Purification and characterization of a putative virulence factor, serine protease, from Vibrio parahaemolyticus.
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 degrees C to 50 degrees 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.
Lee
Chia-Yin
CY
Graduate Institute of Agricultural Chemistry, National Taiwan University, Taipei 106, Taiwan. m477@ccms.ntu.edu.tw
Cheng
Min-Fu
MF
Yu
Mei-Shiuan
MS
Pan
Ming-Jeng
MJ
D000230:Adenocarcinoma / Q000473:pathology; D000818:Animals; D001426:Bacterial Proteins / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology / Q000502:physiology; D016466:CHO Cells / Q000187:drug effects; D002522:Chlorocebus aethiops; D003110:Colonic Neoplasms / Q000473:pathology; D006224:Cricetinae; D003412:Cricetulus; D010450:Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D005759:Gastroenteritis / Q000382:microbiology; D006367:HeLa Cells / Q000187:drug effects; D006460:Hemolysin Proteins / Q000502:physiology; D006801:Humans; D051379:Mice; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014709:Vero Cells / Q000187:drug effects; D014735:Vibrio Infections / Q000382:microbiology*; D014736:Vibrio parahaemolyticus / Q000201:enzymology* / Q000472:pathogenicity; D014774:Virulence
D001426:Bacterial Proteins; D006460:Hemolysin Proteins; D010450:Endopeptidases