Purification of a toxic cysteine protease produced by pathogenic Aeromonas hydrophila isolated from rainbow trout.
An extracellular lethal toxin produced by Aeromonas hydrophila strain RT860715K originally isolated from diseased rainbow trout (Oncorhynchus mykiss) was purified by using Fast Protein Liquid Chromatography system with hydrophobic interaction chromatography and anion exchange columns. The toxin was a cysteine protease, inhibited by L -cysteine, iodoacetic acid, N -ethylamleimide, P-chloromercuibenzene-sulfonic acid and N-α-p-tosyl-1-lysine-chloromethyl ketone (TLCK), and showed maximal activity at pH 6.0. The molecular weight of the purified enzyme proved to be 94 kDa as estimated by SDS-PAGE. In addition, the toxin was also completely inhibited by HgCl(2) but partially inhibited by ethylenediamine tetraacetic acid (EDTA) and CuCl₂. Both the extracellular products of Aeromonas hydrophila RT860715K and the purified protease were lethal to rainbow trout (weighing 18 g) with LD₅₀ values of 2.87 and 0.93 μg protein g⁻¹ fish body weight, respectively. The addition of L-cysteine completely inhibited the lethal toxicity of the purified protease, indicating that this cysteine protease was a lethal toxin produced by the bacterium.
Liu
Ping-Chung
PC
Department of Aquaculture, National Taiwan Ocean University, Keelung, Taiwan.
Chuang
Wen-Hsiao
WH
Tu
Chih-Ching
CC
Lee
Kuo-Kau
KK
D016980:Aeromonas hydrophila / Q000201:enzymology* / Q000302:isolation & purification; D000818:Animals; D001427:Bacterial Toxins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism / Q000633:toxicity; D002853:Chromatography, Liquid / Q000379:methods; D057056:Cysteine Proteases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism* / Q000633:toxicity; D015853:Cysteine Proteinase Inhibitors / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005393:Fish Diseases / Q000382:microbiology; D016905:Gram-Negative Bacterial Infections / Q000382:microbiology / Q000662:veterinary; D006863:Hydrogen-Ion Concentration; D007928:Lethal Dose 50; D008970:Molecular Weight; D017686:Oncorhynchus mykiss / Q000382:microbiology*; D016019:Survival Analysis
D001427:Bacterial Toxins; D015853:Cysteine Proteinase Inhibitors; D057056:Cysteine Proteases