Biochemical Characterization of a Phospholipase A₂ from Photobacterium damselae subsp. piscicida

Abstract

Photobacterium damselae subsp. piscicida (Phdp) is the causative agent of fi sh photobacteriosis (pasteurellosis) in cultured cobia (Rachycentron canadum) in Taiwan. A component was purifi ed from the extracellular products (ECP) of the bacterium strain 9205 by fast protein liquid chromatography (FPLC) and identifi ed as a phospholipase. An N-terminal sequence of 10 amino acid residues, QDQPNLDPGK, was determined by mass spectroscopy (MS) and found to be identical with that of another Phdp phospholipase (GenBank accession no. BAB85814) at positions 21 to 30. The corresponding gene sequence of the phospholipase (GenBank accession no. AB071137) was employed to design primers for amplifi cation of the sequence by the polymerase chain reaction (PCR). The PCR products were transformed into Escherichia coli, and a recombinant protein product was obtained which was purifi ed as a His-tag fusion protein by Ni-metal affi nity chromatography. A single 43-kDa band was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Phosphatidylcholine was degraded by this protein to lysophosphatidylcholine and a fatty acid. These products were characterized by thin-layer (TLC) and gas chromatography (GC), respectively, allowing the identifi cation of the protein as a phospholipase A 2 . The recombinant protein had maximum enzymatic activity between pH 4 and 7, and at 40 °C. The activity was inhibited by Zn 2+ and Cu 2+ , activated by Ca 2+ and Mg 2+ , and completely inactivated by dexamethasone and p-bromophenacyl bromide. A rabbit antiserum against the recombinant protein neutralized the phospholipase A 2 activity in the ECP of Phdp strain 9205 and the recombinant protein itself. The recombinant protein was toxic to cobia of about 5 g weight with an LD 50 value between 2 and 4 μg protein/g fi sh. The results revealed phospholipase A 2 as a fi sh toxin in the ECP of Phdp strain 9205.