title: Purification and partial characterisation of a fish lethal extracellular protease from Vibrio pelagius.
abstract: The purification, characterisation and lethal effect of an extracellular protease present in the extracellular products (ECPs) of a pathogenic Vibrio pelagius (7P) strain are described. The extracellular protease was purified by size-exclusion high-performance liquid chromatography and characterised by enzymatic assays. The lethal effect was evaluated by injection into fish. The native protease had a molecular mass of 39 kDa, was active on casein and L-leucyl-beta-naphthylamide (LNA) and its metalloprotease nature was shown by the LNA inhibition profile. Kinetic studies on the hydrolysis of casein and LNA confirmed a competitive inhibition of one substrate with respect to the other. The temperature assays showed that both aminopeptidolytic and caseinolytic activities were labile at 70 degrees C for 3 min. The N-terminal amino acid sequence of 7P protease revealed a high degree of homology with other metalloproteases of Vibrio species that are implicated in virulence. The purified 7P protease showed an LD(50) of 1.77 microgprotein/g fish for turbot. The quick lethal effect (<24h) and the macroscopic damage (external haemorrhagic areas, principally on fins and mouth, petechial haemorrhages in internal organs, but with no external or internal apparent necrotic areas) detected in the host were similar to those obtained by injection of total ECP and live cells of 7P strain. An extracellular protease with endopeptidolytic and exopeptidolytic activities, responsible for the lethal effect of ECP and clinical signs of vibriosis in turbot was purified from a pathogenic V. pelagius (7P) strain.
authors.affiliation: Departamento de Biología Funcional y Ciencias de la Salud, Facultad de Ciencias, Universidad de Vigo, 36200 Vigo, Pontevedra, Spain.
authors.forename: R
authors.initials: R
authors.lastname: Farto
authors.forename: M J
authors.initials: MJ
authors.lastname: Pérez
authors.forename: A
authors.initials: A
authors.lastname: Fernández-Briera
authors.forename: T P
authors.initials: TP
authors.lastname: Nieto
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001681:Biological Assay / Q000662:veterinary; D002364:Caseins / Q000037:antagonists & inhibitors / Q000378:metabolism; D002850:Chromatography, Gel / Q000662:veterinary; D002851:Chromatography, High Pressure Liquid / Q000662:veterinary; D004591:Electrophoresis, Polyacrylamide Gel / Q000662:veterinary; D010450:Endopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005393:Fish Diseases / Q000382:microbiology; D005412:Flatfishes; D006461:Hemolysis; D007931:Leucyl Aminopeptidase / Q000037:antagonists & inhibitors / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017386:Sequence Homology, Amino Acid; D014733:Vibrio / Q000201:enzymology*; D014735:Vibrio Infections / Q000382:microbiology / Q000662:veterinary
chemical_list: D002364:Caseins; D004791:Enzyme Inhibitors; D010450:Endopeptidases; D007931:Leucyl Aminopeptidase