期刊
PARASITOLOGY
卷 128, 期 -, 页码 541-548出版社
CAMBRIDGE UNIV PRESS
DOI: 10.1017/S0031182004004883
关键词
Philasterides dicentrarchi; fish pathogen; scuticociliatosis; ciliate; cysteine protease; histolytic activity
类别
This study investigated protease activities in a crude extract and in vitro excretion/secretion (E/S) products of Philasterides dicentrorchi, a ciliate fish parasite causing economically significant losses in aquaculture. Gelatin/SDS-PAGE analysis (pH 4, reducing conditions) detected 7 hands with gelatinolytic activity (approximate molecular weights 30-63 kDa) in the crude extract. The banding pattern observed in analysis of E/S products was practically identical, except for 1 low-molecular-weight band detected in the crude extract but not in the E/S products. In assays with synthetic peptide p-nitroanilide substrates, the Crude extract hydrolysed substrates characteristic of cysteine proteases, namely Z-Arg-Arg pNA, Bz-Phe-Val-Arg pNA and Z-Phe-Arg pNA. These activities were strongly inhibited by the cysteine protease inhibitor E-64 and by Ac-Leu-Val-Lys aldehyde, a potent inhibitor of cysteine proteases of the cathepsin B protease subfamily. The proteases present in the crude extract degraded both type-1 collagen and haemoglobin in vitro, consistent with roles in tissue invasion and nutrition respectively. Again, E-64 completely (collagen) or markedly (haemoglobin) inhibited this degradation. Finally, the histolytic activity of the ciliate in turbot fibroblast monolayers was strongly reduced in the presence of E-64, confirming the importance of secreted cysteine proteinases in the biology of Philasterides dicentrarchi.
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