cells and its cytotoxic activity was not blocked by gangliosides. Protease A lysed erythrocytes well but its hemolytic activity was unstable after heat treatment, indicating that protease A is able to cause hemolysis but is a heat‐labile protein. The purified protease caused tissue hemorrhage and death in mice when injected both intraperitoneally and intravenously. In conclusion, this is the first report of a serine protease purified directly from the supernatant of V. parahaemolyticus and identifying it as a potential virulence factor.

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

“…However, the mechanism by which V. vulnificus metalloprotease can convert human zymogens is not clarified yet. Some other human pathogenic Vibrio species including V. parahaemolyticus, an etiologic agent causing watery diarrhea, secretes serine proteases (Ishihara et al, 2002;Lee et al, 2002). Although this serine protease can digest various host proteins, its biological or pathogenic actions are not documented.…”

Isolation and characterization of Vibrio parahaemolyticus from seafoods along the southwest coast of India

“…This weak haemolysis points towards the presence of virulence factors other than TDH/TRH. The capability of the strains to produce few extracellular enzymes may also lead to the weak haemolysis (Lee et al 2002). In contrast Sanjeev (1999) and Sudha et al (2002) reported higher Kanagawa positives from environmental strains of Cochin.…”

Occurrence and distribution of virulent strains of Vibrio parahaemolyticus in seafoods marketed from Cochin (India)

“…The weak haemolysis points towards the presence of other virulence factors other than tdh in V. parahaemolyticus. The capability of the strains to produce few extra cellular enzymes may also lead to the weak haemolysis (Lee et al 2002). There are reports of Kanagawa phenomenon being correlated with the positive detection of urease by urea hydrolysis (Kaysner et al 1994;Iida et al 1998).…”