Purifi cation of a Toxic Metalloprotease Produced by the Pathogenic Photobacterium damselae subsp. piscicida Isolated from Cobia (Rachycentron canadum)

Abstract

The aim of the present study was to purify and characterize a toxic protease secreted by the pathogenic Photobacterium damselae subsp. piscicida strain CP1 originally isolated from diseased cobia (Rachycentron canadum). The toxin isolated by anion exchange chromatography, was a metalloprotease, inhibited by L-cysteine, ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(β-aminoethyl ether)N,N,N’,N’-tetraacetic acid (EGTA), 1,10-phenanthroline, N-tosyl-L-phenylalanine-chloromethyl ketone (TPCK), and N-α-ptosyl- L-lysine-chloromethyl ketone (TLCK), and showed maximal activity at pH 6.0 - 8.0 and an apparent molecular mass of about 34.3 kDa. The toxin was also completely inhibited by HgCl2, and partially by sodium dodecyl sulfate (SDS) and CuCl2. The extracellular products and the partially purified protease were lethal to cobia with LD50 values of 1.26 and 6.8 μg protein/g body weight, respectively. The addition of EDTA completely inhibited the lethal toxicity of the purified protease, indicating that this metalloprotease was a lethal toxin produced by the bacterium.

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

“…Koo et al (2007) identifi ed PLA activity as an important factor in the cytotoxicity and lethality caused by Vibrio vulnificus. In the present study (Table IV), the purifi ed recombinant protein (LD 50 value between 2 and 4 μg protein/g fi sh) was found more virulent than the metalloprotease from the same strain (LD 50 6.8 μg protein/g fi sh) (Liu et al, 2011). As the LD 50 value of the recombinant protein was similar to that of the total ECP (LD 50 3.25 μg protein/g fi sh), it must be considered as an important virulence factor in ECP of the bacterium.…”

“…In previous studies, phospholipase (PL) and protease activities were found in the extracellular products (ECP) of Phdp (Hu, 2005;Liu et al, 2011). The extracellular protease was purifi ed and characterized as a 34.3-kDa toxic metalloprotease (Liu et al, 2011), however, the extracellular PL required further characterization.…”

“…The extracellular protease was purifi ed and characterized as a 34.3-kDa toxic metalloprotease (Liu et al, 2011), however, the extracellular PL required further characterization. PL has been suggested to be an important toxin in the pathogenesis of some bacterial species cau sing different disease syndromes such as massive tissue destruction related to gas gangrene of skin and lung infection caused by Pseudomonas aeruginosa (Schmiel and Miller, 1999).…”

“…According to previous studies (Hu, 2005;Liu et al, 2011), purifi cation of the enzyme was achieved by fast protein liquid chromatography (FPLC) (Pharmacia, Uppsala, Sweden) with anion exchange columns (Q Sepharose high-performance and Resource Q; Pharmacia) equilibrated with 20 mM tris(hydroxymethyl) methylamine (Tris buffer, pH 7.0). Fractions were eluted with a sodium chloride gradient (0 -1.0 M NaCl) at a rate of 1 ml/min.…”

“…For determination of the thermostability of the enzyme, aliquots of the purifi ed PL were incubated separately for 30 min at 4 °C, 10 °C, and then in 10-degree intervals up to 90 °C, respectively, then directly cooled on ice prior to the PL assay, carried out as described above. For both pH and temperature dependence, the highest PL activity was used as a control (100% of relative activity), as previously described (Liu et al, 2011).…”